Use of synthetic peptides to locate novel integrin α2β1-binding motifs in human collagen III
Use of synthetic peptides to locate novel integrin α2β1-binding motifs in human collagen III
复制标题
DOI：
10.1074/jbc.m509818200
复制
发表时间：
2006-02-17
影响因子：
4.8
通讯作者：
Farndale, RW
中科院分区：
文献类型：
--
作者：
Raynal, N;Hamaia, SW;Farndale, RW
一组57个合成肽包围了人III型胶原的整个三螺旋结构域，用来定位胶原结合整合素α(2)和β(1)的结合部位。检测了这些多肽支持几种整合素制剂与镁离子依赖的结合的能力。野生型整合素(重组α(2)I结构域、从血小板膜纯化的重组α(2)β(1)和重组可溶性α(2)β(1)表达为α(2)-Fos/beta(1)-Jun杂二聚体)只与三个肽结合，其中两个含有GXX‘ger基序(GROGER和GMOGER，其中O是羟脯氨酸)，一个含有两个相邻的GXX’gen基序(GLKGEN和GLOGEN)。测试了两个突变的α(2)I结构域：无活性的T221a突变体，它不识别任何多肽；以及成分活性的E318W突变体，它结合了更大的多肽子集。活化的人血小板与含有GER的多肽的粘附性强于静息状态的血小板，HT1080细胞与更多的多肽结合得更好。细胞与重组蛋白的结合可被抗α(2)单抗6F1和镁离子的螯合作用所消除。我们描述了人III型胶原蛋白中两个新的高亲和力整合素结合基序(GROGER和GLOGEN)和第三个显示中等活性的基序(GLKGEN)。每个基序都使用较短的合成多肽进行了验证。
A set of 57 synthetic peptides encompassing the entire triple-helical domain of human collagen III was used to locate binding sites for the collagen-binding integrin alpha(2)beta(1). The capacity of the peptides to support Mg2+-dependent binding of several integrin preparations was examined. Wild-type integrins (recombinant alpha(2) I-domain, alpha(2)beta(1) purified from platelet membranes, and recombinant soluble alpha(2)beta(1) expressed as an alpha(2)-Fos/beta(1)-Jun heterodimer) bound well to only three peptides, two containing GXX'GER motifs (GROGER and GMOGER, where O is hydroxyproline) and one containing two adjacent GXX'GEN motifs (GLKGEN and GLOGEN). Two mutant alpha(2) I-domains were tested: the inactive T221A mutant, which recognized no peptides, and the constitutively active E318W mutant, which bound a larger subset of peptides. Adhesion of activated human platelets to GER-containing peptides was greater than that of resting platelets, and HT1080 cells bound well to more of the peptides compared with platelets. Binding of cells and recombinant proteins was abolished by anti-alpha(2) monoclonal antibody 6F1 and by chelation of Mg2+. We describe two novel high affinity integrin-binding motifs in human collagen III (GROGER and GLOGEN) and a third motif (GLKGEN) that displays intermediate activity. Each motif was verified using shorter synthetic peptides.