Shariat-Madar Z, Mahdi F, Schmaier A H
Division of Hematology and Oncology, University of Michigan, Ann Arbor, Michigan 48109-0640, USA.
J Biol Chem. 1999 Mar 12;274(11):7137-45. doi: 10.1074/jbc.274.11.7137.
Human cytokeratin 1 (CK1) in human umbilical vein endothelial cells (HUVEC) is expressed on their membranes and is able to bind high molecular weight kininogen (HK) (Hasan, A. A. K., Zisman, T., and Schmaier, A. H. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 3615-3620). New investigations have been performed to demonstrate the HK binding domain on CK1. Four overlapping recombinant (r) CK1 proteins were produced in Escherichia coli by a glutathione S-transferase gene fusion system. Biotin-HK specifically bound to rCK128 and rCK131 in the presence of Zn2+ but not to Deleted1-6rCK131. Recombinant CK128 and rCK131 also inhibited biotin-HK binding to HUVEC with IC50 of 0.4 and 0.5 microM, respectively. Alternatively, rCK114 and Deleted1-6rCK131 did not inhibit binding at concentrations >/=1 microM. Seven sequential 20 amino acid peptides of CK1 were prepared to cover the protein coded by exons 1-3. Only the first peptide (GYG20) coded by exon 1 significantly inhibited HK binding to HUVEC with an IC50 of 35 microM. Fine mapping studies isolated two overlapping peptides also coded by exon 1 (GPV15 and PGG15) that inhibited binding to HUVEC with IC50 of 18 and 9 microM, respectively. A sequence scrambled peptide of PGG15 did not block binding to HUVEC and biotin-GPV20 specifically bound to HK. Peptides GPV15 and PGG15 also blocked prekallikrein activation on endothelial cells. However, inhibition of PK activation by peptide PGG15 occurred at 10-fold lower concentration (IC50 = 1 microM) than inhibition of biotin-HK binding to HUVEC (IC50 = 10 microM). These studies indicate that HK binds to a region of 20 amino acids coded by exon 1 on CK1 which is carboxyl-terminal to its glycine-rich amino-terminal globular domain. Furthermore, HK binding to CK1 modulates PK activation on HUVEC.
人脐静脉内皮细胞（HUVEC）中的人细胞角蛋白1（CK1）在其细胞膜上表达，并且能够结合高分子量激肽原（HK）（哈桑，A.A.K.，齐斯曼，T.，和施迈尔，A.H.（1998年）《美国国家科学院院刊》95，3615 - 3620）。已开展新的研究以证明CK1上的HK结合结构域。通过谷胱甘肽S - 转移酶基因融合系统在大肠杆菌中产生了四种重叠的重组（r）CK1蛋白。在Zn2 +存在下，生物素 - HK特异性结合rCK128和rCK131，但不结合缺失1 - 6rCK131。重组CK128和rCK131也抑制生物素 - HK与HUVEC的结合，IC50分别为0.4和0.5微摩尔。或者，rCK114和缺失1 - 6rCK131在浓度≥1微摩尔时不抑制结合。制备了CK1的七个连续的20个氨基酸的肽段以覆盖由外显子1 - 3编码的蛋白质。只有由外显子1编码的第一个肽段（GYG20）以35微摩尔的IC50显著抑制HK与HUVEC的结合。精细定位研究分离出同样由外显子1编码的两个重叠肽段（GPV15和PGG15），它们分别以18和9微摩尔的IC50抑制与HUVEC的结合。PGG15的序列打乱肽段不阻断与HUVEC的结合，并且生物素 - GPV20特异性结合HK。肽段GPV15和PGG15也阻断内皮细胞上的前激肽释放酶激活。然而，肽段PGG15对PK激活的抑制发生在比抑制生物素 - HK与HUVEC结合（IC50 = 10微摩尔）低10倍的浓度（IC50 = 1微摩尔）下。这些研究表明HK结合到CK1上由外显子1编码的20个氨基酸区域，该区域位于其富含甘氨酸的氨基末端球状结构域的羧基末端。此外，HK与CK1的结合调节HUVEC上的PK激活。
