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. 1995 Dec 2;131(6):1623–1633. doi: 10.1083/jcb.131.6.1623

Regulation of CD44 binding to hyaluronan by glycosylation of variably spliced exons

PMCID: PMC2120678  PMID: 8522617

Abstract

The hyaluronan (HA)-binding function (lectin function) of the leukocyte homing receptor, CD44, is tightly regulated. Herein we address possible mechanisms that regulate CD44 isoform-specific HA binding. Binding studies with melanoma transfectants expressing CD44H, CD44E, or with soluble immunoglobulin fusions of CD44H and CD44E (CD44H-Rg, CD44E-Rg) showed that although both CD44 isoforms can bind HA, CD44H binds HA more efficiently than CD44E. Using CD44-Rg fusion proteins we show that the variably spliced exons in CD44E, V8-V10, specifically reduce the lectin function of CD44, while replacement of V8-V10 by an ICAM-1 immunoglobulin domain restores binding to a level comparable to that of CD44H. Conversely, CD44 bound HA very weakly when exons V8-V10 were replaced with a CD34 mucin domain, which is heavily modified by O- linked glycans. Production of CD44E-Rg or incubation of CD44E- expressing transfectants in the presence of an O-linked glycosylation inhibitor restored HA binding to CD44H-Rg and to cell surface CD44H levels, respectively. We conclude that differential splicing provides a regulatory mechanism for CD44 lectin function and that this effect is due in part to O-linked carbohydrate moieties which are added to the Ser/Thr rich regions encoded by the variably spliced CD44 exons. Alternative splicing resulting in changes in protein glycosylation provide a novel mechanism for the regulation of lectin activity.

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Selected References

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  1. Arch R., Wirth K., Hofmann M., Ponta H., Matzku S., Herrlich P., Zöller M. Participation in normal immune responses of a metastasis-inducing splice variant of CD44. Science. 1992 Jul 31;257(5070):682–685. doi: 10.1126/science.1496383. [DOI] [PubMed] [Google Scholar]
  2. Aruffo A., Stamenkovic I., Melnick M., Underhill C. B., Seed B. CD44 is the principal cell surface receptor for hyaluronate. Cell. 1990 Jun 29;61(7):1303–1313. doi: 10.1016/0092-8674(90)90694-a. [DOI] [PubMed] [Google Scholar]
  3. Ashwell G., Harford J. Carbohydrate-specific receptors of the liver. Annu Rev Biochem. 1982;51:531–554. doi: 10.1146/annurev.bi.51.070182.002531. [DOI] [PubMed] [Google Scholar]
  4. Bartolazzi A., Jackson D., Bennett K., Aruffo A., Dickinson R., Shields J., Whittle N., Stamenkovic I. Regulation of growth and dissemination of a human lymphoma by CD44 splice variants. J Cell Sci. 1995 Apr;108(Pt 4):1723–1733. doi: 10.1242/jcs.108.4.1723. [DOI] [PubMed] [Google Scholar]
  5. Bartolazzi A., Peach R., Aruffo A., Stamenkovic I. Interaction between CD44 and hyaluronate is directly implicated in the regulation of tumor development. J Exp Med. 1994 Jul 1;180(1):53–66. doi: 10.1084/jem.180.1.53. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Braesch-Andersen S., Stamenkovic I. Sialylation of the B lymphocyte molecule CD22 by alpha 2,6-sialyltransferase is implicated in the regulation of CD22-mediated adhesion. J Biol Chem. 1994 Apr 22;269(16):11783–11786. [PubMed] [Google Scholar]
  7. Brown T. A., Bouchard T., St John T., Wayner E., Carter W. G. Human keratinocytes express a new CD44 core protein (CD44E) as a heparan-sulfate intrinsic membrane proteoglycan with additional exons. J Cell Biol. 1991 Apr;113(1):207–221. doi: 10.1083/jcb.113.1.207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Culty M., Miyake K., Kincade P. W., Sikorski E., Butcher E. C., Underhill C., Silorski E. The hyaluronate receptor is a member of the CD44 (H-CAM) family of cell surface glycoproteins. J Cell Biol. 1990 Dec;111(6 Pt 1):2765–2774. doi: 10.1083/jcb.111.6.2765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Damle N. K., Klussman K., Aruffo A. Intercellular adhesion molecule-2, a second counter-receptor for CD11a/CD18 (leukocyte function-associated antigen-1), provides a costimulatory signal for T-cell receptor-initiated activation of human T cells. J Immunol. 1992 Feb 1;148(3):665–671. [PubMed] [Google Scholar]
  10. Diamond M. S., Staunton D. E., Marlin S. D., Springer T. A. Binding of the integrin Mac-1 (CD11b/CD18) to the third immunoglobulin-like domain of ICAM-1 (CD54) and its regulation by glycosylation. Cell. 1991 Jun 14;65(6):961–971. doi: 10.1016/0092-8674(91)90548-d. [DOI] [PubMed] [Google Scholar]
  11. Dougherty G. J., Cooper D. L., Memory J. F., Chiu R. K. Ligand binding specificity of alternatively spliced CD44 isoforms. Recognition and binding of hyaluronan by CD44R1. J Biol Chem. 1994 Mar 25;269(12):9074–9078. [PubMed] [Google Scholar]
  12. Dougherty G. J., Landorp P. M., Cooper D. L., Humphries R. K. Molecular cloning of CD44R1 and CD44R2, two novel isoforms of the human CD44 lymphocyte "homing" receptor expressed by hemopoietic cells. J Exp Med. 1991 Jul 1;174(1):1–5. doi: 10.1084/jem.174.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Galandrini R., Galluzzo E., Albi N., Grossi C. E., Velardi A. Hyaluronate is costimulatory for human T cell effector functions and binds to CD44 on activated T cells. J Immunol. 1994 Jul 1;153(1):21–31. [PubMed] [Google Scholar]
  14. Goldstein L. A., Zhou D. F., Picker L. J., Minty C. N., Bargatze R. F., Ding J. F., Butcher E. C. A human lymphocyte homing receptor, the hermes antigen, is related to cartilage proteoglycan core and link proteins. Cell. 1989 Mar 24;56(6):1063–1072. doi: 10.1016/0092-8674(89)90639-9. [DOI] [PubMed] [Google Scholar]
  15. Green S. J., Tarone G., Underhill C. B. Aggregation of macrophages and fibroblasts is inhibited by a monoclonal antibody to the hyaluronate receptor. Exp Cell Res. 1988 Oct;178(2):224–232. doi: 10.1016/0014-4827(88)90393-x. [DOI] [PubMed] [Google Scholar]
  16. Günthert U., Hofmann M., Rudy W., Reber S., Zöller M., Haussmann I., Matzku S., Wenzel A., Ponta H., Herrlich P. A new variant of glycoprotein CD44 confers metastatic potential to rat carcinoma cells. Cell. 1991 Apr 5;65(1):13–24. doi: 10.1016/0092-8674(91)90403-l. [DOI] [PubMed] [Google Scholar]
  17. Hathcock K. S., Hirano H., Murakami S., Hodes R. J. CD44 expression on activated B cells. Differential capacity for CD44-dependent binding to hyaluronic acid. J Immunol. 1993 Dec 15;151(12):6712–6722. [PubMed] [Google Scholar]
  18. He Q., Lesley J., Hyman R., Ishihara K., Kincade P. W. Molecular isoforms of murine CD44 and evidence that the membrane proximal domain is not critical for hyaluronate recognition. J Cell Biol. 1992 Dec;119(6):1711–1719. doi: 10.1083/jcb.119.6.1711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Huet S., Groux H., Caillou B., Valentin H., Prieur A. M., Bernard A. CD44 contributes to T cell activation. J Immunol. 1989 Aug 1;143(3):798–801. [PubMed] [Google Scholar]
  20. Hyman R., Lesley J., Schulte R. Somatic cell mutants distinguish CD44 expression and hyaluronic acid binding. Immunogenetics. 1991;33(5-6):392–395. doi: 10.1007/BF00216699. [DOI] [PubMed] [Google Scholar]
  21. Jackson D. G., Bell J. I., Dickinson R., Timans J., Shields J., Whittle N. Proteoglycan forms of the lymphocyte homing receptor CD44 are alternatively spliced variants containing the v3 exon. J Cell Biol. 1995 Feb;128(4):673–685. doi: 10.1083/jcb.128.4.673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Jackson D. G., Buckley J., Bell J. I. Multiple variants of the human lymphocyte homing receptor CD44 generated by insertions at a single site in the extracellular domain. J Biol Chem. 1992 Mar 5;267(7):4732–4739. [PubMed] [Google Scholar]
  23. Jalkanen S., Bargatze R. F., de los Toyos J., Butcher E. C. Lymphocyte recognition of high endothelium: antibodies to distinct epitopes of an 85-95-kD glycoprotein antigen differentially inhibit lymphocyte binding to lymph node, mucosal, or synovial endothelial cells. J Cell Biol. 1987 Aug;105(2):983–990. doi: 10.1083/jcb.105.2.983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Jalkanen S., Jalkanen M. Lymphocyte CD44 binds the COOH-terminal heparin-binding domain of fibronectin. J Cell Biol. 1992 Feb;116(3):817–825. doi: 10.1083/jcb.116.3.817. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Katoh S., Zheng Z., Oritani K., Shimozato T., Kincade P. W. Glycosylation of CD44 negatively regulates its recognition of hyaluronan. J Exp Med. 1995 Aug 1;182(2):419–429. doi: 10.1084/jem.182.2.419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Koopman G., Heider K. H., Horst E., Adolf G. R., van den Berg F., Ponta H., Herrlich P., Pals S. T. Activated human lymphocytes and aggressive non-Hodgkin's lymphomas express a homologue of the rat metastasis-associated variant of CD44. J Exp Med. 1993 Apr 1;177(4):897–904. doi: 10.1084/jem.177.4.897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kuan S. F., Byrd J. C., Basbaum C., Kim Y. S. Inhibition of mucin glycosylation by aryl-N-acetyl-alpha-galactosaminides in human colon cancer cells. J Biol Chem. 1989 Nov 15;264(32):19271–19277. [PubMed] [Google Scholar]
  28. Lesley J., English N., Perschl A., Gregoroff J., Hyman R. Variant cell lines selected for alterations in the function of the hyaluronan receptor CD44 show differences in glycosylation. J Exp Med. 1995 Aug 1;182(2):431–437. doi: 10.1084/jem.182.2.431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lesley J., He Q., Miyake K., Hamann A., Hyman R., Kincade P. W. Requirements for hyaluronic acid binding by CD44: a role for the cytoplasmic domain and activation by antibody. J Exp Med. 1992 Jan 1;175(1):257–266. doi: 10.1084/jem.175.1.257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lesley J., Howes N., Perschl A., Hyman R. Hyaluronan binding function of CD44 is transiently activated on T cells during an in vivo immune response. J Exp Med. 1994 Jul 1;180(1):383–387. doi: 10.1084/jem.180.1.383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lesley J., Hyman R. CD44 can be activated to function as an hyaluronic acid receptor in normal murine T cells. Eur J Immunol. 1992 Oct;22(10):2719–2723. doi: 10.1002/eji.1830221036. [DOI] [PubMed] [Google Scholar]
  32. Lesley J., Hyman R., Kincade P. W. CD44 and its interaction with extracellular matrix. Adv Immunol. 1993;54:271–335. doi: 10.1016/s0065-2776(08)60537-4. [DOI] [PubMed] [Google Scholar]
  33. Lesley J., Kincade P. W., Hyman R. Antibody-induced activation of the hyaluronan receptor function of CD44 requires multivalent binding by antibody. Eur J Immunol. 1993 Aug;23(8):1902–1909. doi: 10.1002/eji.1830230826. [DOI] [PubMed] [Google Scholar]
  34. Lesley J., Schulte R., Hyman R. Binding of hyaluronic acid to lymphoid cell lines is inhibited by monoclonal antibodies against Pgp-1. Exp Cell Res. 1990 Apr;187(2):224–233. doi: 10.1016/0014-4827(90)90085-o. [DOI] [PubMed] [Google Scholar]
  35. Liao H. X., Levesque M. C., Patton K., Bergamo B., Jones D., Moody M. A., Telen M. J., Haynes B. F. Regulation of human CD44H and CD44E isoform binding to hyaluronan by phorbol myristate acetate and anti-CD44 monoclonal and polyclonal antibodies. J Immunol. 1993 Dec 1;151(11):6490–6499. [PubMed] [Google Scholar]
  36. McCourt P. A., Ek B., Forsberg N., Gustafson S. Intercellular adhesion molecule-1 is a cell surface receptor for hyaluronan. J Biol Chem. 1994 Dec 2;269(48):30081–30084. [PubMed] [Google Scholar]
  37. Miyake K., Kincade P. W. A new cell adhesion mechanism involving hyaluronate and CD44. Curr Top Microbiol Immunol. 1990;166:87–90. doi: 10.1007/978-3-642-75889-8_12. [DOI] [PubMed] [Google Scholar]
  38. Miyake K., Underhill C. B., Lesley J., Kincade P. W. Hyaluronate can function as a cell adhesion molecule and CD44 participates in hyaluronate recognition. J Exp Med. 1990 Jul 1;172(1):69–75. doi: 10.1084/jem.172.1.69. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Murakami S., Shimabukuro Y., Miki Y., Saho T., Hino E., Kasai D., Nozaki T., Kusumoto Y., Okada H. Inducible binding of human lymphocytes to hyaluronate via CD44 does not require cytoskeleton association but does require new protein synthesis. J Immunol. 1994 Jan 15;152(2):467–477. [PubMed] [Google Scholar]
  40. Noble P. W., Lake F. R., Henson P. M., Riches D. W. Hyaluronate activation of CD44 induces insulin-like growth factor-1 expression by a tumor necrosis factor-alpha-dependent mechanism in murine macrophages. J Clin Invest. 1993 Jun;91(6):2368–2377. doi: 10.1172/JCI116469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Peach R. J., Hollenbaugh D., Stamenkovic I., Aruffo A. Identification of hyaluronic acid binding sites in the extracellular domain of CD44. J Cell Biol. 1993 Jul;122(1):257–264. doi: 10.1083/jcb.122.1.257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Puré E., Camp R. L., Peritt D., Panettieri R. A., Jr, Lazaar A. L., Nayak S. Defective phosphorylation and hyaluronate binding of CD44 with point mutations in the cytoplasmic domain. J Exp Med. 1995 Jan 1;181(1):55–62. doi: 10.1084/jem.181.1.55. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Rothman B. L., Blue M. L., Kelley K. A., Wunderlich D., Mierz D. V., Aune T. M. Human T cell activation by OKT3 is inhibited by a monoclonal antibody to CD44. J Immunol. 1991 Oct 15;147(8):2493–2499. [PubMed] [Google Scholar]
  44. Screaton G. R., Bell M. V., Jackson D. G., Cornelis F. B., Gerth U., Bell J. I. Genomic structure of DNA encoding the lymphocyte homing receptor CD44 reveals at least 12 alternatively spliced exons. Proc Natl Acad Sci U S A. 1992 Dec 15;89(24):12160–12164. doi: 10.1073/pnas.89.24.12160. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Simmons D. L., Satterthwaite A. B., Tenen D. G., Seed B. Molecular cloning of a cDNA encoding CD34, a sialomucin of human hematopoietic stem cells. J Immunol. 1992 Jan 1;148(1):267–271. [PubMed] [Google Scholar]
  46. St John T., Meyer J., Idzerda R., Gallatin W. M. Expression of CD44 confers a new adhesive phenotype on transfected cells. Cell. 1990 Jan 12;60(1):45–52. doi: 10.1016/0092-8674(90)90714-p. [DOI] [PubMed] [Google Scholar]
  47. Stamenkovic I., Amiot M., Pesando J. M., Seed B. A lymphocyte molecule implicated in lymph node homing is a member of the cartilage link protein family. Cell. 1989 Mar 24;56(6):1057–1062. doi: 10.1016/0092-8674(89)90638-7. [DOI] [PubMed] [Google Scholar]
  48. Stamenkovic I., Aruffo A., Amiot M., Seed B. The hematopoietic and epithelial forms of CD44 are distinct polypeptides with different adhesion potentials for hyaluronate-bearing cells. EMBO J. 1991 Feb;10(2):343–348. doi: 10.1002/j.1460-2075.1991.tb07955.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Tarone G., Ferracini R., Galetto G., Comoglio P. A cell surface integral membrane glycoprotein of 85,000 mol wt (gp85) associated with triton X-100-insoluble cell skeleton. J Cell Biol. 1984 Aug;99(2):512–519. doi: 10.1083/jcb.99.2.512. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Thomas L., Byers H. R., Vink J., Stamenkovic I. CD44H regulates tumor cell migration on hyaluronate-coated substrate. J Cell Biol. 1992 Aug;118(4):971–977. doi: 10.1083/jcb.118.4.971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Wayner E. A., Carter W. G. Identification of multiple cell adhesion receptors for collagen and fibronectin in human fibrosarcoma cells possessing unique alpha and common beta subunits. J Cell Biol. 1987 Oct;105(4):1873–1884. doi: 10.1083/jcb.105.4.1873. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Yang B., Yang B. L., Savani R. C., Turley E. A. Identification of a common hyaluronan binding motif in the hyaluronan binding proteins RHAMM, CD44 and link protein. EMBO J. 1994 Jan 15;13(2):286–296. doi: 10.1002/j.1460-2075.1994.tb06261.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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