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. 1977 Jan;74(1):168–172. doi: 10.1073/pnas.74.1.168

Protein-sugar interactions: preparation, purification, and properties of rabbit antibodies against di-N-acetylchitobiose.

C M Kieda, F M Delmotte, M L Monsigny
PMCID: PMC393219  PMID: 264671

Abstract

Antibodies against di-N-acetylchitobiose (CB) were raised in rabbits after injection of CB-bovine serum albumin conjugates and were fractionated by columns into two classes: the first bound to a column of Sepharose covalently coupled with N-acetylglucosamine (GlcNAc); the second bound to a column coupled with CB. Active antibodies were eluted by a moderate concentration of a chaotropic agent, but not by high ionic strength buffers or acidic buffers. The active fractions were identified as IgG by ultracentrifugation and immuno-electrophoresis. These antibodies gave precipitation bands with CB-protein conjugates and this reaction could be reversed in the presence of free CB. The GlcNAc-bound fraction quantitatively quenched the fluorescence of O-(4-methylumbelliferyl)-glucosides of GlcNAc and CB, while the CB-bound fraction quenched only the glycoside of CB; other O-(4-methylumbelliferyl)-glucosides were not quenched. Among eleven monosaccharides and oligosaccharides, only GlcNAc, CB, and tri-N-acetylchitotriose were able to inhibit the precipitation of antibodies against CB with CB-protein conjugate. These antibodies failed to agglutinate erythrocytes from various species but did agglutinate transformed cells and mouse lymphocytes. The binding of these antibodies on cell lembranes was reversed by free CB and by CB-protein conjugates. The properties of these antibodies are related to those of lectins with similar specificities and to the structure of glycoconjugates.

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Selected References

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  1. Allen A. K., Neuberger A., Sharon N. The purification, composition and specificity of wheat-germ agglutinin. Biochem J. 1973 Jan;131(1):155–162. doi: 10.1042/bj1310155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anderson B., Jameson A., Hirata A. A., Safford J. W., Tomita J. T. Carcinoembryonic antigen-identification of a tri-N-acetylchitotriosyl type structure as an immunodeterminant group. Immunochemistry. 1975 Jul;12(6-7):577–580. doi: 10.1016/0019-2791(75)90088-9. [DOI] [PubMed] [Google Scholar]
  3. BERGMANN F. H., LEVINE L., SPIRO R. G. Fetuin: immunochemistry and quantitative estimation in serum. Biochim Biophys Acta. 1962 Mar 26;58:41–51. doi: 10.1016/0006-3002(62)90815-6. [DOI] [PubMed] [Google Scholar]
  4. Banjo C., Gold P., Gehrke C. W., Freedman S. O., Krupey J. Preparation and isolation of immunologically active glycopeptides from carcinoembryonic antigen (CEA). Int J Cancer. 1974 Feb 15;13(2):151–163. doi: 10.1002/ijc.2910130202. [DOI] [PubMed] [Google Scholar]
  5. Burger M. M., Goldberg A. R. Identification of a tumor-specific determinant on neoplastic cell surfaces. Proc Natl Acad Sci U S A. 1967 Feb;57(2):359–366. doi: 10.1073/pnas.57.2.359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. CONCHIE J. Beta-Glucosidase from rumen liquor; preparation, assay and kinetics of action. Biochem J. 1954 Dec;58(4):552–560. doi: 10.1042/bj0580552. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Delmotte F., Kieda C., Monsigny M. Protein-sugar interaction: purification by affinity chromatography of Solanum tuberosum agglutinin (STA-lectin). FEBS Lett. 1975 May 15;53(3):324–330. doi: 10.1016/0014-5793(75)80047-0. [DOI] [PubMed] [Google Scholar]
  8. Delmotte F., Kiéda C., Monsigny M. Mise en évidence d'une endo-beta-N-acétyl-glucosaminidase du sérum de lapin. C R Acad Sci Hebd Seances Acad Sci D. 1976 Oct 18;283(9):1121–1124. [PubMed] [Google Scholar]
  9. Hammarström S., Engvall E., Johansson B. G., Svensson S., Sundblad G., Goldstein I. J. Nature of the tumor-associated determinant(s) of carcinoembryonic antigen. Proc Natl Acad Sci U S A. 1975 Apr;72(4):1528–1532. doi: 10.1073/pnas.72.4.1528. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kornfeld R., Kornfeld S. The structure of a phytohemagglutinin receptor site from human erythrocytes. J Biol Chem. 1970 May 25;245(10):2536–2545. [PubMed] [Google Scholar]
  11. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  12. Loor F. Lectin-induced lymphocyte agglutination. An active cellular process? Exp Cell Res. 1973 Dec;82(2):417–425. doi: 10.1016/0014-4827(73)90360-1. [DOI] [PubMed] [Google Scholar]
  13. McCARTY M. Further studies on the chemical basis for serological specificity of Group A streptococcal carbohydrate. J Exp Med. 1958 Sep 1;108(3):311–323. doi: 10.1084/jem.108.3.311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Nagata Y., Burger M. M. Wheat germ agglutinin. Molecular characteristics and specificity for sugar binding. J Biol Chem. 1974 May 25;249(10):3116–3122. [PubMed] [Google Scholar]
  15. Parker D. C., Krause R. M., Wofsy L. Affinity labeling of anti-Group A streptococcal polysaccharide antibodies. Immunochemistry. 1973 Nov;10(11):727–734. doi: 10.1016/0019-2791(73)90174-2. [DOI] [PubMed] [Google Scholar]
  16. Privat J. P., Delmotte F., Mialonier G., Bouchard P., Monsigny M. Fluorescence studies of saccharide binding to wheat-germ agglutinin (lectin). Eur J Biochem. 1974 Aug 15;47(1):5–14. doi: 10.1111/j.1432-1033.1974.tb03661.x. [DOI] [PubMed] [Google Scholar]
  17. Privat J. P., Delmotte F., Monsigny M. Protein-sugar interactions. Association of beta-(1 leads to 4) linked N-acetyl-D-glucosamine oligomer derivatives with wheat germ agglutinin (lectin). FEBS Lett. 1974 Sep 15;46(1):224–228. doi: 10.1016/0014-5793(74)80373-x. [DOI] [PubMed] [Google Scholar]
  18. Privat J. P., Delmotte F., Monsigny M. Protein-sugar interactions. Association of wheat germ agglutinin (lectin) and O-(4-methyl-umbelliferyl)-glycosides. FEBS Lett. 1974 Sep 15;46(1):229–232. doi: 10.1016/0014-5793(74)80374-1. [DOI] [PubMed] [Google Scholar]
  19. Rafestin M. E., Obrenovitch A., Oblin A., Monsigny M. Purification of N-acetyl D-glucosamine-binding proteins by affinity chromatography. FEBS Lett. 1974 Mar 15;40(1):62–66. doi: 10.1016/0014-5793(74)80894-x. [DOI] [PubMed] [Google Scholar]
  20. Roche A. C., Monsigny M. Purification and properties of limulin: a lectin (agglutinin) from hemolymph of Limulus polyphemus. Biochim Biophys Acta. 1974 Nov 5;371(1):242–254. doi: 10.1016/0005-2795(74)90174-3. [DOI] [PubMed] [Google Scholar]
  21. Sela B. A., Wang J. L., Edelman G. M. Antibodies reactive with cell surface carbohydrates. Proc Natl Acad Sci U S A. 1975 Mar;72(3):1127–1131. doi: 10.1073/pnas.72.3.1127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Shier W. T. Preparation of a "chemical vaccine" against tumor progression. Proc Natl Acad Sci U S A. 1971 Sep;68(9):2078–2082. doi: 10.1073/pnas.68.9.2078. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Spik G., Bayard B., Fournet B., Strecker G., Bouquelet S., Montreuil J. Studies on glycoconjugates. LXIV. Complete structure of two carbohydrate units of human serotransferrin. FEBS Lett. 1975 Feb 15;50(3):296–299. doi: 10.1016/0014-5793(75)80513-8. [DOI] [PubMed] [Google Scholar]
  24. Spiro R. G. Glycoproteins. Adv Protein Chem. 1973;27:349–467. doi: 10.1016/s0065-3233(08)60451-9. [DOI] [PubMed] [Google Scholar]
  25. TABACHNICK M., SOBOTKA H. Azoproteins. II. A spectrophotometric study of the coupling of diazotized arsanilic acid with proteins. J Biol Chem. 1960 Apr;235:1051–1054. [PubMed] [Google Scholar]
  26. Tai T., Ito S., Yamashita K., Muramatsu T., Kobata A. Asparagine-linked oligosaccharide chains of IgG: a revised structure. Biochem Biophys Res Commun. 1975 Aug 4;65(3):968–974. doi: 10.1016/s0006-291x(75)80480-3. [DOI] [PubMed] [Google Scholar]
  27. Vrba R., Alpert E., Isselbacher K. J., Jeanloz R. W. Studies on carcino embryonic antigen (CEA): effect of carbohydrate and synthetic glycopeptides on CEA competitive-inhibition radioimmunoassays. Immunochemistry. 1976 Mar;13(3):285–287. doi: 10.1016/0019-2791(76)90228-7. [DOI] [PubMed] [Google Scholar]
  28. Watanabe K., Hakomori S. A glycosphingolipid sharing reactivity with both wheat germ lectin and "carcinoembryonic antisera (Gold)": partial identity of these reactive sites. FEBS Lett. 1973 Dec 1;37(2):317–320. doi: 10.1016/0014-5793(73)80486-7. [DOI] [PubMed] [Google Scholar]

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