Portal de Periódicos da CAPES

Mapping Binding Domains of Kininogens on Endothelial Cell Cytokeratin 1

1999; Elsevier BV; Volume: 274; Issue: 11 Linguagem: Inglês

10.1074/jbc.274.11.7137

1083-351X

Zia Shariat‐Madar, Fakhri Mahdi, Alvin H. Schmaier,

Mast cells and histamine

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 rCK1 28 and rCK1 31 in the presence of Zn 2+ but not to Deleted1–6 rCK1 31 . Recombinant CK1 28 and rCK1 31 also inhibited biotin-HK binding to HUVEC with IC 50 of 0.4 and 0.5 μm, respectively. Alternatively, rCK1 14 and Deleted1–6 rCK1 31 did not inhibit binding at concentrations ≥1 μm. 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 IC 50 of 35 μm. Fine mapping studies isolated two overlapping peptides also coded by exon 1 (GPV15 and PGG15) that inhibited binding to HUVEC with IC 50 of 18 and 9 μm, 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 (IC 50 = 1 μm) than inhibition of biotin-HK binding to HUVEC (IC 50 = 10 μm). 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.