The proteolytic activity and cleavage specificity of fibronectin-gelatinase and fibronectin-lamininase

Human plasma fibronectin contains two latent aspartic proteinases, FN-gelatinase and FN-lamininase. Both enzymes can be generated and activated in the presence of Ca2+ from the purified cathepsin D-produced 190-kDa fibronectin fragment. We investigated the proteolytic activity and cleavage specificity of both enzymes in a range of pH from 3.5 to 9.0 using the B chain of oxidized bovine insulin and chromogenic peptides as substrates. The inhibition of the enzymes by several natural inhibitors from human plasma was also tested. The specificities of FN-gelatinase and FN-lamininase are similar to other major acidic proteinases, including pepsin, renin, cathepsin D, and HIV-proteinases. Both enzymes mainly hydrolyze three peptide bonds in the oxidized insulin B chain, namely Glu-Ala (residues 13-14), Tyr-Leu (residues 16-17), and Phe-Phe (residues 24-25). For the peptide substrates H-Pro-Thr-Glu-Phe-p-nitro-Phe-Arg-Leu-OH and H-Phe-Gly-His-p-nitro-Phe-Phe-Val-Leu-OMe that were cleaved the respective values of k(cat)/K-M were 105.1 and 11.8 mM(-1) sec(-1) for cleavage by FN-gelatinase, and 123.2 and 15.5 mM(-1) sec(-1) for cleavage by FN-lamininase. The maximal activities of both enzymes were observed in a range between pH 5.6 and 6.3 and they became inactivated at a pH value above 8.4. Both FN-gelatinase and FN-lamininase were efficiently inhibited by alpha(2)-macroglobulin.