Identification of the zinc-dependent endothelial cell binding protein for high molecular weight kininogen and factor XII: Identity with the receptor that binds to the globular ''heads'' of C1q (gC1q-R)
Identification of the zinc-dependent endothelial cell binding protein for high molecular weight kininogen and factor XII: Identity with the receptor that binds to the globular ''heads'' of C1q (gC1q-R)
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DOI：
10.1073/pnas.93.16.8552
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发表时间：
1996-08-06
影响因子：
11.1
通讯作者：
Kaplan, AP
中科院分区：
文献类型：
--
作者：
Joseph, K;Ghebrehiwet, B;Kaplan, AP
已知高分子量激肽原（HK）和因子XII以锌依赖性和可饱和的方式与人脐静脉内皮细胞（HUVEC）结合，表明HUVEC表达这些蛋白质的特异性结合位点。然而，识别和免疫化学表征的推定受体位点（S）尚未完成。在这份报告中，我们已经确定了细胞表面糖蛋白，这是一个可能的候选人的HK结合位点的HUVEC。当溶解的HUVEC膜在存在或不存在50 μ M ZnCl 2的情况下经受HK-亲和柱并且结合的膜蛋白被洗脱时，仅在锌的存在下获得单一的主要蛋白峰。SDS/PAGE分析和蛋白峰的银染显示该蛋白为33 kDa，部分序列分析与gC 1 q-R的NH 2末端相匹配，gC 1 q-R是一种与C1 q的球状“头部"结合的膜糖蛋白。还获得了另外两种约70 kDa和45 kDa的次要蛋白质。通过Western印迹分析后，发现33-kDa条带与识别gC 1 q-R上不同表位的几种单克隆抗体（mAb）反应。配体和斑点印迹分析显示，锌依赖性结合的生物素化HK以及生物素化因子XII的分离的33-kDa HUVEC分子以及重组gC 1 q-R。此外，I-125-HK与HUVEC细胞的结合被选定的单克隆抗gC 1 q-R抗体抑制。然而，C1 q不抑制I-125-HK与HUVEC的结合，已知抑制C1 q与gC 1 q-R结合的单克隆抗体也不抑制I-125-HK与HUVEC结合。综上所述，这些数据表明HK（和因子XII）通过一种33 kDa的细胞表面糖蛋白与HUVEC结合，该糖蛋白似乎与gC 1 q-R相同，但与gC 1 q-R上与C1 q结合不同的位点相互作用。
High molecular weight kininogen (HK) and factor XII are known to bind to human umbilical vein endothelial cells (HUVEC) in a zinc-dependent and saturable manner indicating that HUVEC express specific binding site(s) for those proteins. However, identification and immunochemical characterization of the putative receptor site(s) has not been previously accomplished. In this report, we have identified a cell surface glycoprotein that is a likely candidate for the HK binding site on HUVECs. When solubilized HUVEC membranes were subjected to an HK-affinity column in the presence or absence of 50 mu M ZnCl2 and the bound membrane proteins eluted, a single major protein peak was obtained only in the presence of zinc. SDS/PAGE analysis and silver staining of the protein peak revealed this protein to be 33 kDa and partial sequence analysis matched the NH2 terminus of gC1q-R, a membrane glycoprotein that binds to the globular ''heads'' of C1q. Two other minor proteins of approximate to 70 kDa and 45 kDa were also obtained. Upon analysis by Western blotting, the 33-kDa band was found to react with several monoclonal antibodies (mAbs) recognizing different epitopes on gC1q-R. Ligand and dot blot analyses revealed zinc-dependent binding of biotinylated HK as well as biotinylated factor XII to the isolated 33-kDa HUVEC molecule as well as recombinant gC1q-R. In addition, binding of I-125-HK to HUVEC cells was inhibited by selected monoclonal anti-gC1q-R antibodies. C1q, however, did not inhibit I-125-HK binding to HUVEC nor did those monoclonals known to inhibit C1q binding to gC1q-R. Taken together, the data suggest that HK (and factor XII) bind to HUVECs via a 33-kDa cell surface glycoprotein that appears to be identical to gC1q-R but interact with a site on gC1q-R distinct from that which binds C1q.