Skip to main content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
J Cell Biol. 1995 Dec 2; 131(6): 1623–1633.
doi: 10.1083/jcb.131.6.1623
PMCID: PMC2120678
PMID: 8522617

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

Copyright and License information PMC Disclaimer

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.

Full Text

The Full Text of this article is available as a PDF (1.3M).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  • 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. [PubMed] [Google Scholar]
  • Aruffo A, Stamenkovic I, Melnick M, Underhill CB, Seed B. CD44 is the principal cell surface receptor for hyaluronate. Cell. 1990 Jun 29;61(7):1303–1313. [PubMed] [Google Scholar]
  • Ashwell G, Harford J. Carbohydrate-specific receptors of the liver. Annu Rev Biochem. 1982;51:531–554. [PubMed] [Google Scholar]
  • 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. [PubMed] [Google Scholar]
  • 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. [PMC free article] [PubMed] [Google Scholar]
  • 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]
  • Brown TA, Bouchard T, St John T, Wayner E, Carter WG. 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. [PMC free article] [PubMed] [Google Scholar]
  • Culty M, Miyake K, Kincade PW, Sikorski E, Butcher EC, 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. [PMC free article] [PubMed] [Google Scholar]
  • Damle NK, 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]
  • Diamond MS, Staunton DE, Marlin SD, Springer TA. 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. [PubMed] [Google Scholar]
  • Dougherty GJ, Cooper DL, Memory JF, Chiu RK. 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]
  • Dougherty GJ, Landorp PM, Cooper DL, Humphries RK. 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. [PMC free article] [PubMed] [Google Scholar]
  • Galandrini R, Galluzzo E, Albi N, Grossi CE, 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]
  • Goldstein LA, Zhou DF, Picker LJ, Minty CN, Bargatze RF, Ding JF, Butcher EC. 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. [PubMed] [Google Scholar]
  • Green SJ, Tarone G, Underhill CB. Aggregation of macrophages and fibroblasts is inhibited by a monoclonal antibody to the hyaluronate receptor. Exp Cell Res. 1988 Oct;178(2):224–232. [PubMed] [Google Scholar]
  • 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. [PubMed] [Google Scholar]
  • Hathcock KS, Hirano H, Murakami S, Hodes RJ. 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]
  • He Q, Lesley J, Hyman R, Ishihara K, Kincade PW. 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. [PMC free article] [PubMed] [Google Scholar]
  • Huet S, Groux H, Caillou B, Valentin H, Prieur AM, Bernard A. CD44 contributes to T cell activation. J Immunol. 1989 Aug 1;143(3):798–801. [PubMed] [Google Scholar]
  • Hyman R, Lesley J, Schulte R. Somatic cell mutants distinguish CD44 expression and hyaluronic acid binding. Immunogenetics. 1991;33(5-6):392–395. [PubMed] [Google Scholar]
  • Jackson DG, Bell JI, 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. [PMC free article] [PubMed] [Google Scholar]
  • Jackson DG, Buckley J, Bell JI. 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]
  • Jalkanen S, Jalkanen M. Lymphocyte CD44 binds the COOH-terminal heparin-binding domain of fibronectin. J Cell Biol. 1992 Feb;116(3):817–825. [PMC free article] [PubMed] [Google Scholar]
  • Jalkanen S, Bargatze RF, de los Toyos J, Butcher EC. 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. [PMC free article] [PubMed] [Google Scholar]
  • Katoh S, Zheng Z, Oritani K, Shimozato T, Kincade PW. Glycosylation of CD44 negatively regulates its recognition of hyaluronan. J Exp Med. 1995 Aug 1;182(2):419–429. [PMC free article] [PubMed] [Google Scholar]
  • Koopman G, Heider KH, Horst E, Adolf GR, van den Berg F, Ponta H, Herrlich P, Pals ST. 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. [PMC free article] [PubMed] [Google Scholar]
  • Kuan SF, Byrd JC, Basbaum C, Kim YS. 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]
  • 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. [PubMed] [Google Scholar]
  • 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. [PubMed] [Google Scholar]
  • Lesley J, Hyman R, Kincade PW. CD44 and its interaction with extracellular matrix. Adv Immunol. 1993;54:271–335. [PubMed] [Google Scholar]
  • Lesley J, Kincade PW, 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. [PubMed] [Google Scholar]
  • Lesley J, He Q, Miyake K, Hamann A, Hyman R, Kincade PW. 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. [PMC free article] [PubMed] [Google Scholar]
  • 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. [PMC free article] [PubMed] [Google Scholar]
  • 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. [PMC free article] [PubMed] [Google Scholar]
  • Liao HX, Levesque MC, Patton K, Bergamo B, Jones D, Moody MA, Telen MJ, Haynes BF. 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]
  • McCourt PA, 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]
  • Miyake K, Kincade PW. A new cell adhesion mechanism involving hyaluronate and CD44. Curr Top Microbiol Immunol. 1990;166:87–90. [PubMed] [Google Scholar]
  • Miyake K, Underhill CB, Lesley J, Kincade PW. Hyaluronate can function as a cell adhesion molecule and CD44 participates in hyaluronate recognition. J Exp Med. 1990 Jul 1;172(1):69–75. [PMC free article] [PubMed] [Google Scholar]
  • 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]
  • Noble PW, Lake FR, Henson PM, Riches DW. 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. [PMC free article] [PubMed] [Google Scholar]
  • Peach RJ, 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. [PMC free article] [PubMed] [Google Scholar]
  • Puré E, Camp RL, Peritt D, Panettieri RA, Jr, Lazaar AL, 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. [PMC free article] [PubMed] [Google Scholar]
  • Rothman BL, Blue ML, Kelley KA, Wunderlich D, Mierz DV, Aune TM. 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]
  • Screaton GR, Bell MV, Jackson DG, Cornelis FB, Gerth U, Bell JI. 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. [PMC free article] [PubMed] [Google Scholar]
  • Simmons DL, Satterthwaite AB, Tenen DG, 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]
  • St John T, Meyer J, Idzerda R, Gallatin WM. Expression of CD44 confers a new adhesive phenotype on transfected cells. Cell. 1990 Jan 12;60(1):45–52. [PubMed] [Google Scholar]
  • Stamenkovic I, Amiot M, Pesando JM, 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. [PubMed] [Google Scholar]
  • 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. [PMC free article] [PubMed] [Google Scholar]
  • 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. [PMC free article] [PubMed] [Google Scholar]
  • Thomas L, Byers HR, Vink J, Stamenkovic I. CD44H regulates tumor cell migration on hyaluronate-coated substrate. J Cell Biol. 1992 Aug;118(4):971–977. [PMC free article] [PubMed] [Google Scholar]
  • Wayner EA, Carter WG. 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. [PMC free article] [PubMed] [Google Scholar]
  • Yang B, Yang BL, Savani RC, Turley EA. 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. [PMC free article] [PubMed] [Google Scholar]
Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press
-