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

We have analyzed the proteolytic activity of a recombinant form of apo lipoprotein(a) [r-apo(a)], A mutant 17-kringle form of r-apo(a) was en gineered that contained a serine to arginine substitution which reinst ates 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 b y tPA as determined by SDS-PAGE and fluorography and by Western blot a nalysis, However, tPA cleavage did not result in an active protease as both wildtype r-apo(a) and the mutant, either free or incorporated in to r-Lp(a) particles, were uniformly inactive against a variety of chr omogenic serine protease tripeptide substrates. To assess whether the large number of kringle IV repeats present in apo(a) inhibits proteoly tic activity, we generated truncated forms of the Ser-->Arg mutant con taining one or 10 kringle IV repeats. These truncated versions of r-ap o(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 no t 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 puri fied human LDL; no degradation of LDL was observed after 16 h at 37 de grees C. The results of this study suggest that one or more of the sub stitutions present in the protease domain of apo(a), in addition to th e Arg-->Ser substitution, render apo(a) proteolytically inactive.