Joseph K, Ghebrehiwet B, Peerschke E I, Reid K B, Kaplan A P
Department of Medicine, State University of New York, Stony Brook 11794-8161, USA.
Proc Natl Acad Sci U S A. 1996 Aug 6;93(16):8552-7. doi: 10.1073/pnas.93.16.8552.
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 microM 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 approximately 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 125I-HK to HUVEC cells was inhibited by selected monoclonal anti-gC1q-R antibodies. C1q, however, did not inhibit 125I-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.
已知高分子量激肽原（HK）和因子 XII 以锌依赖性和饱和性方式与人脐静脉内皮细胞（HUVEC）结合，这表明 HUVEC 表达这些蛋白质的特异性结合位点。然而，此前尚未完成对假定受体位点的鉴定和免疫化学表征。在本报告中，我们鉴定出一种细胞表面糖蛋白，它可能是 HUVEC 上 HK 结合位点的候选者。当在存在或不存在 50 μM ZnCl₂ 的情况下，将溶解的 HUVEC 膜通过 HK 亲和柱，并洗脱结合的膜蛋白时，仅在存在锌的情况下获得了一个主要的蛋白峰。对该蛋白峰进行 SDS/PAGE 分析和银染显示，该蛋白为 33 kDa，部分序列分析与 gC1q-R 的 NH₂ 末端匹配，gC1q-R 是一种与 C1q 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 印迹分析发现，33 kDa 的条带与几种识别 gC1q-R 上不同表位的单克隆抗体（mAb）发生反应。配体和斑点印迹分析表明，生物素化的 HK 以及生物素化的因子 XII 与分离的 33 kDa HUVEC 分子以及重组 gC1q-R 存在锌依赖性结合。此外，选定的抗 gC1q-R 单克隆抗体可抑制¹²⁵I-HK 与 HUVEC 细胞的结合。然而，C1q 并不抑制¹²⁵I-HK 与 HUVEC 的结合，已知抑制 C1q 与 gC1q-R 结合的那些单克隆抗体也无此作用。综上所述，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 gC1q-R 相同，但与 gC1q-R 上与 C1q 结合的位点不同的位点相互作用。
