GB2225324A - Stabilisation of glycoproteins by periodate oxidation - Google Patents

Abstract

A process for the preparation of stabilized glycoproteins is described. An important feature of the process is that no foreign molecule is introduced into a stabilized glycoprotein. The process consists mainly of periodate oxidation of the susceptible monosaccharides covalently attached to the protein part of the molecule. The oxidized glycoprotein is incubated in a buffer under conditions favorable for the reaction between the aldehyde groups generated in the sugar part and the amino acid residues from the protein part. Thus, the oxidized carbohydrate chains act as a polyaldehyde cross-linker. As demonstrated with yeast invertase, the cross-linking reaction produces intramolecularly and intermolecularly linked derivatives. The amount and size of the intermolecularly linked derivatives can be controlled by degree of oxidation and protein concentration. The thermal stability (at 63 DEG C) of the prepared derivatives was dependent on the degree of oxidation and under optimal conditions it was about 10 times better than the stability of native invertase. The presented results strongly indicate that certain stable bonds are formed in oxidized glycoproteins, because a monohydrazide added to a sample of oxidized invertase could not dissociate invertase subunits. Additional stabilization of less than optimally oxidized invertase can be achieved by reduction with NaCNBH3. These results indicate that stable bonds play a major role in stabilization of the active conformation of oxidized glycoproteins.

Description

Title: Stabilization of Glycoproteins FIELD OF THF, INVENTION This invention concerns a process for preparation of the stabilized glycoproteins.

BACKGROUND OF THE INVENTION Stahility of a protein is very often the critical factor which imposes a limit on practical use of that protein in techological or medical applications.

Therefore, much fort has been directed into understanding of those processes that lead to loss of the biological activity. These processes can conveniently be devided into covalent and conformational process (reference 1). The covalent processes include deamidation of asparagine, destrtiction of disulfide bridges and cleavage of t3e peptide bonds at aspartic acid residues, whereas conformational processes include changes in the spatial structure of the polypeptide backbone (reference 1). It is clear that stabilized proteins will be produced by those methods which diminish covalent and/or conformational processes responsible for the inactivation.

There are two general ways to prepare a protein more stable than the original one. by genetic engineering it is possible to exchange one amino acid with the other which is less susceptible to a reaction deleterious for stability or with a new amino acid which contributes to the stabilizing forces. By chemical cross-linking of. the original protein it is possible to introduce additional covalent links, which then stabilize the active conformation.There are many reports showing that stabilized proteins can he obtained by hoth approaches. With respect to the present invention, it should be noted the one of the most widely used protein cross-linking reagents is glutaraldehyde, which is an efficient cross-linker because it always contains polymeric aldehydes (reference 2).

Glycoproteins are proteins that contain covalontly linked sugar chains. The carbohydrate chains are usually not directly involved in biological activity of a glycoprotein, and particularly not in erizymic activity of glycoenzymes (references 3,4 and 5). We havo shown that glycoenzymes can be specifically cross-linked through their carbohydrate chains (reference 6). Our cross-linking procedure (reference 6) consisted of two steps. In the first step, susceptible monosaccharides were oxidized by periodate.

This resulted in aldehyde groups which in the second step reacted with a bifunctional cross-linker, such as adipic acid dihydrazide.

as demonstrated by electrophoresis, cross-linking with the dihydrazide produces intramolecularly and intermolecularly cross-liked derivatives (reference 7).

We have also shown that the cross-linking of carbohydrate chains improves greatly the stability of a glycoonzyme, most likely by increasing the rigidity of its polypeptide backbone (reference 7).

As a control, in that study we have also examined whether intermolecularly cross-linked oligomers were in part the result of Schiff base formation between sugar aldehyde groups and protein amino groups. The results clearly showed that the intermolecu larly cross-linked oligoirers are mostly the result of adipic acid dihydrazide reaction, although oxidized invertase and acid phosphatase, but not glucose oxidase, without i::}ie cross-linker formcd a very low amount of oligomers (reference 7). We @ave al.so shown that the stabilization effect observed is a result of the cross-linking reaction, since the oxidized invertase and glucose oxidase were essentially as stable as the native enzymes, while oxidized acid phospatase was less stable than the native enzyme (reference 7). such results appeared reasonahle, since the pressumed linkago (Schiff base) between the oxidized sugar and the protein part is reversible. Accordingly, at that time the possibility of glycoprotein stabilization only by periodate oxidation was regarded as unsuitable.

However, after full apprehension of the idea that an oxidized carbohydrate chain of a glycoprotein may act as an efficient intramolecular cross-linker due to its polyaldehyde character, we have done furtho experiments. They demonstrated that certain irreversible linkages were formed after a longer incubation of strongly oxidized glycoproteins. Tn addition, the new derivatives were found to be much more stable than the native enzyme.

OBJECTIVES OF THE INVENTION It is an object of the present invention to provide a process for the preparation of stabilize glycoproteins.

It is another object of the present invention to demonstrate a better performance of a stabilizeci glycoprotein in an application sunder harsh conditions.

It is another object of the present invention to provide procedures suitable for characteriation of the c-ross-linked derivatives.

SUMMARY OF THE INVENTION This invention concerns mostly a process for the preparation of stabilized glycoproteins. The process consists mainly of periodate oxidation of the susceptible monosaccharides covalently attached to the protein part of the molecule. The oxidized glycoprotein is incubated in a buffer under conditions favorable for its stability and for the reaction between the aldehyde groups generated in the sugar part and the amino acid residues from the protein part. At the end of incubation, if desired, the modified glycoprotein is reduced with a suitable reducing agent, such as NaCNBH3 or Naflil4.

DETAILED DESCRIPTION OF THE INVENTION Various aspects of the present invention are illustrated by 4 examples and 10 figures.

As a model glycoprotein we have taken yeast irivertase because this is an industrially interestillg enzyme and because we have already studied the cross-linkig of this enzyme (references 6 and 7).

Example 1. Influence of degree of oxidation on the oligomer formation and stability.

invertase (from Sigma) at concentration of 1.5 mg/ml in 0.1 M sodium acetate pH 4.6, was oxidized with different amounts of the freshly prepared sodium periodate solutions. The quantity of sodium periodate added is expressed as a molar percentage to the neutral sugars present in invertase. Neutral sugars we-ra quantitated by the orcinol-sulfuric acid method (reference 8), with annose as a standard.

The enzyme was oxidied at 40C for 24 h in the dark. The samples were then desalted by gel filtration ort a small column (1.5 X 8 cm), filled with Sephadex C-25 and equillibrated in the same acetate buffer. T.1ic desalted samples were concentrated to the original protein concentration and stored at roo3n temperature or two days. They were then analyzed by electrophoresis in the 3-30% polyacrylamide gradient gels under nondenaturing (figure 1) or denaturing (SDS) conditions (figure 2). Electrophoresis buffer was 0.1 Tris-borate pH 8,3, with 1 mM EDTA.For SDS electrophoresis, 0,1% SDS was added to the buffer and electrophoretically introduced into 3-30% gradient gels hefore application of tho samples.

Figure 1 shows untreated dimeric invertasc' (lane 1), and invertase oxidized with 56 periodate (lane 2), 10% (lane 3), 20% (lane 4), 50% (lane 5), 100% (lane 6) and 200% (lane 7). As can be seen, the quantity of oligomers (produced by intermolecular cross-linking) increases up to 100% added periodate. No further increase is noticed at 200% periodate. These results indicate that, under the conditions specified, no additional and reactive aldehyde groups are produced when reriodate is added in a molar amount exceeding tilat of mannose, because maximal formation of oligomers is achieved at 100% or less periodate.

Figure 2 shows the invertase subunit (lane 1) and the oxidized invertase derivatives in the same order as described under figure 1. There is very little intersubunit cross-linking (leading to the dimor represented hy the upper band) at 5 and 1 0e periodate oxidation (lanes 2 and 3). However, at 100% (and 200%) periodate oxidation most invertase subunits are cross-linked, a larger fraction of subunits into the dimer and the smaller into the high molecular weight oliors (lanes 6 and 7).

The results presented in figures 1 and 2 thus demonstrate that an oxidized glycoprotein molecule can react with another such molecule and form oligomers (figure 1). These results also demonstrate that an oxidized sugar chain from one polypeptide (subunit) can react with the other polypeptide (subunit) present in the same dimeric invertase molocule (figure 2).We assume that, due to even more favorable steric conditions, an oxidized sugar chain reacts also with tile polypeptide to which it is bound (this would be intrasubunit cross-linking), but such derivatives cannot be detected by the methods we have used We have investigated the thermal stability of invertase oxidized to various degrees by sodium periodate. invertase derivatives were incubated at 63 C and at the times indicated, portions of the enzyme solution were removed, cooled by dilution in I chilled buffer and the remaining invertase activity was measured at 30 C, as described in reference 9, The remaining activity was plotted against time intervals (figure 3).The curves represent native invertase (1), invertase oxidized with 104 periodate (2), 20% (3), 50t (4), 100% (5) and 200% (6). As can be seen, the thermal stability of invertaso increases with the degree of oxidation, from 10 to 1008 periodate added (curvos 2-5). At 2008 oxidation (curve 6) the stability is lower. Under the best oxidation conditions (curve 5), tlie enzyme is about 1 0 times more stable than the native invertase, as determined from the time required for 50% inactivation.

Note 1. In our previous work, during investigation of the thermal stability of adipic acid dihydriazide cross-linked enzyme, the thermal stability ot 30} oxidized invertase was measured as a control (reference 7). The weak stabilization effect, as compared to the strong stabilization effect of the same enzyme preparation after cross-linked with adipic acira dihydrazide (reference 7), has suggested that this approach is not promising. Since the enzyme oxidized with more than 30% periodate usually fore, uon cross-linking with adipic acid dihydrazide, partially insoluble derivatives which were difficult to handle, strongly oxidized invertase was not investigated at that time, Example 2.Influence of the protein concentration the formation and stability of the cross-linked derivatives.

Invertase at different concentrations was oxidized with 103t periodate and further treated under conditions specified in Example 1. The samples were analyzed $by polyacrylamide gradient (3-30%) gel electrophoresis under nondenaturing (figure 4) and denaturing (figure 5) conditions. Untreated invertase was applied to this gel (lane 2), as was the enzyme oxidized at 0,33 mg/ml (lane 3), 1.0 mg/ml (lane 4), 2.0 rng/ml (lane 5), 4.5 mg/ml (lane 6) and 9.0 tng/ml (lane 7). Standard proteins were applied to lane 1.

As expected, tulle amount of oligomers increases with protein concentration, and at the concentrations above 2 mg/ml there is very little of the native dimer left anti at 9 mg/ml partially insoluble derivatives are formed.

SDS electrophoresis (figure 5) of the same samples as in figure 4, shows that practically every invertase subunit is linked to another one at protein concentrations above 2 mg/ml. At higher protein concentrations (4.5 and 9.0 mg/ml) some oligomers are so large that they cannot enter the gel (figures 4 and 5).

We have further studied the thermal stability of the cross-linked invertase derivatives, prepared at different protein concentrations as described above.

The measurements of the remaining invertase activity were done as given in Example 1. Figure 6 shows the stability of native invertase (curve 1) and invertase that was oxidized and after desalting incubated at 0.33 mg/ml (curve 2), 1 mg/ml (curve 3) and 4.5 mg/r: (curve 4).

All cross-linked derivatives were much more stable than the native invertase, Moreover, there is vc-ry little difference between the stability ot derivatives prepared at various protein concentrations (curves 2-4). This finding indicates that the oligomers formed at higher protein concentrations (figures 4 and 5) are not much more therrnally stable than the cross-linked dimar. Accordingly, the results of figures 4, 5 and 6 indicate that intramolecular (including intrasubunit) crcss-li.nking is of primary importance in stabilization of the glycoprotein conformation.

Example 3. Influence of addition of hexanoic acid hydrazide on formation of the cross-linked invertase derivatives.

Invertase (2 mg/ml) in 0.1 M sodium acetate buffer pH 4.6 was oxidized with 100% periodate. After desalting one part was treated with hexanoic acid hydrazide (twice the molar amount of periodate) and, as the other part, left for two days at room tempera Lure.

The samples were then analyzed by electrophoresis in 3-30% gradient gels under nondenaturing (figure 7) and denaturing conditions (figure 8).

In figure 7, native invertase was appiiec' to lane 1, the oxidized enzyme without the monohydrazide to lane 2 and the oxidized enzyme with the monohydrazide t lane 3. Standard proteins were applied to Jane 4.

As can he seen, addition of hexanoic acid hydrazide almost completely prevented the formation of irivertase oligomers (lane 2 versus lane 3), and the derivative with hexanoic acid hydrazide migrated to a similar distance as did the native invertase (lane 3 versus lane 1).

Figure 8 shows the same samples as those applied to ire 7 but run under denaturing conditions.

Untreated invertase was applied to lane 1, standard proteins to lane 2, oxidized invertase to lane 3 and oxidized invertase treated with hexanoic acid hydrazide to lane 4.

In hoth oxidized samples (lanes 3 and t), there is one band migrating the same distance as invertase subunit (lane 1). The. intensity of this and is stronger in lane 4, demonstrating that hexanoic acid hydrazide prevented formation of some intramolecular cross-links between invertase subunits. However, at the concentration used, this monohydrazide was not ale to completely prevent the formation of cross-links between the two subunits, because some derivatives migrated as a dimer (lane 4).An alternative explanation is that hexanoic acid monohydrazide was not able to reverse certain linkages that were formed before it could react with the free aldehyde groups (or the Schiff bases).

we have further attempted to see whether hexanoic acid hydrazide could reverse the intermolecular cross-links. Figure 9 shows a native 3-30% polyacrylamide gradient gel, containing the following samples. tane 1 contains standard proteins; lane 2, native invertase; lane 3, oxidized invertase (100z, left for two days); lane 4, oxidized invertase to which hoxasloic acid hydrazide was added beforo the desalting step; lane 5, oxidized invertase to which hexanoic acid hydrazide was added immediately after the desalting step and lane 6, oxidized invertase to which hexanoic acid hydrazide was added 48 h after the desalting step.

As shown also in the previous figures, the oxidized invertase formed oligomers (lane 3). The adition of hexanoic acid hydrazide before desalting prevented partially and after desalting almost completely the formation of intermolecular cross-links (lane 4 versus' lane 5).This is apparently a contradicting result because earlier addition should have a better preventive effect. however, unreacted periodate reacts with a hydrazide group (our unpublished observation), and the resulting lower concentration of the monohydrazide in the non-desalted sample is a likely explanation for the weaker preventive effect (lane 4 versus lane 5). moreover, hexanoic acid hydrazide added after 48 h was able to partially reverse the cross-links (lane 6 versus lane 3), but some oligomers still persisted (lane 6 versus lane 5).

Note 2. The results presented in figures 7, 8 and 9 strongly indicate that certain stable bonds are formed after a prolonged incubation of the oxidized glycoproteins. Such bonds may be similar to ketoamine linkages resulting from the Amadory rearrangment of the Schiff base, which is initially formed from a sugar aldehyde and a primary amino group (reference 10).

However, wo do not know which bonds lead to the stable invertase derivatives. They may mostly come from the Ainadory or another rearrangmcnt of Schiff bases or from completely new type of reactions involving the aldehydes and other amino acid side chains.

Example 4. Influence of the addition of a monohydrazide and a reducing reagent on stability of the oxidized invertase.

Figure 10 shows the thermal stability (63 C) of various invertase derivatives. Native invertase is represented by curve 1. Invertase (2 mg/ml) was oxidized with 50% periodate, desalted and left for two days (curve 2). The same sample of oxidized invertase after two days was treated with hexanoic acid hydrazide (curve 3). The oxidized invertasc that was cross-linked with adipic acid dihydrazide (reference 7) is represented by curve 4, The oxidized invertase was reduced (20 mM NaCNBH3, pH 6.5, 4 h) after two days (curve 5). To the reduced invertase, hexanoic acid hydrazide was added (curve 6).

The oxidized enzyme is more stable than the native one (curve 2 versus 1). Further, tbc- oxidized enzyme becomes less stable after treatment with a monohydrazide (curve 2 versus 3), indicating that towards hydrazide reversible bonds also participate in stabilization of the active conformation. In accordance with our previous findinys (reference 7), the cross-linking of the oxidized enzyme with a dihydrazide increases its thermal stability (curve 4 versus 2). The treatment with NaCNBH3 (resulting in reduction of Schiff bases to the stable secondary amines) greatly improved the thermal stability (curve 5 versus 2).

Subsequent addition of the monohydrazide to the reduced sample showed practically no effect (curve. 6 versus S).

These results indicate that thermal stability is highly dependent on the number of newly formed stable bonds. This number can be increased by a subsequent treatment, such ascross-linking with a dihydrazide or reduction with NaCNBH3. However, it should be be noted that 100% oxidized invertase, without any further treatment, showed a similar thermostability as did the subsequently treated enzyme (figure 3 versus 10) The results presented herein (figures 3, 6 and 10) clearly dertionstrate that a stabilized glycoprotein shows a better performance under harsh conditions (high temperature) than tho original one.Thus, duc to its improved stability much more invert sugar will ha produced by the cross-linked invertase under conditions (about 50-60 C) often used in industrial applicatios.

The stabilization of glycoproteins only by period ate oxidation and eventually by reduction hut not cross-linking with a dihydraide, is more suited for those glycoproteins that do not lose much activity upon a strong oxidation. This approach is also advantageous in those applications of the stabilized derivatives in which the presence of a foreign molecule, such as an dihydrazide cros-linker, is not desirable.

REFERENCE 1. Ahern, T. J., Klibanov, A. M. (1986) in Protein Structure, Folding and Design, Oxender, D. L., Editor, Alan R. Liss, Inc. New York, pp. 283 2. Peters, K., and Richards, F. M. (1977) Ann. Rev.

Biochem. 46, 523 3. Tarentino, A. L., Plumer, T. H., and Maley, F.

(1974) J. Biol. Chem. 249, 818 4. Chu, F. K., Trimble, R. B., and Maley, F. (197n) J.

Riol. Chef. 253, 8691 5. Barbaric, S., Mrsa, V., Ries, B., and Mildner, P.

(1984) Arch. Biochem. niophys. 234, 567 6. Kozulic, B., Barbaric, S., Ries, B., and Mildner, P.

(1984) Biochem. Biophys, Res. Commun. 122, 1083 7. Kozulic, B., Leustek, I., Pavlovic, B Mildner, P., and Barbaric, S. (1987) Appl. Biochem, Biotech. 15, 265 8. Francois, C., Marshall, R. D., and Neuberger, A.

(1962) Riochem. J. 83, 335 9. Bernfeld, P. (1951) Adv. Enymol. 12, 3/9 10. Mori, N., and Manning, J. M. (1986) Anal. Biochem.

152, 396

Claims (14)

1. CLAIMS 1. A process for the preparation of a stabilized glycoprotein, which process includes periodate oxidation and which process introduces no foreign molecule into an oxidized glycoprotein.
2. 2. A process according to claim 1, in which the molar amount of added periodate is preferentially in the range from 5 to 200% wit respect to the monosaccharides attached to the protein.
3. 3. A process according to claim 2, in which the periodate oxidation is performed preferentially in this dark in a nonbuffered solution or in a buffered solution with a p value from 2 to 11.
4. 4. A process according to claim 2, in which periodate oxidation is performed preferentially for up to 24 hours.
5. 5. A process according to claim 2, in which periodate oxidation is performed preferentially at room temperature or temperatures below room temperature.
6. 6. A process according to claim 2, in which the low molecular weight molecules are removed or not removed after the periodate oxidation.
7. 7. A process according to any one of the claims 1 to G, in which an oxidized protein is incubated under conditions favorable for a reaction of the alde1yda groups generelted hy periodatc oxidation.
8. 8. A process according to claim 7, in which an oxidized protein is incubated in a solution with a pH value Crom 2 to 11.
9. 9. A process according to claim 7, in which an oxidized protein is incubated preferentially at temperatures from 0 to 80 C,
10. 10. A process according to claim 7, in which an oxidized protein is not further treated before an a cation.
11. 11. A process according to claim 7, in which an oxidized protein is further treated before an application.
12. 12. A process according to claim 11, in which further treatment includes a reduction.
13. 13. A process according to claim 11, in which further treatment includes a reduction with NaCNBH3 or NaBH4.
14. 14. A stahilized glycoprotein, prepared according to claims 1-13 and essentially as described herein.