CONFORMATIONAL-CHANGES IN LOW-MOLECULAR-WEIGHT KININOGEN ALTERS ITS ABILITY TO BIND TO ENDOTHELIAL-CELLS

The plasma kininogens, high (HK) and low (LK) molecular weight kininog ens, are the parent proteins for bradykinin, a potent vasoactive pepti de that locally influences vascular biology. Binding of both HK and LK to the endovascular wall contributes to bradykinin delivery. Recently , we found one preparation of LK (LKd) which had reduced inhibition of biotin-HK binding to endothelium. The functional defect in LKd was no t merely due to bradykinin loss because two preparations of bradykinin -free LK blocked biotin-HK binding. However, using two different parti cular monoclonal antibodies to bradykinin, LKd, but no other preparati on of LK, had its epitope to bradykinin exposed on non-reduced samples on immunoblot. These data suggested that LKd had an altered conformat ion which exposed the amino terminal arginine of bradykinin to antigen ic detection. The altered conformation of LKd allowed it to be more su sceptible to trypsin proteolysis. On circular dichroism, the percentag e of alpha-helix was significantly increased, indicating an alteration in the protein. This alteration in LKd was not due to a loss of molec ular mass of the protein. On laser desorption mass spec troscopy, the molecular mass of LKd was similar to the other preparations of LK. Inv estigations were performed to ascertain the mechanism by which LKd had altered ability to bind to cells. LKd was found to be proteolyzed by an unknown protease at the beginning of domain 2 between threonine(119 ) and alanine(120). Reduction of functional LK with dithiothreitol to expose its bradykinin epitope did not produce the LKd defect. Proteoly sis of functional LK with plasma kallikrein, elastase followed by plas ma kallikrein, chymotrypsin, or bromelain also did not produce the def ect seen in LKd These combined data indicated that LK maintains a part icular conformation that allows the protein to orient itself such that it can bind to endothelial cells. Proteolysis in the surface exposed region between domains 1 and 2 probably allows for the protein to unfo ld and contributes to its lost ability to bind to endothelial cells.