DE10053785A1 - Test systems and their use to identify and characterize compounds - Google Patents

Abstract

The invention relates to test systems that are based on transmembrane receptors from helminths and arthropods and to their use for identifying and characterizing substances that are effective against helminths, arthropods or that have an effect on the calcium metabolism of organisms and/or cells. The invention further relates to the use of a specific ligand in said test system and to the use thereof as an anthelminthic or arthropodan active substance.

Description

The present invention relates to transmembrane receptors from helminths and Arthropod based test systems and their use for identification and Characterization of substances with effects against helminths, arthropods or with an effect on the calcium balance of organisms and / or cells. The he The invention further relates to the use of a specific ligand in this Test system and its use as an anthelmintic or arthropodicidal effect material.

Parasitic helminths and arthropods pose a major problem for humans and animals health problem. In the area of agriculture alone, the annually to combat or prevent such parasites Damages worldwide more than DM 7 billion (as of 1997). For the treatment of by the endoparasitosis caused mainly by helminths, as well as in Ectoparasitosis caused primarily by arthropods is a major one Number of active substances from a number of active substance classes available. Over the past two decades, such active ingredients in particular have: at the same time a very good effect against several parasite species within one Tribe (e.g. helminths), or even across different zoological tribes away (e.g. helminths and insects), gained in importance. The former count among other things the broad spectrum helmets such as B. the benzimidazoles or the imidazothiazoles, while the macrocyclic lactones are among the latter counting.

The macrocyclic lactones were last added about two decades ago new, broadly effective group of active substances launched on the market. Meanwhile ever but a multitude of endo and ectoparasite species are resistant to ver tents of individual, sometimes also several classes of active substances developed simultaneously.  

Therefore, there is a constant and increasingly urgent need, new anti-parasitic develop effective substances.

To the few new promising groups of active substances, at which currently ge the cyclic depsipeptides, which are the subject of extensive work rich patent (WO 93/19053, EP-OS 626 376, WO 94/19334, WO 95/07272, EP-OS 626 375, EP-OS 657 171, EP-OS 657 172, EP-OS 657 173) and research shares vities (Conder et al. 1995; Martin et al. 1996; Sasaki et al. 1992; Terada M. 1992; Samson-Himmelstjerna et al. 2000).

Several studies have already been described to elucidate the mechanism of action of a representative of this group of active substances, the PF1022A (cyclo (-D-Lac-L-MeLeu-D-PhLac-L-MeLeu-) ₂ ) (cf. information in DE-A -197 04 024). This also included the identification and characterization of a specific binding protein for cyclic depsipeptides and the DNA sequence coding for the protein from the sheep parasite Haemonchus contortus (DE-A-197 04 024). In the context of the present invention, the functional interaction of the above-mentioned protein, called HC110-R, is inter alia with the ligand BAY44-4400 (cyclo (-D-Lac-L-MeLeu- Dp-morpholinyl-PhLac-L-MeLeu-) , as a further representative of the cyclic depsipeptides for this purpose, recombinant eukaryotic cell lines were constructed in which the HC110-R, based on the sequence ID No. 2 described in the earlier application DE-A-197 04 024, is expressed.

The present invention is therefore based in particular on the object Base of transmembrane receptors of helminths and arthropods, preferred from nematodes and acarina, particularly preferably from Trichostrongylidae, quite be particularly preferred from Haemonchus spp. and especially based on the receptor HC110-R from H. contortus, test systems with a high throughput of test ver bindings (high throughput screening assays; HTS assays).  

Proteins which are at least one are considered homologous proteins 70% identity, preferably 80% identity, particularly preferably 90% Identity, very particularly preferably 95% identity, with a sequence according to SEQ ID NO. 2 of document DE-A-197 04 024, the content of which is expressly part of the present application should be preferred over a length of at least 20 have at least 25, particularly preferably at least 30 consecutive amino acids and very particularly preferably have over their total lengths.

The degree of identity of the amino acid sequences is preferably determined using Help of the program GAP from the program package GCG, version 9.1 under standard settings (Devereux et al. 1984).

The object is achieved by providing polypeptides, which at least exert a biological activity of a GPCR receptor as well as through the ready provision of a method for the production of these polypeptides and by the ready provision of methods for identifying nematicidal and arthropodicidal activity seed connections.

Test systems based on recombinant microorganisms have already achieved a great deal used extensively to identify pharmaceutically active substances, un ter also using microorganisms that recombinantly para express sitic genes (Klein and Geary 1997). So far, however, there are no systems known in which parasitic transmembrane receptors as recombinant, functio nale proteins can be used as targets in eukaryotic cells. By in Test systems described in this invention can be used in high-throughput Screening (HTS), or new ultra-HTS agonists and antagonists of these new ones Identify receptor class.

The recombinant expression of receptors from nematodes has been shown to be regular proven difficult. So far, it has generally not been possible to pair G-protein pelte receptors (GPCR) from nematodes to express their functional  Properties (e.g. sensitivity to inhibitors) to those more natural Corresponds to receptors.

It is of great practical importance, for example when looking for new ones Anthelmintics or arthropodicides, helminth receptors, in particular Functional design of nematodes and arthropods in eukaryotic systems shape.

The present invention is therefore also based on the object, one possibility for the expression of transmembrane receptors, especially of GPC receptors To provide nematodes and arthropods and based on this a test system develop that the identification of new nematicidal and arthropodicidal effective Substances.

The present invention thus relates in particular to expression and ver using an orphan G protein-coupled receptor from helminths and Arthropods, preferably from nematodes and acarina, particularly preferably from Trichostrongylidae, most preferred from Haemonchus and most preferred from the parasitic nematode H. contortus as the target protein for the effective search for nematicidal active ingredients.

This receptor was identified in a cDNA library that was derived from the gastro intestinal nematodes H. contortus was obtained. The cDNA codes for a heptahelical transmembrane protein with a size of 110 kDa, which as HC110-R be was drawn. The protein belongs to the secretin family of G-protein coupled Receptors (GPCR) and shows a high similarity to latrophilin.

The HC110-R receptor as a target

The GPCR latrophilin was originally isolated from the mammalian brain. Latrophilin has a molecular mass of 210 kDa and is post-translational  The remaining 18 split upstream of the first transmembrane segment, therefore consists of two noncovalently linked subunits. The sub-unit p120 contains the N- terminal, hydrophilic extracellular portion, the p85 subunit contains the seven transmembrane domains and the intracellular C-terminal region of the Latrophilins, which is unusually large for GPCRs. Recently two were close Homologues identified latrophilin-2 and latrophilin-3. The latter is preferred like latrophilin-1, expressed in the brain, while latrophilin-2 ubiquitous with a preference for placenta, kidney, spleen, ovaries, heart and lungs of mammals is expressed (Ichtchenko et al. 1999; Sugita et al. 1998).

Although HC110-R is only about half the size of the 210 kDa latrophilin, he the similarity of the sequence also extends to a functional similarity. For Both receptors, the endogenous ligand is still unknown, but both latrophilin as well as HC110-R are by the artificial ligand α-LTX (alpha-Latro toxin).

Are z. B. HEK-293 cells transiently transfected with latrophilin, the addition of α-LTX causes the influx of external Ca ²⁺ , as can be shown from an experimental approach with radioactive ⁴⁵ Ca ²⁺ . α-LTX also causes such a Ca ²⁺ influx in HEK-293 cells that have been transiently trans fected with HC110-R, which can be observed, for example, by Ca ²⁺ imaging.

However, this Ca ²⁺ inflow is very complex. If the HC110-R is in the form of a construct with a C-terminal green fluorescent protein (GFP) attachment, it is biphasic, ie there is a change after about 3 and another after about 22 minutes. However, if the HC110-R is preceded only by a Myc-His-Tag N-terminal, the main inflow is already observed approx. 2-3 minutes after the addition of α-LTX. The reason for this is still unknown, but the reaction to the α-LTX addition is specific, as the later explanations show:

• 1. The influx of Ca ²⁺ cannot be observed in non-transfected cells and in cells transiently transfected with a β ₂ -adrenergic receptor from the mouse.
• 2. The changes in [Ca ²⁺ ] _i are dose-dependent on α-LTX.
• 3. The biphasic change requires the influx of Ca ²⁺ that occurs through Ca ²⁺ channels that can be blocked by Cd ²⁺ , especially those of the L type, as can be seen from their sensitivity to nifedipine.

The exact mechanism of the interaction of α-LTX with HC110-R and the forwarding of signals still has to be clarified. Using the example of latrophilin-1, it could be shown that only a single transmembrane domain is necessary for the Ca ²⁺ influx caused by α-LTX in HEK-293 cells (Kraspernov et al. 1999).

The fact that nifedipine has the effect of α-LTX in that described here System blocked, shows that the system is also used to identify new ones specific calcium channel blocker is suitable. Nifedipine belongs to a class of Calcium channel agonists and antagonists that work according to their binding a specific location of a calcium channel can be classified, e.g. B. based on their Binding to L-type channels. The top three classes of calcium Channel blockers (L type) are benzoacetonitriles (e.g. verapamil, WO 91/02497), benzothiazepinones (e.g. diltiazem) and 1,4-dihydropyridine derivatives such as nifedipine, nivaldipine, nimodipine, nicardipine, isradipine, amlodipine, nitrene dipine, felodipine or nisoldipine (Bacon et al. 1989; WO 90/09792). Another Class of blockers of calcium channels of the L type are 1,3-diphosphonates, e.g. B. Belfosodil.  

This invention therefore also relates to the use of α-LTX binding Transmembrane receptors for the identification of new calcium channel blockers. Heptahelical transmembrane receptors are particularly preferred uses, particularly preferably G-protein binding receptors. The use of transmembrane receptors is very particularly preferred Secretin family. Most preferred is the use of the HC110-R Receptor according to SEQ ID. NO. 2 as well as 70%, preferably 80%, particularly preferably 90% and particularly preferably 95% homologous receptor proteins.

The present data in particular show that HC110-R is a target for the new anthelmintic depsipeptide BAY44-4400. BAY44-4400 interferes with signal transmission by α-LTX in HEK-293 cells that have been transfected with HC110-R. This disorder may be due to the ionophoric activity of the BAY44-4400, as is typical for other depsipeptides, e.g. B. Beauvericin, Enniatin etc. (Geßner et al. 1996). However, BAY44-4400 does not cause changes in non-transfected cells. In addition, BAY44-4400 does not interfere with isoproterenol-induced signal transmission through HEK-293 cells transfected with the mouse β ₂ -adrenergic receptor. In addition, BAY44-4400 acts as an antagonist of α-LTX, while BAY44-4400 alone does not affect the Ca ²⁺ concentration in HC110-R transfected HEK-293 cells.

The HC110-R protein

The membrane protein described in DE 197 04 024 A1 has been identified as a possible target protein for the anthelmintic active ingredient PF1022A. To identify possible target proteins, a λZAPII cDNA expression library of the parasitic nematode Haemonchus contortus was prepared and the cDNA library was searched using a conjugate of PF1022A and KLH (keyhole limpet hemocyanin), PF1022A-KLH, and polyclonal antibodies against this conjugate. Examination of approximately 1.5 × 10 ⁶ unamplified, recombinant clones revealed a 3036 bp long cDNA clone that hybridized to a 3.6 kb H. contortus mRNA. The RACE PCR technique was used to complete the 3 'and 5' ends. Finally, a 3539 bp cDNA was obtained which was designated HC110-R. This cDNA codes for 986 amino acids (110 kDa), the open reading frame beginning with the ATG ¹⁰⁰ . The start kodon is surrounded by a Kozak sequence for optimal initiation of translation. The derived molecular weight of the protein was confirmed by the in vitro translation of the in vitro transcribed HC110-R RNA.

The amino acid sequence of the HC110-R protein has some special features. The extracellular N-terminal part of the protein consists of a total of 535 residues. The N-terminus contains a signal peptide with a length of 21 amino acids and a cleavage site at position 18. This is followed by a lectin-like sequence (AS 22-125) and a so-called "thr stretch" (AS 128-147), the is only interrupted by a serine in position 144. Downstream of the "thr-stretch" follows a cysteine-rich motif of the structure CX ₉ WX ₁₂ CX ₉ WXCX ₅ WX ₉ CX ₃ W (AS 166-221). In addition, the protein HC110-R contains seven hydrophobic, α-helical transmembrane domains between residues 536 and 772. Directly upstream of the transmembrane region there is another 4-Cys region of the structure CXWWX ₆ WX ₄ CX ₄ CX ₁₁ CXC (AS 478-524). In the transmembrane region there are three extracellular loops containing residues 587-597, 654-673 and 743-749 and three intracellular loops (positions 559-569, 627-636, 696-724). The C-terminus is 214 residues long (24 kDa) and contains a proline-rich part (AS 845-861) and a PEST region (AS 915-933). Finally, there are three putative N-glycosylation sites at residues 26, 499 and 862 and 14 putative phosphorylation sites in the derived intracellular domains.

Analysis of databases showed 48% identity and 76% similarity the HC110-R protein with an unknown transmembrane protein (1014 AS), which is derived from the genomic clone B0457 (GenBank ™ Accession Number Z54306) of the nematode Caenorhabditis elegans.  

The comparison of both sequences shows common features of both proteins, see above for example the lectin-like sequence, the "thr stretch", the Cys motifs and the PEST sequence. The highest identity is in the area of the transmembrane region too find (62%), whereas a less pronounced identity in the N-terminal (44%) and C-terminal (50%) region can be found.

In addition, the protein HC110-R has a 20-30% identity heptahelical G protein-coupled transmembrane receptors (GPCR), be especially with the secretary subfamily. Comparisons of seven transmembrane Domains show high identity and similarity in structure and sequence between different GPCR of the secretin subfamily and HC110-R.

Latrophilin, a member of the secretin subfamily, from mammals like humans (Gen Bank ™ Accession No .: E1360690), beef (e.g. G416021, G416053 and G4185804), and the rat (U78105 or U72487) shows a slightly higher identity (31%) to HC110-R than other secretin receptors. Especially the transmembrane regions show an identity of 45-48%. HC110-R and the 1466 amino acids large rat latrophilin-1 GPCR (U78105) have common features on, e.g. B. the lectin domain, the cysteine-rich region and a conserved 4-cys- Motif in front of the transmembrane region. The latter was recently used as a proteolysis site proposed by Latrophilin and other major GPCR secretin. In contrast, contains the N-terminus of the HC110-R not the olfactomedin region and the Pro / Thr Region of latrophilin, while latrophilin in turn does not "thr stretch" the HC110-R.

Cellular localization

Transient transfection experiments with an HC110-R-GFP fusion protein have been carried out in various mammalian cell lines, for example COS-7- Cells or HEK-293 cells. The heterologous expression was chosen because so far no H. contortus cell lines have yet been established.  

The green fluorescent protein (GFP) from the Pacific jellyfish Aequorea victoria can be used for protein localization in living cells. At the cellular level, GFP serves as an in vivo reporter to show the frequency of a transient or stable transfection and at the subcellular level to localize proteins. Wild-type GFP is a 27 kD monomer made of 238 amino acids, which after excitation with UV light (360-400 nm; max. At 395 nm) or blue light (440-480 nm; max. At 475 nm) has green light a maximum of 509 nm without the need for exogenous substrates or cofactors (Chalfie et al. 1994). GFP can thus be detected directly by fluorescence microscopy in vivo and its fluorescence behavior remains essentially unchanged even as part of a fusion protein. EGFP ("enhanced" GFP) is a genetic variant of wild-type GFP and is used to transfect mammalian cells (Yang et al. 1996). The excitation maximum of EGFP was shifted to only one peak at 490 nm by substitution of Ser ⁶⁵ by Thr. The vectors pEGFP-C1 (GenBank Accession No .: U55763) and pEGFP-N3 (GenBank Accession No .: U55762) [Clontech, Palo Alto, CA, USA] express EGFP under the control of the strong constitutive CMV promoter and can be used for fusion of other proteins at the N- or C-terminus of EGFP.

Stable cell lines offer the advantage over transient expression that each Cell permanently expresses the desired protein and isolates the protein after localization becomes possible. To fluoresce the protein microscopically or with the help of a Western blot, an antibody against the required protein, or it is advisable to use an expression vector choose, for example, a Myc or His-Tag C-terminal with the actual Protein fused in the correct reading frame, against which it is then commercially available, usually even monoclonal antibodies there.  

Effect of alpha-latrotoxin on the HC110-R protein

To further substantiate the similarity between HC110-R and latrophilin and to check the functionality of the recombinantly expressed HC110-R HEK-293 cells became transient and stable with HC110-R-GFP fusion protein transfected and stimulated with alpha-latrotoxin (α-LTR).

Alpha-Latrotoxin is a presynaptic neurotoxin that can be obtained from the poison of the Black Widow (Latrodectus mactans). It is known for its toxicity to the central nervous system of vertebrates, where it increases the depolarization of neurons by increasing the [Ca ²⁺ ] _i and stimulating the uncontrolled exocytosis of neuro transmitters. It has also become known that the effect of α-latrotoxin is at least partially mediated by latrophilin. It is believed that the toxicity of α-latrotoxin is based on its ability to interact with receptors that are coupled to GTP-binding protein (GPCR). These receptors normally mediate the effects of endogenous hormones or neuropeptides (Holz and habener 1998).

In the present invention, the so-called "Ca ²⁺ imaging" technique was used to test the response of transfected HEK-293 cells to α-LTX by changing the intracellularly present Ca ²⁺ [Ca ²⁺ ] _i is determined.

α-LTX causes a biphasic increase in [Ca ²⁺ ] _i . At a concentration of 75 nM, α-LTX initially induces a very small increase of only 5 ± 0.2 nM 2 minutes after α-LTX addition and a stronger, delayed increase of approximately 220 ± 14.9 nM Ca ²⁺ after 22 minutes. As the concentration of α-LTX (7.5 nM-120 nM) increases, the first increase increases, while the second increase decreases. At a concentration of 120 nM α-LTX, the first increase reached values of approximately 135 ± 13.6 nM Ca ²⁺ , while the second increase decreased to a value of 50 ± 7.1 nM Ca ²⁺ . The same course when using 90 nM and 120 nM α-LTX indicates saturation.

Transfecting HEK-293 cells with an N-terminal GFP-tagged HC110 R construct and stimulates it with 75 nM α-LTX there is a slight re induced 2nd peak. Finally, one was only slightly reduced Response to α-LTX was observed when the HC110-R protein with an Nth minimum GFP tag was provided. It can therefore be assumed that the Appending a GFP tag N- or C-terminal has no significant impact on the α-LTX binding and the following signal transduction by HC110-R.

Cells that had not been transiently transfected or only transfected with GFP show no response to α-LTX. Are HEK-293 cells transiently transfected with other C-terminal GFP-tagged G protein-coupled receptors, e.g. B. the β ₂ - adrenergic receptor of the mouse, or the human muscarinic H1 acetylcholine receptor, causes α-LTX in a concentration of 75 nM only a slight (from about 40 ± 10.4 nM after about 20 minutes) to no increase the [Ca ²⁺ ] _i .

The increase in [Ca ²⁺ ] _i due to α-LTX can result in both the inflow of extracellular Ca ²⁺ and the outflow of intracellular Ca ²⁺ . If extracellular Ca ²⁺ is removed with 2 mM EGTA before or after the addition of α-LTX, there is only a small increase in the [Ca ²⁺ ] _i of HEK-293 cells which express the HC110-R-GFP fusion protein , From this it can be seen that α-LTX primarily causes the influx of extracellular Ca ²⁺ . This Ca ²⁺ inflow is not based on simple diffusion, but takes place with the help of Ca ²⁺ channels in the plasma membrane.

The majority of the Ca ²⁺ channels involved in the Ca ²⁺ inflow are of the L type, since 15 µM nifedipine is already sufficient to increase the increase in [Ca ²⁺ ] induced by α-LTX _i to suppress significantly. The first increase is completely inhibited, the second increase drops from 267 ± 12.7 nM to 30 ± 5.4 nM.

The stable or transient HEK-293 cell line expressing the C-terminal Myc / His-tagged HC110-R receptor also responds to α-LTX in a dose-dependent manner. 7.5 nM α-LTX are still too low to generate an α-LTX-induced Ca ²⁺ inflow. But 25 nM α-LTX are sufficient to produce an increase in the [Ca ²⁺ ] _i of 130 ± 38.0 nM after only 2 min. When 75 nM α-LTX is added, there is a Ca ²⁺ inflow of 296 ± 91.5 nM, which likewise increases sharply 2 minutes after administration of α-LTX and only returns to its original Ca ²⁺ content after 27 minutes reached. This Ca ²⁺ signal is similar to the second delayed Ca ²⁺ peak HC110-R-GFP of transfected HEK-293 cells at the same α-LTX concentration (75 nM) in the level of the signal and in its course, the answer is only given - as with higher concentrations (90 nM and 120 nM) HC110-R-GFP of transfected cells - immediately after α-LTX addition.

The invention therefore also relates to the use of α-LTX as Agonist of transmembrane receptors of the secretin family from nematodes. before α-LTX is added as an agonist of the HC110-R receptor according to SEQ ID NO. 2 ver and 70%, preferably 80%, particularly preferably 90% and very particularly preferably 95% homologous receptor proteins.

The present invention also relates to the use of α-LTX as Nematicide.

The present invention also relates to the use of α-LTX in a method for identifying nematicidal or arthropodicidal Compounds, the compounds as agonists or antagonists of the Trans membrane receptors can be effective.

The present invention also relates to the use of α-LTX in a method of identifying compounds that have calcium channels To block.  

Influence of BAY44-4400 on the effect of α-LTX

PF1022A exerts its effect on nematodes depending on the species at concentrations in the range of 100-800 ng / ml (Terada, 1992). In order to investigate a possible interaction between PF1022A with HC110-R and the signal transmission mediated by HC110-R, the following Ca ²⁺ imaging experiments were carried out in HEK-293- loaded with FURA-2 and transfected with HC110-R-GFP. Cells using the BAY44-4400, which is a more soluble derivative of PF1022A.

At a concentration of 400 ng / ml, neither the very effective nematicide BAY44-4400 nor the almost ineffective antipode PF1022-001 cause a Ca ²⁺ response in HEK-293 cells transfected in HC110-R-GFP, even if the cells are for Had been preincubated with the active ingredient for 90 minutes. In contrast, both substances influence the signal transmission caused by α-LTX, albeit to different degrees. In the presence of 4 ng / ml BAY44-4400, α-LTX causes only a slight increase in Ca ²⁺ with a maximum of 44 ± 6.0 nM Ca ²⁺ after 14 minutes. In the presence of PF1022-001, however, α-LTX causes a larger increase of 103 ± 11.5 nM Ca ²⁺ after 6 minutes ( Fig. 10B). In another approach, the cells were preincubated with either 4 ng / ml or 400 ng / ml BAY44-4400 and PF1022A for 90 minutes, the drugs were then removed before the cells were stimulated with α-LTX. HEK-293 cells preincubated with PF1022A showed no significant change in their response to α-LTX. Only BAY44-4400 affected the sensitivity of the cells to α-LTX. At a concentration of BAY44-4400 of 4 ng / ml, α-LTX induced an increase in Ca ²⁺ (95 ± 20.5 nM Ca ²⁺ ) 19 minutes before a stable Ca ²⁺ level was reached. At 400 ng / ml BAY44-4400, α-LTX only caused the Ca ²⁺ concentration to increase to about 65 ± 7.5 nM Ca ²⁺ . In addition, this minor increase was delayed by 12 minutes.

To underpin the specificity of these results, the effect of 1 mM carbachol on the endogenous, natural M1-R in non-transfected HEK-293 cells was also measured. The presence of 400 ng / ml BAY44-4400 did not change the response of the cells. The stimulation of the endogenous, natural β ₂ -R or the nicotinic cholinergic receptor in HEK-293 cells induced by isoproterenol and arecolin was in no way influenced by BAY44-4400.

Finally, in the context of the present invention, the influence of BAY44-4400, a more soluble variant of PF1022A, on the HC110-R protein in HEK-293 cells, which is transiently or stably a C-th, was investigated by Ca ²⁺ imaging Expression of HC110-R protein provided with GFP. When HEK-293 cells are incubated with only 400 ng / ml BAY44-4400, the cells do not react in any comparable way with a change in the intracellular Ca ²⁺ concentration for 50 minutes. However, an influence of the BAY44-4400 can be determined in the presence of the α-LTX. If cells are incubated 6 minutes before the addition of 75 nM α-LTX with BAY44-4400, the influence of the α-LTX on the Ca ²⁺ concentration is reduced. The first small increase in [Ca ²⁺ ] _i disappears and the second is reduced by 85% 15 minutes after the addition of α-LTX. The response to α-LTX increases even more if the cells are preincubated with BAY44-4400 for 60 minutes before FURA is added to the cells for 30 minutes. There is a complete disappearance of the first Ca ²⁺ increase and only a very slight second increase of 70 nM [Ca ²⁺ ] _i 30 minutes after the addition of 75 nM α-LTX.

Two control experiments were carried out to show the specific influence of BAY44-4400 on the effect of α-LTX. First, HEK-293 cells were transiently or stably transfected from the mouse with a β- ₂ adrenergic receptor provided with a GFP tag at the C-terminal and isoproterenol was used as the ligand.

In fact, isoproterenol also causes a significant Ca ²⁺ response: immediately after adding isoproterenol, there is only an increase in Ca ²⁺ . However, this increase is not affected by BAY44-4400.

Furthermore, the specificity of the interaction between BAY44-4400 and HC110-R is shown with the help of the optical antipode, which take the place of the BAY44-4400 as a ligand. After preincubation of the cells with 400 ng / ml of a derivative of PF1022A of PF1022-001 that is 100 times weaker in anthelmintic: cyclo (-L-Lac-D-MeLeu-L-PhLac-D-Me-Leu-) ₂ (also as an optical antipode be designated) for 90 minutes there is only a Ca ²⁺ increase of 110 nM 6 minutes after the addition of 75 nM α-LTX, i.e. a decrease of 59%. If 4 ng / ml of the optical antipode is added 6 minutes before the addition of 75 nM α-LTX, there is an increase of 67 nM [Ca ²⁺ ] _i immediately after the addition of the α-LTX. With increasing concentration of the optical antipode, the increase decreases to 20 nM Ca ²⁺ .

The term "polypeptides" as used herein refers to both short amino acid chains, commonly called peptides, oligopeptides, or oligomers are referred to, as well as on longer amino acid chains, commonly called prote be referred to. It includes amino acid chains that are either natural Processes, such as post-translational processing, or by chemical processes that State of the art, can be modified. Such modifications can occur in different places and several times in a polypeptide, such as on the peptide backbone, on the amino acid side chain, on the amino and / or at the carboxy terminus. They include, for example, acetylations, acylations, ADP ribosylations, amidations, covalent linkages with flavins, heme Shares, nucleotides or nucleotide derivatives, lipids or lipid derivatives or Phosphatidylinositol, cyclizations, disulfide bridges, demethylations, Cystine formation, formylation, gamma carboxylation, glycosylation, Hydroxylations, iodinations, methylations, myristoylations, oxidations,  proteolytic processing, phosphorylation, selenoylation and tRNA mediated additions of amino acids.

According to the invention, the polypeptides can be in the form of "mature" proteins or as Parts of larger proteins, e.g. B. can be used as fusion proteins. Furthermore, they call secreting or "leader" sequences, pro-sequences, sequences that allow easy cleaning, such as multiple histidine residues, or additional Liche stabilizing amino acids.

Such proteins or polypeptides are called homologous proteins or polypeptides considered that be at least 70% identity, preferably 80% identity particularly preferably 90% identity, very particularly preferably 95% identity, with a sequence according to SEQ ID NO. 2 of document DE-A-197 04 024, the In is expressly intended to be part of the present application, over a length of at least 20, preferably at least 25, particularly preferably at least 30 running amino acids and very particularly preferably over their total lengths point.

The degree of identity of the amino acid sequences is preferably determined using Help of the program GAP from the program package GCG, version 9.1 under standard settings (Devereux et al. 1984).

The polypeptides do not have to be complete for their use according to the invention Represent receptors, but can only be fragments of them as long as they have at least one biological activity of the complete receptors. The polypeptides do not have to be from transmembrane receptors from H. contortus be derivable. Polypeptides that trans membrane receptors from other helminth or arthropod species speak or fragments of it that still have the biological activity of this Can exert receptors.  

For their use according to the invention, the polypeptides can have deletions or amino acid substitutions in comparison to the corresponding region of naturally occurring GPCR receptors, as long as they still exert at least a biological activity of the complete receptors. Conservative substitutions are preferred. Such conservative substitutions include variations in which one amino acid is replaced by another amino acid from the following group:

• 1. Small aliphatic, non-polar or slightly polar residues: Ala, Ser, Thr, Pro and Gly;
• 2. Polar, negatively charged residues and their amides: Asp, Asn, Glu and Gln;
• 3. Polar, positively charged residues: His, Arg and Lys;
• 4. Large aliphatic, non-polar residues: Met, Leu, Ile, Val and Cys; and
• 5. Aromatic residues: Phe, Tyr and Trp.

The following list shows preferred conservative substitutions:

The above and in DE-A-197 04 024 under SEQ ID NO. 1 and SEQ ID NO. 3 be Written sequences can also be used to find genes which code for polypeptides which are based on the construction of functionally similar Transmembrane receptors in helminths or arthropods are involved. Under functionally similar receptors according to the present invention goren understood that include polypeptides, which are different in terms of Distinguish amino acid sequence from the polypeptides described herein, however have essentially the same biological function.  

The term "essentially the same biological function" as used herein used means participation in the construction of functional G-protein coupled heptahelical transmembrane receptor receptors, especially those Receptors of the secretin subfamily, or one of the HC110-R receptors from H. corresponding function to contortus. Such a function also includes the front described properties of the receptor, such as sensitivity to α-LTX as agonists or nifedipine as antagonists of α-LTX.

The term "hybridize" as used herein describes the pre gang, in which a single-stranded nucleic acid molecule with a complete base pair. In this way, starting from the Sequence information disclosed herein, for example, DNA fragments from others Nematodes as H. contortus and arthropods, which are used for Encode polypeptides with the biological activity of GPCR receptors.

With regard to a suitable probe, the amino-terminal and carboxyter minimal cDNA sections preferred. The hybridization conditions are like this chosen to also detect less similar sequences from other organisms are cash. The hybridization conditions under reduced stringency can For example, look like this: In 6 × SSC / 0% formamide as a hybridization solution is hybridized between 40-55 ° C. The conditions of the specific 2nd wash steps must be tested, e.g. B. initially 2 × SSC at 50 ° C, then estimation the signal intensities. This is followed by the modification of the washing conditions.

Preferred hybridization conditions are given below:
Hybridization solution: 6 × SSC / 0% formamide, preferred hybridization solution: 6 × SSC / 25% formamide
Hybridization temperature: 34 ° C, preferred hybridization temperature: 42 ° C

• 1st washing step: 2 × SSC at 40 ° C,
• 2nd washing step: 2 × SSC at 45 ° C; preferred 2nd washing step: 0.6 × SSC at 55 ° C; particularly preferred 2nd washing step: 0.3 × SSC at 65 ° C.

Hybridization conditions are approximately calculated using the following formula:
The melting temperature Tm = 81.5 ° C + 16.6 log {c (Na ⁺ )] + 0.41 (% G + C)) - 500 / n (Lottspeicher and Zorbas 1998).

Here, c is the concentration and n is the length of the hybridizing sequence section in base pairs. The expression 500 / n is omitted for a sequence <100 bp. It is washed with the highest stringency at a temperature of 5-15 ° C below Tm and an ionic strength of 15 mM Na ⁺ (corresponds to 0.1 × SSC). If an RNA sample is used for hybridization, the melting point is 10-15 ° C higher.

The in the inventive method for identifying nematicide and arthro podicidally active compounds are used by the in DE-A-197 04 024 under SEQ ID NO. 1 and SEQ ID NO. 3 described nucleic acids coded.

Furthermore, such nucleic acids are recorded for the use according to the invention, the be at least 70% identity, preferably 80% identity, particularly be preferably 90% identity, very particularly preferably 95% identity with a Sequence according to SEQ ID NO. 1 or SEQ ID NO. 3 over a length of at least 20, preferably at least 100, particularly preferably at least 500 consecutive Nucleotides and very particularly preferably have over their entire length.  

The nucleic acid according to the invention can also be used to produce transgenic In vertebrates can be used. These can be used in test systems based on an expression of the receptors according to the invention which differs from the wild type or Variants of this are based. This also includes all transgenic invertebrates, in which by the modification of other genes or gene control sequences (promo toren) a change in the expression of the receptors according to the invention or their Variants occurs.

The transgenic invertebrates are produced, for example, in Drosophila melanogaster by P-element mediated gene transfer or in Caenorhabditis elegans by transposon-mediated gene transfer (e.g. by Tc1, Plasterk 1996).

The invention thus also relates to transgenic invertebrates which have at least one of the nucleic acids according to the invention preferably contain transgenic invertebrates the species Drosophila melanogaster or Caenorhabditis elegans, and their trans descendants. The transgenic invertebrates preferably contain them receptors according to the invention in a form which differs from the wild type.

The nucleic acid according to the invention can be produced in the usual way. For example, the nucleic acid molecule can be completely chemically synthesized become. You can also chemically only use short pieces of the sequence according to the invention synthesize and such oligonucleotides radioactive or with a fluorescence mark dye. The labeled oligonucleotides can be used to cDNA libraries made from nematode mRNA or insect mRNA to search. Clones to which the labeled oligonucleotides hybridize selected to isolate the DNA in question. After characterizing the isolated DNA, the nucleic acid according to the invention is obtained in a simple manner.

The nucleic acid according to the invention can also be used by means of PCR methods under Ver using chemically synthesized oligonucleotides.  

The term "oligonucleotide (s)" as used herein means DNA- Molecules consisting of 10 to 50 nucleotides, preferably 15 to 30 nucleotides, be stand. They are chemically synthesized and can be used as probes.

The nucleic acid according to the invention can be used to isolate and characterize the regulatory regions that are naturally adjacent to the coding region occur. Such regulatory regions are therefore also Subject of the present invention.

Vectors are also used in the method according to the invention, the a nucleic acid to be used according to the invention or a nucleic acid according to the invention contain using DNA construct. As vectors, all in molecular bio logical laboratories used phages, plasmids, phagmids, phasmids, Cosmids, YACs, BACs, artificial chromosomes or particles that work for you Particle bombardment are suitable.

Preferred vectors are pBIN and its plant cell derivatives, pFL61 for yeast cells, pBLUESCRIPT vectors for bacterial cells, lamdaZAP (Fa. Stratagene) for phages.

Various vectors were used in the context of the present invention and created several constructs. The vectors used for the transient or stable Transformation of cell lines and for stable expression of the HC110-R receptor were also the subject of the present invention.

EGFP constructs were used for transient expression in eukaryotic cells asked to express fusion proteins with an N-terminal EGFP tag (EGFP-HC110-R) or a C-terminal EGFP tag (HC110-R-EGFP). Those of these vectors and the conventional GFP vectors as well as Rotshift and Blue shift variants encoded polypeptides are also the subject of lying invention.  

Another construct, the vector pMyc6xHis, used, which is also the subject of the present invention. The vector pMyc6xHis is derived from the vector pSecTagA [Invitrogen, Leek, NL] by Dop Pel digest with the restriction enzymes NhiI and SfiI, followed by "blunting" the Ends and religation of the vector.

The present invention also relates to host cells that are a fiction nucleic acid to be used according to or one to be used according to the invention Vector included. In particular, the stably transformed cell line HEK-293 is included HC110-R-Myc / His is the subject of this invention, numbered DSM ACC2464 at the international depository DSMZ-German Collection of microorganisms and cell cultures GmbH, Mascheroder Weg 1b in 38124 Braunschweig was deposited.

The present invention also relates to host cells which contain a nucleic acid to be used according to the invention or a vector to be used according to the invention, and a vector which enables the cells to express aequorin, a luminescent protein which in the presence of Ca ²⁺ - Light emitted. Appropriate host cells make it possible to change the Ca ²⁺ concentration and thus the effect of substances such. B. on HC110-R with the help of the indicator Aequorin. In particular, the stable transformed cell line HEK-293 with HC110-R-Myc / His capable of expressing aequorin is the subject of the invention, which is available under the number DSM ACC2465 from the international depository DSMZ-German Collection of Microorganisms and Cell Cultures GmbH, Mascheroder Weg 1b in 38124 Braunschweig.

The term "host cell" as used herein refers to cells that do not naturally contain the nucleic acids according to the invention.  

Eukaryotic cells such as yeast, mammalian, Amphibian, insect or plant cells. Preferred eukaryotic host cells are HEK-293-, Schneider S2-, Spodoptera Sf9-, Kc-, CHO-, HepG-2-, Kl-, COS-1-, COS-7, HeLa, C127, 3T3 or BHK cells and in particular Xenopus oocytes, HEK-293 or COS-7 cells are particularly suitable.

This invention also relates to the use of DNA accordingly the sequence ID No. described in application DE-A-197 04 024 2 for detection of DNA from helminths, preferably from the Nematoda strain, especially before migrates from the Trichostrongylidae family, most particularly of the genus Haemonchus and most preferred the species Haemonchus contortus.

The invention relates to oligonucleotides which correspond to a region of the above-described DNA sequence or its complementary strand and can hybridize to it. The invention relates to the use of these oligonucleotides or parts thereof as

• a) samples in Northern or Southern blot assays,
• b) PCR primer in a diagnostic method for the detection of the above ge called nematodes, the DNA of the helminths in question specific amplified using the primer and the PCR technique and then identified becomes.

The invention also relates to a method for the detection of helminths preferably from the Nematoda tribe, particularly preferably from the Tri family chostrongylidae, very particularly preferred of the genus Haemonchus and am most preferred of the species Haemonchus contortus, oligonucleotides as above hybridize specifically to DNA sequences described from the above Organisms originate, and which are then amplified using the PCR technique.  

The detection of organisms as mentioned above can e.g. B. done by

• a) an oligonucleotide sample or primer is available that matches the above said DNA coding for HC110-R or strands complementary thereto hydride or to the 5 'or 3' flanking regions thereof,
• b) the oligonucleotide sample or the primer with a correspondingly riding a sample containing DNA,
• c) the hybridization of the oligonucleotide or primer is detected (e.g. with Polymerase chain reaction),
• d) the detected sequence of the HC110-R gene is sequenced, and
• e) compares the sequence with the DNA sequences according to the invention, the were described above, preferably with that in application DE-A-197 04 024 described sequence ID No. Second

The invention also relates to a diagnostic test kit for the detection of von Helminthen, preferably from the Nematoda strain, particularly preferably from the Trichostrongylidae family, very particularly preferred of the genus Haemonchus and most preferred is the species Haemonchus contortus, which includes oligo provides nucleotides as described above that are used in detection methods can be used by species from the systematic groups mentioned.

The invention also relates to a diagnostic test kit as above wrote, with the oligonucleotides provided in this kit with are provided with a detectable marker. Such detectable markers can u. a. Enzymes include, enzyme substrates, coenzymes, enzyme inhibitors, fluorescence Zen markers, chromophores, luminescent markers and radioisotopes.  

This invention also relates to the use of the above HC110-R polypeptides or fragments thereof as well as 70%, preferably 70% thereof 80%, particularly preferably 90% and particularly preferably 95% more homologous Receptor proteins from helminths, preferably from the Nematoda strain, in particular preferably from the family Trichostrongylidae, very particularly preferably of the genus Haemonchus and most preferred the species Haemonchus contortus provision of vaccines containing at least one HC110-R polypeptide or fragment or one to 70%, preferably 80%, particularly preferably 90% and particularly preferably containing 95% homologous receptor protein thereof. The vaccine is there able to elicit an immune response that is specific for one above described HC110-R protein.

In a preferred embodiment, the vaccine contains an antigenic detergent minante, e.g. B. a single determinant of a polypeptide with an amino acid sequence according to the sequence ID No. described in application DE-A-197 04 024 2 or a polypeptide derived from the above DNA or fragments of which is encoded.

The present invention furthermore relates to methods for producing the Polypeptides to be used according to the invention. To make the polypeptides, which are encoded by the known nucleic acids, can host cells that these Contain nucleic acids, can be cultivated under suitable conditions. The ge desired polypeptides can then be removed from the cells or the cell in the usual manner Culture medium can be isolated. The polypeptides can also be used in in vitro systems getting produced.

For example, as described in the examples, a rapid method for isolating the polypeptides according to the invention, which are synthesized by host cells using a nucleic acid according to the invention, begins with the expression of a fusion protein, whereby the fusion partner can be affinity-purified in a simple manner. The fusion partner can be, for example, glutathione S-transferase. The fusion protein can then be purified on a glutathione affinity column. The fusion partner can be separated by partial proteolytic cleavage, for example on the linker between the fusion partner and the polypeptide according to the invention to be purified. The linker can be designed to include target amino acids such as arginine and lysine residues that define sites for trypsin cleavage. Standard cloning techniques using oligonucleotides can be used to create such linkers. Another possible method is based on the use of histidine fusion proteins and their purification using Ni ²⁺ talon columns.

Other possible cleaning processes are based on preparative electrophoresis, FPLC, HPLC (e.g. using gel filtration, reverse phase or light hydrophobic columns), gel filtration, differential precipitation, ion exchange Chromatography and Affinity Chromatography.

Since receptor-like protein kinases are membrane proteins, the Cleaning process preferably carried out detergent extractions, for example using detergents that cover the secondary and tertiary structures of the Do not or little affect polypeptides, such as non-ionic detergents.

The isolation of the polypeptides to be used according to the invention can be purified of membranes starting from host cells which contain the nucleic acids according to the invention express, include. Such cells preferably express the polypeptides in a sufficient number of copies so that the amount of polypeptides in one Membrane fraction is at least 10 times higher than that in comparable Membranes are found from cells that naturally express the HC110-R gene mieren; the amount is particularly preferably at least 100 times, very particularly preferably preferably at least 1000 times higher.  

The terms "insulation or cleaning" as used herein are indicate that the polypeptides are from other proteins or other macromolecules from the cell or tissue. Preferably, is a fiction moderate composition containing the polypeptides in terms of protein content compared to a preparation from the host cells at least 10 times and especially be preferably enriched at least 100 times.

The polypeptides according to the invention can also be used without fusion partners using Antibodies that bind to the polypeptides are affinity purified.

The invention furthermore relates to antibodies which are specific to the above bind mentioned polypeptides or receptors. The production of such antibodies he follows in the usual way. For example, such antibodies can be produced are injected with a substantially immunocompetent host with a for the antibody production effective amount of a transmememplate according to the invention bran receptor such as the HC110-R receptor from H. contortus or a fragment thereof and by subsequently obtaining this antibody. Furthermore, in receive an immortalized cell line known per se, the monoclonal Produces antibodies. The antibodies can optionally be detected reagent must be marked. Preferred examples of such a detection reagent are Enzymes, radio-labeled elements, fluorescent chemicals or biotin. Instead of the complete antibody, fragments can also be used, which have the desired specific binding properties.

The term "agonist" as used herein refers to a molecule activated the transmembrane receptors.

The term "antagonist" as used herein refers to a mole cool that displaces an agonist from its binding site or the function of the Inhibits agonists.  

The term "modulator" as used herein provides the preamble Agonist or antagonist. Modulators can be small organic chemical Molecules, peptides or antibodies that bind to the polypeptides according to the invention tie. Furthermore, modulators can contain small organic chemical molecules, Peptides or antibodies that bind to a molecule, which in turn binds to the binds polypeptides according to the invention, and thereby their biological activity influenced. Modulators can be mimetics of natural substrates and ligands represent.

The modulators are preferably small organic chemical Links.

The binding of the modulators to the polypeptides can affect the cellular processes change a way leading to the death of the helminths treated with it or Arthropods leads.

Therefore, the present invention extends to the use of Modulators of the polypeptides as anthelmintics and arthropodicides.

In particular, the use of the α-LTX as an anthelmintic is also counter stood this invention.

The inventive use of the nucleic acids or polypeptides in a he The method according to the invention also makes it possible to find compounds connected to bind the receptors of the invention. These can also be used as anthelmintics, z. B. as nematicides in plants or as anthelmintic agents in animals be turned. For example, host cells that contain the nucleic acids and express the corresponding receptors or polypeptides or the gene products even with a compound or a mixture of compounds under Be brought into contact with the interaction of at least one compound with the host cells, the receptors or the individual polypeptides.  

In particular, a method is the subject of the present invention that for Identification of nematicidal active substances is appropriate, based on transmembrane prescription bind gates from helminths or arthropods, preferably to GPCR of the secretin sub family, particularly preferred to the receptor HC110-R from H. contortus and 70%, preferably 80%, particularly preferably 90% and very particularly preferably bind to 95% sequence-identical receptors. The proceedings can, however, also be used with an HC110-R homologous receptor from another than of the species mentioned here. Procedures other than the one invented HC110-R receptor according to the invention are of the present invention fully embraced.

The methods of the invention include high throughput screening (e.g. high throughput screening; HTS). Both host cells and cell-free ones can do this Preparations are used which contain the nucleic acids according to the invention and / or contain the polypeptides according to the invention.  

Cell-free test systems

Many test systems that test compounds and natural extracts are aimed at high throughputs to reduce the number of under sought to maximize substances in a given period of time. Test systems that rely on cell-free work, need purified or semi-purified protein. They are suitable for a "first" exam, which is primarily aimed at taking one to detect possible influence of a substance on the target protein.

Effects such as cell toxicity are usually ignored in these in vitro systems. The test systems check both inhibitory and suppressive effects of Substances, as well as stimulatory effects. The effectiveness of a substance can be checked by concentration-dependent test series. Control approaches without Test substances can be used to evaluate the effects.

Test systems based on cells

Due to the availability of the present invention, stable with HC110-R transformed cell lines, but also by means of the present invention identifiable corresponding homologous receptors from other species the development of test systems based on cells for the identification of Substances that allow the activity of HC110-R and homologous receptors modulate.

Likewise, the identification of further ver enables bonds that are effective as calcium channel blockers.

To find modulators, a synthetic reaction mix (e.g. products in vitro translation) or a cellular component such as a membrane or any other preparation containing the polypeptide together with one labeled substrate or ligand of the polypeptides in the presence and absence  of a candidate molecule, which can be an agonist or antagonist become. The ability of the candidate molecule, the activity of the invention Increasing or inhibiting polypeptides is recognizable by an increased or ver reduced binding of the labeled ligand or to an increased or decreased Implementation of the marked substrate. Molecules that bind well and become one lead to increased activity of the polypeptides according to the invention are agonists. pier cool, which bind well, but not the biological activity of the invention Triggering polypeptides are probably good antagonists.

Scintillation Proximity Assay (SPA)

A way of identifying substances that affect the activity of HC110-R or modulate receptor proteins homologous to this is the so-called "Scintillation Proximity Assay" (SPA), see EP 015 473. This test system uses the interaction of a receptor (e.g. HC110-R) with a radiolabeled ligand (e.g. a small organic molecule or a second, radioactively labeled Protein molecule). The receptor is on small beads ("microspheres") or Tied beads, which are provided with scintillating molecules. in the The course of the fall in radioactivity becomes the scintillating substance in the bead and excited by the subatomic particles of the radioactive marker detectable photon emitted. The test conditions are optimized so that only those particles originating from the ligand lead to a signal, those from one the receptor or HC110-R bound ligand.

In one possible embodiment, HC110-R is bound to the beads, either together or without interacting or binding test substances. Who used fragments of the HC110-R receptor could also be used. A radioactive mar cated ligand could e.g. B. labeled α-LTX, nifedipine or a labeled Depsipeptide. When binding a binding ligand to the immobilized HC110-R receptor, this ligand would have an existing interaction between the inhibit or cancel immobilized HC110-R and the labeled ligand,  to bind yourself in the area of the contact surface. A binding to the immobilized HC110-R receptor can then be detected using a flash of light become. Accordingly, an existing complex between an immobili based and a free, labeled ligand by binding a test substance destroyed, which leads to a drop in the detected light flash intensity. The test system then corresponds to a complementary inhibition system.

Two hybrid system

An example of such a test system is the so-called "two hybrid system". A specific example of this is the so-called "Interaction Trap", the Inter ON SALE case. It is a genetic selection of inter acting proteins in yeast (see e.g. Gyuris et al. 1993). The test system is designed to detect and describe the interaction of two proteins, by an interaction that leads to a detectable signal.

Such a test system can also be used to test large numbers of test substances be adjusted in a given period.

The system is based on the construction of two vectors, the "bait" - and the "Prey" vector. One for an HC110-R according to the invention or fragments thereof coding gene is cloned into the bait vector and then as a fusion protein the LexA protein, a DNA binding protein. A second gene, ko for an interaction partner of HC110-R, for example for an α-LTX cloned into the Prey vector, where it functions as a fusion protein with the B42-Prey protein is expressed. Both vectors are in a Saccharomyces cerevisiae host, copies of LexA binding DNA on the 5 'side of a lacZ or HIS3 reporter gene contains. Finds an interaction between the two Fusion proteins take place, the transcription of the reporter is activated gens. Does the presence of a test substance inhibit or disrupt the  Interaction, the two fusion proteins can no longer interact and that Reporter gene product is no longer produced.

displacement test

Another example of a method with which modulators of the fiction polypeptides can be found is a displacement test in which the polypeptides according to the invention and a potential modulator with a molecule known to invent binds polypeptides according to the invention, such as a natural substrate or ligand or a substrate or ligand mimetic. Is preferred the α-LTX used for this in the manner according to the invention.

In the context of molecular interaction studies using the fully permanent HC110-R protein or the N- and / or C-terminal deletion mutants of HC110-R, or with by in vitro mutagenesis or other known Methods altered HC110-R molecule variants can be a known analysis system are used, e.g. B. from Biacore AB, Uppsala, Sweden. It can on the one hand (i) the HC110-R protein or fragments thereof via known chemical Methods (coupling via amines, thiols, aldehydes) or affinity binding (e.g. Streptavidin-Biotin, IMAC) coupled to a biochip or, on the other hand, (ii) α-LTX or another modulator z. B. BAY44-4400 or other possible ligands such as described under (i) are coupled to the chip. Binding one in solution ligands (HC110-R protein, or any modulator e.g. BAY44-4400 etc.) on the immobilized molecules is physically measurable. In which Biacore instrument, the ligand is immobilized on a sesor chip has a thin gold layer. The analyte solution is separated by a microflow cell perfused the chip. Binding of the analyte to the immobilized ligand increases the local concentration on the surface, the refractive index of the Medium near the gold layer is gradually increased. This has an impact on the Interaction between free electrons (plasmons) in the metal and photons that  be emitted by the instrument. These physical changes are proportions tional to the mass and number of molecules on the chip, the ligand-analyte bond is in Real time registered, thereby determining the apparent association / dissociation rate is possible (Fivash et al. 1998). The specificity is determined by competition tests validated the bond.

Corresponding measurements also serve to determine the binding of Ligands important HC110-R protein domains as well as the identification of new ones unknown ligands of HC110-R.

Calcium imaging

Another method for the detection of those interacting with the HC110-R Calcium imaging or signaling can be regarded as substances. Come here Calcium indicators for use with the help of changes in the intracellular Calcium levels can be made detectable. Come under these procedures HC110-R expressing cells are used, which are loaded with calcium indicators become. With UV excitation, it then comes from an HC110-R Calcium influx caused by agonists, or when released intracellularly Calcium to a change in absorption depending on the calcium loading of the Indicator. An antagonist can be identified in such a system by the complete or partial suppression of the cal induced by the agonist (e.g. α-LTX) recognize ciumsignals. The Fura-2 is another possible calcium indicator (Sigma) or Indo-1 (Molecular Probes).

Additional calcium indicators can be stimulated by visible light and changed to Depending on their calcium load, their fluorescence behavior is detectable. The Fluo-3 and Fluo-4 indicators have a high affinity for calcium. Fluo-4 is suitable with its stronger fluorescence signal, especially for measurements Test systems in which the cells are only used in low density, as in the Trap of the HEK293 cells. Other indicators are Rhod-2, x-Rhod-1, Fluo-5N, Fluo-  5F, Mag-Fluo-4, Rhod-5F, Rhod-5N, Y-Rhod-5N, Mag-Rhod-2, Mag-X-Rhod-1, Calcium Green-1 and 2, Calcium Green-5N, Oregon Green 488 BAPTA-1, Orgeon Green 488 BAPTA-2 and -5N, Fura Red, Calcein and the like.

An alternative to loading cells with calcium indicators is the right combined expression of photoproteins in the target cells. These photoproteins then react in the form of a light emission after being combined with calcium ions Complex. One in numerous examinations and test systems Aequorin is already widely used photoprotein. The the target protein and cells expressing the aequorin at the same time are used in this test procedure next load with the luminophore coelenterazine. The one made by the cells Apoaequorin forms a complex with the coelenterazine and carbon dioxide. Come on when calcium is added to the cell and binds to the complex, it is released Settlement of carbon dioxide and blue light (maximum emission 6466 nm). The Light emission correlates with the intracellular calcium concentration tration. This invention relates to the use of HC110-R, fragments thereof or similar proteins from other organisms in a test procedure in which the function of the receptor or the binding to the receptor by means of a Aequorin or similar photoproteins mediated light signal is detected.

Using host cells or transgenic invertebrates, which he Containing nucleic acid according to the invention, it is also possible to absorb substances find that change the expression of the receptors.

The active substances found with the aid of the method according to the invention are correspondingly suitable for controlling animal pests, in particular insects, arachnids and nematodes, which occur in agriculture, in forests, in the protection of stored goods and materials and in the hygiene sector. The active compounds found with the process according to the invention are particularly suitable for controlling nematodes and arachnids. The pests mentioned above include:
From the order of the Isopoda z. B. Oniscus asellus, Armadillidium vulgare, Porcellio scaber.
From the order of the Diplopoda z. B. Blaniulus guttulatus.
From the order of the Chilopoda z. B. Geophilus carpophagus, Scutigera spp.
From the order of the Symphyla z. B. Scutigerella immaculata.
From the order of the Thysanura z. B. Lepisma saccharina.
From the order of the Collembola z. B. Onychiurus armatus.
From the order of Orthoptera z. B. Acheta domesticus, Gryllotalpa spp., Locusta migratoria migratorioides, Medanoplus spp., Schistocerca gregaria.
From the order of the Blattaria z. B. Blatta orientalis, Periplaneta americana, Leucophaea maderae, Blattella germanica.
From the order of the Dermaptera z. B. Auricular Forficula.
From the order of the Isoptera z. B. Reticulitermes spp.
From the order of the Phthiraptera z. B. Pediculus humanus corporis, Haematopinus spp., Linognathus spp., Trichodectes spp., Damalinia spp.
From the order of the Thysanoptera z. B. Hercinothrips femoralis, Thrips tabaci, Thrips palmi, Frankliniella accidentalis.
From the order of Heteroptera z. B. Eurygaster spp., Dysdercus intermedius, Piesma quadrata, Cimex lectularius, Rhodnius prolixus, Triatoma spp.
From the order of Homoptera z. B. Aleurodes brassicae, Bemisia tabaci, Trialeurodes vaporariorum, Aphis gossypii, Brevicoryne brassicae, Cryptomyzus ribis, Aphis fabae, Aphis pomi, Eriosoma lanigerum, Hyalopterus arundinis, Phylloxera vastatrix, Pemphigus sppe, Phrosiphumumonpp., Macrosiphumum padi, Empoasca spp., Euscelis bilobatus, Nephotettix cincticeps, Lecanium corni, Saissetia oleae, Laodelphax striatellus, Nilaparvata lugens, Aonidiella aurantii, Aspidiotus hederae, Pseudococcus spp., Psylla spp.
From the order of the Lepidoptera z. B. Pectinophora gossypiella, Bupalus piniarius, Cheimatobia brumata, Lithocolletis blancardella, Hyponomeuta padella, Plutella xylostella, Malacosoma neustria, Euproctis chrysorrhoea, Lymantria spp., Bucculatrix thurberiisisppa, Fpp , Heliothis spp., Mamestra brassicae, Panolis.flammea, Spodoptera spp., Trichoplusia ni, Carpocapsa pomonella, Pieris spp., Chilo spp., Pyrausta nubilalis, Ephestia kuehniella, Galleria mellonella, Tineola bisselliecella, Hofia pellellaella, Hofea pellellaella, Tinea pellella , Capua reticulana, Choristoneura fumiferana, Clysia ambiguella, Homona magnanima, Tortrix viridana, Cnapha / ocerus spp., Oulema oryzae.
From the order of the Coleoptera z. B. Anoblum punctatum, Rhizopertha dominica, Bruchidius obtectus, Acanthoscelides obtectus, Hylotrupes bajulus, Agelastica alni, Leptinotarsa decemlineata, Phaedon cochleariae, Diabrotica spp., Psylliodes chrysocephis spp., Or. , Otiorrhynchus sulcatus, Cosmopolites sordidus, Ceuthorrhynchus assimilis, Hypera postica, Dermestes spp., Trogoderma spp., Anthrenus spp., Attagenus spp., Lyctus spp., Meligethes aeneus, Ptinus spp., Giptusibbium psol. Tenebrio molitor, Agriotes spp., Conoderus spp., Melolontha melolontha, Amphimallon solstitialis, Costelytra zealandica, Lissorhoptrus oryzophilus.
From the order of the Hymenoptera z. B. Diprion spp., Hoplocampa spp., Lasius spp., Monomorium pharaonis, Vespa spp.
From the order of the Diptera z. B. Aedes spp., Anopheles spp., Culex spp., Drosophila melanogaster, Musca spp., Fannia spp., Calliphora erythrocephala, Lucilia spp., Chrysomyia spp., Cuterebra spp., Gastrophilus spp., Hyppobosca spp., Stomox ., Oestrus spp., Hypoderma spp., Tabanus spp., Tannia spp., Bibio hortulanus, Oscinella frit, Phorbia spp., Pegomyia hyoscyami, Ceratitis capitata, Dacus oleae, Tipula paludosa, Hylemyia spp., Liriomyza spp.
From the order of the Siphonaptera z. B. Xenopsylla cheopis, Ceratophyllus spp.
From the class of the Arachnida z. B. Scorpio maurus, Latrodectus mactans, Acarus siro, Argas spp., Ornithodoros spp., Dermanyssus gallinae, Eriophyes ribis, Phyllocoptruta oleivora, Boophilus spp., Rhipicephalus spp., Amblyomma spp., Hyalommod spp., I ., Chorioptes spp., Sarcoptes spp., Tarsonemus spp., Bryobia praetiosa, Panonychus spp., Tetranychus spp., Hemitarsonemus spp., Brevipalpus spp.

The plant parasitic nematodes include z. B. Pratylenchus spp., Radopholus similis, Ditylenchus dipsaci, Tylenchulus semipenetrans, Heterodera spp., Globodera spp., Meloidogyne spp., Aphelenchoides spp., Longidorus spp., Xiphinema spp., Trichodorus spp., Bursaphelenchus spp.

The active substances found with the method according to the invention act not only against plant, hygiene and stored-product pests, but also in the veterinary sector against animal parasites (ectoparasites) such as shield ticks, leather ticks, space mites, running mites, flies (stinging and licking), parasitic fly larvae, Lice, hair lice, featherlings and fleas. These parasites include:
From the order of the Anoplurida z. B. Haematopinus spp., Linognathus spp., Pediculus spp., Phtirus spp., Solenopotes spp.
From the order of the Mallophagida and the subordinates Amblycerina and Ischnocerina z. B. Trimenopon spp., Menopon spp., Trinoton spp., Bovicola spp., Werneckiella spp., Lepikentron spp., Damalina spp., Trichodectes spp., Felicola spp.
From the order Diptera and the subordinates Nematocerina and Brachycerina z. B. Aedes spp., Anopheles spp., Culex spp., Simulium spp., Eusimulium spp., Phlebotomus spp., Lutzomyia spp., Culicoides spp., Chrysops spp., Hybomitra spp., Atylotus spp., Tabanus spp., Haematopota spp., Philipomyia spp., Braula spp., Musca spp., Hydrotaea spp., Stomoxys spp., Haematobia spp., Morellia spp., Fannia spp., Glossina spp., Calliphora spp., Lucilia spp., Chrysomyia spp ., Wohlfahrtia spp., Sarcophaga spp., Oestrus spp., Hypoderma spp., Gasterophilus spp., Hippobosca spp., Lipoptena spp., Melophagus spp.
From the order of the Siphonapterida z. B. Pulex spp., Ctenocephalides spp., Xenopsylla spp., Ceratophyllus spp.
From the order of the Heteropterida z. B. Cimex spp., Triatoma spp., Rhodnius spp., Panstrongylus spp.
From the order of the Blattarida z. B. Blatta orientalis, Periplaneta americana, Blattela germanica, Supella spp.
From the subclass of Acaria (Acarida) and the orders of the Meta and Mesostigmata z. B. Argas spp., Ornithodorus spp., Otobius spp., Ixodes spp., Amblyomma spp., Boophilus spp., Dermacentor spp., Haemophysalis spp., Hyalomma spp., Rhipicephalus spp., Dermanyssus spp., Raillietia spp. Pneumonyssus spp., Sternostoma spp., Varroa spp.
From the order of the Actinedida (Prostigmata) and Acaridida (Astigmata) z. B. Acarapis spp., Cheyletiella spp., Ornithocheyletia spp., Hyobia spp., Psorergates spp., Demodex spp., Trombicula spp., Listrophorus spp., Acarus spp., Tyrophagus spp., Caloglyphus sph., Hypodectes spp. Pterolichus spp., Psoroptes spp., Chorioptes spp., Otodectes spp., Sarcoptes spp., Notoedres spp., Knemidocoptes spp., Cytodectes spp., Laminosioptes spp.

For example, they show excellent effectiveness against Sarcoptes scabei, S. suis, S. bovis, S. ovis and S. equi.

The active compounds of the formula (I) according to the invention are also outstandingly effective against mites, especially house dust mites e.g. B. Dermatophagoides pteronyssinus and D. farinae.  

The active compounds of the formula (I) according to the invention are also suitable for combating of arthropods, the farm animals, such as. B. cattle, sheep, goats, Horses, pigs, donkeys, camels, buffaloes, rabbits, chickens, turkeys, ducks, geese, Bees, other pets such as B. dogs, cats, house birds, aquarium fish as well as so-called experimental animals, such as. B. hamsters, guinea pigs, rats and Infest mice. By fighting these arthropods, deaths and Reductions in performance (for meat, milk, wool, skins, eggs, honey, etc.) can be reduced so that by using the active compounds according to the invention more economical and easier animal husbandry is possible.

Compounds found using the described methods and polypeptides are also valuable for the treatment of animals and people who use pathogenic endoparasites of humans or of farm animals, pets, zoo animals and laboratory and laboratory animals are infected.

The compounds are active against all developmental stages of normal, sensitive strains and also resistant strains. Treatment with agents containing one or more of these compounds can prevent or treat both economic losses in farm animals and diseases in humans and animals. The following parasites are of particular interest as targets of the active compounds found:
Enoplida, e.g. B. Trichuris spp., Capillaria spp., Trichomosoldes spp., Trichinella spp.
Rhabditia, e.g. B. Micronema spp., Strongyloides spp.
Strongylida, e.g. B. Strongylus spp., Triodontophorus spp., Oesophagodontus spp., Trichonema spp., Gyalocephalus spp., Cylindropharynx spp., Poteriostomum spp., Cyclococercus spp., Cylicostephanus spp., Oesophagostomum spp., Stephanab. Ancylostoma spp., Uncinaria spp., Bunostomum spp., Globocephalus spp., Syngamus spp., Cyathostomum spp., Cylicocyclus spp., Neostrongylus spp., Cystocaulus spp., Pneumostrongylus spp., Spicocaulusostppyl. Spp., Spicocaulusost ., Crenosoma spp., Paracrenosoma spp., Angiostrongylus spp., Aelurostrongylus spp., Filaroides spp., Parafilaroides spp., Trichostrongylus spp., Haemonchus spp., Ostertagia spp., Marshallagia spp., Cooperia sirus., Hyostrongylus spp., Obeliscoides spp., Amidostomum spp., Ollulanus spp. Dictyocaulus spp., Muellerius spp., Protostrongylus spp.
Oxyurida, e.g. B. Oxyuris spp., Enteroblus spp., Passalurus spp., Syphacia spp., Aspi culuris spp., Heterakis spp.
Ascaridia, e.g. B. Ascaris spp., Toxascaris spp., Toxocara spp., Parascaris spp., Anisa kis spp., Ascaridia spp.
Spirurida, e.g. B. Gnathostoma spp., Physaloptera spp., Thelazia spp., Gongylonema spp., Habronema spp., Parabronema spp., Draschia spp., Dracunculus spp.
Filariida, e.g. B. Stephanofilaria spp., Parafilaria spp., Setaria spp., Loa spp., Dirofilaria spp., Litomosoides spp., Brugia spp., Wuchereria spp., Onchocerca spp.
Gigantorhynchida, e.g. B. Filicollis spp., Moniliformis spp., Macracanthorhynchus spp., Prosthenorchis spp.
Mastigophora (Flagellata).
Trypanosomatidae, e.g. B. Trypanosoma b. brucei, T. b. gambiense, T. b. rhodesiense, T. congolense, T. cruzi, T. evansi, T. equinum, T. lewisi, T. percae, T. simiae, T. vivax, Leishmania brasiliensis, L. donovani, L. tropica.
Trichomonadidae, e.g. B. Giardia lambilia, G. canis.
Sarcomastigophora (Rhizopoda), e.g. B. Entamoeba histolytica.
Hartmanellidae, e.g. B. Acanthamoeba sp., Hartmanella spp.
Apicomplexa (Sporozoa), e.g. B. Eimeria acervulina, E. adenoides, E. alabahmensis, E. anatis, E. anseris, E. arloingi, E. ashata, E. auburnensis, E. bovis, E. brunetti, E. canis, E. chinchillae, E. elupearum, E. columbae, E. contorta, E. crandalis, E. debliecki, E. dispersa, E. ellipsoidales, E. falciformis, E. faurei, E. labbeana, E. leucarti, E. magna, E. maxima , E. media, E meleagridis. E meleagrimitis, E. mitis, E necatrix, E. ninakohlyakimovae, E ovis, E. parva, E. pavonis, E. perforans, E. phasani, E. piriformis, E. praecox, E. residua, E. scabra, E. spec., E. stiedai, E. suis, E. tenella, E. truncata, E. truttae, E. zuernii, Globidium spec., Isospora belli, I. canis, I. felis, I. ohioensis, I. rivolta , L spec., I. suis, Neospora caninum, Cystisospora spec., Cryptosporidium spec.
Toxoplasmadidae, e.g. B. Toxoplasma gondii.
Sarcocystidae, e.g. B. Sarcocystis bovicanis, S. bovihominis, S. neuvona, S. ovicanis, S. ovifelis, S. spec., S. suihominis.
Leucozoids, e.g. B. Leucozytozoon simondi.
Plasmodiidae, e.g. B. Plasmodium berghei, P. falciparum, P. malariae, P. ovale, P. vivax, P. spec.
Piroplasmea, e.g. B. Babesia argentina, B. bovis, B. canis, B. spec., Theileria parva, T. spec.
Adeleina, e.g. B. Hepatozoon canis, H. spec.

Are still important
Myxospora and Microspora, e.g. B. Glugea spec. and Nosema spec., and Pneumocystis carinii, Ciliophora (Ciliata), e.g. B. Balantidium coli, Ichthiophthirius spec., Trichondina spec. or Epistylis spec.

The compounds and agents found are also effective against Proto zoos of insects, such as those of the Microsporidia strain, especially those of the Order Nosema, especially those of the species Nosema apis, the parasites of the Honeybee are.  

Examples 1. Construction and search of the cDNA library

The construction of the cDNA library, the production of KLH-PF1022A- Conjugate and antiserum against PF1022A and the immune screening of the cDNA Library and DNA analysis was carried out as described in WO 98/15625.

2. Isolation of RNA

Total RNA was extracted and isolated from adult Haemonchus contortus nematodes according to the GTC / CsCl cushion method (Sambrook et al. 1989) or obtained by GTC / phenol / chloroform extraction in a single step (Chomczynski and Sacchi 1987). Poly (A) ⁺ RNA was isolated by chromatography on oligo (dT) cellulose (Aviv and Leder, 1972).

3. Northern blotting

Glyoxylated total RNA from Haemonchus contortus (20 µg per lane) was separated in an agarose gel (Sambrook et al. 1989; McMaster and Carmicheal 1977) and transferred to a Hybond N membrane (Amersham) using a basic capillary transfer method (Chomczynski, 1992). Radiolabeled samples were prepared by randomized labeling of linearized plasmid DNA (HC110-R) using a megaprime kit (Amersham, Braunschweig, Germany) and 50 µCi [α- ³² P] dCTP (3000 Ci / mmol, ICN, Meckenheim, Germany ) were used. Hybridization was carried out overnight at 65 ° C in 6 × SSC (1 × SSC: 0.15 M NaCl, 0.015 M Na citrate), 5 × Denhardt's reagent (0.1% polyynylpyrrolidone, 0.1% BSA, 0 , 1% Ficoll 400), 0.1% SDS and 100 µg / ml herring sperm DNA. The filters were washed with high stringency in 0.1 × SSC and 0.1% SDS at 65 ° C and exposed (Kodak BioMax MS films at -80 ° C).

4. 5 'and 3' RACE PCR

The 5 'and 3' ends missing in the cDNA clone identified were isolated the procedure of the 5 '/ 3' RACE applied.

The 5'-RACE is based on the specific amplification of the 5'-end of a gene from mRNA. With the help of a sequence-specific primer and the AMV reverse The first strand of cDNA is synthesized using transcriptase. A will be added to the product poly (A) tail attached, so that in the subsequent PCR an oligo dT anchor primer and a nested sequence-specific primer can be used. In a second PCR, another "nested" primer can be used to measure specificity to ensure.

In the 3'-RACE the cDNA first strand synthesis takes place with an oligo dT primer and the subsequent PCR reactions with sequence-specific primers.

cDNA was synthesized from 1 µg of total H. contortus RNA with the primer 5'-GGT CAC CGT CGT CCC AGA AA-3 'using the 5'-RACE kit from GIBCO BRL (Eggenstein, Germany). Superscript reverse transcriptase was used for this. The C-tailing of the C-terminus of the cDNA was carried out using the deoxynucleotidyltransferase terminals. The first amplification was carried out with 400 nM of an oligo-deoxy-inosyl anchor primer (5'-CUA CUA CUA CUA GGC CAC GCG TCG ACT AGT ACG GGI IGG GII GGG IIG-3 ') and 400 nM of the first "nested" primer 5'-CCA TTC GAT TCC TCT TCT CG-3 '(Birsner and Grob, Denzlingen, Germany) in 50 µl containing 200 µM each dNTP, 1.5 mM MgCl ₂ , a fifth of the "tailed" cDNA and 2.5 U the native Taq polymerase. The first denaturation was at 94 ° C for 5 minutes, followed by 35 cycles of 1 minute each at 94 ° C, 1 minute at 53 ° C and 2 minutes at 72 ° C, again followed by a final synthesis step of 10 minutes at 72 ° C. The reaction conditions for the "nested" PCR were the same as described above, with the exception that 1 μl of the first amplification product was used as a template, and the gene-specific second "nested" primer 5'-GTC GAT GGT GCA GAT TTC GC-3 ' , a shortened form of the anchor primer (5'-CUA CUA CUA CUA GGC CAC GCG TCG ACT AGT AC-3 ') and only 25 cycles were carried out at an annealing temperature of 53 ° C.

The 3'-RACE-PCR (GIBCO BRL, Eggenstein, Germany) was carried out with 1 µg of the total RNA adult H. contortus and 500 nM of the oligo dT adapter primer 5'-GGC CAC GCG TCG ACT AGT ACT TTT TTT TTT TTT TTT T-3 'was performed, starting with preincubation for 10 minutes at 70 ° C and 2 minutes at 4 ° C. After adding 2.5 mM MgCl ₂ , 500 μM each dNTP, 10 μM DTT and 200 U Super-Script II reverse transcriptase in 20 μl, the cDNA was at 50 ° C. for 50 minutes and in a final step at 70 for 15 minutes ° C and incubated for 10 minutes at 4 ° C. The RNA was removed by 2 U E. coli RNase H and incubation at 37 ° C for 10 minutes. The first amplification was carried out with 400 nM of a universal adapter primer (5'-CUA CUA CUA CUA GGC CAC GCG TCG ACT AGT AC-3 ') and 400 nM of the sequence-specific primer 5'-TTTGTTCTT CCT TGG TAT CC-3' in 50 µl containing 200 mM each dNTP, 1.5 mM MgCl ₂ , one tenth of the "tailed" cDNA and 2.5 U native Taq DNA polymerase. The first denaturation step was carried out at 94 ° C. for 3 minutes, followed by 35 cycles of 1 minute each at 94 ° C., 1 minute at 51 ° C. and 2 minutes at 72 ° C. and a final synthesis step of 15 minutes at 72 ° C. , For the "nested" PCR, 2 ul of the first amplification were used under otherwise identical conditions, with the shorter adapter primer 5'-GGC CAC GCG TCG ACT AGT AC-3 'and the "nested" primer 5'-ACA CTC TAA TCT CCA ACT G-3 'were now used for 30 cycles. The PCR products were analyzed on 2% agarose gels, eluted and cloned into the TA vector pMosBlue (Amersham, Braunschweig, Germany).

5. Transfer of RNA

The transfer of the separated RNA from the glyoxal gel or genome. DNA from a TBE agarose gel on a neutral Hybond-N nylon membrane [Amersham, Braunschweig] is carried out according to the "Downward Alkaline Capillary Transfer" in alkaline transfer solution using the Chomczynski 1992 method for 2 h. Then the membrane is neutralized for 20 min in 0.2 M sodium phosphate buffer (pH 6.8), dried and baked at 80 ° C. for 20-60 min. For the hybridization against radioactively labeled probes, the membrane is sealed with 5 ml / cm ² prehybridization solution in plastic bags and incubated for 3 h at 65 ° C. For hybridization, the buffer is replaced by a hybridization solution. When using Rapid Hyb. Solution [Amersham, Braunschweig], prehybridization takes place in 30 min at 65 ° C without additional prehybridization solution. After addition of the probe radioactively labeled by "random priming", the hybridization takes place at 65 ° C. overnight. The membrane is then washed once with 65 ° C. warm 2 × SSC / 0.1% SDS for 20 min and three times with 0.1 × SSC / 0.1% SDS for 1 h each. The membrane is exposed on Kodak X-OMAT or Kodak BIOMAX-MS X-ray films with the appropriate intensifying screen at -80 ° C.

6. In vitro transcription and translation

The TNT T7 / T3 coupled reticulocyte lysate system (Promega) was used to generate the full length of the coding sequence of HC110-R in the presence of ³⁵ S-labeled methionine and cysteine (ICN, Eschwege, Germany) according to the manufacturer's instructions transcribe and translate. The RNA was translated using a rabbit reticulocyte lysate system (Promega, Serva, Heidelberg), using a 40 μCi ³⁵ S-labeled mixture (<1000 Ci / mmol, ICN, Meckenheim, Germany), 1 μg circularized HC110-R plasmid DNA and 10 U T3 RNA polymerase were used. The reaction was carried out at 30 ° C for 90 minutes. A positive control contained 1 µg luciferase control DNA.

The reaction products were by SDS polyacrylamide gel electrophoresis (Lämmli 1970) separated and using the "Amplify Fluorography Solution" (Amersham Pharmacia Biotech) or 1 M sodium salicylate (pH 7) fluorographed (Chamberlain 1979), the gels were then dried and exposed 86413 00070 552 001000280000000200012000285918630200040 0002010053785 00004 86294 (Kodak BioMax MR Film with Ver stronger at -80 ° C).

Alternatively, the "MAXIscript In Vitro Transcription Kit" from Ambion [Heidelberg] was used for the in vitro synthesis of an RNA transcript from HC110-R cDNA cloned in pBluescript SK. 4 µg of HC110-R plasmid DNA were linearized with the restriction enzyme Hind III or Sal I behind the stop codon, extracted with phenol-chloroform, precipitated in 3 vol. Ethanol and dissolved in DEPC-H ₂ O. In a 50 µl reaction mixture, 2 µg HC110-R cDNA with 2.5 µl 200 mM DTT, 2.5 µl 10 mM ATP, CTP, GTP and UTP, 5 µl 10 × transcription buffer, 1.6 µl RNasin inhibitor ( 40 U / µl) [Promega, Heidelberg] and 2 µl T3 phage polymerase (10 U / µl) were added and incubated for 2 h at 37 ° C. After 1 h, 2 ul T3 phage polymerase (10 U / ul) were added again. 1.5 µl RNase-free DNase I (2 U / µl) and 1 µl RNasin inhibitor (40 U / µl) were added, incubated for 15 min at 37 ° C, extracted with phenol-chloroform, in 3 vol. Ethanol precipitated and resuspended in 25 µl DEPC-H ₂ O. 1/5 vol. Of the sample was analyzed in a glyoxal gel.

7. DNA analysis

The clones were removed by the dideoxynucleotide chain termination method Automated laser fluorescence sequencing (LICOR 4000; MWG, Ebersberg, Germany) and the "Thermo Sequenase fluorescent-labeled cycle sequencing kit "(Amersham, Braunschweig, Germany). The sequences of both strands were determined and the sequence data with the GCG Software (Genetics Computer Group Inc., Madison, WI, U.S.A.) and the PC / GENE- Software (Intelligenetics, Mountain View, CA, U.S.A.). The FASTA programs  (Pearson and Lipman 1988), BLITZ (Smith and Waterman 1981) and BLAST (Altschul and Lipmann 1990) were used to measure the EMBL and Swiss Prot Protein databases according to sequence similarities of the derived protein sequence search. The protein sequences were analyzed with SAPS (Brendel et al. 1992), as well as with PROSITE and Prot-Param (Appel et al. 1994).

8. Production of protein extracts from Haemonchus contortus

50-100 mg of H. contortus worms frozen in liquid nitrogen were in 1 ml of TRIZOL [Gibco, Karlsruhe] added. The nematodes were put together homogenized with the TRIZOL 3 × for 15 sec in a glass potter and for 5 min RT incubated. After adding 200 µl chloroform per ml TRIZOL and shaking the sample for 15 sec, the mixture was incubated for a further 2-3 min at RT before it was centrifuged for 10 min at 7000-12000 rpm and 4 ° C. The top watery Phase contains the RNA, the interphase the genomic DNA, the red organic Phase the proteins (Coombs et al. 1990; Chomczynski 1993). The aqueous phase was removed and processed separately. The interphase and organic phase were mixed with 300 µl 100% ethanol per ml TRIZOL, mixed well and for 2-3 min incubated at RT. After centrifugation for 2000 rpm for 5 min and 4 ° C the protein supernatant was carefully transferred to a new Eppendorf tube and with 1 ml Precipitated isopropanol for 10 min at RT. It was again for 10 min at 12000 rpm and centrifuged at 4 ° C., the supernatant was discarded and the protein pellet was 3 × with 2 ml each 0.3 M guanidine hydrochloride solution in 95% ethanol, "vortexed", for Incubated for 20 min at RT and centrifuged for 5 min at 7500 rpm and 4 ° C. After that the pellet was dissolved in 2 ml of 100% ethanol, precipitated for 20 min at RT and for Pelleted for 5 min at 7500 rpm and 4 ° C. The pellet was briefly vacuum dried and resuspended in urea lysis buffer (8 M). Insoluble material has been removed centrifugation for 10 min at 10000 rpm and 4 ° C, the supernatant in a transferred to a new Eppendorf tube and after determining the protein concentration up to stored at -20 ° C for further processing.  

9. Production of protein extracts from yeast cells

To produce protein extracts from a yeast culture, 5 ml of YPAD medium were inoculated with a single yeast colony and shaken to saturation at 30 ° C. (2 days). In each case 2 ml of such a yeast culture were centrifuged for 2 min at 3000 rpm and 4 ° C. (Heraeus Biofuge 15 R), washed once with H ₂ O, resuspended in 250 μl yeast lysis buffer, transferred to a small test tube, with glass beads (∅ 0.5 mm) to just below the liquid level and "vortexed" for 5 min at the highest level. Then 4 × RotiLoad buffer [Roth, Karlsruhe] was added and the mixture was incubated at 95 ° C. for 5 min. The disrupted cells were transferred to an Eppendorf tube and the cell debris was centrifuged for 5 min at 14000 rpm (Eppendorf centrifuge 5415 C). 20 µl each of the samples prepared in this way were applied to an SDS polyacrylamide gel.

10. Production of protein extracts from E. coli cells

To produce protein extracts, 1 × 10 ⁷ E. coli cells were pelleted from a 5 ml overnight culture for 5 min at 5000 rpm and 4 ° C. in a Heraeus undersink, washed with 5 ml PBS and centrifuged again. The pellet was then resuspended in 1 ml TRIZOL [Gibco, Karlsruhe] and processed further.

Alternatively, the cell pellet washed with PBS was resuspended in PBS dated and the cells with the help of several short ultrasonic pulses [Sonifier B- 12, Branson Sonic Power Company, Danbury, U.S.A.], liquid nitrogen and destroyed by adding lysozyme.

Following the protein determination, 4 × RotiLoad buffer [Roth, Karlsruhe] added and the fraction by application to an SDS polyacrylamide gel analyzed. A total amount of protein of 10-20 µg was found per lane carried.  

11. Production of protein extracts from cell culture

A confluent 35 mm cell culture dish of adherent cells or 5-10 × 10 ⁶ non-adherent mammalian cells in FCS-containing medium had previously been trypsinized with a trypsin-EDTA solution or removed mechanically after exposure to cold with a cell scraper, transferred to an Eppendorf vessel, Pelleted for 10 sec at 13000 rpm and RT, washed twice with PBS and centrifuged again. After the cells had been taken up in 1 ml of TRIZOL, they were lysed by vigorous "vortexing" or by repeatedly pulling the cells through the cannula of a disposable syringe and incubating for 5 min at RT. Alternatively, the cell pellet can also be resuspended in 1 ml PBS or urea lysis buffer (8 M) and subjected to a brief ultrasound treatment [Sonifier B-12, Branson Sonic Power Company, Danbury, USA]. Then the protein content according to Bradford (1976) or Lowry et al. (1951).

12. Inducible protein expression in E. coli

For the production of polyclonal antibodies against the HC110-R protein three HC110-R fragments - the complete HC110-R cDNA, the N-terminal end without TM domains and the C-terminal end after the 7th TM domain - in Ex The pressure vector pRSET B (Invitrogen, Leek, NL) is cloned and thus N-terminally a 6xHis tag merged taking into account the reading grid.

The complete coding region of HC110-R was with the P84_ATG BamHI 5'-primer 5'-CTG CCG GAT CCT CGG TTT AAT ACC AAC ATG AGG-3 'and the P3121_TGA HindIII 3'-primer 5'-GCA CTA AGC TTG ACT GAA GCG CAC AAC CTC G-3 ', the N-terminus up to the 1st transmembrane domain was with the primers P84_ATG BamHI 5' primer (see above) and the P1434_TGA HindIII 3'-primer 5'-GGC TCA AGC TTA TCA GAG AAC AAG CGA CAC GGC-3 ', and the C-Ter minus starting after the 7th transmembrane domain was treated with the P2486_ATG BamHI 5'-primer 5'-CTA TCG GAT CCC AAC ATG GCT GGC TCC CGT GAT ACC TCT AGG-3' and the P3121_TGA HindIII 3 'primer (see above). The annealing temperature in all three plasmid PCR was first 56 ° C over 5 cycles and then 62 ° C over 30 cycles. The PCR products were digested with the enzymes BamHI and HindIII and ligated directionally into the pRSET B expression vector, which was also linearized by BamHI / HindIII.

Expression in the pRSET B vector is via a viral promoter of B7 bacteriophage controlled. The cloning was therefore carried out in the XL1-Blue E. coli strain that does not contain a T7 RNA polymerase gene. The recombinant plasma mid was then expressed in BL7 (De3) pLysS E. coli cell expressing T7 polymerase len transformed, which additionally contain the plasmid pACYC 184, which via a Chloramphenicol resistance is stabilized, and in small amounts the T7 Express lysozyme, a natural inhibitor of T7 RNA polymerase. There these cells are under the control of the lacUVS promoter takes place at IPTG- Induction of the expression of the T7 RNA polymerase and thus that of the fusion protein.

A single colony with the desired HC110-R plasmid was inoculated from a fresh LB plate with 50 µg / ml ampicillin and 35 µg / ml chloramphenicol in 50 ml LB medium under the same selection pressure and overnight at 37 ° C and 280 rpm until stationary Phase shaken. This ÜN culture was then diluted to OD ₆₀₀ = 0.3 and 100 ml of the culture at 37 ° C and 280 rpm shaken to an OD ₆₀₀ = 0.6-0.5.

1 OD ₆₀₀ unit was removed, the uninduced cells were briefly centrifuged and taken up in 150 μl of 8 M urea lysis buffer, pH 8.0, and 50 μl of 4 × RotiLoad buffer [Roth, Karlsruhe]. The non-induced sample was denatured for 2 min, the genomic DNA was sheared by short ultrasonic pulses of a few seconds in Sonifier B-12 [Branson Sonic Power Company, Danbury, USA] and insoluble particles were pelleted by centrifugation at 14,000 rpm for 3 minutes.

Expression of the fusion protein was induced by adding 1 mM IPTG and incubated the 100 ml culture for a further 3-4 h at 37 ° C. After incubation the induced cells for 15 min at 5000 rpm and 4 ° C [Heraeus Centrifuge], the pellet washed in PBS and then resuspended in 8 ml of 8 M urea lysis buffer. The cells were disrupted by immersing the cells 3 times in liquid nitrogen and then Thaw at 37 ° C. After the first nitrogen treatment, 0.75 mg / ml Lysozyme added. After the last incubation in liquid nitrogen there was a Incubation for 20 min at 16 ° C, followed by short ultrasonic pulses of 10 sec each and cooling in an ice water bath until the solution has a water-like viscosity acted. After centrifugation at 13000 rpm and 4 ° C for 10 minutes [Beckmann J2-21; JS 13.1 rotor], an induced 150 µl sample was taken with 50 µl 4 × RotiLoad buffer [Roth] added and the induction of the respective HC110-R Protein fragment together with the uninduced sample using SDS-PAGE subsequent Coomassie staining or Western blot analysis with a mouse anti His antibody checked.

The remaining supernatant was used for further purification using affinity Chromatography transferred to a fresh vessel and stored at -20 ° C.

13. Protein purification by metal affinity chromatography

The enrichment was carried out under denaturing conditions via the N-terminal 6 × His tag using the IMAC system ( "I mmobilized M et al A ffinity C hromato graphy") on Talon spin columns from Clontech [Palo Alto, USA]. The resin of the column was first equilibrated separately with 5 vol. 8 M urea lysis buffer, pH 8.0, sedimented for 4 min at 3000 rpm and 4 ° C and together with the HC110-R protein supernatant for 20 min incubated at RT with gentle shaking.

After centrifugation again, the resin was 3 times with 10 times the volume 8 M urea lysis buffer, pH 8.0, for 10 min at RT with gentle shaking wash and centrifuged again. After the last wash, the pellet became taken up in 1 ml of 8 M urea lysis buffer and loaded onto the column; this was subsequently made twice with 3 times the volume of 8 M urea lysis buffer washed before elution with imidazole-containing 8 M urea Lysis buffer was carried out in several fractions of 150 µl each. The content of mergers Protein in the fractions is checked by gel analysis and protein determination Bradford determined.

14. Protein purification by affinity chromatography

At this point, protein purification by metal affinity chromatography is to be described as an example. The enrichment was carried out of denaturation anti-cross conditions on the N-terminal 6 × His tag using the IMAC system ( "I mmobilized M et al A ffinity C hromatography") on Talon spin columns from Clontech [Palo Alto, USA]. The resin of the column was first equilibrated separately with 5 vol. 8 M urea lysis buffer, pH 8.0, sedimented for 4 min at 3000 rpm and 4 ° C. and together with the HC-1 protein supernatant for 20 min RT incubated with gentle shaking. After a renewed centrifugation, the resin was washed 3 times with 10 times the volume of 8 M urea lysis buffer, pH 8.0, for 10 min at RT with gentle shaking and centrifuged again. After the last washing step, the pellet was taken up in 1 ml of 8 M urea lysis buffer and loaded onto the column; This was subsequently washed twice with 3 times the volume of 8 M urea lysis buffer before elution with imide azole-containing 8 M urea lysis buffer was carried out in several fractions of 150 μl each. The content of fusion protein in the fractions is determined by gel analysis and protein determination according to Bradford.

15. Amino acid sequence analysis

To check the deduced amino acid sequence starting from the cDNA full length clone HC110-R, the 3 'end consisting of 688 bp (pos. 2486-3182) with a leading start codon and Kozak sequence in the pRSET B- Expression vector cloned and in competent BL21 (DE3) pLysS E. coli cells Expression of the 21 kD protein (189 AA) induced by adding 1 mM IPTG. The fusion protein provided with an N-terminal HisTag was transferred via a Talon matrix enriched and concentrated and removed using centricon tubes salted. Because of the N-terminal HisTag a C-terminal protein An sequencing 1 nM of the 21 kD HC110-R protein after the stepwise Slag-Kumpf mining (Schlack et al. 1926) in a modification by Boyd (Boyd et al. 1992) by TopLab (Martinsried). The sequencing the amino acids split off were carried out in an amino acid sequencer PROCISE 492 (PE Applied Biosystems, Weiterstadt) and was awarded the PROCISE C Reversed phase HPLC system, consisting of an ABI 140C micro gradient System and an ABI 785A UV / VIS detector, analyzed and with the PROCISE C Control software and the ABI Data Analysis Software identified.

25 µmol of the 21 kD HC110-R protein was removed by 2% trypsin (Roche Molecular Biochemicals, Mannheim) split internally at 37 ° C overnight. Overall, were four peptide fragments by comparing the HC110-R standard chromato grams after trypsin digestion with the chromatogram of the trypsin blank digest selected to sequence from tryptic autolysis products close. These four peptide fragments became an N-terminal protein An sequencing using the gradual Edman degradation process in one Modification according to Hunkapiller et al. (1983) by TopLab (Martinsried) subjected. The cleaved peptide fragments were separated by means of HPLC and fractionated. The peptide fraction blotted on Immobilon was converted into the Reaction chamber of the PROCISE 492 amino acid sequencer (Applied Bio systems, Weiterstadt) and the amino acids in a 140 C-PTH  Analyzer and UV detector 785 A (Applied Biosystems, Weiterstadt) separated. The Amino acids were quantitated by "Reversed Phase" HPLC and over Retention time comparison with a standard created before the sequence analysis chromatogram identified.

16. Cell culture lines

The following cell culture lines were obtained from the German Collection of Microorganisms and Cell Cultures GmbH [Braunschweig] [Drexler et al. 1995]:

COS-7 cells (DSM: ACC 60) - monkey, kidney

HEK-293 cells (ATCC: CRL 1573) - Human, embryonic, kidney.

17. Cultivation of the Different Cell Culture Lines

The adherent cell lines COS-7 and HEK-293 were cultivated in 110 mm tissue culture dishes [Greiner, Solingen] in a volume of 10 ml medium at 37 ° C., 5% CO ₂ and 95% atmospheric humidity. In order to maintain the cell culture, COS-7 and HEK-293 cells were cultivated in DMEM medium. The media contained 3.024 g / l NaHCO ₃ , 10% FCS, 50 U / ml penicillin and 50 µg / ml streptomycin and were warmed to 37 ° C before use. To subculture the COS-7 cells, they were stored at 4 ° C. for 2 h and then mechanically removed from the culture dish using a cell scraper. The HEK-293 cells could be detached directly from the bottom of the culture dish using a glass pipette.

18. Transient and stable transfection of eukaryotic cells

The non-liposomal transfection reagent FuGENE 6 from Roche Molecular Biochemicals [Mannheim] was used to introduce foreign DNA transiently into mammalian cells (Kurachi et al. 1998). For this purpose, approximately 0.5-1.5 × 10 ⁵ cells in 2 ml of medium were applied to 35 mm tissue culture dishes into which a sterile glass slide coated with 1% gelatin was placed for the later confocal laser scanning microscopy. The cells were cultured overnight at 37 ° C, 5% CO ₂ and 95% humidity. The medium was changed again before the transfection. For the transfection, 3 µl FuGENE 6 [Roche Molecular Biochemicals] were diluted in 97 µl serum-free medium per reaction mixture and left for 5 min. incubated at RT. 100 µl of the diluted FuGENE 6 per batch were pipetted into 1-2 µg plasmid DNA (0.5-1 µg / µl) per batch, mixed carefully and incubated for a further 15 min at RT. The complete reaction mixture was then added dropwise to the cells and the transfection mixture was evenly distributed by gently swirling the dish. The cells were cultured for 1-2 days without changing the medium. The following 3 negative controls were always carried out with each transfection: A 35 mm tissue culture dish with 0.5-1.5 × 10 ⁵ cells was not transfected, DNA was added to a second dish but no FuGENE 6 in the transfection batch and a third Shell was added to FuGENE 6, but no DNA.

For a stable transfection, the desired plasmid HC110-R was used in the right one Reading frame in the slightly modified expression vector pSecTag A [Invitrogen, Leek, NL] and pIRESneo [Clontech, Palo Alto, U.S.A.]. These vectors carry a resistance gene as a marker, so that when adding Zeozin or G418 or Bleomycin 48 72 h after transient transfection and with each subsequent medi change only successfully transfected cells that last the resistance gene product express, survive. The optimal concentration of the Zeozin- or G418- containing selection medium was previously by applying a dilution series for the respective cell line is determined.

19. Cellular localization of recombinant proteins in mammalian cells

The complete coding region of the HC110-R-DNA was translated into the HindIII / SalI- Site of pEGFP-N3 cloned to the HC110-R protein C-terminal with GFP (Green Fluorescent protein). The 137 kDa fusion protein is in  transiently transformed recipient cell lines, which is indicated by Western blot Analysis can be demonstrated. The CLSM (Confocal Laser Scanning Micros copy) shows that the HC110-R-GFP fusion protein in COS-7 and HEK-293 cells is localized in the cytoplasm and to a lesser extent also on the plasma membrane.

A Zeiss IM 35 microscope was used for confocal laser scanning microscopy [Zeiss, Oberkochen] with a Leica CLSM attachment TCS NT ["Confocal Laser Scanning Microscope Unit ", Leica Lasertechnik, Heidelberg], version 1.5.451, ver applies. The fluorescence of the GFP protein and the dye fluorescein iso thiocyanate (FITC) was measured at 488 nm using an argon laser, which is for rhodamine, Texas Red, Phycoerythrin, Alexa 568, LysoTracker ™ Red DND-99, MitoTracker ™ Red CMX Ros or Propidium iodide at 568 nm with a Krypton Laser excited. Z series of optical sections through the cell were made with a Resolution of 1024 × 1024 pixels and a layer depth of 0.5 µm scanned (Giese et al. 1995). The evaluation was carried out with the AVS software [Advanced Visual Systems Inc., Waltham, M.A., U.S.A.] and later with Adobe Photoshop 5.0 and Corel Draw 8.0 for Windows.

The fusion protein is mainly found in acidic lysosomes, as the colocalization with the help of Lysotracker ™, a sample for labeling acidic organelles, shows. The vesicles containing the HC110-R protein were increasingly observed in the vicinity of the nucleus. As a control, a fusion protein from GFP and the β ₂ -adrenergic GPCR was produced from the mouse (Accession Number X155643, Nakada et al. 1989), the transfection of which resulted in the same distribution pattern within the cell as in the case of the HC110-GFP fusion protein ,

20. EGFP constructs for transient expression

The vectors pEGFP C1 and pEGFP N3 [Clontech, Palo Alto, CA, USA] were cut with the restriction enzymes HindIII and SalI. The HC110-R "full length" cDNA was amplified using the P83EGFP_ATG HindIII 5'-primer 5'-GGT AGA AGC TTT TCG GTT TAA TAC CAA C AT G AG G-3 'and the P3057EGFP_o.TGA SalI 3'-primer 5'-CTG TGT CGA CAA CAT TTC GCC AAT AGT TAG G-3 'at an annealing temperature of 65 ° C. The PCR product was then also cut with the enzymes HindIII and SalI and ligated and transformed to produce an open reading frame between the HindIII and SalI cleavage sites of the respective vector. The resulting fusion protein with the N-terminal GFP tag was named GFP-HC110-R , that with the C-terminal EGFP tag HC110-R-GFP . A mouse β ₂ adrenergic receptor (Gen Bank Accession No .: P18762; Nakada et al. 1989) was fused to a C-terminal EGFP tag by combining the "full length" cDNA with the P117Mausβ ₂ AR XhoI 5 ' - Primer 5'-TAC CTC GAG CTG CTA ACC TGC CAG CC A TG -3 'and the P1349Mausβ ₂ AR EcoRI 3' primer 5'-TGT AGA ATT CTT CCT TCC TTG GGA GTC AAC GCT-3 'with an annealing temperature of 55/60 ° C amplified, cut with the restriction enzymes XhoI and EcoRI and linearized in the XhoI-EcoRI pEGFP N3 ligated. The "full length" cDNA of a human muscarinic receptor 1 (huM1Rez; Gen Bank Accession No .: Y00508; Allard et al. 1987), which had previously been ligated with the P70HumM1Rez XhoI 5 ', was also ligated into the pEGFP N3 vector cut from XhoI-EcoRI. -Primer 5'-ATA TCT CGA GAG CCC CAC CTA GCC ACC ATG AAC A-3 'and the P1465HumM1Rez EcoRI 3'- primer 5'-GAC GAA TTC CAT TGG CGG GAG GGA GTG CGG T-3' at 55/60 ° C was amplified. The resulting open-frame fusion proteins were named Mausβ₂AR-EGFP and huM1Rez-EGFP .

21. HC110-R-MycHis-Tag constructs for the stable or transient transfection

The vector pMyc6xHis is derived from the vector pSecTagA [Invitrogen, Leek, NL] by double digestion with the restriction enzymes NhiI and SfiI, followed by blunting of the ends and religation of the vector. The coding region of HC110-R was determined using the PCR primers P83MycTag_ATG BamHI 5'-primer 5'-ATA GGA TCC TTC GGT TTA ATA CCA AC A TG A GG-3 'and P3058MycTag_o.TGA XbaI 3'-Primer 5' -CCT GTC TAG AAA CAT TTC GCC AAT AGT TAG G-3 'amplified at an annealing temperature of 56/60 ° C, cut with the enzymes BamHI and XbaI and ligated into the BamHI-XbaI likewise linearized pMyc6xHis vector and in E. coli DH5α cells transformed. In the following, COS-7 cells were stably transfected with the construct and kept under Zeocin selection pressure.

In parallel, the HC110-RMycHis cDNA with the primers P83_ATGNotI- 5 '5'-ATA TTG CGG CCG CTT CGG TTT AAT ACC AAC ATG -3' and pMycHis_TGABamHI-3 '5'-CGC GGA TCC TAG AAG GCA CAG TCG AGG -3 'amplified, cut and ligated into the bicistronic expression vector pIRES1neo (GenBank Accession No .: U89673) [Clontech, Palo Alto, USA], which was also restricted by BamHI and NotI. The pIRES1neo vector also contains an internal ribosome binding site (IRES) of the encephalomyocarditis virus (ECMV) shortly before the start ATG of the neomycin resistance gene (Rees et al. 1996). In this way, two open reading frames, that of the HC110-R and that of the antibiotic resistance marker, are translated from only one mRNA with a human cytomegalovirus (CMV) promoter (Jackson et al. 1990; Jang et al. 1988). The selection was made by G418 [Calbiochem-Novabiochem, La Jolla, CA, USA] in COS-7 and HEK-293 cells.

22. Confocal laser scanning microscopy

A Zeiss IM 35 microscope was used for confocal laser scanning microscopy [Zeiss, Oberkochen] with a Leica CLSM attachment TCS NT ["Confocal Laser Scanning Microscope Unit ", Leica Lasertechnik, Heidelberg], version 1.5.451, ver applies. The fluorescence was at 488 nm with an argon laser, at 568 nm with excited with a krypton laser. Z series of optical sections through the cell were with a resolution of 1024 × 1024 pixels and a layer depth of 0.5 µm scanned [Giese et al. 1995]. The evaluation was carried out with the AVS software  [Advanced Visual Systems Inc., Waltham, M.A., U.S.A.] and later with Adobe Photoshop 5.0 and Corel Draw 8.0 for Windows.

23. Binding studies of α-LTX on HC110-R fragments

Proteins (20 µg / lane) were separated by SDS-PAGE (Lammli et al., 1970) and electroblotted on nitrocellulose membranes. In the case of α-LTX binding experiments were carried out at room temperature with α-LTX at a Concentration of 20 nM incubated in TST for 2 h. After washing three times with TST became an anti-α-LTX antibody (from rabbit, Calbiochem-Novabiochem) at a concentration of 0.1 µg / ml and a conjugated with peroxidase and Antibody directed against IgG from rabbits (from goat, dilution: 1: 25000) used. The antibodies were detected in all experiments with ECL (Amersham-Pharmacia).

24. Calcium imaging

The intracellular free Ca ²⁺ concentration [Ca ²⁺ ] _i was transiently transfected with the HC110-R-EGFP or β ₂ -AR-EGFP construct COS-7 and HEK293 cells with the aid of calcium imaging Method measured. 2 × 10 ⁵ COS-7 and HEK293 cells each were applied to a 42 mm cover glass coated with 1% gelatin in 55 mm tissue culture dishes with 5 ml DMEM medium (with 10% FCS and pen / strep). The transfection was carried out with the aid of the non-liposomal transfection reagent FuGENE 6 [Roche Molecular Bio chemicals, Mannheim]. 6 μl of FuGENE 6 were incubated in 200 μl of DMEM medium without FCS first for 5 min at RT and after adding 4 μg plasmid DNA for a further 15 min at RT before the entire transfection batch was pipetted dropwise into the cells. Constructs of the HC110-R "full length" clone were transfected with a GFP tag fused in the correct reading frame and the complete cDNA of the β ₂ -adrenergic receptor, which had also been provided with a GFP tag at the 3 'end. 4 µg of plasmid DNA of the pure pEGFP-N3 expression vector [Clontech, Heidelberg] and non-transfected cells to which the transfection reagent FuGENE 6 without DNA was added were used as controls.

The cells were loaded with 1 μM fura-2 / acetoxymethyl ester (Fura-2 / AM) [Sigma, Deisenhofen] 48 h after the transfection in a Na ⁺ HBS solution (150 mM NaCl; 5.4 mM KCl; 1 , 8 mM CaCl ₂ ; 0.8 mM MgSO ₄ .7 H ₂ O; 20 mM glucose; 20 mM Hepes in H ₂ O, pH 7.3) for 30 min at 37 ° C, 5% CO ₂ and 95% Humidity. After the incubation, the cells loaded with Fura-2 / AM were placed in an inverted microscope [Zeiss, Aciovert; Germany] and viewed in parallel in a digital "imaging" fluorescence microscope (PTI) and at a wavelength of 440 or 490 nm into transfected and non-transfected cells. On average, 5-7 transfected and non-transfected cells were selected by setting an ROI ("region of interest"). The absorbance was measured at 340 nm (calcium-bound Fura-2 / AM) and 380 nm (free Fura-2 / AM), the emission at 510 nm. The evaluation was carried out with the Image Master 1.x software Formation of the quotient of 340: 510 nm, 380: 510 nm and the ratio of 340/380: 510 nm ("background corrected images") as a function of the added agent.

Agents were always added 6 min after the start of the measurement by taking a corresponding volume of Na ⁺ -HBS buffer and pipetting the agents directly into the sample vessel for a further 30 to 50 min. The total volume in the sample vessel was a constant 1.5 ml during the entire experiment. The COS-7 cells expressing the fusion protein HC110-R-GFP were initially 30 nM and 75 nM α-latrotoxin [α-LTX, RBI. Natick, USA], different concentrations of α-LTX (7.5 nM; 25 nM; 50 nM, 75 nM, 90 nM and 120 nM) were used to determine the dose dependence on the HEK-293 cells expressing HC110-R-GFP. , or to determine the optimal active concentration, various concentrations of the cyclic depsipeptide BAY44-4400 (100 ng / ml (89.3 nM), 333 ng / ml (297 nM); 400 ng / ml (357 nM), 1 µg / ml (893 nM), 10 µg / ml (8.9 µM) in 0.1% DMSO) added 6 min after the start of the experiment. In some experiments, the HEK-293 cells were treated with 4 or 400 ng / ml BAY44-4400 or with 4 or 400 ng / ml of the anthelmintically inactive optical antipode PF1022-001 in Na ⁺ -HBS at 37 for 90 min ° C, 5% CO ₂ and 97% humidity before and the cells loaded after 60 min by adding 1 µM Fura-2 / AM to the BAY44-4400 solution or to the PF1022-001 for the remaining 30 min. After the cells had been introduced into the apparatus, 75 nM α-LTX were also added here 6 minutes after the start of the experiment. The optimal α-LTX concentration of 75 nM was tested on HEK-293 cells which express the β ₂ -adrenergic receptor with C-terminal GFP-Tag (β ₂ -R-GFP). CdCl ₂ (1 µM and 10 µM each) were dissolved in Na ⁺ -HBS, nifedipine (15 µM) and EGTA (2 mM) in 0.1% DMSO. CdCl ₂ and nifedipine were added directly at the start of the experiment and before the addition of α-LTX, EGTA was added both at the start of the experiment and 4 minutes after the addition of α-LTX. Α-LTX dissolved at a concentration of 300 nM in 50 mM Tris-HCl, pH 8.0; BAY44-4400 was first dissolved in pure DMSO (stock solutions between 0.004-10 µg / ml). The stock solutions were adjusted to the desired concentration in the Na ⁺ -HBS buffer. The maximum detachable amount of the active ingredient BAY44-4400 at a final concentration of 0.1% DMSO was determined by applying a dilution series in Na ⁺ -HBS. Neither 0.1% DMSO nor any other test component interacted with Fura-2 / AM at the selected concentrations.

The data was generated using the Excel 98 spreadsheet program evaluates. The results result from at least 2-4 reproduced experiments 4-8 transfected and non-transfected cells (see standard deviations).

The intracellular calcium concentration was determined according to Grynkiewicz et al. (1985) after previous calibration according to McCormack et al. (1991).  

25. Binding of PF1022A to HC110-R

In the following, the binding of PF1022A and the morpholine derivative BAY44-4400 to HC110-R is to be shown by SDS-PAGE, ligand precipitation and analytical flow cytometry. In addition, it should be checked whether PF1022A and its derivatives - similar to α-LTX - lead to changes in the [Ca ²⁺ ] _i in HC110-R transfected HEK-293 cells or whether the HC110-R mediated α-LTX "signaling " influence.

Occasionally show antibodies or proteins that are evident in immunofluorescence Give signals, no reaction when using the immunoblot procedure. There denaturation of the proteins is necessary to carry out SDS-PAGE, Conformation-dependent epitopes may be destroyed. Antibodies or proteins that specifically recognize such epitopes can therefore no longer bind. So neither with the biotinylated active ingredient PF1022A, even with the morpholine derivative, which is more soluble than the PF1022A BAY44-4400 identified an HC110-R specific binding in a Western blot become. BAY44-4400 differs from PF1022A in that it has 2 morpholine residues covalently bound to the phenyl rings of the two D-phenyllactyl residues of PF1022A they are. However, if the proteins in the SDS separating gel are renatured with the help of Urea, which is a partial ent by eliminating hydrophobic interactions If the SDS is removed, an HC110-R specific can be used in the Western blot Binding can be demonstrated. For this, total protein from untransfected, from HC110-R-Myc / His transiently expressing HEK-293 cells and from the in E. coli expressed 54 kDa N- and 21 kDa C-terminus of HC110-R separated by SDS-PAGE, renatured and blotted as usual. The Immunodetection was carried out after the incubation with the ligand BAY44-4400, with a rabbit anti-PF1022A-KLH immune serum and a goat anti-rabbit Chen IgG-HRP antibodies. Furthermore, a partially renatured SDS polyacrylic was used amide gel with the protein fractions not transfected, HC110-R-Myc / His transient transfected HEK-293 cells and the N-terminus of  HC110-R and total protein from H. contortus blotted with PF1022A biotin incubated and detected via streptavidin-HRP. Both blots each had a distinct one 116 kDa band in the HC110-R-Myc / His stably expressing HEK-293 Cells, whereas the traces with untransfected cells have no band in of this height. Furthermore, both the morpholine derivative BAY44-4400, as well as the biotinylated PF1022A at the 54 kDa N-terminus of HC110-R, the 21 kDa C-terminus showed no specific PF1022A- Binding. Through the biotinylated PF1022A in the total protein fraction adult H. contortus nematodes a total of 2 bands are detected: one 110 kDa band and another approximately 88 kDa band. The latter acts it’s most likely a biotinylated protein, as it’s already known for Nematode Heterakis spumosa with 83 kDa has been described, especially since it is in the opposite set to the 110 kDa band is detected even if the detection only via streptavidin-HRP. HC110-R-Myc / His stably transfected In contrast, HEK-293 cells do not have a size of 116 kDa under streptavidin-HRP alone Tie up more. Furthermore, it could be excluded that the used Goat anti-rabbit IgG-HRP secondary antibody alone unspecific signals caused.

To further verify these results, the following ligand precipitation was performed carried out. Magnetic Dynal M-280 streptavidin-coupled 2 mg and 4 mg, respectively Beads were mixed with 500 µg of biotinylated PF1022A. A control approach with 4 mg Dynal beads, TST buffer was added instead of PF1022A. about Shot PF1022A biotin was removed by magnetic separation before one Mixture of those expressed in E. coli and previously via affinity chromatography purified HC110-R N and C terminus was added. After renewed likes netic separation to separate unbound HC110-R fragments An aliquot based on 2 mg and 4 mg Dynal-Beads was added via SDS-PAGE separates and blots. The precipitate was detected via the N-terminal His-Tag fused with a mouse anti-His IgG and a rabbit anti mouse IgG-HRP antibody. Only the N-terminal 54 kDa region of HC110-R will  precipitated by PF1022A, consequently the C- not bound to PF1022A Terminus of HC110-R previously removed by magnetic separation. nonspecific Binding of the N- or C-terminus of HC110-R to the streptavidin-coupled Beads were excluded by not using a biotinylated reaction PF1022A was added.

The binding of the ligand PF1022A in vivo to HC110-R was also checked using the FACS analysis ( Fig. 13). For this purpose, HEK-293 cells were transiently transfected with HC110-R-GFP or GFP-HC110-R in triplicate or else transiently co-transfected with HC110-R-Myc / His and GFP. For control purposes, HEK-293 cells that were not transfected and transiently transfected with the β ₂ -R-GFP or the M1-R-GFP were used. 24 h after the transfection, the cells were incubated with biotinylated PF1022A and PF1022A-bound cells were detected via strep tavidin-phycoerythrin and finally fixed. A negative control HC110-R-GFP of transfected cells without PF1022A-biotin was only incubated with strepta vidin-phycoerythrin.

The fluorescence analysis of 10,000 HEK-293 cells each was carried out with the FACScan at an excitation wavelength of 488 nm with regard to their cell size, granules rity and fluorescence staining. Like TRITC, phycoerythrin has one with EGFP overlapping absorption spectrum with sufficiently separated emissions spectra so that both fluorochromes are excited at only one wavelength can. Cell debris was created by placing a "gate" on the main cell popu ration excluded from the measurement of the fluorescence intensity. For quantitative Evaluation was limited to negative, undyed and positive, GFP-fluores ornamental cells based on the non-transfected and GFP-transfected cells established. The 4.6% green fluorescent, non-transfected HEK-293 cells (Autofluorescence) were scanned from the other GFP fluorescent samples pulled; and the value for the non-specific staining of the cells by means of Phyco erythrin-coupled streptavidin (5.4%) was from all red fluorescent cells deducted (Tab. 1).  

Table 1

Detection of PF1022A binding to HC110-R transfected HEK-293 cells by FACScan

If one calculates the percentage of green fluorescent cells that were also red fluorescent after deduction of the autofluorescence or the unspecific red staining by streptavidin-phycoerythrin, 41.2% of the cells expressing HC110-R-GFP bind, 26.8% of the GFP-HC110 -R expressing cells and 44.5% of the cells co-transfected with HC110-R-Myc / His and GFP and PF1022A, since only 7.5% of the GFP expressing cells bind, 9.9% of the β ₂ -R-GFP expressing cells and 19.2% of the cells expressing M1-R-GFP PF1022A ( Fig. 13).

26. Interactions of PF1022A derivatives with HC110-R mediated α-LTX signal transduction

PF1022A shows neuropharmacological in vitro activity between 10 ^-9 -10 ^-3 mg / ml depending on the type of nematode. In order to determine a possible interference of PF1022A and its derivatives with HC110-R and the α-LTX "signaling" mediated by HC110-R, the influence of BAY44-4400, a more soluble one, was determined with the help of the Ca ²⁺ imaging method Morpholine variant of PF1022A, examined for HC110-R transiently or stably expressing HEK-293 cells. The optical antipode to PF1022A, PF1022-001, which was more than 100 times less effective in vitro and in vivo, was used as a control. BAY44-4400 and PF1022-001 were initially dissolved in pure DMSO due to their hydrophobicity. The strain concentration was chosen so that only 0.1% DMSO was present in the test batch. In control experiments for DMSO concentrations up to and including 0.1%, it could be excluded that they would impair the test results in untransfected HEK-293 cells transfected like HC110-R-GFP ( Fig. 14A). Fura-2-loaded, non-transfected cells and cells transfected with HC110-R-GFP were stimulated with 100, 300 or 400 ng / ml and 1 or 10 µg / ml BAY44-4400 on the one hand and with the same concentrations PF1022-001 on the other. At no concentration, a Ca ²⁺ -mediated response could be determined over the entire period of 50 min. Fig. 14B and C show examples of the stimulation of HC110-R-GFP and M1-R-GFP of transfected cells with 400 ng / ml BAY44-4400 and PF1022-001. Since the PF1022A derivatives may also be absorbed by the cells and thus can interfere with signal transduction pathways and because the PF1022A derivatives remain in the worm for a longer period of time depending on the parasite, the cells were preincubated with the derivatives for 90 min in some experiments. Cells preincubated for 90 min with 4 ng / ml or 400 ng / ml BAY44-4400 or PF1022-001 showed no change in the [Ca ²⁺ ] _i ( Fig. 14D). With direct addition of concentrations from 300 ng / ml BAY44-4400 - in contrast to the optical antipode - a slight, but reversible cell swelling as a direct effect on the anthelmintic in HC110-R-GFP transfected HEK-293 cells could be achieved can be observed on a monitor which shows the cells enlarged 40 times.

According to the latest experiments, piperazine is known to synergistically enhance the effect of PF1022A / BAY44-4400 in in vitro and in vivo experiments with Heterakis spumosa. With simultaneous administration of 400 ng / ml BAY44-4400 with 1 or 10 µM piperazine ( Fig. 14F), a change in the [Ca ²⁺ ] was not found in HEK-293 cells that were not transfected or transiently transfected in HC110-R-GFP. _{i are} found. The control batch with 10 μM piperazine likewise did not lead to any change in the Ca ²⁺ household in untransfected and HC110-R-GFP-transfected cells ( FIG. 14E).

In contrast, both BAY44-4400 and the optical antipode PF1022-001, in the presence of 75 nM α-LTX, influenced α-LTX-induced signal transduction from HEK-293 cells expressing HC110-R-GFP, albeit in different ways extent (Fig. 14B-e). In a control experiment in which HC110-R-GFP or M1-R-GFP transfected cells were added 6 min before stimulation with 75 nM α-LTX 0.1% DMSO, it could be excluded that this was true for BAY44-4400 and also PF1022-001 solvents used in this concentration had an influence on the α-LTX-induced change in [Ca ²⁺ ] _i . In contrast to the cells expressing M1-R-GFP, HC110-R-GFP showed the biphasic course already described with comparable values for [Ca ²⁺ ] _i ( Fig. 15A) after addition of α-LTX. Incubating cells expressing HC110-R-GFP 6 min before the addition of α-LTX with 4 ng / ml BAY44-4400 reduced the described influence of the α-LTX on the Ca ²⁺ concentration: that of α-LTX induced first small increase in [Ca ²⁺ ] _i disappeared and the second delayed peak was reduced to 44 ± 60 nM Ca ²⁺ 14 min after the addition of α-LTX ( Fig. 15B). In the presence of 4 ng / ml PF1022-001 - added 6 min before stimulation with α-LTX - there was a rapid immediate increase of 103 ± 11.5 nM Ca ²⁺ ( Fig. 15B). If 400 ng / ml BAY44-4400 was added to cells expressing HC110-R-GFP 6 min before the addition of α-LTX, there was a delayed increase in - similar to 4 ng / ml BAY44-4400 ( Fig. 15B) [Ca ²⁺ ] _i by 49 ± 4.2 nM after 23 min, which dropped 8 min later to a slightly increased plateau compared to the initial values by 11 ± 1.4 nM ( Fig. 15C). If 400 ng / ml PF1022-001 were added 6 min before the incubation with 75 nM α-LTX, there was a rapid Ca ²⁺ increase by 112 ± 14.3 nM Ca ²⁺ ( Fig. 15C), as it could already be observed with the addition of 4 ng / ml PF1022-001 ( Fig. 15B). In another experiment, HC110-R-GFP-expressing HEK-293 cells were preincubated with 4 ng / ml ( Fig. 15D) or 400 ng / ml BAY44-4400 or PF1022-001 ( Fig. 15E) for 90 min.

Unbound or ingested drug was carefully washed away after the cells were incubated and loaded with Fura-2 / AM before the cells were stimulated with 75 nM α-LTX. HC110-R transfected cells preincubated with the optical antipode PF1022-001 showed an increase in the [Ca ²⁺ ] _{i of} 4 ng / ml PF1022-001 by 102 ± 6.4 nM ( Fig. 15D) and an increase of 400 ng / ml compare bare increase of 109 ± 8.4 nM Ca ²⁺ (Fig. 15E). This Ca ²⁺ increase observed just a few minutes after α-LTX addition was similar to the rapid changes in [Ca ²⁺ ] _i already observed in Fig. 15B and C when PF1022-001 was added immediately before stimulation with α-LTX , BAY44-4400, on the other hand, showed different answers regarding the α-LTX-induced Ca ²⁺ influx: If cells expressing HC110-R-GFP were incubated with 4 ng / ml BAY44-4400 for 90 min before, it was caused by α-LTX induction to a maximum increase in [Ca ²⁺ ] _i by 95 ± 20.5 nM at 10 min before it dropped to an extended plateau increased by 23 ± 2.6 nM Ca ²⁺ ( Fig. 15D). In pre-incubation with 400 ng / ml BAY44-4400 there was a Ca ^{2+ increase in} the [Ca ²⁺ ] _i by a maximum of 65 ± 7.5 nM ( Fig. 15D) after α-LTX stimulation ( Fig. 15D) min delayed (Fig. 15C) versus occurred before incubation with 4 ng / ml BAY44-4400.

If HC110-R-Myc / His expressly added HEK-293 cells 6 min before the addition of α-LTX 400 ng / ml BAY44-4400, the observed direct increase in [Ca ²⁺ ] _i by 267 becomes ± 45.8 nM completely inhibited (Fig. 15F). In contrast, the addition of 400 ng / ml PF1022-001 with a Ca ²⁺ increase of 209 ± 33.4 nM Ca ²⁺ does not lead to a significant change in the [Ca ²⁺ ] _i ( Fig. 15F).

To show the specific influence of BAY44-4400 on the α-LTX-mediated Ca ²⁺ signaling in cells expressing HC110-R, the following control experiments were carried out: First, non-transfected HEK-293 cells with 1 mM carbamylcholine chloride (Carbachol), a ligand for muscarinic acetylcholine receptors, stimulated in the presence and absence of 400 ng / ml BAY44-4400 ( Fig. 15G). In both cases there was a comparable immediate increase in [Ca ²⁺ ] _i by endogenous, natural muscarinic acetylcholine receptors by 58 ± 8.1 nM Ca ²⁺ for HEK-293 cells in the presence of BAY44-4400 and by 53 ± 6.8 nM Ca ²⁺ in the absence of the anthelmintic. The additional transient transfection of HEK-293 cells with the C-terminal GFP fused, human M1 muscarinic acetylcholine receptor (M1-R-GFP) resulted in over non-transfected cells (Fig. 15G) slight increase in [Ca ²⁺ ] _i by 89 ± 5.5 nM with on and around 85 ± 6.1 nM Ca ²⁺ in the absence of 400 ng / ml BAY44-4400 ( Fig. 15H). No significant influence of BAY44-4400 on the M1-R-GPCR was observed. If non-transfected HEK-293 cells were stimulated, the endogenously present natural β ₂ -adrenergic receptors with 1 mM isoproterenol or the nicotinic receptors with 1 mM arecolin - as already described for carbachol - in the presence and absence of 400 ng / ml BAY44- 4400, there was no significant difference in the [Ca ²⁺ ] _i ( Fig. 15I). Isoproterenol induced an increase in [Ca ²⁺ ] _i of 45 ± 4.7 nM in on and an increase of 48 ± 5.7 nM Ca ²⁺ in the absence of BAY44-4400. As a result of the arecolin stimulation, there is an increase of 28 ± 4.1 nM Ca ²⁺ in the presence of BAY44-4400, and in the absence of the active ingredient by 27 ± 3.5 nM Ca ²⁺ ( Fig. 15I).

27. Obtaining antibodies

Female NMRI mice and rabbits (chinchilla bastards) were used to obtain antibodies. For the immunization of the NMRI mice, 3 × 15 µg of the purified 21 kD C-terminal HC110-R protein fragment were dissolved in 100 µl PBS ^- together with 100 µl FCA and on day 1, 8 and 15 two naive NMRI- Mice injected subcutaneously. Blood was drawn and serum obtained on day 23.

The blood obtained by cardiac puncture was initially used for serum production Incubate for 1 h at 37 ° C and then for at least 2 h at 4 ° C. Then were the cellular components twice for 10 min at 3000 rpm and 4 ° C in the Beckman GPKR centrifuge pelleted and then the supernatant for 45 min  56 ° C inactivated. After another 10 minutes centrifugation at 13000 rpm and 4 ° C in the Heraeus Biofuge 15R the serum could be removed and up to stored for further use at -20 ° C.

Two rabbits were initially given 50 µg of the C-terminal 21 kD HC110-R protein and the 54 kD N-terminal HC110-R protein in an equal proportion of PBS ^- and FCA after the first blood was drawn and the pre-immune serum was obtained Suspension injected subcutaneously. Two further immunizations with 100 µg antigen each took place on day 31 and day 79 after the first immunization. A first blood sample was taken on day 43. A second blood draw and serum collection were made on day 98 after the first immunization.

Description of the pictures Fig. 1 HC110-R mRNA and protein

• A) Northern blot analysis using total RNA Haemonchus contortus and the 3.6 kbp cDNA from HC110-R.
• B) 10% SDS-PAGE fluorogram of ³⁵ S-labeled HC110-R protein translated in vitro. In vitro translation of 1 µg in vitro transcribed HC110-R mRNA (HC110-R), negative control without HC110-R mRNA (control), 1 µg luciferase mRNA as positive control (luciferase).

Fig. 2 Full length cDNA sequence of HC110-R and derived amino acid sequence

The Kozak motif (Kozak, 1989) and the polyadenylation signal of HC110-R (GenBank Accession No .: AJ272270) are underlined; the start codon in position 100 is printed in bold. Signal peptide (residues 1-21, bold), lectin-like sequence (residues 22-125, dotted), thr-stretch (residues 128-147, gray background); Cysteine-rich area of the structure CX ₉ WX ₁₂ CX ₉ WXCX ₅ WX ₉ CX ₃ W (residues 166-221, curved line, Cys and Trp residues additionally bold); 4-Cys region of the structure CXWWX ₆ WX ₄ CX ₁₁ CXC (residues 478-524, dashed, Cys and Trp residues additionally bold); the 7 transmembrane regions (between residues 563-772, bold and underlined); the proline-rich stretch (residues 845-861, shaded gray), the PEST region (residues 915-933, shaded gray); the N-glycosylation site (residues 26, 499 and 862, bold); the highly conserved putative disulfide bond of the Cys-Cys pair between secretin GPCRs (positions 595 and 666, bold and double underlined).

Fig. 3 Alignment of the Derived Amino Acid Sequences of H. contortus HC110-R and Caenorhabditis elegans Cosmid clones B0457 (CE- B0457: GenBank Accession No .: Z54306)

Signal peptide (SP, residues 1-21, bold), lectin-like sequence (lectin, residues 22-125, dotted); Thr-Stretch (T-rich, remnants 128-147, gray background); Cys-rich region of the structure CX ₉ WX ₁₂ CX ₉ WXCX ₅ WX ₉ CX ₃ W (C signature, residues 166-221, curved line, Cys and Trp residues additionally bold); 4-Cys motif of the structure CXWWX ₆ WX ₄ CX ₁₁ CXC (4 C region, residues 478-524, dashed, Cys and Trp residues additionally bold); the 7 transmembrane regions (between residues 536-772, bold and underlined); the pro-rich stretch (P-rich, remnants 845-861, highlighted in gray); the PEST region (PEST, remains 915-933, highlighted in gray); the N-glycosylation sites (residues 26, 499 and 862, bold); the highly conserved putative disulfide bond of the Cys-Cys pair between secretin GPCRs (positions 595 and 666, bold and double underlined). Identical amino acids are marked with asterisks, very closely related amino acids with a colon, related amino acids with a single point.

Fig. 4 Alignment of the 7-transmembrane receptor HC110-R and several other receptors of the secretin subfamily

HC110-R: Haemonchus contortus heptahelical orphan transmembrane receptor (Accession No .: AJ272270); BTLAT3: Bos taurus latrophilin-3 (multiple splicing Variants, accession no .: G4164053-G4164075); RNLAT1: Rattus norvegicus Latrophilin-1 (Accession No .: U78105, U72487); MMEMR1: Mus musculus EMR1 Hormone receptor precursor (Accession No .: Q61549); HSCD97: Homo sapiens Leukocyte activation antigen CD97 (Accession No .: P48960); MMCADH: M. musculus cadherin 7 transmembrane receptor precursor (accession no .: G3800738); XLXRF1: Xenopus laevis corticotropin releasing receptor precursor (Accession No .: O42602); RNVIP1: R. norvegicus vasoactive intestinal polypeptide receptor Forerunner 2 (Accession no .: Q02643). The seven transmembrane domains are gray  (I-VII) and the highly conserved putative disulfide bond is shown in bold provides.

Fig. 5 Alignment of the Derived Amino Acid Sequences of H. contortus HC11-R and R. norvegicus Latrophilin-1 (GenBank Acc. No. U78105, U72487)

Signal peptide (SP, residues 1-21, bold), lectin-like sequence (lectin, residues 22-125, dotted); Thr-Stretch (T-rich, remnants 128-147, gray background); Cys-rich region of the structure CX ₉ WX ₁₂ CX ₉ WXCX ₅ WX ₉ CX ₃ W (C signature, residues 166-221, curved line, Cys and Trp residues additionally bold); 4-Cys motif of the structure CXWWX ₆ WX ₄ CX ₁₁ CXC (4 C region, residues 478-524, dashed, Cys and Trp residues additionally bold); the 7 transmembrane regions (between residues 536-772, bold and underlined); the pro-rich stretch (P-rich, remnants 845-861, highlighted in gray); the PEST region (PEST, remains 915-933, highlighted in gray); the N-glycosylation sites (residues 26, 499 and 862, bold); the highly conserved putative disulfide bond of the Cys-Cys pair between secretin GPCRs (positions 595 and 666, bold and double underlined). Identical amino acids are marked with asterisks, very closely related amino acids with a colon, related amino acids with a single point.

Fig. 6 Structure of rat HC110-R protein and latrophilin-1

• A) Hydrophobicity plot of HC110-R with the 7-transmembrane domain.
• B) Protein structure of HC110-R with the following characteristic motifs:
  Signal sequence (SP), lectin domain (lectin), thr-stretch (T-stretch), Cys motif (signature), 4-Cys region (4C region), 7-transmembrane domain (1-7, black), the pro-rich motif (P-rich) and the PEST sequence (PEST). The putative glycosylation sites are shown below the diagram (N), the Cys residues of the 4C region and the two conserved Cys residues of the putative disulfide bond are also shown (thin line without capital letters).
• C) Protein structure of latrophilin-1. Latrophilin-1 has no thr stretch, but contains an additional olfactomedin binding motif (olfactomedin), a Pro-Thr region (P-T region), a linker domain (left) and one long region containing several repeats (long).

Fig. 7 Expression of HC110-R-GFP in HEK-293 and COS-7 cells

• A) Total protein from transient with GFP (GFP) or HC110-R-GFP (HC110-R) transfected and non-transfected (n.d.) HEK-293 and COS-7 cells was isolated 24 h after transfection using the Trizol method.
• B) Total protein from transiently transfected with β ₂ -R-GFP (β ₂ -R) or M1-R-GFP (M1-R) as well as non-transfected (nt) HEK-293 cells was transferred to the trizole 24 h after the transfection Method isolated. SDS marker bands at 116, 90, 70 and 55 kDa (marker).

20 µg of total protein per lane were each on a 10% SDS polyacrylamide gel separated and blotted onto a nitrocellulose membrane. After blocking in Rotisslock solution overnight was immunodetected with the monoclonal Mouse anti-GFP IgG primary (0.4 µg / ml) and the monoclonal rabbit anti Mouse IgG-HRP secondary antibody (1: 25000) by the ECL system.  

Fig. 8 Cellular localization of HC110-R and β ₂ -R in transfected COS-7 cells by CLSM

COS-7 cells were transfected with HC110-R-GFP and β ₂ -R-GFP, both of which carry a C-terminal GFP tag. The CSLM was performed 24 hours after the transfection. The bar corresponds to 10 µm.

• A) HC110-R-GFP is on the plasma membrane and in cytoplasmic Compartments expressed. In the vicinity of the nucleus there may be a compaction of cytoplasmic vesicles.
• B) CSLM colocalization of the green HC110-R-GFP protein with acid Lysosomes stained with LysoTracker Red DND-99 at 37 ° C for 1 h.
• C) β ₂ -R-GFP transfected cells show a GFP fluorescence pattern similar to HC110-R-GFP, or as described under point (A). The receptor can be located on the plasma membrane and in vesicles and can be partially localized with acid lysosomes, stained with LysoTracker Red DND-99 at 37 ° C for 1 h.
• D) Expressed β ₂ -R-GFP partially colocalized with the endoplasmic reticulum, stained with monoclonal anti-KDEL antibodies.

Fig. 9 Binding of α-latrotoxin to HC110-R

• A) 5 µg total protein of isolated Latrodectus revivensis poison glands (1) and pure 130 kDa α-LTX (2), also 40 µg total protein stable with the HC110-R-Myc / His construct in the pIRES1neo vector HEK-293 cells (3), untransfected cells (4) and that in E. coli in reduced 54 kDa N-terminus (6) and 21 kDa C-terminus (7) of HC110-R were separated in a 10% SDS polyacrylamide gel,  blotted and blocked overnight. The denatured proteins were kept for 2 h incubated with 20 nM α-LTX and possible binding of α-LTX with a Rabbit anti-α-LTX IgG- (1: 5000), a goat anti-rabbit IgG- HRP antibody (1: 25000) and the ECL system detected.
• B) 40 µg total protein of the C- (1) and N-terminus (2) induced in E. coli of HC110-R as well as non-transfected (3) and stable with HC110-R-Myc / His transfected HEK-293 cells (4) and adult H. contortus nematodes (5) was separated in a 10% SDS polyacrylamide gel and with a Partially renatured 4 M urea-containing renaturation buffer. The blot was blocked overnight, incubated with 20 nM pure α-LTX and α-LTX binding proteins with a rabbit anti-α-LTX IgG antibody (1: 5000), biotin protein A (1: 100), streptavidin peroxidase (1: 3000) and proven by the ECL system.

Fig. 10 Dose dependence of α-LTX on the HC110-R mediated Ca ²⁺ signaling

HEK-293 cells were exposed 48 h after transient transfection with HC110-R-GFP load with 1 µM Fura-2 / AM for 30 min. The cells were stimulated for 6 min start the measurement with different α-LTX concentrations for more 44 min. The quotient from 340/380 nm (ratio) was measured as a function of currently. n indicates the number of selected cells.

• A) Cells expressing HC110-R-GFP were exposed to 7.5 nM (black line) or 25 nM α-LTX (gray line) stimulated.
• B) HC110-R-GFP expressing cells were 50 nM (gray line) or 75 nM α-LTX (black line) added.  
• C) HC110-R-GFP expressing cells were with 90 nM (gray line) or 120 nM α-LTX (black line) stimulated.
• D) On the abscissa is the [Ca ²⁺ ] _i in nM, on the ordinate are the HEK-293 cells used to stimulate HC110-R-GFP expressing α-LTX concentrations (0, 7.5, 25 , 50, 75, 90 and 120 nM) for the first fast peak (1st peak, gray line) and the second delayed peak (2nd peak, black line).

Fig. 11 Ca ²⁺ imaging of HC110-R transfected HEK 293 cells

• A) C-terminally tagged HC110-R (HC110-R-GFP): black line.
  HC110-R (GFP-HC110-R) tagged N-terminal with GFP: gray line.
• B) 75 nM α-LTX with untransfected HEK-293 cells (n.d., black line) and GFP transfected cells (GFP, gray line).
• C) 75 nM α-LTX with cells which are labeled with the C-terminally tagged human M1 muscarinic acetylcholine receptor (M1-R-GFP, gray line) and with the C-terminally tagged β ₂ -adrenergic acetylcholine receptor from the mouse (β ₂ - R- GFP, black line) were transfected.
• D) HC110-R-GFP transfected cells were 6 minutes before α-LTX addition (75 nM) treated with 2 mM EGTA (+ EGTA, gray line). In control EGTA was omitted (-EGTA, black line).
• E) 2 mM EGTA was added 10 min after α-LTX addition (75 nM) (+ EGTA, gray line). No EGTA was added to the control batch (-EGTA, black line).  
• F) As a control, non-transfected (n.d., gray line) and HC110-R- Cells expressing GFP (black line) over 30 minutes at 2 nM EGTA offset.

Fig. 12 Interference in signal transmission caused by α-LTX BAY44-4400

HEK-293 cells were transiently transfected with GFP-tagged HC110-R protein and stimulated 48 hours after the transfection (arrows). Ca ²⁺ imaging was carried out for 50 minutes ( Fig. AD), control experiments with various endogenous natural receptors of untransfected HEK-293 cells for 20 minutes. n = number of cells.

• A) Add 400 ng / ml BAY44-4400 (black line) or PF1022-001 (gray line) to the cells. As a control, the cells were treated with Na ⁺ -HBS-HEPES buffer (HBS) after the addition of BAY44-4400 or PF1022-001, which also served as a solvent for the respective substances.
• B) Signal transmission due to α-LTX (75 nM) in the presence of 4 ng / ml BAY44-4400 (black line) and PF1022-001 (gray line).
• C) α-LTX-related signal transmission in cells that are used for 90 minutes 4 ng / ml BAY44-4400 (black line) or PF 1022-001 (gray line) were incubated. The substances were reapplied before the addition of α-LTX away.
• D) α-LTX-related signal transmission transfected in HC110-R-GFP Cells for 90 minutes with 400 ng / ml BAY44-4400 (black line) or PF1022-001 (gray line) were incubated. The substances were before the addition of α-LTX removed.  
• E) Non-transfected HEK-293 cells were treated with 1 mM carbamylcholine chloride (carbachol) for 100 s in An (400 ng / ml BAY44-4400, black Line) or absence of 400 ng / ml BAY44-4400 (gray line, -BAY44-4400) stimulated.
• F) Non-transfected HEK-293 cells were treated with 1 mM isoproterenol and with 1 mM arecoline for 100 s each in An (400 mg / ml BAY44-4400, black Line) or absence of 400 ng / ml BAY44-4400 (-BAY44-4400, gray Line) stimulated.

Fig. 13 Detection of PF1022A binding to HC110-R transfected HEK-293- Cells by FACScan

5 × 10 ⁵ each not transfected, transiently transfected with GFP, HC110-R-GFP, GFP-HC110-R, β ₂ -R-GFP and M1-R-GFP and transiently co-transfected with HC110-R-Myc / His and GFP HEK-293 cells were incubated 24 h after the transfection with 0.5 μg / ml PF1022A biotin and streptavidin-phycoerythrin (1: 300) and then fixed. As a negative control, HC110-R-GFP-transfected cells without PF1022A biotin were only incubated with streptavidin-phycoerythrin. After deducting the autofluorescence, i.e. the 4.6% non-transfected cells (nt) in the green channel from all green fluorescent cells and the 4.4% unspecific staining in the red channel due to streptavidin-phycoerythrin, the percentage of GFP-fluorescent cells (GFP , HC110-R-GFP, GFP-HC110-R, β ₂ -R-GFP, M1-R-GFP and HC110-R-Myc / His + GFP) that bound PF1022A (PF1022A binding in%). The standard deviation results from the determination of the mean values of triple approaches.

Fig. 14 Influence of BAY44-4400 and the optical antipode PF1022-001 on the HC110-R mediated Ca ²⁺ - "signaling"

The cells were loaded with 1 μM Fura-2 / AM in non-transfected HEK 293 cells and 48 h after transient transfection with HC10-R-GFP or M1-R-GFP. The quotient from 340/380 nm (ratio) was measured depending from the time. n indicates the number of selected cells.

• A) cells that were not transfected (nt, gray line) and HC110-R-GFP transfected (HC110-R-GFP, black line) were treated with 0.1% of the solvent DMSO in Na ⁺ -HBS buffer after 6 min as a control ,
• B) HC110-R-GFP (black line) and M1-R-GFP (gray line) transfected Cells were stimulated with 400 ng / ml BAY44-4400 after 6 min.
• C) Like (B), HC110-R-GFP (black line) and M1-R-GFP (gray line) transfected cells were stimulated with 400 ng / ml PF1022-001 after 6 min.
• D) HC110-R-GFP expressing cells were at 90 ng / ml for 90 min BAY44-4400 (black line) or PF1022-001 (gray line) pre-incubated and washed away unbound substances before measurement.
• E) Non-transfected (n.t., gray line) and expressing HC110-R-GFP Cells (HC110-R-GFP, black line) became 10 µM piperazine after 6 min added.
• F) Like (E), but was not transfected (n.t., gray line) and HC110-R-GFP expressing cells (HC110-R-GFP, black line) after 6 min Mixture of 10 µM piperazine and 400 ng / ml BAY44-4400 administered.

Fig. 15 Interactions of the HC110-R mediated α-LTX "signaling" through PF1022A derivatives

The cells were loaded with 1 μM Fura-2 / AM in non-transfected HEK-293 cells and 48 h after the transient transfection with HC110-R-GFP or M1-R-GFP. The amount of α-LTX used was always 75 nM. For the experiments with the control substances, the cells were washed around with a flow rate of 1.6 ml Na ⁺ -HBS / min. The quotient from 340/380 nm (ratio) was measured as a function of time. n indicates the number of selected cells.

• A) Cells expressing HC110-R-GFP (black line) and M1-R-GFP (gray line) were treated with 0.1% DMSO 6 min before the α-LTX stimulation in order to rule out that DMSO influences α- LTX induced changes in [Ca ²⁺ ] _i decreases.
• B) HC110-R-GFP cells were transfected 6 min before the α-LTX addition 4 ng / ml BAY44-4400 (black line) or PF1022-001 (gray line) ver puts.
• C) Like (B), only HC110-R-GFP expressing cells was 6 min before Stimulation with α-LTX 400 ng / ml BAY44-4400 (black line) or PF1022-001 (gray line) added.
• D) HC110-R-GFP transfected cells were at 90 ng / ml for 90 min BAY44-4400 (black line) or PF1022-001 (gray line) pre-incubated and unbound substances removed before measurement. The stimulation the cells were carried out after 6 min with α-LTX.
• E) As (D), except that HC110-R-GFP were transfected with cells for 90 min 400 ng / ml BAY44-4400 (black line) or PF1022-001 (gray line) pre-incubated, unbound substances removed before measurement and the  Cells stimulated with α-LTX after 6 min.
• F) HC110-R-Myc / His stably expressing cells were 6 min before the addition of α-LTX with 400 ng / ml BAY44-4400 (black line) or PF1022-001 (gray line) offset.
• G) As a control, non-transfected cells in the presence (400 ng / ml BAY44-4400, black line) or in the absence (-BAY44-4400, gray line) of 400 ng / ml BAY44-4400 after 6 min 1 mM carbamylcholine chloride (Carbachol) added for 100 s and washed out again with Na ⁺ -HBS.
• H) Like (G), except that M1-R-GFP became transfected cells in the presence (400 ng / ml BAY44-4400, black line) or absence (-BAY44-4400, gray line) of 400 ng / ml BAY44-4400 after 6 min stimulated with 1 mM carbachol for 100 s and washed out again with Na ⁺ -HBS.
• I) As a control, non-transfected cells in presence (400 ng / ml BAY44-4400, black line) or absence (-BAY44-4400, gray line) of 400 ng / ml BAY44-4400 after 3 min for 100 s with 1 mM iso proterenol and after 12 min for 100 s stimulated with 1 mM arecolin. The substances were washed out again with Na ⁺ -HBS after the 100 s.

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Claims (43)

1. Use of calcium channel transmembrane receptors from helminths to identify anthelmintically active substances. 2. Use of calcium channel transmembrane receptors from arthropods to identify arthropodicidal substances. 3. Use of transmembrane receptors according to claim 1 or 2, because characterized by that it is G-protein coupled transmembrane receptors with seven transmembrane domains. 4. Use of transmembrane receptors according to one of claims 1 to 3, characterized in that it is transmembrane receptors of Secretin subfamily. 5. Use of transmembrane receptors according to one of claims 1, 3 or 4, characterized in that they are transmembrane receptors is about nematodes. 6. Use of transmembrane receptors according to one of claims 2 to 4, characterized in that it is transmembrane receptors Akarina acts. 7. Use of transmembrane receptors according to one of claims 1 and 3 to 5, characterized in that it is the transmembrane receptor HC110-R from Haemonchus contortus. 8. Use of transmembrane receptors according to one of claims 1 to 6, characterized in that it is the transmembrane receptor HC110-R are homologous.   9. Use of transmembrane receptors according to one of claims 1 to 8 to identify calcium channel blockers. 10. Use of transmembrane receptors according to one of claims 1 to 8 for identifying calcium channel blockers, characterized in that they are transmembrane receptors that bind alpha-latrotoxin. 11. Use of alpha-latrotoxin as an agonist of transmembrane receptors according to one of claims 1, 3 to 5 and 7. 12. Use of alpha-latrotoxin as a nematicide. 13. Use of alpha-latrotoxin in a method of identifying nematicidal and / or arthropodicidal compounds. 14. Process for obtaining the HC110-R receptor and homologous to it Proteins comprising expression of the polypeptide or fragments thereof in a prokaryotic or eukaryotic expression system. 15. The method according to claim 14, characterized in that it is a eukaryotic expression system. 16. The method according to claim 15, characterized in that for expression HEK 293 or COS7 cells can be used. 17. Host cells showing transient expression of the HC110-R receptor as well enable homologous proteins. 18. Host cells that have stable expression of the HC110-R receptor as well enable homologous proteins.   19. Host cells according to claim 18, characterized in that they are aequorin express. 20. Host cells according to claim 18, characterized in that it is the Stably transformed cell line with the accession number DSM ACC2464 is. 21. Host cells according to claim 18 or 19, characterized in that it the stable transformed cell line of the deposit number DSM ACC2465 acts. 22. Vectors for the stable transformation of host cells according to claim 18 or 19 and for the transient transformation of host cells according to claim 17th 23. Method for identifying agonists and / or antagonists of Calcium channel transmembrane receptors from helminths and / or arthro poden. 24. The method according to claim 23, characterized in that it is G- Protein-coupled transmembrane receptors of the secretin subfamily or Fragments of it. 25. The method according to claim 23 and 24, characterized in that it is to the transmembrane receptor HC110-R or fragments thereof and to it homologous proteins. 26. The method according to any one of claims 23 to 25, characterized in that the test substance is brought into contact with the transmembrane receptor under conditions which allow the receptor molecules to interact with the test substance,

• a) the binding of the test substance is detected, and
• b) comparing the activity of the receptor molecule in the presence of the test substance with its activity in the absence of a test substance.

27. The method according to any one of claims 23 to 26, characterized in that that you use a cell-based test system. 28. The method according to claim 27, characterized in that cells ge used according to one of claims 17 to 21. 29. The method according to any one of claims 22 to 26, characterized in that that you use a cell-free test system. 30. The method according to any one of claims 23 to 29, characterized in that the interaction of a test substance with the transmembrane receptor through the displacement of bound alpha-latrotoxin is detected. 31. The method according to any one of claims 23 to 29, characterized in that that the interaction of a test substance with the transmembrane receptor the displacement of bound nifedipine is detected. 32. Use of nifedipine in a process according to one of the claims 23 to 29. 33. Substances used in a method according to any one of claims 23 to 32 be identified.   34. Use of substances according to claim 33 for the manufacture of a To combat helminths and / or arthropods. 35. Use of modulators of the HC110-R receptor and for this homologous proteins as anthelmintics and / or arthropodicides. 36. Use of the DNA coding for the receptor HC110-R and for this homologous DNA for the production of transgenic invertebrates. 37. Transgenic invertebrates which have the receptor HC110-R or homologous to it Contain proteins. 38. Transgenic invertebrates according to claim 37, characterized in that it is Drosophila melanogaster and Caenorhabditis elegans. 39. Use of DNA oligonucleotides that are specific to those for the Hybridize receptor HC110-R coding DNA for the detection of DNA, which comes from Helminthen. 40. Method for the detection of DNA from helminths, characterized in that

• a) provides DNA oligonucleotides which hybridize to the DNA coding for the receptor HC110-R or to strands complementary thereto or to the 5 'or 3' flanking regions thereof,
• b) bringing the DNA oligonucleotides into contact with a sample containing DNA,
• c) the hybridization of the DNA oligonucleotide is detected,
• d) the detected sequence is sequenced, and
• e) the sequence is compared with the DNA sequence coding for the receptor HC110-R.

41. Diagnostic test kit comprising a coding for the receptor HC110-R DNA sequence or fragment thereof or DNA sequences homologous thereto. 42. Diagnostic test kit according to claim 41, characterized in that the DNA sequences are provided with a detectable marker. 43. Use of the HC110-R receptor or fragments thereof, and therefor homologous proteins for the production of vaccines.