RABBIT ALPHA-1-ANTIPROTEINASE-E - A NOVEL RECOMBINANT SERPIN WHICH DOES NOT INHIBIT PROTEINASES

A cDNA coding for the E isoform of alpha-1-antiproteinase (also called alpha-1-antitrypsin or alpha-1-proteinase inhibitor) was isolated by oligonucleotide hybridization following immunochemical screening of th e rabbit liver cDNA library. The deduced amino acid sequence of the E isoform showed 96.4% identity in 413 residues of the F and S-1 isoform s of rabbit alpha-1-antiproteinase, The N-terminal half of the amino a cid residues of the three isoforms was almost identical, but the putat ive reactive-site loop structure (P8-P'8) was significantly different in the various forms, the P1 site of the E form being glutamic acid. I nteraction of the recombinant E form with the various proteinases was investigated by SDS/PAGE, followed by immunoblot analysis. The recombi nant protein and trypsin formed a 62 kDa equimolar complex, which grad ually became graded to the 37 kDa fragment through several intermediat es. The E form also formed a complex of a similar size with elastase a nd became degraded to the 31 kDa fragment. Several proteinases which c leaved the E form without forming a detectable complex on SDS/PAGE are chymotrypsin, protease V8, pancreas kallikrein, thermolysin, papain a nd ficin. Other proteinases, with a stringent substrate specificity, s uch as thrombin, factor Xa, plasmin, plasma kallikrein and cathepsin G , did not attack the E form. Unlike the F and S-1 forms of rabbit plas ma alpha-1-antiproteinase, the recombinant E form did not inhibit the amidolytic and proteolytic activities of trypsin. Neither elastase nor protease V8 was inhibited by the E form. Thus the change in the amino acid residues in the reactive-site loop, probably in the P1 site, is responsible for the loss of inhibitory activity of rabbit alpha-1-anti proteinase E. The novel character of the E form could provide a new in sight into the interaction of serpin and proteinases.