Urinary Oligosaccharides of GM,-Gangliosidosis

Transcription

1 ~~ THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol No. 16, Issue of August 25. pp , 1981 Prcnted m U. S.A. Urinary Oligosaccharides of GM,-Gangliosidosis STRUCTURES OF OLIGOSACCHARIDES EXCRETED IN THE URINE OF TYPE 1 BUT NOT IN THE URlNE OF TYPE 2 PATIENTS* (Received for publication, February 18, 1981, and in revised form, April 28, 1981) Takashi Ohkura, Katsuko Yamashita, and Akira Kobata From the Department of Biochemistry, Kobe University School of Medicine, Chuo-ku, Kobe, Japan Among the seven oligosaccharide fractions obtained by Bio-Gel P-4 column chromatography of urine of Most of the Results in detail are presented in miniprint gangliosidosis Type 1 patients, three fractions (peaks and only the synopsis is described here. V, VI, and W) were completely missing in the urine of All oligosaccharides in peaks V, VI, and VI1 are composed Gxl-gangliosidosis Type 2 patients. Structural study of of mannose, N-acetylglucosamine, and galactose. They all these oligosaccharide fractions by sequential exogly- contain three mannoses, but differ in their contents of galaccosidasedigestionincombinationwithmethylation tose and N-acetylglucosamine (Table X). These oligosacchaanalysis and periodate oxidation has shown that peaks rides were completely converted to Manal + G(Mancu1 4 V, VZ, and Vn are mixtures of 16, 30, and 49 isomeric S)Ma@l+ 4GlcNAco~ by incubation with a mixture of jack oligosaccharides. All these 95 oligosaccharides contain Galpl-+ 4GlcNAcp13 3 repeating structures in their bean,%galactosidase and jack bean,b-n-acetylhexosaminiouter chain moieties, indicating that tissues the of Gwldase. Therefore, oligosaccharides in peaks V, VI, and VI1 gangliosidosis Type 2 patients do contain p-galactosidase activity which releases readily galactose residue from such repeating sugar chains. As already shown in many papers (1-61, structural study of the oligosaccharides excreted in the urine of exoglycosidase deficiency patients gives an useful information of the structures of asparagine-linked sugar chains produced within human body. Oligosaccharide patterns obtained by Bio-Gel P-4 column chromatography of the urine of GMl-gangliosidosis Type 1 patients are quite different from those of Gul-gangliosidosis Type 2 (7). The amount of total oligosaccharides excreted in the urine of Type 2 patients is 10-20% of that in the urine of Type 1 patients. Not only quantitative difference, but qualitative difference was also found between the oligosaccharides excreted in the urine of two GMl-gangliosidosis subgroups: urine from Type 2 patients do not contain the peaks V, VI, and VI1 which were always found in the urine of Type 1 patients. In order to disclose the whole feature of the biochemical basis of GMl-gangliosidosis subgroups, and also to obtain further information about the sugar chain structures of human glycoproteins, the structural study of the oligosaccharides in peaks V, VI, and VI1 was performed. EXPERIMENTAL PROCEDURES, * This work was supported in part by research grants from the Yamanouchi Foundation for Research on Metabolic Disorders and the Scientific Research Funds of the Ministry of Education, Science, and Culture of 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 aduertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Portions of this paper (including Experimental Procedures, most of the Results, Tables I and 11, Figs. 1-7 and 9, and Scheme 1) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD Request Document No. 81M-359, cite author(s), and include a check or money order for $11.20 per set ofphotocopies. Full size photocopies are also included RESULTS AND DISCUSSION should have 5, 6, and 7 each of,&galactosyl and P-N-acetylglucosaminyl residues linked to the tetrasaccharide core. That the two monosaccharide residues occur as Gal/3l+ 4GlcNAc unit was confirmed by sequential digestion with jack bean,bgalactosidase and jack bean P-N-acetylhexosaminidase and by methylation analysis. The sequential exoglycosidase digestion also revealed that peaks V, VI, and V1I were mixtures of triantennary and tetraantennary oligosaccharides. The tetraantennary oligosaccharides in peaks V, VI, and VII were named as V-1, VI-1, and VII-1 and the triantennary oligosaccharides in peaks V, VI, and VI1 as V-2, VI-2, and VII-2, respectively. Detailed structural analysis of each oiigosaccharides by sequential exoglycosidase digestion combination with methylation analysis and periodate oxidation disclosed that all these oligosaccharides are isomeric mixtures with the structures as shown in Fig. 8. That Gwl -+ 4GlcNAc,B repeating units are randomly distributed on the outer chains was confirmed by using the substrate specificity of diplococcal,b-n-acetylhexosaminidase (26). For example, the radioactive oligosaccharide V-1 was subjected to a sequential digestion with jack bean,bgalactosidase and jack bean -&-N-acetylhexosaminidase. Since this oligosaccharide has 3 GalBl4 4GlcNAc and 1 Gal GlcNAcPl * 3GalPl 4GlcNAc outer chains, a single Gal61 --i 4GlcNAc group should remain at its outer chain moiety by the enzymatic digestion. The radioactive product was then incubated with a mixture of diplococcal P-galacto- sidase and diplococcal j3-n-acetylhexosaminidase. Analysis of the radioactive oligosaccharides in the reaction mixture (as described in detail in the Miniprint) revealed the presence of Man3-GlcNAco~, GlcNAcPl -+ 6Manal 3 G(Mana1 -+ 3)Man,Bl -f 4GlcNAco~, and Manal -+ 6 (GlcNAcPl -+ 4Manal-+ B)Man,Bl-+ 4GlcNAco~. This result indicated that the tetrasaccharide outer chain was distributed at all four in the microfilm edition of the Journal that is available from Waverly Press. * The abbreviation used is: XylNAc, N-acetylxylosamine. Subscript OT is used in this paper to indicate NaBt3H)+-reduced oligosaccharides. All sugars mentioned in this paper were of D-configuration.

2 8486 Oligosaccharides in GMI - Gangliosidosis Urine v-l Cal6l+4ClcNAc6kg Mamk6 Cal@+4ClCNAC& 3Man8b4ClcNAc Cali?J+llClCNAC6h,, f Manal Cal8+4ClCNAC68 V-7a V- 2b VI-la VI-lb VI-2a VI-2b FIG. 8. Proposed structures of oligosaccharides excreted in the urine of Gm-gangliosidosis Type 1 patients. In order to save space, GalPl+ 4GlcNAcP1+ 3 repeating structures are shown by parentheses. For example, (Galpl- 4GlcNAc~I-+ 3)z stands for Gal@ + 4GlcNAcj31+ 3GalB1+ 4 GlcNAcPlj 3. VIF2C possible positions of the tetraantennary structure as shown in Fig. 8. Results of the analysis of other five oligosaccharide fractions as described in the Miniprint also indicated that the GalPl -+ 4GlcNAcP1 + 3 repeating units are distributed randomly in all cases. Therefore, peaks V, VI, and VI1 are mixtures of 16, 30, and 49 isomeric oligosaccharides, respectively. The oligosaccharides shown in Fig. 8, which are excreted in the urine of GM,-gangliosidosis Type 1 patients but not in the urine of Type 2 patients, all contain GalBl+ 4GlcNAcPlj 3 repeating structures in their outer chain moieties. This result supports the inference made in the preceding paper (7) that the tissues of GMl-gangliosidosis Type 2 patients have a P- galactosidase activity which readily releases galactose residue from outer chains with GalPl -+ 4GlcNAcP repeating structures, but act very slowly on GalPl -+ 4GlcNAc outer chain. All 95 oligosaccharides reported in this paper have ManPl

3 Gim-Gangliosidosis Oligosaccharides Urine -+ 4GlcNAc at their reducing termini, indicating that they are REFERENCES released from the asparagine-linked sugar chains of glycopro- 1. Nishigaki, M., Yamashita, K., Matsuda, I., Arashima, S., and teins by the action of endo-p-n-acetylglucosaminidase (15- Kobata, A. (1978) J. Biochem. (Tokyo) 84, ). Therefore, the structures of these oligosaccharides should 2. Yamashita, K., Tachibana, Y., Takada, S., Matsuda, I., Arashima, reflect those of the asparagine-linked sugar chains produced - S., and Kobata, A. (1979) J. Biol. Chem. 254, Strecker, G., Peers, M.-C., Michalski, J.-C., Hondi-Assah, J., within human body. The structural rule that GlcNAcPl -+ Farriaux, J.-P., Maroteau, P., and Durand, P. (1977) Eur. J. 4(GlcNAcPl-+ 2)Man group is formed only on Manal 3 Biochem. 75, side and GlcNAcPl 4 G(GlcNAcPl-+ 2)Man group only on 4. Strecker, G., Peers, M.-C., Fournet, B., and Montreuil, J. (1977) the Mand + 6 side in the triantennary and tetraantennary Eur. J. Biochem. 81, asparagine-linked sugar chains of human glycoproteins, as 5. Nordin, N. E., Lundblad, A., Svensson, S., and Autio, S. (1974) suggested in the previous paper (7), canbe applied to all Biochemistry 13, Yamashita, K., Tachibana, Y., Mihara, K., Okada, S., Yabuuchi, oligosaccharides reported in this paper. H., and Kobata, A. (1980) J. Biol. Chem. 255, We have recently found that some of the bovine plasma 7. Yamashita, K., Ohkura, T., Okada, S., Yabuuchi, H., and Kobata, glycoproteins contain an unusual disaccharide, GalPl.+ A. (1981) J. Biol. Chem. 256, GlcNAc, in the outer chain moieties of their asparagine- 8. Tai, T., Ito, S., Yamashita, K., Muramatsu, T., and Kobata, A. linked sugar chains (18-20). The plasma membrane glycopro- (1975) Biochem. Biophys. Res. Commun. 65, teins of bovine thymocytes have been shown to contain an- 9. Li, Y.-T., and Li, S.-C. (1972) Methods Enzymol. 28, Sugawara, K., Okumura, T., and Yamashina, I. (1972) Biochim. other unusual GalPl+ 3G@l+ 4GlcNAc group in the outer Biophys. Acta 268, chain moieties of their asparagine-linked sugar chains (21-23). 11. Glasgow, L. R., Paulson, J. C., and Hill, R. L. (1977) J. Biol. Complete absence of oligosaccharides with such outer chains Chem. 252, in the urine of Type 1 patients indicates that the unusual 12. Takasaki, S., and Kobata, A. (1978) Methods Enzymol. 50,50-54 asparagine-linked sugar chains found in bovine tissue are not 13. Endo, Y., Yamashita, K., Tachibana, Y., Tojo, S., and Kobata, A. formed within human body. Our results on this point is, (1979) J. Biochem. (Tokyo) 85, Mizuochi, T., Yonemasu, K., Yamashita, K., and Kobata, A. however, inconsistent with the result reported by Strecker et (1978) J. Biol. Chem. 253, a2. (24), that GalPl -+ 3Gal group does exist in some of the 15. Nishigaki, M., Muramatsu, T., and Kobata, A. (1974) Biochem. urinary oligosaccharides obtained from GM,-gangliosidosis pa- Biophys. Res. Commun. 59, tients. 16. Pierce, R., Spik, G., and Montreuil, J. (1979) in Glycoconjugate The evidence that the Gal -+ 4GlcNAcPl-+ 3 repeating (Schauer, R., Boer, P., Buddecke, E., Kramer, M. F., Vilegenunits are randomly distributed in the tri- and tetraantennary thart, J. F. C., and Wiegandt, H., eds) pp , Georg Thieme Publishers, Stuttgardt, West Germany outer chains of oligosaccharides reported in this paper indi- 17. Tachibana, Y., Yamashita, K., and Kobata, A. (1980) Seikuguku cates that the N-acetylglucosaminyl transferase which elon- 52,554 gate the disaccharide outer chains of complex type asparagine- 18. Mizuochi, T., Yamashita, K., Fujikawa, K., Kisiel, W., and Kobata, linked sugar chains can add N-acetylglucosamine to any P- A. (1979) J. Biol. Chem. 254, galactosyl residue of the sugar chains. This enzyme may also 19. Mizuochi, T., Yamashita, K., Fujikawa, K., Titani, K., and Kobe responsible for the formation of G@l -f 4GlcNAcPl- 3 bata, A. (1980) J. Biol. Chem. 255, repeating units found in the mucin-type sugar chains such as 20. Takasaki, S., Yamashita, K., Suzuki, K., Iwanaga, S., and Kobata, A. (1979) J. Biol. Chem. 254, in blood group substances. Asparagine-linked sugar chains 21. Kornfeld, R. (1978) Biochemistry 17, with GalPl -+ 4GlcNAcP repeating unit in their outer 22. Yoshima, H., Takasaki, S., and Kobata, A. (1980) J. Biochem. chain moieties have already been found in band 3 glycoprotein (Tokyo) 88, of human erythrocyte (25). 23. Yoshima, H., Takasaki, S., and Kobata, A. (1980) J. Biol. Chem. 255, Montreuil, J. (1980) in Adu. Curbohydr. Chem. Biochem. 37, Acknowledgments-We wish to thank Dr. S. Okada, Department 25. Jiirnefelt, J., Rush, J., Li, Y.-T., and Laine, R. A. (1978) J. Biol. of Paediatrics, Osaka University Hospital, for providing us the urine Chem. 253, samples of GMl-gangliosidosis Type i patients. Thank; are also due 26. Yamashita, K., Ohkura, T., Yoshima, H., and Kobata, A. (1981) to Junk0 Fujii for her expert secretarial assistance. Biochem. Biophys. Res. Commun. 100, &1a7

4 Oligosaccharides in GM1-Gangliosidosis Urine

5 Oligosaccharides in Gm,-Gangliosidosis Urine 8489 UreS 2A ELUTION VOLUME (MU F I E] ELUTION VOLUME (MU 2 1 " ELUTION VOLUME IMLI

6 8490 Oligosaccharides in GM1-Gangliosidosis Urine le A DISTANCE FRM ORIGIN (CM) treated peak b I" FL~. 70. E and F. from "-2: e, that from VI-2; F, that from VII ELUTION 225 VOLUME (ML) Fig.5A Fig.5C