Ha Cam T, Waterhouse Roseann, Warren James, Zimmermann Wolfgang, Dveksler Gabriela S
Department of Pathology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814, USA.
Am J Reprod Immunol. 2008 Mar;59(3):251-8. doi: 10.1111/j.1600-0897.2007.00573.x.
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.
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.
PSG19 binds to the tetraspanin CD9, specifically to extra cellular loop 2 and can induce secretion of TGFbeta1 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.
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.
小鼠妊娠特异性糖蛋白（PSG）由17个不同基因编码。不同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功能并结合相同受体。此外，这些高度糖基化蛋白活性所需的翻译后修饰仍未确定。
通过亲和层析产生并纯化重组PSG17和PSG19。筛选表达文库以鉴定小鼠PSG19的受体。通过下拉试验分析不同表达系统中产生的蛋白质与受体的结合以及结合结构域的定位。使用DIG聚糖分化试剂盒对受体结合结构域的碳水化合物组成进行分析。
PSG19与四跨膜蛋白CD9结合，特异性结合细胞外环2，并可诱导巨噬细胞系分泌TGFβ1。PSG17和PSG19的受体结合结构域通过添加N-连接碳水化合物进行翻译后修饰，当在CHO细胞中表达时，可检测到末端唾液酸。细菌中产生的PSG不与CD9结合。
如先前对PSG17的测定，PSG19与四跨膜蛋白CD9的第二个细胞外环2结合。PSG19的第一个免疫球蛋白可变样结构域足以实现受体结合和功能。对其余15种小鼠PSG的受体使用情况进行分析很可能需要在真核表达系统中产生这些蛋白质，因为我们已经证明碳水化合物的添加对于PSG-受体相互作用至关重要。
