Bartolazzi A, Nocks A, Aruffo A, Spring F, Stamenkovic I
Department of Pathology, Harvard Medical School,, Charlestown Navy Yard, Boston, Massachusetts 02129, USA.
J Cell Biol. 1996 Mar;132(6):1199-208. doi: 10.1083/jcb.132.6.1199.
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.
CD44介导的细胞与透明质酸的黏附受一些尚不清楚的机制控制。在本研究中，我们研究了N-连接糖基化和丝氨酸-甘氨酸基序在调节CD44与透明质酸相互作用中的作用。我们的结果表明，用N-连接糖基化抑制剂衣霉素处理一组组成性表达CD44的人细胞系，会导致这些细胞与透明质酸包被底物的附着丧失。相反，用抑制高甘露糖寡糖向复杂N-连接碳水化合物转化的脱氧甘露基野尻霉素处理相同的细胞，CD44介导的与透明质酸的黏附要么没有变化，要么增加，这表明复杂N-连接寡糖可能不是CD44与透明质酸相互作用所必需的，甚至可能抑制这种相互作用。使用稳定转染了CD44 N-连接糖基化位点特异性突变体的人黑色素瘤细胞，我们表明CD44透明质酸识别域内五个潜在N-连接糖基化位点的完整性对于透明质酸结合至关重要。这些潜在N-连接糖基化位点中的任何一个发生突变都会消除CD44介导的黑色素瘤细胞与透明质酸包被表面的附着，这表明所有五个位点对于将透明质酸识别域维持在适当构象都是必需的。我们还证明，构成膜近端结构域中四个丝氨酸-甘氨酸基序并为糖胺聚糖侧链附着提供潜在位点的丝氨酸残基发生突变会损害透明质酸结合。综上所述，这些观察结果表明CD44糖基化的变化可对其与透明质酸的相互作用产生深远影响，并表明糖基化可能为CD44功能提供重要的调节机制。
