N-glycosylation is required for binding of murine pregnancy-specific glycoproteins 17 and 19 to the receptor CD9
N-glycosylation is required for binding of murine pregnancy-specific glycoproteins 17 and 19 to the receptor CD9
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DOI：
10.1111/j.1600-0897.2007.00573.x
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发表时间：
2008-03-01
影响因子：
3.6
通讯作者：
Dveksler, Gabriela S.
中科院分区：
文献类型：
--
作者：
Ha, Cam T.;Waterhouse, Roseann;Dveksler, Gabriela S.
问题小鼠妊娠特异性糖蛋白（PSG）由17个不同的基因编码。不同的家族成员在胚胎发育的不同阶段具有不同的表达水平。目前尚不清楚这个胎盘分泌蛋白家族的所有成员是否具有相同的功能并与相同的受体结合。此外，这些高度糖基化的蛋白质的活性的翻译后修饰的要求仍然undetermined.Method的研究重组PSG 17和PSG 19产生和纯化通过亲和层析。筛选表达文库以鉴定小鼠PSG 19的受体。通过下拉测定分析在不同表达系统中产生的蛋白质与受体的结合和结合结构域的作图。使用DIG聚糖分化试剂盒分析受体结合结构域的碳水化合物组成。结果PSG 19与四跨膜蛋白CD 9结合，特别是与细胞外环2结合，并可诱导巨噬细胞系分泌TGF β（1）。PSG 17和PSG 19的受体结合结构域通过添加N-连接的碳水化合物进行后修饰，并且当在CHO细胞中表达时，检测到末端唾液酸。结论细菌产生的PSG 19与四跨膜蛋白CD 9的第二胞外环2结合。PSG 19的第一免疫球蛋白可变样结构域足以用于受体结合和功能。剩余的15个鼠PSG的受体使用的分析将最有可能需要在真核表达系统中产生的蛋白质，因为我们已经证明，添加碳水化合物是必不可少的PSG-受体相互作用。
Problem Murine pregnancy-specific glycoproteins (PSGs) are encoded by 17 different genes. Different family members have different expression levels at different stages of embryonic development. It is currently unknown whether all members of this family of placentally secreted proteins have the same function and bind to the same receptor. Furthermore, the requirement of post-translational modifications for the activity of these highly glycosylated proteins remains undetermined.Method of study Recombinant PSG17 and PSG19 were generated and purified by affinity chromatography. An expression library was screened to identify the receptor for mouse PSG19. Binding to the receptor by proteins generated in different expression systems and mapping of the binding domain were analyzed by pull-down assays. Analysis of the carbohydrate composition of the receptor-binding domain was performed with the DIG glycan differentiation kit.Results PSG19 binds to the tetraspanin CD9, specifically to extra cellular loop 2 and can induce secretion of TGF beta(1) by a macrophage cell line. The receptor-binding domain of PSG17 and PSG19 is post-translationally modified by the addition of N-linked carbohydrates and, when expressed in CHO cells, terminal sialic acids are detected. PSGs produced in bacteria do not bind CD9.Conclusion PSG19, as previously determined for PSG17, binds to the second extracellular loop 2 of the tetraspanin CD9. The first immunoglobulin variable-like domain of PSG19 is sufficient for receptor binding and function. Analysis of receptor usage by the remaining 15 murine PSGs will most likely require that the proteins be generated in eukaryotic expression systems, as we have demonstrated that the addition of carbohydrates is essential for PSG-receptor interaction.