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 from of r-apo(a) was engineered that contained a serine to arginine substitution which reinstates the tissue-type plasminogen activator (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 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 diisopropylflurophosphate (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.
D000595:Amino Acid Sequence; D001053:Apolipoproteins / Q000235:genetics / Q000378:metabolism*; D053241:Apoprotein(a); D001120:Arginine / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D005341:Fibrinolysin / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007531:Isoflurophate / Q000494:pharmacology; D018082:Kringles; D017270:Lipoprotein(a); D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000235:genetics; D010959:Tissue Plasminogen Activator / Q000378:metabolism; D014316:Tritium
D001053:Apolipoproteins; D017270:Lipoprotein(a); D011994:Recombinant Proteins; D014316:Tritium; D007531:Isoflurophate; D012694:Serine; D001120:Arginine; D053241:Apoprotein(a); D010959:Tissue Plasminogen Activator; D005341:Fibrinolysin