English N M, Lesley J F, Hyman R
Cancer Biology Laboratory, The Salk Institute, San Diego, California 92186-5800, USA.
Cancer Res. 1998 Aug 15;58(16):3736-42.
CD44 is a cell surface receptor for the glycosaminoglycan hyaluronan (HA). Not all CD44-positive cells bind HA, and binding ability is strictly regulated. Three different HA binding states have been defined: inactive, inducible (by certain CD44-specific monoclonal antibodies), and constitutively active. The observation that sets of genetically related cell lines representing different HA binding states showed correlated differences in N-glycosylation of CD44, and that inhibition of N-glycosylation enhanced HA binding (Lesley et al., J. Exp. Med., 182: 431-437, 1995) led us to examine directly whether specific N-glycosylation site modifications were involved in regulating the HA binding function. CD44-negative, -active, and inducible cell lines were stably transfected with mutant constructs in which each of the five N-glycosylation sites of murine CD44 had been separately inactivated. Ability to bind soluble HA was examined over a range of CD44 expression levels. For the active cell line, AKR1, transfectants for all N-glycosylation mutants bound HA as well as did transfectants for wild type CD44. No inhibitory effects of inactivating specific N-glycosylation sites were observed. HA binding was activated when two of the mutant constructs were transfected into a novel CD44-negative inducible cell line. Inactivation of N-glycosylation sites at residues 25 or 120 converted the inducible cell line to constitutively active, whereas inactivation of other sites had little or no effect. Fusion proteins secreted from inactive, inducible, or active cell lines were purified, bound to beads, and assayed for HA binding activity by flow cytometric analysis. Fusion proteins derived from inactive, inducible, and constitutively active cells exhibited three distinguishable "threshold" densities required for HA binding ability. The results imply that the CD44 molecules produced in cells in these three activation states have intrinsic differences in HA binding function. Treatment of the fusion proteins with neuraminidase altered the HA binding state, and glycosylation mutations that affected the phenotype of the inducible cell line lowered the threshold required for HA binding of CD44-immunoglobulin fusion proteins derived from the inducible cell line. Thus, alterations of glycosylation of CD44 itself can affect HA binding ability as manifested by a change in HA binding state.
CD44是糖胺聚糖透明质酸（HA）的细胞表面受体。并非所有CD44阳性细胞都能结合HA，且结合能力受到严格调控。已定义了三种不同的HA结合状态：无活性、可诱导性（由某些CD44特异性单克隆抗体诱导）和组成型活性。代表不同HA结合状态的一组遗传相关细胞系在CD44的N-糖基化方面表现出相关差异，并且N-糖基化的抑制增强了HA结合（莱斯利等人，《实验医学杂志》，182: 431 - 437，1995），这使我们直接研究特定的N-糖基化位点修饰是否参与调节HA结合功能。用突变构建体稳定转染CD44阴性、活性和可诱导性细胞系，其中小鼠CD44的五个N-糖基化位点中的每一个都已分别失活。在一系列CD44表达水平上检测结合可溶性HA的能力。对于活性细胞系AKR1，所有N-糖基化突变体的转染子与野生型CD44的转染子一样能结合HA。未观察到失活特定N-糖基化位点的抑制作用。当将两个突变构建体转染到一种新的CD44阴性可诱导细胞系中时，HA结合被激活。第25位或120位残基处的N-糖基化位点失活将可诱导细胞系转变为组成型活性，而其他位点的失活几乎没有影响。从无活性、可诱导性或活性细胞系分泌的融合蛋白被纯化，与珠子结合，并通过流式细胞术分析检测HA结合活性。源自无活性、可诱导性和组成型活性细胞的融合蛋白表现出HA结合能力所需的三种可区分的“阈值”密度。结果表明，在这三种激活状态下细胞中产生的CD44分子在HA结合功能上存在内在差异。用神经氨酸酶处理融合蛋白改变了HA结合状态，影响可诱导细胞系表型的糖基化突变降低了源自可诱导细胞系的CD44 - 免疫球蛋白融合蛋白HA结合所需的阈值。因此，CD44自身糖基化的改变可通过HA结合状态的变化影响HA结合能力。
