ANALYSIS OF THE PROTEOLYTIC ACTIVITY OF A RECOMBINANT FORM OF APOLIPOPROTEIN(A)

We have analyzed the proteolytic activity of a recombinant form of apolipoprotein(a) [r-apo(a)], A mutant 17-kringle form of r-apo(a) was engineered that contained a serine to arginine substitution which reinstates the tissue-type plasminogen activator (tPA) cleavage site in the protease domain of r-apo(a), The mutant form of r-apo(a) was cleaved by tPA as determined by SDS-PAGE and fluorography and by Western blot analysis, However, tPA cleavage did not result in an active protease as both wildtype r-apo(a) and the mutant, either free or incorporated into r-Lp(a) particles, were uniformly inactive against a variety of chromogenic serine protease tripeptide substrates. To assess whether the large number of kringle IV repeats present in apo(a) inhibits proteolytic activity, we generated truncated forms of the Ser-->Arg mutant containing one or 10 kringle IV repeats. These truncated versions of r-apo(a) were susceptible to cleavage by tPA but were inactive against the plasmin substrate S-2251. Treatment of the Ser-->Arg mutant of the 17-kringle r-apo(a) with tPA and diisopropylfluorophosphate (DFP) did not result in modification of the mutant protease domain by DFP. Finally, we incubated r-apo(a) or r-Lp(a) particles formed in vitro with purified human LDL; no degradation of LDL was observed after 16 h at 37 degrees C. The results of this study suggest that one or more of the substitutions present in the protease domain of apo(a), in addition to the Arg-->Ser substitution, render apo(a) proteolytically inactive.