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Identification of CD44 Residues Important for Hyaluronan Binding and Delineation of the Binding Site*

Open AccessPublished:January 02, 1998DOI:https://doi.org/10.1074/jbc.273.1.338
      CD44 is a widely distributed cell surface protein that plays a role in cell adhesion and migration. As a proteoglycan, CD44 is also implicated in growth factor and chemokine binding and presentation. The extracellular region of CD44 is variably spliced, giving rise to multiple CD44 isoforms. All isoforms contain an amino-terminal domain, which is homologous to cartilage link proteins. The cartilage link protein-like domain of CD44 is important for hyaluronan binding. The structure of the link protein domain of TSG-6 has been determined by NMR. Based on this structure, a molecular model of the link-homologous region of CD44 was constructed. This model was used to select residues for site-specific mutagenesis in an effort to identify residues important for ligand binding and to outline the hyaluronan binding site. Twenty-four point mutants were generated and characterized, and eight residues were identified as critical for binding or to support the interaction. In the model, these residues form a coherent surface the location of which approximately corresponds to the carbohydrate binding sites in two functionally unrelated calcium-dependent lectins, mannose-binding protein and E-selectin (CD62E).
      CD44 is a type I transmembrane protein encoded by a gene containing 19 exons (
      • Jackson D.G.
      • Buckley J.
      • Bell J.I.
      ,
      • Screaton G.R.
      • Bell M.V.
      • Jackson D.G.
      • Cornelis F.B.
      • Gerth U.
      • Bell J.I.
      ). Ten of these exons are variably spliced (V1–V10), giving rise to multiple CD44 isoforms. All CD44 isoforms contain at their amino terminus a domain of ∼100 residues, which is homologous to cartilage link protein domains (
      • Stamenkovic I.
      • Amiot M.
      • Pesando J.M.
      • Seed B.
      ,
      • Goldstein L.A.
      • Zhou D.F.
      • Picker L.S.
      • Minty C.N.
      • Bargatze R.F.
      • Ding J.F.
      • Butcher E.C.
      ). The link homology domain of CD44 has been implicated in the hyaluronan (HA)
      The abbreviations used are: HA, hyaluronan; HS, heparan sulfate; mAb, monoclonal antibody; MBP, mannose-binding protein; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; HRP, horseradish peroxidase.
      1The abbreviations used are: HA, hyaluronan; HS, heparan sulfate; mAb, monoclonal antibody; MBP, mannose-binding protein; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; HRP, horseradish peroxidase.
      binding activity of CD44 (
      • Aruffo A.
      • Stamenkovic I.
      • Melnick M.
      • Underhilll C.B.
      • Seed B.
      ,
      • Culty M.
      • Miyake K.
      • Kincade P.W.
      • Silorski E.
      • Butcher E.C.
      • Underhilll C.
      ). In different CD44 isoforms, polypeptides encoded by the variably spliced exons are inserted following exon E5. The functional relevance of different CD44 isoforms is still under investigation, but the following observations have been made. (a) Inclusion of exon V3 results in the modification of CD44 with heparan sulfate (HS) added to an SGSG site contained in this exon (
      • Jackson D.G.
      • Bell J.I.
      • Dickinson R.
      • Timans J.
      • Shields J.
      • Whittle N.
      ,
      • Bennett K.L.
      • Jackson D.G.
      • Simon J.C.
      • Tanczos E.
      • Peach R.
      • Modrell B.
      • Stamenkovic I.
      • Plowman G.
      • Aruffo A.
      ). These CD44 isoforms can interact with HS-binding growth factors and chemokines. (b) Inclusion of exon V6 renders tumor cells expressing this CD44 isoform aggressively metastatic (
      • Günthert U.
      • Hofmann M.
      • Rudy W.
      • Reber S.
      • Zöller M.
      • Haußmann I.
      • Matzku S.
      • Wenzel A.
      • Ponta H.
      • Herrlich P.
      ,
      • Arch R.
      • Wirth K.
      • Hofmann M.
      • Ponta H.
      • Matzku S.
      • Herrlich P.
      • Zoller M.
      ). (c) Inclusion of variably spliced exons results in a increase in the number ofO-linked carbohydrates in CD44 (
      • Bennett K.L.
      • Modrell B.
      • Greenfield B.
      • Bartolazzi A.
      • Stamenkovic I.
      • Peach R.
      • Jackson D.G.
      • Spring F.
      • Aruffo A.
      ). This change in glycosylation has been proposed to modulate the ability of CD44 to bind HA and is consistent with the finding that N-linked glycosylation can also modulate HA binding (
      • Lesley J.
      • English N.
      • Perschl A.
      • Gregoroff J.
      • Hyman R.
      ,
      • Katoh S.
      • Zheng Z.
      • Oritani K.
      • Shimozato T.
      • Kincade P.W.
      ). The variably spliced region of CD44 is followed by a stalk encoded by exons E15 and E16, a hydrophobic transmembrane domain, and a cytoplasmic domain that can engage in intracellular signaling pathways (
      • Screaton G.R.
      • Bell M.V.
      • Jackson D.G.
      • Cornelis F.B.
      • Gerth U.
      • Bell J.I.
      ).
      CD44 is expressed by a large number of different cell types. Leukocytes predominantly express the standard form of CD44 (CD44H). This isoform contains no variably spliced exons and binds HA on activated leukocytes (
      • Stamenkovic I.
      • Amiot M.
      • Pesando J.M.
      • Seed B.
      ,
      • Aruffo A.
      • Stamenkovic I.
      • Melnick M.
      • Underhilll C.B.
      • Seed B.
      ,
      • Culty M.
      • Miyake K.
      • Kincade P.W.
      • Silorski E.
      • Butcher E.C.
      • Underhilll C.
      ). This interaction has been shown to play an important role in leukocyte adhesion and migration at sites of inflammation (
      • DeGrendele H.C.
      • Estess P.
      • Picker L.J.
      • Siegelman M.H.
      ). Activated macrophages and dendritic cells express CD44 isoforms containing exon V3 (
      • Bennett K.L.
      • Jackson D.G.
      • Simon J.C.
      • Tanczos E.
      • Peach R.
      • Modrell B.
      • Stamenkovic I.
      • Plowman G.
      • Aruffo A.
      ). Thus, CD44 is modified with HS and can bind and present HS-binding growth factors and chemokines. This allows these antigen-presenting cells to more efficiently amplify an ongoing immune response.
      Although the three-dimensional structure of the ligand binding domain(s) of CD44 is currently unknown, attempts have been made previously to map the HA binding site. In an initial mutagenesis study on CD44, only one residue in the link homology domain, Arg-41, could be identified as critical for the interaction with HA (
      • Peach R.J.
      • Hollenbaugh D.
      • Stamenkovic I.
      • Aruffo A.
      ). Recently, the solution structure of TSG-6 link domain was determined and found to be similar to the calcium-dependent (C-type) lectin fold (
      • Kohda D.
      • Morton C.J.
      • Parkar A.A.
      • Hatanaka H.
      • Inagaki F.M.
      • Campbell I.D.
      • Day A.J.
      ) which was first determined for rat mannose-binding protein (MBP) (
      • Weis W.I.
      • Kahn R.
      • Fourme R.
      • Drickamer K.
      • Hendrickson W.A.
      ,
      • Weis W.I.
      • Drickamer K.
      • Hendrickson W.A.
      ). TSG-6 provides a prototypic fold for the link protein superfamily to which CD44 belongs and allowed the generation of a comparative molecular model of CD44. This model was used to support a more extensive analysis of the CD44 ligand binding site.
      Here, we report the generation of the CD44 model and its application in a mutagenesis analysis of the HA binding site. Twenty-four site-specific mutant proteins were generated, and eight residues were identified as important for HA binding. Together with the previously identified Arg-41, residues Tyr-42, Arg-78, and Tyr-79 form a cluster of residues critical for HA binding. In the model, these residues form a coherent surface with additional residues that support binding. The HA binding surface is extensive, consistent with the size of the ligand, and its location approximately corresponds to the carbohydrate binding sites in MBP and E-selectin.

      Acknowledgments

      We thank Gary Carlton for help in generating Fig. 1 and Debby Baxter for help in the preparation of the manuscript.

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