Sun X, Skorstengaard K, Mosher D F
Department of Medicine, University of Wisconsin, Madison 53706.
J Cell Biol. 1992 Aug;118(3):693-701. doi: 10.1083/jcb.118.3.693.
Thrombospondin (TSP) contains the Arg-Gly-Asp (RGD) sequence that is thought to be important for cell adhesion mediated by several cell-surface integrin receptors. The RGD sequence is located in the type 3 repeat region of TSP that has multiple Ca2+ binding sites and is subject to a complex intramolecular thiol-disulfide isomerization. TSP that we isolated from thrombin-activated human platelets using buffers containing 0.1 mM Ca2+, in which Cys974 is the major labeled cysteine, did not have RGD-inhibitable adhesive activity. However, one of our preparations of TSP and TSP purified following alternative procedures using greater than or equal to 0.3 mM Ca2+ did have RGD-inhibitable adhesive activity. Reduction of TSP with DTT, either before or after adsorption to surfaces, enhanced its adhesive activity. Reduced TSP supported robust cell spreading when coated at concentrations as low as 1 micrograms/ml, whereas "adhesive" TSP not treated with DTT was active at coating concentration of greater than 20 micrograms/ml and supported only modest cell spreading. Lower DTT concentrations were required for enhancement of the adhesive activity of TSP if Ca2+ was chelated with EDTA. Cellular adhesion to DTT-treated TSP was inhibited by RGD-containing peptide and by mAb to a functional site of the alpha v beta 3 integrin. Cell blots of reduced proteolytic fragments of TSP localized the adhesive activity to the RGD-containing type 3 repeat region. These results suggest a novel mechanism for regulation of integrin-ligand interactions in which the ligand can isomerize between inactive and active forms.
血小板反应蛋白（TSP）含有精氨酸-甘氨酸-天冬氨酸（RGD）序列，该序列被认为对由几种细胞表面整合素受体介导的细胞黏附很重要。RGD序列位于TSP的3型重复区域，该区域有多个Ca2+结合位点，并经历复杂的分子内硫醇-二硫键异构化。我们使用含0.1 mM Ca2+的缓冲液从凝血酶激活的人血小板中分离出的TSP，其中Cys974是主要的标记半胱氨酸，不具有RGD抑制性黏附活性。然而，我们制备的一种TSP以及采用大于或等于0.3 mM Ca2+的替代方法纯化的TSP确实具有RGD抑制性黏附活性。在吸附到表面之前或之后用二硫苏糖醇（DTT）还原TSP，可增强其黏附活性。还原后的TSP在低至1微克/毫升的浓度下包被时能支持强劲的细胞铺展，而未用DTT处理的“黏附性”TSP在包被浓度大于20微克/毫升时才有活性，且仅支持适度的细胞铺展。如果用乙二胺四乙酸（EDTA）螯合Ca2+，则需要较低浓度的DTT来增强TSP的黏附活性。细胞对经DTT处理的TSP的黏附受到含RGD肽和针对αvβ3整合素功能位点的单克隆抗体的抑制。TSP还原蛋白水解片段的细胞印迹将黏附活性定位到含RGD的3型重复区域。这些结果提示了一种整合素-配体相互作用调节的新机制，即配体可在无活性和活性形式之间异构化。
