Disulfides modulate RGD-inhibitable cell adhesive activity of thrombospondin.
Disulfides modulate RGD-inhibitable cell adhesive activity of thrombospondin.
复制标题
二硫化物调节血小板反应蛋白的 RGD 抑制细胞粘附活性。
DOI：
10.1083/jcb.118.3.693
复制
发表时间：
1992-08
影响因子：
7.8
通讯作者：
Mosher, D F
中科院分区：
文献类型：
--
作者：
Sun, X;Skorstengaard, K;Mosher, D F
血栓反应蛋白（TSP）含有Arg-Gly-Asp （RGD）序列，该序列被认为对几种细胞表面整合素受体介导的细胞粘附很重要。RGD序列位于TSP的3型重复区域，该区域具有多个Ca2+结合位点，并受到复杂的分子内硫醇二硫异构化的影响。我们使用含有0.1 mM Ca2+的缓冲液从凝血酶激活的人血小板中分离出TSP，其中Cys974是主要的标记半胱氨酸，不具有rgd抑制的粘附活性。然而，我们的一种TSP制剂和使用大于或等于0.3 mM Ca2+的替代程序纯化的TSP确实具有抑制rgd的粘附活性。用DTT还原TSP，无论是在表面吸附之前还是之后，都能增强其粘附活性。当包被浓度低至1微克/毫升时，减少的TSP支持细胞强劲的扩散，而未经过DTT处理的“粘附”TSP在包被浓度大于20微克/毫升时具有活性，仅支持适度的细胞扩散。如果Ca2+与EDTA螯合，则需要较低的DTT浓度来增强TSP的粘附活性。含有RGD的肽和针对α v β 3整合素功能位点的单抗抑制了dtt处理的TSP的细胞粘附。细胞印迹显示，还原的TSP蛋白水解片段将黏附活性定位在含有rgd的3型重复区域。这些结果提示了整合素-配体相互作用调节的新机制，其中配体可以在非活性和活性形式之间异构化。
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.