- Email: [email protected]
[31] Attachment of oligosaccharide-asparagine derivatives to proteins: Activation of asparagine with ninhydrin and coupling to protein by reductive amination
[31]
NEOGLYCOPROTEINS FROM GLYCOSYL-Asn DERIVATIVES
409
cells. Similarly, Gal-derivatized poly(D-lysine) and poly(L-lysine) both actively bind and are taken up by mammalian hepatocytes, but only the poly(L-lysine) derivatives are degraded. In both cases, the amount of poly(o-lysine) derivatives taken up by the cells far exceeds the total amount of receptor molecules, and thus renders support to the notion of receptor recycling, and that receptor recycling does not require degradation of ligands.
[31] A t t a c h m e n t o f O l i g o s a c c h a r i d e - A s p a r a g i n e D e r i v a t i v e s to P r o t e i n s : A c t i v a t i o n o f A s p a r a g i n e w i t h N i n h y d r i n a n d C o u p l i n g to P r o t e i n b y R e d u c t i v e A m i n a t i o n
By A R L E N E
J. MENCKE,
DAVID
T.
CHEUNG,
and
FINN WOLD
It is frequently most convenient to liberate naturally occurring Nlinked oligosaccharides from glycoproteins by exhaustive digestion of the protein with proteases, producing oligosaccharides with a single asparagine residue still attached. Although there are glycoproteins for which the peptide bonds adjacent to the glycosylated Asn are quite resistant to proteolysis, and for which the product is a glycosylated di- or tripeptide, the glycosylated Asn appears to be a likely product if an effort is made to make the proteolytic digestion go to completion. Since the Asn moiety represents a convenient site for chemical manipulations which will not alter the oligosaccharide structure, such glycosyl-Asn derivatives are attractive starting materials for the preparation of neoglycoproteins, and some methods have been devised for derivatizing the Asn as a means of incorporating the oligosaccharide unit into proteins. ~-3 Surprisingly, however, this does not appear to be a common strategy. One such method, based on the direct activation of the Asn moiety by oxidative deamination-decarboxylation with ninhydrin, is the subject of this article. The reaction is illustrated in Scheme 1, which also shows the subsequent reaction of the resulting aldehyde with protein amino groups and the NaCNBH3 reduction of the Schiff base to the stable secondary amine link in the neoglycoprotein derivative. This method of activation and coupling was used successfully to incorporate six different ovalbumin I j. C. Rogers and S. Kornfeld, Biochem. Biophys Res. Commun. 45, 622 (1971). 2 S.-C. B. Yan, this volume [32]. 3 V. A. Chen, this volume [33].
METHODS IN ENZYMOLOGY, VOL. 138
Copyright © 1987by AcademicPress, Inc. All rights of reproduction in any form reserved.
410
PREPARATIONS
[31]
H2N-CH-CH2-CONH-[GE-~ I
COO-
t/f Ninhydrin
O=CH-CH2-CONH-I-G-E~ ~
Protein--N H2
Protein-N=CH-CH2-CONH-
~
NaCNBH3
Protein-NH-CH2-CH2-CONHSCHEME 1.
glycosyl-Asn derivatives into serum albumin. 4 Since the desired product was albumin containing a single oligosaccharide, the procedures described involve a relatively large excess of protein in the coupling step. In fact, the proteon concentration is so high that we assume that the rate of the coupling reaction is zero order with respect to protein. If this is correct, lower protein to oligosaccharide ratios should be acceptable, but this has not been explored in a systematic manner. The product of the activation step, presumed to be the N-glycosylated malonamide semialdehyde, has not been characterized, but the rapid loss of coupling activity as a function of pH and temperature, the well-established properties of the ninhydrin reaction, 5 and the successful use of the product in the reductive amination with the protein are all consistent with the reactions outlined in Scheme I. Materials. The different oligosaccharide-Asn derivatives were prepared from ovalbumin by the method of Huang et al. 6 with modifications suggested by other workers.7,8 After fractionation of the oligosaccharide4 A. J. Mencke and F. Wold, J. Biol. Chem. 257, 14799 (1982). 5 M. Friedman and L. D. Williams, Bioorg. Chem. 3, 267 (1974). 6 C. C. Huang, H. E. Mayer, and R. Montgomery, Carbohydr. Res. 13, 127 (1970). 7 K. Yamashita, Y. Tachibana, and A. Kobata, J. Biol. Chem. 253, 3862 (1978). 8 j. Conchie and I. Strachan Carbohydr. Res. 63, 193 (1978).
[31]
NEOGLYCOPROTEINS FROM GLYCOSYL-Asn DERIVATIVES
411
Asn mixture by chromatography on Dowex 50H +, each fraction was further purified by affinity chromatography on Sepharose-concanavalin A (Sigma). Only the fractions established to bind to Con A and to subsequently be eluted with a-methylmannoside were used in subsequent reactions. Chemical reagents, resins, and adsorbants used were all obtained from commercial sources. Methods Because of the instability of the product of the ninhydrin reaction, a rather rigid protocol for the reaction was developed. The main variables tested were temperature and pH in both the activation and the coupling reactions. The following procedure represents the empirical derivative of these tests. The methods are described for the activation and coupling of 1 /zmol of oligosaccharide to 5/zmol of serum albumin. Prereaction Procedures. (1) Preparation of gel filtration column for the separation of activated oligosaccharide and ninhydrin. A 12 ml (1 x 16 cm) column of BioGel P-2 was poured and equilibrated with 0.17 M potassium phosphate buffer (pH 7.5) in the cold room (4°). The column was carefully calibrated to establish accurately the elution volumes of the oligosaccharide, ninhydrin, and its reaction product, Ruheman's purple. We have found that the appropriate oligosaccharide-Asn derivative labeled at the a-amino group with either radioactive propionate (using the hydroxysuccinimide ester) or acetate (using acetic anhydride) makes a convenient reference compound for this purpose. A mixture of ninhydrin and Ruheman's purple was produced by reacting a 2 : 1 mixture of ninhydrin and asparagine at 100°, pH 6.0, for 30 min. The oligosaccharide elutes well ahead of ninhydrin. After calibration and reequilibration with buffer, the column was made ready for use by clamping it off just as the buffer meniscus reached the top of the packing. (2) Preparation of the serum albumin suspension for the coupling reaction. Serum albumin (350 mg) was weighed into a screw-capped vial and 500/zl 0.17 M phosphate buffer (pH 7.5) was added to uniformly wet and suspend the protein. Later, during the activation reaction, 100/zl of a solution containing 1.3 mmol N a C N B H J m l in the same phosphate buffer was added to the protein suspension, and gentle agitation was applied to achieve a homogeneous mixture. Activation and Coupling. The glycosyl-Asn derivative, 1 /zmol, was added to a small reaction vial containing 200/zl of a solution containing 50 /zmol ninhydrin/ml of 0.1 M acetate buffer, pH 6 (the buffer was prepared by titrating a 0.1 M sodium acetate solution to pH 6.0 with acetic acid). The mixture was rapidly mixed, and the vial was immediately placed in a
412
PREPARATIONS
[31 ]
boiling water bath. After exactly 5 min in the water bath, the vial was transferred to an ice bath and agitated manually to bring the temperature down to about 5° as rapidly as possible. The reaction mixture was next transferred to the prepared BioGel P-2 column, the vial was rinsed with 200/zl of the pH 7.5 phosphate buffer, and the rinse was added to and gently mixed with the reaction mixture before the elution was started using the pH 7.5 buffer. Based on the data from the column calibration, the first 160-200 drops, corresponding to the void volume of the column were discarded, and the next 180-200 drops (5-6 ml), corresponding to the total elution volume of the oligosaccharide, were collected manually with continuous agitation directly into the reaction vial containing the protein-NaCNBH3 mixture. At this time the elution was continued with a fraction collector to permit subsequent monitoring of the remaining fractions to ensure, if necessary, that all the monsaccharide had been collected in the reaction vial. Purification of Neoglycoprotein. The homogeneous reaction mixture was left overnight at room temperature, and then applied directly to a 140ml Sepharose-Con A column, equilibrated and eluted with a 0.01 M Tris0.15 M NaCI buffer (pH 7.5). The unreacted albumin was carefully removed by extensive washing with the equilibration buffer before the bound fraction was eluted with 0.1 M ~-methylmannoside in the same buffer. The bound fractions absorbing at 280 nm were pooled, concentrated if needed, and subjected to gel filtration on a 2.5 × 30 cm column of BioGel P-10, equilibrated and eluted with 0.01 M ammonium acetate. On this column, the neoglycoprotein fractions (phenol-sulfuric acid positive and 280 nm absorbing) were well separated from unreacted oligosaccharide (phenol-sulfuric acid positive), and the excluded fractions could be pooled, lyophilized, and characterized as the final neoglycoprotein product. It may be of interest to note that the order of the purification steps does not appear to be important. We have obtained equivalent results if the gel filtration step precedes the affinity chromatography step.
Conclusions The direct activation of the asparagine moiety of glycoprotein-derived oligosaccharide-Asn derivatives can be accomplished by treatment with ninhydrin at pH 6.0 at 100° for 5 rain. The activated product can be coupled to proteins by reductive amination with NaCNBH3. The activated intermediate appears to be quite unstable, and it is clear that various side reactions compete effectively with the desired reaction. Under the procedures described it was possible to consistently incorporate 15-
[32]
PREPARATION OF NEOGLYCOPROTEIN
413
20% of the starting oligosaccharide into serum albumin in the reaction of six different ovalbumin-derived oligosaccharide-Asn derivatives. The resuiting neoglycoproteins showed several characteristic features of stable glycoproteins in binding to the appropriate lectin and being preferentially cleared from circulation of rats.
[32] C o v a l e n t A t t a c h m e n t o f O l i g o s a c c h a r i d e - A s p a r a g i n e D e r i v a t i v e s : I n c o r p o r a t i o n into G l u t a m i n e R e s i d u e s w i t h t h e Enzyme Transglutaminase
By
SAU-CHI BETTY Y A N
Introduction l
The information encoded in oligosaccharides lies in the primary sequence, the chemistry of the linkage of the monosaccharide units, and the degree of branching. For oligosaccharides in glycoproteins, additional information may be encoded in the number and topological arrangements of the oligosaccharide units on the three-dimensional matrix of the polypeptide. Moreover, as a result of the interaction of the protein with the carbohydrate, information encoded in the oligosaccharide(s) can be modulated. The encoded information is decoded and translated into biological signals by lectins or processing enzymes interacting with the sugar moieties. Unfortunately, it is difficult to study the biological functions of oligosaccharide with naturally occurring glycoproteins due to the high degree of heterogeneity of the oligosaccharides in a glycoprotein. To remedy this problem, one approach is to prepare synthetic glycoprotein (neoglycoprotein) with a specific number of homogeneous oligosaccharides in specific positions on the polypeptide backbone. To accomplish this chemically is a monumental task, because it is difficult to control the extent and sites of reaction in chemical modification of proteins. The reaction specificity required to prepare the desired neoglycoprotein, however, fits the descriptions of the very nature of enzymatic reactions. This article describes the use of an enzyme, guinea pig liver transglutaminase (protein-glutamine 3,-glutamyltransferase), to prepare neoglycoproteins. Work done in Professor Finn Wold's laboratory, Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston, Texas. Supported by U.S. Public Health Science Grant GM31305 and by a grant from Robert A. Welch Foundation.
METHODS IN ENZYMOLOGY, VOL. 138
Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved,
NEOGLYCOPROTEINS FROM GLYCOSYL-Asn DERIVATIVES
409
cells. Similarly, Gal-derivatized poly(D-lysine) and poly(L-lysine) both actively bind and are taken up by mammalian hepatocytes, but only the poly(L-lysine) derivatives are degraded. In both cases, the amount of poly(o-lysine) derivatives taken up by the cells far exceeds the total amount of receptor molecules, and thus renders support to the notion of receptor recycling, and that receptor recycling does not require degradation of ligands.
[31] A t t a c h m e n t o f O l i g o s a c c h a r i d e - A s p a r a g i n e D e r i v a t i v e s to P r o t e i n s : A c t i v a t i o n o f A s p a r a g i n e w i t h N i n h y d r i n a n d C o u p l i n g to P r o t e i n b y R e d u c t i v e A m i n a t i o n
By A R L E N E
J. MENCKE,
DAVID
T.
CHEUNG,
and
FINN WOLD
It is frequently most convenient to liberate naturally occurring Nlinked oligosaccharides from glycoproteins by exhaustive digestion of the protein with proteases, producing oligosaccharides with a single asparagine residue still attached. Although there are glycoproteins for which the peptide bonds adjacent to the glycosylated Asn are quite resistant to proteolysis, and for which the product is a glycosylated di- or tripeptide, the glycosylated Asn appears to be a likely product if an effort is made to make the proteolytic digestion go to completion. Since the Asn moiety represents a convenient site for chemical manipulations which will not alter the oligosaccharide structure, such glycosyl-Asn derivatives are attractive starting materials for the preparation of neoglycoproteins, and some methods have been devised for derivatizing the Asn as a means of incorporating the oligosaccharide unit into proteins. ~-3 Surprisingly, however, this does not appear to be a common strategy. One such method, based on the direct activation of the Asn moiety by oxidative deamination-decarboxylation with ninhydrin, is the subject of this article. The reaction is illustrated in Scheme 1, which also shows the subsequent reaction of the resulting aldehyde with protein amino groups and the NaCNBH3 reduction of the Schiff base to the stable secondary amine link in the neoglycoprotein derivative. This method of activation and coupling was used successfully to incorporate six different ovalbumin I j. C. Rogers and S. Kornfeld, Biochem. Biophys Res. Commun. 45, 622 (1971). 2 S.-C. B. Yan, this volume [32]. 3 V. A. Chen, this volume [33].
METHODS IN ENZYMOLOGY, VOL. 138
Copyright © 1987by AcademicPress, Inc. All rights of reproduction in any form reserved.
410
PREPARATIONS
[31]
H2N-CH-CH2-CONH-[GE-~ I
COO-
t/f Ninhydrin
O=CH-CH2-CONH-I-G-E~ ~
Protein--N H2
Protein-N=CH-CH2-CONH-
~
NaCNBH3
Protein-NH-CH2-CH2-CONHSCHEME 1.
glycosyl-Asn derivatives into serum albumin. 4 Since the desired product was albumin containing a single oligosaccharide, the procedures described involve a relatively large excess of protein in the coupling step. In fact, the proteon concentration is so high that we assume that the rate of the coupling reaction is zero order with respect to protein. If this is correct, lower protein to oligosaccharide ratios should be acceptable, but this has not been explored in a systematic manner. The product of the activation step, presumed to be the N-glycosylated malonamide semialdehyde, has not been characterized, but the rapid loss of coupling activity as a function of pH and temperature, the well-established properties of the ninhydrin reaction, 5 and the successful use of the product in the reductive amination with the protein are all consistent with the reactions outlined in Scheme I. Materials. The different oligosaccharide-Asn derivatives were prepared from ovalbumin by the method of Huang et al. 6 with modifications suggested by other workers.7,8 After fractionation of the oligosaccharide4 A. J. Mencke and F. Wold, J. Biol. Chem. 257, 14799 (1982). 5 M. Friedman and L. D. Williams, Bioorg. Chem. 3, 267 (1974). 6 C. C. Huang, H. E. Mayer, and R. Montgomery, Carbohydr. Res. 13, 127 (1970). 7 K. Yamashita, Y. Tachibana, and A. Kobata, J. Biol. Chem. 253, 3862 (1978). 8 j. Conchie and I. Strachan Carbohydr. Res. 63, 193 (1978).
[31]
NEOGLYCOPROTEINS FROM GLYCOSYL-Asn DERIVATIVES
411
Asn mixture by chromatography on Dowex 50H +, each fraction was further purified by affinity chromatography on Sepharose-concanavalin A (Sigma). Only the fractions established to bind to Con A and to subsequently be eluted with a-methylmannoside were used in subsequent reactions. Chemical reagents, resins, and adsorbants used were all obtained from commercial sources. Methods Because of the instability of the product of the ninhydrin reaction, a rather rigid protocol for the reaction was developed. The main variables tested were temperature and pH in both the activation and the coupling reactions. The following procedure represents the empirical derivative of these tests. The methods are described for the activation and coupling of 1 /zmol of oligosaccharide to 5/zmol of serum albumin. Prereaction Procedures. (1) Preparation of gel filtration column for the separation of activated oligosaccharide and ninhydrin. A 12 ml (1 x 16 cm) column of BioGel P-2 was poured and equilibrated with 0.17 M potassium phosphate buffer (pH 7.5) in the cold room (4°). The column was carefully calibrated to establish accurately the elution volumes of the oligosaccharide, ninhydrin, and its reaction product, Ruheman's purple. We have found that the appropriate oligosaccharide-Asn derivative labeled at the a-amino group with either radioactive propionate (using the hydroxysuccinimide ester) or acetate (using acetic anhydride) makes a convenient reference compound for this purpose. A mixture of ninhydrin and Ruheman's purple was produced by reacting a 2 : 1 mixture of ninhydrin and asparagine at 100°, pH 6.0, for 30 min. The oligosaccharide elutes well ahead of ninhydrin. After calibration and reequilibration with buffer, the column was made ready for use by clamping it off just as the buffer meniscus reached the top of the packing. (2) Preparation of the serum albumin suspension for the coupling reaction. Serum albumin (350 mg) was weighed into a screw-capped vial and 500/zl 0.17 M phosphate buffer (pH 7.5) was added to uniformly wet and suspend the protein. Later, during the activation reaction, 100/zl of a solution containing 1.3 mmol N a C N B H J m l in the same phosphate buffer was added to the protein suspension, and gentle agitation was applied to achieve a homogeneous mixture. Activation and Coupling. The glycosyl-Asn derivative, 1 /zmol, was added to a small reaction vial containing 200/zl of a solution containing 50 /zmol ninhydrin/ml of 0.1 M acetate buffer, pH 6 (the buffer was prepared by titrating a 0.1 M sodium acetate solution to pH 6.0 with acetic acid). The mixture was rapidly mixed, and the vial was immediately placed in a
412
PREPARATIONS
[31 ]
boiling water bath. After exactly 5 min in the water bath, the vial was transferred to an ice bath and agitated manually to bring the temperature down to about 5° as rapidly as possible. The reaction mixture was next transferred to the prepared BioGel P-2 column, the vial was rinsed with 200/zl of the pH 7.5 phosphate buffer, and the rinse was added to and gently mixed with the reaction mixture before the elution was started using the pH 7.5 buffer. Based on the data from the column calibration, the first 160-200 drops, corresponding to the void volume of the column were discarded, and the next 180-200 drops (5-6 ml), corresponding to the total elution volume of the oligosaccharide, were collected manually with continuous agitation directly into the reaction vial containing the protein-NaCNBH3 mixture. At this time the elution was continued with a fraction collector to permit subsequent monitoring of the remaining fractions to ensure, if necessary, that all the monsaccharide had been collected in the reaction vial. Purification of Neoglycoprotein. The homogeneous reaction mixture was left overnight at room temperature, and then applied directly to a 140ml Sepharose-Con A column, equilibrated and eluted with a 0.01 M Tris0.15 M NaCI buffer (pH 7.5). The unreacted albumin was carefully removed by extensive washing with the equilibration buffer before the bound fraction was eluted with 0.1 M ~-methylmannoside in the same buffer. The bound fractions absorbing at 280 nm were pooled, concentrated if needed, and subjected to gel filtration on a 2.5 × 30 cm column of BioGel P-10, equilibrated and eluted with 0.01 M ammonium acetate. On this column, the neoglycoprotein fractions (phenol-sulfuric acid positive and 280 nm absorbing) were well separated from unreacted oligosaccharide (phenol-sulfuric acid positive), and the excluded fractions could be pooled, lyophilized, and characterized as the final neoglycoprotein product. It may be of interest to note that the order of the purification steps does not appear to be important. We have obtained equivalent results if the gel filtration step precedes the affinity chromatography step.
Conclusions The direct activation of the asparagine moiety of glycoprotein-derived oligosaccharide-Asn derivatives can be accomplished by treatment with ninhydrin at pH 6.0 at 100° for 5 rain. The activated product can be coupled to proteins by reductive amination with NaCNBH3. The activated intermediate appears to be quite unstable, and it is clear that various side reactions compete effectively with the desired reaction. Under the procedures described it was possible to consistently incorporate 15-
[32]
PREPARATION OF NEOGLYCOPROTEIN
413
20% of the starting oligosaccharide into serum albumin in the reaction of six different ovalbumin-derived oligosaccharide-Asn derivatives. The resuiting neoglycoproteins showed several characteristic features of stable glycoproteins in binding to the appropriate lectin and being preferentially cleared from circulation of rats.
[32] C o v a l e n t A t t a c h m e n t o f O l i g o s a c c h a r i d e - A s p a r a g i n e D e r i v a t i v e s : I n c o r p o r a t i o n into G l u t a m i n e R e s i d u e s w i t h t h e Enzyme Transglutaminase
By
SAU-CHI BETTY Y A N
Introduction l
The information encoded in oligosaccharides lies in the primary sequence, the chemistry of the linkage of the monosaccharide units, and the degree of branching. For oligosaccharides in glycoproteins, additional information may be encoded in the number and topological arrangements of the oligosaccharide units on the three-dimensional matrix of the polypeptide. Moreover, as a result of the interaction of the protein with the carbohydrate, information encoded in the oligosaccharide(s) can be modulated. The encoded information is decoded and translated into biological signals by lectins or processing enzymes interacting with the sugar moieties. Unfortunately, it is difficult to study the biological functions of oligosaccharide with naturally occurring glycoproteins due to the high degree of heterogeneity of the oligosaccharides in a glycoprotein. To remedy this problem, one approach is to prepare synthetic glycoprotein (neoglycoprotein) with a specific number of homogeneous oligosaccharides in specific positions on the polypeptide backbone. To accomplish this chemically is a monumental task, because it is difficult to control the extent and sites of reaction in chemical modification of proteins. The reaction specificity required to prepare the desired neoglycoprotein, however, fits the descriptions of the very nature of enzymatic reactions. This article describes the use of an enzyme, guinea pig liver transglutaminase (protein-glutamine 3,-glutamyltransferase), to prepare neoglycoproteins. Work done in Professor Finn Wold's laboratory, Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston, Texas. Supported by U.S. Public Health Science Grant GM31305 and by a grant from Robert A. Welch Foundation.
METHODS IN ENZYMOLOGY, VOL. 138
Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved,