Variant cell lines selected for alterations in the function of the hyaluronan receptor CD44 show differences in glycosylation.
Variant cell lines selected for alterations in the function of the hyaluronan receptor CD44 show differences in glycosylation.
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DOI：
10.1084/jem.182.2.431
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发表时间：
1995-08-01
期刊：
影响因子：
--
通讯作者：
Hyman R
中科院分区：
文献类型：
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作者：
Lesley J;English N;Perschl A;Gregoroff J;Hyman R
CD 44是细胞外基质糖胺聚糖透明质酸（HA）的主要细胞表面受体。然而，CD 44结合配体的能力受到严格的调节。已经证明了CD 44的三种活化状态：（a）无活性;（B）可诱导（通过某些CD 44特异性mAb）;和（c）组成型活性。从两个表达非活性状态的CD 44的亲本细胞系，前B细胞（RAW 253）和成纤维细胞（L细胞）开始，我们在诱导mAb存在下使用荧光素缀合的透明质酸进行荧光激活细胞分选，以获得具有可诱导状态的CD 44的变体细胞系。在不存在诱导抗体的情况下，通过另一轮荧光激活细胞分选从诱导型变体中分离组成型活性衍生物。然而，组成型活性变体不能直接从表达非活性状态的CD 44的亲本细胞中分离。这些结果表明，两个遗传事件必须发生，以获得一个活跃的CD 44-HA受体从一个非活性受体。变体和亲本细胞衍生的CD 44分子在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上表现出迁移差异，这部分归因于N-连接糖基化的差异。此外，在衣霉素中培养2-3天将亲本和诱导型细胞系转化为显示组成型CD 44介导的HA结合的细胞。此外，通过在对硝基苯基β-D-吡喃木糖苷中培养细胞或用软骨素酶ABC处理来去除细胞表面糖胺聚糖链，导致具有失活的CD 44受体的细胞转化为可诱导状态。这些结果表明，CD 44的碳水化合物侧链和/或细胞表面上与CD 44相互作用的其他分子可能参与调节细胞表面上CD 44的HA结合功能。
CD44 is a major cell surface receptor for the extracellular matrix glycosaminoglycan hyaluronan (HA). However, the ability of CD44 to bind ligand is strictly regulated. Three activation states of CD44 have been demonstrated: (a) inactive; (b) inducible (by certain CD44-specific mAb); and (c) constitutively active. Starting with two parental cell lines expressing CD44 in the inactive state, a pre-B cell (RAW 253) and a fibroblast (L cells), we used fluorescence-activated cell sorting with fluorescein-conjugated hyaluronan in the presence of inducing mAb to derive variant cell lines with CD44 in the inducible state. Constitutively active derivatives were isolated from the inducible variants by a further round of fluorescence-activated cell sorting in the absence of inducing antibody. However, constitutively active variants could not be isolated directly from parental cells expressing CD44 in the inactive state. These results suggest that two genetic events must occur to obtain an active CD44-HA receptor from an inactive receptor. Variant and parental cell-derived CD44 molecules exhibited differences in migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis that were partly attributable to differences in N- linked glycosylation. Furthermore, culture in tunicamycin for 2-3 d converted parental and inducible cell lines into cells showing constitutive CD44-mediated HA binding. Also, removal of cell surface glycosaminoglycan chains by culture of cells in p-nitrophenyl beta-D- xylopyranoside or treatment with chondroitinase ABC resulted in conversion of cells with an inactive CD44 receptor to an inducible state. These results indicate that carbohydrate side chains of CD44 and/or other molecules on the cell surface that interact with CD44 are potentially involved in regulating the HA-binding function of CD44 on the cell surface.