THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 985 by The American Society of Biological Chemists, Inc. Vol. 260, No. 8, Issue of April 25, pp. 46884693, 985 Printed in U.S.A. Fractionation of L-Fucose-containing Oligosaccharides on Immobilized Aleuria aurantia Lectin (Received for publication, October 5, 984) Katsuko Yamashita, Naohisa KochibeS, Takashi Ohkura, Ikuko Ueda, and Akira KobataOT From the Department of Biochemistry, Kobe University School of Medicine, Chuo-ku, Kobe 650, Japan, the $Department of Biology, Faculty of Education, Gunma University, Maebashi 37, Japan, and the Department of Biochemistry, the Institute of Medical Science, the University of Tokyo, Minato-ku, Tokyo 08, Japan The carbohydrate-binding specificity of Aleuria aurantia lectin was investigated by analyzing the behavior of a variety of fucose-containing oligosaccharides on an A. aurantia lectin-sepharose column. Studies with complex-type oligosaccharides obtained from various glycoproteins by hydrazinolysis and their partial degradation fragments indicated that the presence of the a-fucosyl residue linked at the C-6 position of the proximal N-acetylglucosamine moiety is indispensable for binding to the lectin column. Binding was not affected by the structures of the outer chain moieties nor by the presence of the bisecting N-acetylglucosamine residue. These results indicated that A. aurantia lectin-sepharose is useful for the group separation of mixtures of complex-type asparagine-linked sugar chains. Studies of glycosylated Bence Jones proteins indicated that this procedure is also applicable to intact glycoproteins. The behavior of oligosaccharides isolated from human milk and the urine of patients with fucosidosis indicated that the oligosaccharides with Fucal+ 2Galp~4GlcNAc and Galfil+4(Fucal~3)GcNAc groups interact with the lectin, but less strongly than complex-type sugar chains with a fucosylated core. Lacto-N-fucopentaitol, which has a Galj3-3(Fuca-4)GlcNAc group, interacts less strongly than the above two groups with the matrix. Oligosaccharides with Fucal+2Ga~-+3GlcNAc and Gal/3+ 4GlcNAcfi~3Gal~l+4(Fuccul~3)GlcNAc groups showed almost no interaction with the matrix. a-l-fucopyranosyl residues are widely distributed in the sugar chains of glycoconjugates: glycoproteins, glycolipids, and proteokeratan sulfate. In many cases, the fucosyl residues constitute parts of important antigenic determinants such as the blood group antigens H, Lea, and Leb () and the stagespecific embryonic antigens (2). The recent establishment of controlled hydrazinolysis (3) and Bio-Gel P-4 column chromatography (4) has facilitated analysis and structural studies of the asparagine-linked sugar chains of glycoproteins. However, the presence or absence of an a-l-fucosyl residue together with the bisecting N-acetylglucosamine makes the resultant fractionation patterns of these oligosaccharides difficult to interpret. *This work has been supported by Grants-in-Aid for Special Project Research, Cancer-Bioscience from the Ministry of Education, Science and Culture, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 8 U.S.C. Section 734 solely to indicate this fact. 9 To whom requests for reprint should be addressed. 4688 Fractionation methods for oligosaccharides based on their content of a-fucosyl residues may be useful for purification and structural studies of the sugar chains of glycoproteins. Lectin-Sepharose columns have afforded conventional and useful methods for group separations and for structural studies of soluble and membrane-bound glycoproteins according to their specific carbohydrate structure (5-8). Among various lectins, those of Ulex europeus (UEA-I) (9), Lotus tetragonolobus (asparagus pea) (lo), Anguilla japonica (eel serum) (ll), Euonymus europeus (32), Griffonia Simplicifolia (GS IV) (33), and Aleuria aurantia (3) are known to bind fucose residues. A. aurantia lectin (AAL ) was found by Kochibe and Furukawa (3). They purified the lectin by affinity chromatography using blood group H-active glycopeptides coupled to Sepharose 4B. It has a molecular weight of 72,000 and is composed of two identical subunits. Each subunit has one combining site for a fucose residue. The lectin agglutinates human erythrocytes irrespective of their AB0 and Lewis blood types. Bombay phenotype erythrocytes and blood group 0 erythrocytes treated with Bacillus fulminans a-fucosidase, which specifically cleaves Fucal-2Gal linkages, were also agglutinated by the lectin. These results indicated that AAL binds not only to the fucosyl residue in the blood type H determinant but also to other fucosyl residues on the human erythrocytes. The detailed investigation of the sugar binding specificity of AAL described in this paper, revealed that the AAL- Sepharose column can be used as an effective tool for the fractionation of glycoproteins as well as oligosaccharides according to their content of a-fucosyl linkages. EXPERIMENTAL PROCEDURES Materials-AAL was purified from the fruiting bodies of A. aurantia according to the method reported previously (3). Purified AAL was coupled to Sepharose 4B by the procedure of March et al. (4). The amount of AAL bound to Sepharose was approximately 3 mg/ ml of the gel. The structures of oligosaccharides used in this study are summarized in Tables -. Oligosaccharides X, XI, XXIII, and XXIV were obtained from the urine of patients with known fucosidosis (5, 6). Oligosaccharides XII, XIV, XV, XVIII-XXI, XXV, and XXVI were isolated from human milk as described previously (7,8) and labeled by reduction with NaB[3H]4 (3). Oligosaccharide XXII was isolated from the N-3 fraction (7) of milk obtained from an Le(a-b-), nonsecretor individual. Oligosaccharides XIII, XVI, and XVII were kindly provided by Dr. Elvin A. Kabat, Columbia University. The three oligosaccharides werelabeled with tritium by the galactose ~xidase-nab[~h], method (2). The Bence Jones proteins: Nei X, Sm X, and Wh X, which contain The abbreviations used are: AAL, Aleuria aurantia lectin; subscript OT is used to indicate NaB[3H]r-reduced oligosaccharides (all sugars mentioned in this paper were of D-configuration except for fucose which has an L-configuration). K. Yamashita and A. Kobata, unpublished results.
4 Oligosaccharides Aleuria aurantia Lectin Specificity TABLE I Structures of typical oligosaccharides widely distributed in various glycoproteins, and their behavior on an AAL-Sepharose column. Structures Galpl4Gl~NAcpl+2Manal~~ Manpl4R" Galpl4Gl~NAc~l+2Manal~~ Gal/34GlcNAc/3+2Manal Galpl4GlcNAc~+2Manal~ Glc NAcPl Galpl4GIcNAc~l+2Manal, ~ "+ f Manp4R IV Gal@4GlcNAc@l -., 2 Manal R Galpl4GlcNAcpl- W Manpl4 V ManalP3 Gal(34GlcNAcpl R VI Gal/34GlcNAc/3 L6 Galpl4GlcNAcpl ~2 Manalh6 R Manpl4 VI P3 Galp4GlcNAc/3 -t 2 Manal R Galpl4GlcNAcpl VI b6 Manalu6 R IX Man04 Galpl4GlcNAc~l" Galpl4GlcNAcpl- Gal/34GlcNAcpl A P3 Manal R, X Fucal4GlcNAcoT XI Mana4Manpl4R R, GIcNAc~~~GIcNAc~T; R, GlcNAcP4(Fucal4)GlcNAcoT. Elution profiles on an AAL-Sepharose column (Fig. ) Panel line) A (solid Panel B (solid line) Panel C (solid line) Same as A Same as B Same as A Same as B Same as A Same as B Panel D Panel E TABLE I Structures of oligosaccharides containing the Fucal+2Gal group, and their behavior on an AAL-Sepharose column Oligosaccharides Structures Elution profiles on an AAL- Sepharose column (Fig. 2) XI Fucal+2GaI~l4Glc0~ Panel A XI (2'-fucosyllactitol) Fuc~l-.2[6-3H]Ga~l4GlcNAc~l+3hexene-l,2,5,6-tetrol Panel B XIV Fucal+2Gal~l+3GlcNAc~l+3Gal~l4Glc~~ (lacto-n-fucopentaitol I).Panel C XV Galpl4GlcNAcpl Panel D "Galpl4GlcoT Fucal+2Galpl-+3GlcNAcpl~ (monofucosyl lacto-n-hexaitol I) Fucal 2 XVI [6-3H]GalNAcal+3Gal~4GlcNAc~l+3hexene-,2,5,6-tetrol Panel E Fucoll 2 XVII [6-3H]Galal-.3Gal~4GlcNAc~l~3hexene-,2,5,6-tetrol Panel F 4689 one biantennary complex-type asparagine-linked sugar chain/mole- 4(Fucal-*G)GlcNAc, respectively (9). Oligosaccharides I,, and cule, were kindly provided by Dr. T. Isobe, Kobe University School were obtained by hydrazinolysis of these Bence Jones proteins folof Medicine. The structures of their carbohydrate moieties have been lowed by NaB[3H]4 reduction and sialidase digestion (9). confirmed as ~NeuAca2+6Galpl+4GlcNAc~l~2Manal+6 Oligosaccharides IV-IX were obtained from hamster melanoma (fneuaca2+6galfll+4glcnacfll+2manal-+3)man~l+ tyrosinase by hydrazinolysis (20) and each of these oligosaccha- 4GlcNAcfll+4GlcNAc, fneuaca2+6gal~l+4glcnacp-, rides was purified by Bio-Gel P-4 (<400 mesh) column chromatog 2Mancrl+6(fNeuAco2~6Gal~l~4GlcNAc~l+2Manal+ raphy. Oligosaccharides IV and VI and oligosaccharides V and VII, 3)Man~+4GlcNAc~+4(Fuca-*6)GlcNAc, and +NeuAca2+ which could not be separated by gel permeation, were separated by 6Gal~l+4GlcNAcfll-*2Manal+6(fNeuAca2+6Galfll+ paper chromatography using ethyacetate:pyridine:acetic acidwater 4GlcNAc@+2Manal+3) (GlcNAc/3~4)Man~l+4GlcNAc~+(5:5::3) as solvent. After development for 20 h, oligosaccharides IV,
4690 Aleuria aurantia Lectin Specificity -& " "' I - e"- ',-.+D- l i i C I,\ I D 5 IO 5 FRACTION NUMBER FIG.. Chromatography of oligosaccharides obtained from asparagine-linked sugar chains by hydrazinolysis and glycoproteins containing an asparagine-linked sugar chain on AAL-Sepharose. Radioactive oligosaccharides Z, II, ZIZ, X, and XZ in Table I were chromatographed on AAL-Sepharose as described under "Experimental Procedures," and their elution patterns are shown as solid lines in A, B, C, D, and E, respectively. The dotted lines in A, B, and C indicate the elution patterns of Bence Jones proteins, Nei X, Sm X, and Wh X, respectively. Proteins were detected by the method of Lowry et al. (30). Arrows indicate the positions where the elution buffer was switched to buffer containing fucose. VI, V, and VI were located at 33, 23, 29, and 20 cm from the origin, respectively. Affinity Chromatography on AAL-Sepharose Columns-Tritiumlabeled oligosaccharides (2-400 X lo3 cpm, 0.-20 nmol) or glycoproteins (0. mg) dissolved in 50 ~l of phosphate-buffered saline (6.7 mm KH2P04, 0.5 M NaCI, ph 7.4) were applied to AAL-Sepharose columns (2.0 ml) previously equilibrated with buffered saline and allowed to stand at room temperature (20-25 "C) for 30 min. Columns were then eluted with 0 ml of the same buffer, followed by buffer containing 0.5 mm L-fucose. Fractions (.0 ml) were collected at 5 ml/h and the radioactivity in each fraction was determined by liquid scintillation spectrometry. Protein content was measured by the method of Lowry et al. (2). Recoveries of oligosaccharides based on radioactive content ranged from 98% to 00%. RESULTS AND DISCUSSION Behavior of Oligosaccharides Related to Complex-type Asparagine-linked Sugar Chains-When each of the 9 tritiumlabeled oligosaccharides (I-IX) liberated from the glycoproteins by hydrazinolysis was applied to an AAL-Sepharose column, all of those with an a-fucosyl residue on their reducing terminal N-acetylglucosamine residue were bound to the column and eluted with buffer containing 0.5 mm fucose. Oligosaccharides lacking this fucose residue passed through the column unretarded (Table I). The actual elution profiles of the three biantennary oligosaccharides, I,, and are shown as solid lines in Fig., A, B, and C, respectively. These results indicated that the binding of oligosaccharides is not FIG. 2. Chromatography of oligosaccharides containing the Fucd-rZGal group on AAL-Sepharose. Radioactive oligosaccharides XZI-XVZI in Table I were chromatographed on AAL-Sepharose as described under "Experimental Procedures," and their elution patterns are shown in A-F, respectively. The sample was eluted with 3 ml of phosphate-buffered saline only in the case of oligosaccharide XZII. altered by the number of outer chain moieties nor by the presence of the bisecting N-acetylglucosamine residue. Although the data are not shown, the elution profiles were not changed by the presence of sialic acid residues linked at the C-6 position of the terminal galactoses of their outer chain moiety. Since oligosaccharides I,, and I were obtained from three Bence Jones proteins: Nei X, Sm X and Wh X, respectively, the behavior of these glycoproteins on AAL-Sepharose chromatography was also studied to evaluate the effect of polypeptide moieties on the binding. All these glycoproteins contain asparagine-linked sugar chain in molecule (22-24). As shown by the dotted lines in Fig., A, B, and C, each glycoprotein was eluted at exactly the same position as that of the sugar it contained. The results indicated that AAL- the Sepharose column can be used to fractionate and characterize intact glycoproteins by their carbohydrate moiety. Since oligosaccharide X (Fig. D) and XI (Fig. E) were bound to the AAL-Sepharose column, the presence of outer chain moieties and even the trimannosyl portion are not required for binding. It is worth noting that this is the first case in which a disaccharide binds to a lectin column. These characteristics of AAL are extremely different from those of lentil lectin and pea lectin. These lectins not only require a fucose residue on the proximal N-acetylglucosamine, but also
Oligosaccharides XVIII Aleuria Lectin aurantia Specificity 469 TABLE Structures of oligosaccharides containing either Galp4(Fucal4)GlcNAc group or Galp4(Fucal4)GkNAc group, and their behavior on an AAL-Sepharose column Fucalb 3 Gal~l+4GlcNAc~l+3Gal~l+4GlcoT (lacto-n-fucopentaitol ) Fucal 3 Structures Elution Profiles in an AAL- Sepharose column (Fig. 3) Panel A XIX xx XXI XXII XXIII XXIV xxv XXVI Galpl4GlcNAcpl b6galpl+4glcot Gap+3(4)GlcNAcfllA3 (monofucosyl lacto-n-hexaitol ) Fucal -y3 Galp4GlcNAcpl6 3Galp4Gl~ Fuca+2Gal~l~3GlcNAc~ (difucosyl lacto-n-hexaitol I) Fucolll 3 Gal~4(3)GlcNAc~+3Gal~l4GlcNAcpl L6Galpl+4GlcoT Galpl-3(4)Gl~NAcpl~~ (monofucosyl lacto-n-(neo)-octaitol) FucalL3 Galpl-4GlcNAcpl L6Galpl+4Glcm Gal~+4GlcNA~pl~~ F ~ ~ ~ I ~ ~ (difucosyl lacto-n-neohexaitol) Fucal 3 Gal~l+4GlcNAc~l+2(4)Mana-~3(6)Man~4GlcNA~ Fucal b3 Gal~l+4GlcNAc~l-+2(4)Manal b6manp4glcnacm Gal~+4Gl~NAcpl+2(4)Manal~~ Fuca r3 Fucal, 4 Gal~l-*3GlcNAc~l-~3Galpl4Gl~ (lacto-n-fucopentaitol ) Fucal 63 Galpl+4Glcm (3-fucosyllactitol) Panel B Panel C Panel D Panel E Panel F Panel G Panel H Panel I the presence of the trimannosyl core, the two a-mannosyl residues of which are either free or substituted only at its C- 2 or C-2,6 positions (6). Accordingly, AAL-Sepharose column chromatography can be a useful tool for the group separation of a series of heterogeneous asparagine-linked sugar chain mixtures by virtue of the presence or absence of the a-fucosyl residue linked to the trimannosyl core. The fucosylated sugar chain group may be further fractionated by using either lentil or pea lectin-sepharose. Behavior of Oligosaccharides Containing the Fucal *2Gal Group-Because the Fucal+2Gal group is widely found in the sugar chains of glycoproteins, we attempted to define the role of that group in binding to the ligand by subjecting 6 radioactive oligosaccharides shown in Table I to AAL-Sepharose column chromatography. Oligosaccharides XI (Fig. 24) and XI (Fig. 2B) interacted with the column, although the binding of the latter was weaker than that of the former oligosaccharide and was eluted in buffer without fucose. Oligosaccharide XIV showed slight retardation (Fig. 2C) but XV (Fig. 20) passed through the column without apparent interaction. These results indicated that the Type 2 blood group H determinant interacts weakly with AAL, while the Type interacts even more weakly with the lectin. The reason that oligosaccharide XI binds strongly to the column is probably because the sorbitol residue allows more access to the Fuccul- 2Gal group than the glucopyranose residue. It is interesting that oligosaccharides XVI (Fig. 2E) and XVII (Fig. 2F) did not bind to the column. These results indicated that the Fuca-2Gal group must be free from substitution by other sugars.
4692 Akuria aumntia Lectin Specificity FIG. 3. Chromatography of oligosaccharides containing either the Gal@~4(Fucal"r3)GlcNAc group or the Gal@~3(Fuc~~l~4)GlcNAc group on AAL-Sepharose. Radioactive oligosaccharides XVZZZ-XXVZ in Table I were chromatographed on AAL-Sepharose and their elution patterns are shown in A-Z, respectively. 5 FRACTION NUMBER Behavior of Oligosaccharides Containing Either Gal@I+ located at the nonreducing terminal as illustrated by oligosac- 4(Fucal+3)GlcNAc or Gal@l4(Fucal+4)GlcNAc Group on charide XXI (Fig. 30). Since oligosaccharide XXV was re- AAL-Sepharose Chromatography-The trisaccharide group: tarded on this column (Fig 3H), the Lea-antigenic structure Gal@l+4(Fucal+3)GlcNAc, which is arbitrarily called the shows almost the same binding as the X-antigenic structure "X-antigenic determinant" occurs in the outer chain moieities with AAL. Gal@4(Fucal+3)Glc0~ was firmly bound to the of the asparagine-linked sugar chains of many glycoproteins column and eluted by buffer containing 0.5 mm L-fucose (Fig. such as human parotid a-amylase (25), human al-acid glyco- 30. The stronger affinity might result from the fact that the protein (26), human lactoferrin (27), and human secretory sorbitol residue affords more accessibility to the fucose residue component (28). Many structural differences are detected in as in the case of 2'-fucosyllactitol. sugar chains containing the X-antigenic determinant. For The results so far described indicate that AAL-Sepharose example, Gal@l4GlcNAc/3+3Gal@l+4(Fucal+3)GlcNAc column can be a useful tool for the fractionation and characand Gal@l+4(Fucal+3)GlcNAc@+ repeating structures terization of a variety of fucose-containing oligosaccharides were found in the sugar chains of human secretory component and glycoproteins. The column will be especially useful for (28) and Gal@l+4(Fucal+3)GlcNAc@l+3Gal@l+4GlcNAc structural studies of asparagine-linked sugar chains of glycoin human lactoferrin (27). The distribution of the X-antigenic proteins. As already discussed in the Introduction, establishdeterminant in complex-type asparagine-linked sugar chains ment of hydrazinolysis (3) and fractionation of oligosacchais also different in glycoproteins. For example, the trisaccharrides obtained by Bio-Gel P-4 (<400 mesh) column chromaide group is included in the tri- and tetraantennary sugar chains of human al-acid glycoprotein but not in the biantentography (4) are now routinely used for structural studies of nary sugar chain (26). In contrast, it occurs in the biantennary sugar chains. Since the effective size of each oligosaccharide sugar chains of human parotid a-amylase (25). In order to expressed in glucose units is now known (4), one can estimate obtain information about the behavior of these oligosacchaalmost all sugar chains included in a glycoprotein from the rides on AAL-Sepharose columns, the radioactive oligosaccharides in Table I were subjected to affinity chromatography. Oligosaccharides XVIII (Fig. 3A), XIX (Fig. 3B), XX (Fig. 3C), and XXIII (Fig. 3F) were retarded indicating that the X-antigenic sugar chain shows weak interaction with AAL. The eluting positions of these oligosaccharides are faster than oligosaccharide XIV (Fig. 2B) indicating that the interaction of the X-antigenic structure with AAL is even weaker than that of the Type 2 blood group H determinant. Oligosac- charides with two X-antigenic sugar chains interact far more strongly because oligosaccharides XXII (Fig. 3E) and XXIV (Fig. 3G) were bound to the column and were eluted with buffer containing 0.5 mm L-fucose. Two reactive residues are required for the firm binding of oligosaccharides to lectin- Sepharose column in the case of concanavalin A (5). Enhancement of binding by the co-existence of two a-fucosyl groups might also explain the evidence that oligosaccharide XX (Fig. 3C) is eluted after oligosaccharide XIX (Fig. 3B). The X- antigenic determinant within the repeating N-acetyllactosamine showed much less interaction with AAL than that elution pattern of an oligosaccharide mixture. However, one of the drawbacks of this method is that the presence or absence of a fucose residue linked to the proximal N-acetylglucosamine residue makes the elution profile hard to interpret. Therefore, fractionation of an oligosaccharide mixture by AAL-Sepharose column chromatography before application to Bio-Gel P-4 will facilitate the interpretation of the fractionation patterns. A lectin with similar characteristics to AAL has been iso- lated by Guillot et al. (29) from the mushroom Laccaria amethystina which contains two isolectins. Separation was achieved by affinity chromatography and incorporation of the stromas of human blood group "0" erythrocytes in polyacrylamide gel. The lectin, which was eluted by L-fucose, agglutinated the erythrocytes of phenotype 0 and AZ more strongly than those of the AI and the Bombay phenotype which lacks the blood type H determinant. It might be interesting to see if this lectin has the same binding specificity as AAL. Acknowledgments-We wish to express our gratitude to Dr. Alistair
G. C. Renwick for his critical reading of this paper and to Yumiko Kimizuka for her skilled secretarial assistance. REFERENCES. Hakomori, S., and Kobata, A. (974) in The Antigens (Sela, M., ed) Vol. 2, pp. 79-40, Academic Press, New York 2. Solter, D., and Knowles, B. B. (978) Proc. Natl. Acad. Sci. U. S. A. 75,5565-5569 3. Takasaki, S., Mizuochi, T., and Kobata, A. (982) Methods Enzymol. 83,263-268 4. Yamashita, K., Mizuochi, T., and Kobata, A. (982) Methods Enzymol. 83, 05-26 5. Ogata, S., Muramatsu, T., and Kobata, A. (975) J. Biochem. (Tokyo) 78,687-696 6. Kornfeld, K., Reitman, M. L., and Kornfeld, R. (98) J. Biol. Chem. 256,6633-6640 7. Cummings, R. D., and Kornfeld, S. (982) J. Biol. Chem. 257, 235-240 8. Yamashita, K., Hitoi, A., and Kobata, A. (983) J. Biol. Chem. 258,4753-4755 9. Boyd, W. C. (963) Vox Sang. 8, -32 0. Morgan, W. T. J., and Watkins, W. M. (953) Br. J. Exp. Pathol. 34,94-03. Watkins, W. M., and Morgan, W. T. J. (952) Nature (Lond.) 69,825-826 2. Takasaki, S., and Kobata, A. (976) J. Biol. Chem. 25, 3603-3609 3. Kochibe, N., and Furukawa, K. (980) Biochemistry 9, 284-2846 4. March, S. C., Parikh, I., and Cuatrecasas, P. (974) Anal. Biochem. 60, 49-52 5. Nishigaki, M., Yamashita, K., Matsuda, I., Arashima, S., and Kobata, A. (978) J. Biochem. (Tokyo) 84,823-834 6. Yamashita, K., Tachibana, Y., Takada, S., Matsuda, I., Arashima, Specificity Aleuria aurantia Lectin 4693 S., and Kobata, A. (979) J. Biol. Chem. 254,4820-4827 7. Kobata, A. (972) Methods Enzymol. 28, 262-27 8. Kobata, A., Yamashita, K., and Tachibana, Y. (978) Methods Enzymol. 50, 26-220 9. Ohkura, T., Isobe, T., Yamashita, K., and Kobata, A. (985) Biochemistry, in press 20. Ohkura, T., Yamashita, K., Mishima, Y., and Kobata, K. (984) Arch. Biochem. Bwphys. 235.63-77 2. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (95) J. Biol. Chem. 93, 265-275 22. Gamer, F. A., and Hilschmann, N. (972) Eur. J. Biochem. 26, 0-32 23. Gamer, F. A., Chang, L. S., Kiefer, C. R., Mendicino, J., Chandrasekaran, E. V., Isobe, T., and Osserman, E. F. (98) Eur. J. Biochem. 5,643-652 24. Kiefer, C. R., Patton, H. M., Jr., McGuire, B. S., Jr., and Gamer, F. A. (980) J. Immunol. 24, 30-306 25. Yamashita, K., Tachibana, Y., Nakayama, T., Kitamura, M., Endo, Y., and Kobata, A. (980) J. Bwl. Chem. 255, 5635-5642 26. Yoshima, H., Matsumoto, A., Mizuochi, T., Kawasaki, T., and Kobata, A. (98) J. Biol. Chem. 256,8476-8484 27. Matsumoto, A., Yoshima, H., Takasaki, S., and Kobata, A. (982) J. Biochem. (Tokyo) 9, 43-55 28. Mizoguchi, A., Mizuochi, T., and Kobata, A. (982) J. Biol. Chem. 257,962-962 29. Guillot, J., Genaud, L., Gueugnot, J., and Damez, M. (983) Biochemistry 22,5365-5369 30. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (95) J. Biol. Chem. 93, 265-275 3. Tanasaki, S., and Kobata, A. (978) Methods Enzymol. 50, 50-54 32. Petryniak, J., Pereira, M. E. A., and Kabat, E. A. (977) Arch. Biochem. Biophys. 78, 8-34 33. Shibata, S., Goldstein, I. J., and Baker, D. A. (982) J. Biol. Chem. 257,9324-9329