GLYCOSYLATION OF CD44 IS IMPLICATED IN CD44-MEDIATED CELL-ADHESION TOHYALURONAN

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-hyalur onate 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 conversio n of high mannose oligosaccharides to complex N-linked carbohydrates, results in either no change or an increase in CD44-mediated adhesion t o hyaluronate, suggesting that complex N-linked oligosaccharides may n ot be required for and may even inhibit CD44-HA interaction. Using hum an melanoma cells stably transfected with CD44 N-linked glycosylation site-specific mutants, we show that integrity of five potential N-link ed glycosylation sites within the hyaluronate recognition domain of CD 44 is critical for hyaluronate binding, Mutation of any one of these p otential N-linked glycosylation sites abrogates CD44-mediated melanoma cell attachment to hyaluronate-coated surfaces, suggesting that all f ive sites are necessary to maintain the HA-recognition domain in the a ppropriate conformation, We also demonstrate that mutation of serine r esidues which constitute the four Ser-Gly motifs in the membrane proxi mal domain, and provide potential sites for glycosaminoglycan side cha in attachment, impairs hyaluronate binding. Taken together, these obse rvations indicate that changes in glycosylation of CD44 can have profo und effects on its interaction with hyaluronic acid and suggest that g lycosylation may provide an important regulatory mechanism of CD44 fun ction.