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. 1994 Mar 1;179(3):1035–1040. doi: 10.1084/jem.179.3.1035

Identification of amino acids at the junction of exons 3 and 7 that are used for the generation of glycosylation-related human CD45RO and CD45RO-like antigen specificities

PMCID: PMC2191423  PMID: 7509359

Abstract

The CD45 transmembrane protein tyrosine phosphatase plays an essential role in lymphocyte activation. In humans, CD45 is composed of five isoforms that are generated by alternative splicing of three exons of a common precursor mRNA. Expression of the smallest molecular mass 180-kD CD45 isoform (CD45-O) results from splicing out of exons 4(A), 5(B), and 6(C), which encode peptide regions near the NH2 terminus, and is regulated during T cell maturation and activation. Two monoclonal antibodies (mAb), UCHL1 (anti-CD45RO) and A6 (anti-CD45RO-like), were studied that selectively bind to murine transfectant cells expressing the human CD45-O isoform. The anti-CD45RO-like A6 mAb, but not the anti- CD45RO UCHL1 mAb, also weakly reacted with transfectant cells expressing the human CD45 isoforms that contained exons 4 and 5(AB), or exon 5(B) encoded sequences. The structural basis of the antigen specificities of these two different human anti-CD45RO mAbs was investigated at the molecular level by using potential glycosylation- defective CD45-O isoform variants containing amino acid substitutions at the junction of exons 3 and 7. Replacement of the threonine residue at position 8 (last amino acid encoded in exon 3 and a putative O- linked carbohydrate anchorage site) by an alanine, completely abrogated the reactivity of the UCHL1 mAb, but did not affect that of the A6 mAb. Conversely, replacement of either the asparagine at position 174 or the serine at position 176 (the first two putative carbohydrate anchorage sites in exon 7) by alanine, abrogated the reactivity of the A6 mAb, but not that of the UCHL1 mAb. Both the UCHL1 and A6 epitopes were dependent on the presence of O-linked carbohydrates; and the UCHL1, but not the A6 epitope, was dependent on the presence of sialic acid. These results demonstrate a pivotal role for the amino acids encoded at the junction of exons 3 and 7 for the generation of glycosylation-related CD45RO epitopes that are expressed in a cell lineage- and activation- regulated fashion.

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Selected References

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  1. Aversa G. G., Hall B. M. Memory T cells express high levels of a novel leukocyte common antigen epitope identified by the A6 MAb. Transplant Proc. 1991 Feb;23(1 Pt 1):300–301. [PubMed] [Google Scholar]
  2. Berti E., Aversa G. G., Soligo D., Cattoretti G., Delia D., Aiello A., Parravicini C., Hall B. M., Caputo R. A6--a new 45RO monoclonal antibody for immunostaining of paraffin-embedded tissues. Am J Clin Pathol. 1991 Feb;95(2):188–193. doi: 10.1093/ajcp/95.2.188. [DOI] [PubMed] [Google Scholar]
  3. Chang H. L., Lefrancois L., Zaroukian M. H., Esselman W. J. Developmental expression of CD45 alternate exons in murine T cells. Evidence of additional alternate exon use. J Immunol. 1991 Sep 1;147(5):1687–1693. [PubMed] [Google Scholar]
  4. Desai D. M., Sap J., Schlessinger J., Weiss A. Ligand-mediated negative regulation of a chimeric transmembrane receptor tyrosine phosphatase. Cell. 1993 May 7;73(3):541–554. doi: 10.1016/0092-8674(93)90141-c. [DOI] [PubMed] [Google Scholar]
  5. Dianzani U., Redoglia V., Malavasi F., Bragardo M., Pileri A., Janeway C. A., Jr, Bottomly K. Isoform-specific associations of CD45 with accessory molecules in human T lymphocytes. Eur J Immunol. 1992 Feb;22(2):365–371. doi: 10.1002/eji.1830220212. [DOI] [PubMed] [Google Scholar]
  6. Goldman S. J., Uniyal S., Ferguson L. M., Golan D. E., Burakoff S. J., Kiener P. A. Differential activation of phosphotyrosine protein phosphatase activity in a murine T cell hybridoma by monoclonal antibodies to CD45. J Biol Chem. 1992 Mar 25;267(9):6197–6204. [PubMed] [Google Scholar]
  7. Justement L. B., Campbell K. S., Chien N. C., Cambier J. C. Regulation of B cell antigen receptor signal transduction and phosphorylation by CD45. Science. 1991 Jun 28;252(5014):1839–1842. doi: 10.1126/science.1648262. [DOI] [PubMed] [Google Scholar]
  8. Kishihara K., Penninger J., Wallace V. A., Kündig T. M., Kawai K., Wakeham A., Timms E., Pfeffer K., Ohashi P. S., Thomas M. L. Normal B lymphocyte development but impaired T cell maturation in CD45-exon6 protein tyrosine phosphatase-deficient mice. Cell. 1993 Jul 16;74(1):143–156. doi: 10.1016/0092-8674(93)90302-7. [DOI] [PubMed] [Google Scholar]
  9. Lokeshwar V. B., Bourguignon L. Y. Tyrosine phosphatase activity of lymphoma CD45 (GP180) is regulated by a direct interaction with the cytoskeleton. J Biol Chem. 1992 Oct 25;267(30):21551–21557. [PubMed] [Google Scholar]
  10. Mustelin T., Pessa-Morikawa T., Autero M., Gassmann M., Andersson L. C., Gahmberg C. G., Burn P. Regulation of the p59fyn protein tyrosine kinase by the CD45 phosphotyrosine phosphatase. Eur J Immunol. 1992 May;22(5):1173–1178. doi: 10.1002/eji.1830220510. [DOI] [PubMed] [Google Scholar]
  11. Ostergaard H. L., Shackelford D. A., Hurley T. R., Johnson P., Hyman R., Sefton B. M., Trowbridge I. S. Expression of CD45 alters phosphorylation of the lck-encoded tyrosine protein kinase in murine lymphoma T-cell lines. Proc Natl Acad Sci U S A. 1989 Nov;86(22):8959–8963. doi: 10.1073/pnas.86.22.8959. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Pingel J. T., Thomas M. L. Evidence that the leukocyte-common antigen is required for antigen-induced T lymphocyte proliferation. Cell. 1989 Sep 22;58(6):1055–1065. doi: 10.1016/0092-8674(89)90504-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Pulido R., Cebrián M., Acevedo A., de Landázuri M. O., Sánchez-Madrid F. Comparative biochemical and tissue distribution study of four distinct CD45 antigen specificities. J Immunol. 1988 Jun 1;140(11):3851–3857. [PubMed] [Google Scholar]
  14. Pulido R., Sánchez-Madrid F. Glycosylation of CD45: carbohydrate composition and its role in acquisition of CD45R0 and CD45RB T cell maturation-related antigen specificities during biosynthesis. Eur J Immunol. 1990 Dec;20(12):2667–2671. doi: 10.1002/eji.1830201221. [DOI] [PubMed] [Google Scholar]
  15. Rothstein D. M., Saito H., Streuli M., Schlossman S. F., Morimoto C. The alternative splicing of the CD45 tyrosine phosphatase is controlled by negative regulatory trans-acting splicing factors. J Biol Chem. 1992 Apr 5;267(10):7139–7147. [PubMed] [Google Scholar]
  16. Schraven B., Kirchgessner H., Gaber B., Samstag Y., Meuer S. A functional complex is formed in human T lymphocytes between the protein tyrosine phosphatase CD45, the protein tyrosine kinase p56lck and pp32, a possible common substrate. Eur J Immunol. 1991 Oct;21(10):2469–2477. doi: 10.1002/eji.1830211025. [DOI] [PubMed] [Google Scholar]
  17. Smith S. H., Brown M. H., Rowe D., Callard R. E., Beverley P. C. Functional subsets of human helper-inducer cells defined by a new monoclonal antibody, UCHL1. Immunology. 1986 May;58(1):63–70. [PMC free article] [PubMed] [Google Scholar]
  18. Streuli M., Hall L. R., Saga Y., Schlossman S. F., Saito H. Differential usage of three exons generates at least five different mRNAs encoding human leukocyte common antigens. J Exp Med. 1987 Nov 1;166(5):1548–1566. doi: 10.1084/jem.166.5.1548. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Streuli M., Morimoto C., Schrieber M., Schlossman S. F., Saito H. Characterization of CD45 and CD45R monoclonal antibodies using transfected mouse cell lines that express individual human leukocyte common antigens. J Immunol. 1988 Dec 1;141(11):3910–3914. [PubMed] [Google Scholar]
  20. Streuli M., Saito H. Regulation of tissue-specific alternative splicing: exon-specific cis-elements govern the splicing of leukocyte common antigen pre-mRNA. EMBO J. 1989 Mar;8(3):787–796. doi: 10.1002/j.1460-2075.1989.tb03439.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Takebe Y., Seiki M., Fujisawa J., Hoy P., Yokota K., Arai K., Yoshida M., Arai N. SR alpha promoter: an efficient and versatile mammalian cDNA expression system composed of the simian virus 40 early promoter and the R-U5 segment of human T-cell leukemia virus type 1 long terminal repeat. Mol Cell Biol. 1988 Jan;8(1):466–472. doi: 10.1128/mcb.8.1.466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Thomas M. L. The leukocyte common antigen family. Annu Rev Immunol. 1989;7:339–369. doi: 10.1146/annurev.iy.07.040189.002011. [DOI] [PubMed] [Google Scholar]
  23. Torimoto Y., Dang N. H., Vivier E., Tanaka T., Schlossman S. F., Morimoto C. Coassociation of CD26 (dipeptidyl peptidase IV) with CD45 on the surface of human T lymphocytes. J Immunol. 1991 Oct 15;147(8):2514–2517. [PubMed] [Google Scholar]
  24. Trowbridge I. S. CD45. A prototype for transmembrane protein tyrosine phosphatases. J Biol Chem. 1991 Dec 15;266(35):23517–23520. [PubMed] [Google Scholar]

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