Mapping binding domains of kininogens on endothelial cell cytokeratin 1

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 pe rformed to demonstrate the HK binding domain on CK1, Four overlapping recom binant (r) CK1 proteins were produced in Escherichia coli by a glutathione S-transferase gene fusion system. Biotin-HK specifically bound to rCK1(28) and rCK1(31) in the presence of Zn2+ but not to (Deleted1-6)rCK1(31). Recom binant CK1(28) and rCK1(31) also inhibited biotin-HK binding to HUVEC with IC50 of 0.4 and 0.5 mu M, respectively. Alternatively, rCK1(14) and (Delete d1-6)rCK1(31) did not inhibit binding at concentrations greater than or equ al to 1 mu M. Seven sequential 20 amino acid peptides of CK1 were prepared to cover the protein coded by exons 1-3. Only the first peptide (GYG20) cod ed by exon 1 significantly inhibited HK binding to HUVEC with an IC50 of 35 mu M. Fine mapping studies isolated two overlapping peptides also coded by exon 1 (GPV15 and PGG15) that inhibited binding to HUVEC with IC50 of 18 a nd 9 mu M, respectively. A sequence scrambled peptide of PGG15 did not bloc k binding to HUVEC and biotin-GPV20 specifically bound to HK. Peptides GPV1 5 and PGG15 also blocked prekallikrein activation on endothelial cells. How ever, inhibition of PK activation by peptide PGG15 occurred at 10-fold lowe r concentration (IC50 = 1 mu M) than inhibition of biotin-HK binding to HUV EC (IC50 = 10 mu M). 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 glyci ne-rich amino-terminal globular domain. Furthermore, HK binding to Chi modu lates PK activation on HUVEC.