, Volume 16, Issue 1, pp 11–22 | Cite as

Primary structure of the H-2Db alloantigen

II. Additional amino acid sequence information, localization of a third site of glycosylation and evidence for K and D region specific sequences
  • W. Lee Maloy
  • John E. Coligan
Original Investigations


The complete amino acid sequence of the CNBr fragment comprising residues 229–284 of the murine major histocompatibility complex antigen H-2Db has been determined using radiochemical methodology. The sequence was determined by N-terminal sequence analysis of the intact CNBr fragment and by sequence determinations of peptides derived from this fragment by trypsin and staphylococcal V8 protease cleavage. In addition to the amino acid assignments for H-2Db, it was possible to assign the linkage position of the third N-linked glycosyl unit to the asparagine at residue 256. Additional amino acid sequence assignments have also been made for three other CNBr fragments that span residues 99–138, 139–228, and 308–331 of the H-2Db molecule. The total protein sequence information available (222 of 338 residues) agrees in every comparable position with the protein sequence derived from the cDNA clone (pH203) isolated by Reyes and co-workers (1982b), which strongly suggests that this clone encodes H-2Db. Combination of the protein sequence with that deduced from the cDNA clone provides the complete H-2Db protein sequence. Comparison of this sequence with other available protein sequence information for murine class I molecules has revealed protein sequences that may be unique to either K or D region molecules.


Major Histocompatibility Complex Protease Cleavage Additional Amino Acid Sequence Assignment Complete Amino Acid Sequence 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Abbreviations used in this paper


high performance liquid chromatography


Staphylococcus aureus V8 protease


major histocompatibility complex


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Brègègére, F., Abastado, J. P., Kvist, S., Rask, L., Lalanne, J. L., Garoff, H., Cami, B., Wiman, R., Larhammar, D., Peterson, P. A., Gachelin, G., Kourilsky, P., and Dobberstein, B.: Structure of C-terminal half of two H-2 antigens from cloned mRNA. Nature 292: 78–81, 1981Google Scholar
  2. Brown, J. L., Kato, K., Silver, J., and Nathenson, S. G.: Notable diversity in peptide composition of murine H-2K and H-2D alloantigens. Biochemistry 13: 3174–3178, 1974Google Scholar
  3. Coligan, J. E., Kindt, T. J., Nairn, R., Nathenson, S. G., Sachs, D. H., and Hansen, T. H.: Primary structural studies of an H-2L molecule confirm that it is a unique gene product with homology to H-2K and H-2D antigens. Proc. Natl. Acad. Sci. U.S.A. 77: 1134–1138, 1980Google Scholar
  4. Coligan, J. E. and Kindt, T. J.: Determination of protein primary structure by radiochemical techniques. J. Immunol. Methods 37: 287–299, 1981Google Scholar
  5. Coligan, J. E., Kimball, E. S., Kindt, T. J., Maloy, W. L., Martinko, J. M., Nairn, R., Nathenson, S. G., and Uehara, H.: Primary structural analyses of murine major histocompatibility antigens. Protides of the Biological Fluids 29: 237–242, 1981aGoogle Scholar
  6. Coligan, J. E., Kindt, T. J., Uehara, H., Martinko, J., and Nathenson, S. G.: Primary structure of a murine transplantation antigen. Nature 291: 35–39, 1981bGoogle Scholar
  7. Coligan, J. E., Gates, F. T., III, Kimball, E. S., and Maloy, W. L.: Radiochemical sequence analysis of biosynthetically labeled proteins. In C.H.W. Hirs and S. N. Timasheff (eds.) Methods in Enzymology: in press, 1982Google Scholar
  8. Evans, G. A., Margulies, D. H., Camerini-Otero, D., Ozato, K., and Seidman, J. G.: Structure and expression of a mouse major histocompatibility antigen gene, H-2Ld. Proc. Natl. Acad. Sci. U.S.A.: 79: 1994–1998, 1982Google Scholar
  9. Ewenstein, B. M., Nisizawa, T., Uehara, H., Nathenson, S. G., Coligan, J. E., and Kindt, T. J.: Primary structure of murine major histocompatibility complex alloantigens: Isolation, biochemical characterization and preliminary alignment of CNBr fragments from the H-2Kb glycoprotein. Proc. Natl. Acad. Sci. U.S.A.: 2909–2913, 1978Google Scholar
  10. Gates, F. T., III, Coligan, J. E., and Kindt, T. J.: Complete amino acid equence of rabbit β2-microglobulin. Biochemistry 18: 2267–2272, 1979.Google Scholar
  11. Hansen, T. H., Ozato, K., Melino, M. R., Coligan; J. E., Kindt, T. J., Jandinski, J., and Sachs, D. H.: Immunochemical evidence in two haplotypes for at least three D region-encoded molecules D, L and R. J. Immunol. 126: 1713–1716, 1981Google Scholar
  12. Iványi, D. and Démant, P.: Complex genetic effect of B10.D2 (M504)(H-2dm1) mutation. Immunogenetics 8: 539–550, 1979Google Scholar
  13. Kimball, E. S., Maloy, W. L., Martinko, J. M., Nathenson, S. G., and Coligan, J. E.: Structural studies on H-2Db and H-2Kd molecules indicate that murine major histocompatibility b and d haplotype alloantigens share structural features. Mol. Immunol. 17: 1283–1291, 1980Google Scholar
  14. Kimball, E. S., Nathenson, S. G., and Coligan, J. E.: Amino acid sequence of residues 1–98 of the H-2Kd murine major histocompatibility alloantigen: Comparison with H-2Kb and H-2Db reveals extensive localized differences. Biochemistry 20: 3301–3308, 1981aGoogle Scholar
  15. Kimball, E. S., Maloy, W. L., and Coligan, J. E.: Evidence for three carbohydrate prosthetic groups on mouse histocompatibility antigens H-2Kd and H-2Db. Mol. Immunol. 18: 677–680, 1981bGoogle Scholar
  16. Krakauer, T., Hansen, T. H., Camerini-Otero, R. D., and Sachs, D. H.: Analysis of the heterogeneity of the mouse H-2K, D, and L gene products. J. Immunol. 124 : 2149–2156, 1980Google Scholar
  17. Maloy, W. L., Hämmerling, G., Nathenson, S. G., and Coligan, J. E.: Comparison of alloantisera and hybridoma antibody for purification of the H-2Db murine histocompatibility antigen and preliminary molecular characterization of this antigen. J. Immunol. Methods 37: 287–299, 1980Google Scholar
  18. Maloy, W. L., Nathenson, S. G., and Coligan, J. E.: Primary structure ofmurine major histocompatibility complex alloantigens. Amino acid sequence of the NH2-terminal ninety-eight residues of the H-2Db glycoprotein. J. Biol. Chem. 256: 2863–2872, 1981Google Scholar
  19. Margulies, D. H., Evans, G. A., Flaherty, L., and Seidman, J. G.: H-2-like genes in the Tla region of mouse chromosome 17. Nature 295: 168–170, 1982Google Scholar
  20. Marshall, R. D.: Glycoproteins. Annu. Rev. Biochem. 41: 673–702, 1972Google Scholar
  21. Martinko, J. M., Uehara, H., Ewenstein, B. M., Kindt, T. J., Coligan, J. E., and Nathenson, S. G.: Primary structure of murine major histocompatibility complex alloantigens: completion of the sequence of the amino terminal two hundred eighty-four residues of H-2Kb. Biochemistry 19: 6188–6193, 1980Google Scholar
  22. Moore, K. W., Sher, B. T., Sun, Y. H., Eakle, K. A., and Hood, L.: DNA sequence of a gene encoding a BALB/c mouse Ld transplantation antigen. Science 215: 679–682, 1982Google Scholar
  23. Nathenson, S. G., Uehara, H., Ewenstein, B. M., Kindt, T. J., and Coligan, J. E.: Primary structural analysis of the transplantation antigens of the murine H-2 major histocompatibility complex. Annu. Rev. Biochem. 50: 1025–1052, 1981Google Scholar
  24. Nairn, R., Nathenson, S. G., and Coligan, J. E.: Isolation, characterization and amino acid sequence studies of the cyanogen bromide fragments of the H-2Dd glycoprotein. Eur. J. Immunol. 10: 495–503, 1980Google Scholar
  25. Nairn, R., Nathenson, S. G., and Coligan, J. E.: Amino acid sequence of cyanogen bromide fragment CNC (residues 24–98) of the mouse histocompatibility antigen H-2Dd: A comparison of the aminoterminal 100 residues of H-2Dd, Db, Kd and Kb reveals discrete areas of diversity. Biochemistry 20: 4739–4745, 1981Google Scholar
  26. O'Neill, H. C. and Parish, C. R.: Monoclonal antibody detection of two classes of H-2Kk molecules. Mol. Immunol. 18: 713–722, 1981Google Scholar
  27. Ploegh, H. L., Orr, H. T., and Strominger, J. L.: Major histocompatibility antigens: The human (HLA-A, -B, -C) and murine (H-2K, H-2D) class I molecules. Cell 24: 287–299, 1981Google Scholar
  28. Reyes, A. A., Schold, M., Itakura, K., and Wallace, R. B.: Isolation of a cDNA clone for the murine transplantation antigen H-2Kb. Proc. Natl. Acad. Sci. U.S.A.: In press, 1982aGoogle Scholar
  29. Reyes, A. A., Schold, M., and Wallace, R. B.: The complete amino acid sequence of the murine transplantation antigen H-2Db as deduced by molecular cloning. Immunogenetics 16: 1–9, 1982bGoogle Scholar
  30. Rothbard, J. B., Hopp, T. P., Edelman, G. M., and Cunningham, B. A.: Structure of the heavy chain of the H-2Kk histocompatibility antigen. Proc. Natl. Acad. Sci. U.S.A. 77: 4239–4243, 1980Google Scholar
  31. Schwartz, B. D., Kato, K., Cullen, S. E., and Nathenson, S. G.: H-2 histocompatibility alloantigens. Some biochemical properties of the molecules solubilized by NP-40 detergent. Biochemistry 12: 2157–2164, 1973Google Scholar
  32. Shearer, G. M. and Schmitt-Verhulst, A. M.: Major histocompatibility complex restricted cell-mediated immunity. Adv. Immunol. 25: 55–91, 1977Google Scholar
  33. Steinmetz, M., Frelinger, J. G., Fisher, D., Hunkapiller, T., Pereira, S., Weissman, S. M., Uehara, H., Nathenson, S., and Hood, L.: Three cDNA clones encoding mouse transplantation antigens: Homology to immunoglobulin genes. Cell 24: 125–134, 1981aGoogle Scholar
  34. Steinmetz, M., Moore, K. W., Frelinger, J. G., Sher, B. T., Shen, F-W., Boyse, E. A., and Hood, L.: A pseudogene homologous to mouse transplantation antigens: transplantation antigens are encoded by eight exons that correlate with protein domains. Cell 25: 683–692, 1981bGoogle Scholar
  35. Tarr, G. E., Beecher, J. F., Bell, M., and McKean, D. J.: Polyquarternary amines prevent peptide loss from sequenators. Anal. Biochem. 84: 622–627, 1978Google Scholar
  36. Uehara, H., Ewenstein, B. M., Martinko, J. M., Nathenson, S. G., Coligan, J. E., and Kindt, T. J.: Primary structure of murine major histocompatibility complex alloantigens: amino acid sequence of the amino-terminal one hundred and seventy-three resudues of the H-2Kb glycoprotein. Biochemistry 19: 306–315, 1980Google Scholar
  37. Waterfield, M. D. and Bridgen, J.: The automatic sequencer. In R. N. Perham (ed.): Instrumentation in Amino Acid Sequence Analysis, pp. 41–71, Academic Press, London, 1975Google Scholar
  38. Wu, T. T. and Kabat, E. A.: An analysis of the sequences of the variable regions of Bence Jones proteins and myeloma light chains and their implications for antibody complementarity. J. Exp. Med. 132: 211–250, 1970Google Scholar
  39. Zinkernagel, R. M. and Doherty, P. C.: MHC-restricted cytotoxic T cells: studies on the biological role of polymorphic major transplantation antigens determining T-cell restriction-specificity, function and responsiveness. Adv. Immunol. 27: 51–177, 1979Google Scholar

Copyright information

© Springer-Verlag 1982

Authors and Affiliations

  • W. Lee Maloy
    • 1
  • John E. Coligan
    • 1
  1. 1.Laboratory of Immunogenetics, National Institute of Allergy and Infectious DiseasesNational Institutes of HealthBethesda

Personalised recommendations