Cytokeratin 1 and gC1qR mediate high molecular weight kininogen binding to endothelial cells.
High molecular weight kininogen (HK) attaches to endothelial cells by separate sites on the heavy and light chains and requires 15-50 microM zinc. Previously identified binding proteins include gC1qR, cytokeratin 1, and the urokinase plasminogen activator receptor; however, their relative contributions to binding are not yet clarified. We have prepared affinity columns to which were coupled either cleaved HK or peptide LDCNAEVYVVPWEKKIYPTVNCQPLGM derived from heavy-chain domain 3. Endothelial cell membranes were solubilized and chromatographed in the presence or absence of zinc ion, the bound proteins were eluted, and active fractions were identified by dot blot using biotinylated HK, SDS/PAGE, and Western blot analysis. The peptide containing column eluate revealed but one band at 68 kDa if zinc ion was present which was identified as cytokeratin 1 by amino acid sequencing of an internal peptide. The HK affinity column revealed bands at 68 kDa (cytokeratin 1), 33 kDa (gC1qR), and 66 kDa (unidentified). HK or domain 3-derived peptide bound to the 68 kDa band; prekallikrein and Factor XII did not. HK or Factor XII bound to the 33-kDa band if zinc was present while no binding to the 66 kDa band was observed. Antibody to cytokeratin 1 inhibited HK binding to endothelial cells by 30%, antibody to gC1qR inhibited HK binding to endothelial cells by 72%, and a mixture of both inhibited binding by 86%. Our data suggest HK binding by interaction of the heavy-chain domain 3 with cytokeratin 1 and the light chain with gC1qR.
Joseph
K
K
Division of Pulmonary, Critical Care, Asthma and Allergy, Medical University of South Carolina, 171 Ashley Avenue, Charleston, South Carolina, 29425, USA.
Ghebrehiwet
B
B
Kaplan
A P
AP
D000595:Amino Acid Sequence; D019921:Biotinylation; D002352:Carrier Proteins; D015922:Complement C1q / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology*; D006801:Humans; D018960:Hyaluronan Receptors; D007457:Iodine Radioisotopes; D007633:Keratins / Q000737:chemistry / Q000494:pharmacology*; D019679:Kininogen, High-Molecular-Weight / Q000378:metabolism*; D008024:Ligands; D008562:Membrane Glycoproteins; D024101:Mitochondrial Proteins; D010446:Peptide Fragments / Q000737:chemistry; D011485:Protein Binding / Q000187:drug effects; D011951:Receptors, Complement / Q000502:physiology*; D014471:Umbilical Veins / Q000166:cytology; D015032:Zinc / Q000378:metabolism
C087299:C1QBP protein, human; D002352:Carrier Proteins; D018960:Hyaluronan Receptors; D007457:Iodine Radioisotopes; D019679:Kininogen, High-Molecular-Weight; D008024:Ligands; D008562:Membrane Glycoproteins; D024101:Mitochondrial Proteins; D010446:Peptide Fragments; D011951:Receptors, Complement; C032402:complement 1q receptor; D007633:Keratins; D015922:Complement C1q; D015032:Zinc