CD44-mediated cell adhesion to hyaluronate is controlled by mechanisms which are poorly understood. In the present work we examine the role of N-linked glycosylation and Ser-Gly motifs in regulating CD44-hyaluronate interaction. Our results show that treatment of a panel of human cell lines which constitutively express CD44 with the inhibitor of N-linked glycosylation tunicamycin results in the loss of attachment of these cells to hyaluronate-coated substrate. In contrast, treatment of the same cells with deoxymannojirimycin, which inhibits the conversion of high mannose oligosaccharides to complex N-linked carbohydrates, results in either no change or an increase in CD44-mediated adhesion to hyaluronate, suggesting that complex N-linked oligosaccharides may not be required for and may even inhibit CD44-HA interaction. Using human melanoma cells stably transfected with CD44 N-linked glycosylation site-specific mutants, we show that integrity of five potential N-linked glycosylation sites within the hyaluronate recognition domain of CD44 is critical for hyaluronate binding. Mutation of any one of these potential N-linked glycosylation sites abrogates CD44-mediated melanoma cell attachment to hyaluronate-coated surfaces, suggesting that all five sites are necessary to maintain the HA-recognition domain in the appropriate conformation. We also demonstrate that mutation of serine residues which constitute the four Ser-Gly motifs in the membrane proximal domain, and provide potential sites for glycosaminoglycan side chain attachment, impairs hyaluronate binding. Taken together, these observations indicate that changes in glycosylation of CD44 can have profound effects on its interaction with hyaluronic acid and suggest that glycosylation may provide an important regulatory mechanism of CD44 function.

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.

Glycosaminoglycan-modified isoforms of CD44 have been implicated in growth factor presentation at sites of inflammation. In the present study we show that COS cell transfectants expressing CD44 isoforms containing the alternatively spliced exon V3 are modified with heparan sulfate (HS). Binding studies with three HS-binding growth factors, basic-fibroblast growth factor (b-FGF), heparin binding-epidermal growth factor (HB-EGF), and amphiregulin, showed that the HS-modified CD44 isoforms are able to bind to b-FGF and HB-EGF, but not AR. b-FGF and HB-EGF binding to HS-modified CD44 was eliminated by pretreating the protein with heparitinase or by blocking with free heparin. HS-modified CD44 immunoprecipitated from keratinocytes, which express a CD44 isoform containing V3, also bound to b-FGF. We examined whether HS-modified CD44 isoforms were expressed by activated endothelial cells where they might present HS-binding growth factors to leukocytes during an inflammatory response. PCR and antibody-binding studies showed that activated cultured endothelial cells only express the CD44H isoform which does not contain any of the variably spliced exons including V3. Immunohistological studies with antibodies directed to CD44 extracellular domains encoded by the variably spliced exons showed that vascular endothelial cells in inflamed skin tissue sections do not express CD44 spliced variants. Keratinocytes, monocytes, and dendritic cells in the same specimens were found to express variably spliced CD44. 35SO4(-2)-labeling experiments demonstrated that activated cultured endothelial cells do not express detectable levels of chondroitin sulfate or HS-modified CD44. Our results suggest that one of the functions of CD44 isoforms expressing V3 is to bind and present a subset of HS-binding proteins. Furthermore, it is probable that HS-modified CD44 is involved in the presentation of HS-binding proteins by keratinocytes in inflamed skin. However, our data suggests that CD44 is not likely to be the proteoglycan principally involved in presenting HS-binding growth factors to leukocytes on the vascular cell wall.

CD44 is a polymorphic glycoprotein expressed on the surface of many tissues and cell lines which has been implicated in a number of cellular functions including lymphocyte homing to mucosal lymphoid tissue (Peyers patches), leukocyte activation, lymphopoiesis, and tumor metastasis. The predominant isoform found on human leukocytes, CD44H, is a receptor for hyaluronic acid. Because of the prominent role CD44 plays in diverse biological processes, we set out to identify the hyaluronic acid binding site(s) in the extracellular domain of CD44H. Using truncation and site-directed mutagenesis we identified two regions containing clusters of conserved basic residues which are important in hyaluronic acid binding. One of these regions is situated near the NH2 terminus and is homologous to other hyaluronic acid binding proteins including cartilage link protein. The other more membrane proximal region lies outside the link protein homologous domain. Mutagenesis of basic residues within these regions established their role as determinants in hyaluronic acid binding. Mutation of Arg 41, a position where a basic residue is conserved in all hyaluronic acid binding proteins, completely abolished binding suggesting that this residue plays a critical role in hyaluronic acid binding.

CD44 is a broadly distributed cell surface glycoprotein expressed in different isoforms in various tissues and cell lines. One of two recently characterized human isoforms, CD44H, is a cell surface receptor for hyaluronate, suggesting a role in the regulation of cell-cell and cell-substrate interactions as well as of cell migration. While CD44H has been shown to mediate cell adhesion, direct demonstration that CD44H expression promotes cell motility has been lacking. In this work we show that a human melanoma cell line, stably transfected with CD44H, displays enhanced motility on hyaluronate-coated surfaces while transfectants expressing an isoform that does not bind hyaluronate, CD44E, fail to do so. Migration of CD44H-expressing transfectants is observed to be blocked by a soluble CD44-immunoglobulin fusion protein as well as by anti-CD44 antibody, and to depend on the presence of the cytoplasmic domain of CD44. However, cells expressing CD44H cytoplasmic deletion mutants retain significant binding capacity to hyaluronate-coated substrate. Taken together, our results provide direct evidence that CD44H plays a major role in regulating cell migration on hyaluronate-coated substrate.