title: Rabbit alpha-1-antiproteinase E: a novel recombinant serpin which does not inhibit proteinases.
abstract: 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 the 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 isoforms of rabbit alpha-1-antiproteinase. The N-terminal half of the amino acid residues of the three isoforms was almost identical, but the putative reactive-site loop structure (P8-P'8) was significantly different in the various forms, the P1 site of the E form being glutamic acid. Interaction of the recombinant E form with the various proteinases was investigated by SDS/PAGE, followed by immunoblot analysis. The recombinant protein and trypsin formed a 62 kDa equimolar complex, which gradually became graded to the 37 kDa fragment through several intermediates. The E form also formed a complex of a similar size with elastase and became degraded to the 31 kDa fragment. Several proteinases which cleaved the E form without forming a detectable complex on SDS/PAGE are chymotrypsin, protease V8, pancreas kallikrein, thermolysin, papain and ficin. Other proteinases, with a stringent substrate specificity, such 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 plasma 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-antiproteinase E. The novel character of the E form could provide a new insight into the interaction of serpin and proteinases.
authors.affiliation: Department of Biochemistry, School of Medicine, Kinki University, Osaka, Japan.
authors.forename: A
authors.initials: A
authors.lastname: Saito
authors.forename: H
authors.initials: H
authors.lastname: Sinohara
mesh_terms: D000577:Amides / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018076:DNA, Complementary; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D000515:alpha 1-Antitrypsin / Q000737:chemistry / Q000494:pharmacology*
chemical_list: D000577:Amides; D018076:DNA, Complementary; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D000515:alpha 1-Antitrypsin