CHARACTERIZATION AND FUNCTIONAL-ANALYSIS OF 12 NATURALLY-OCCURRING REACTIVE-SITE VARIANTS OF SERPIN-1 FROM MANDUCA-SEXTA

Serpin gene-1 from the tobacco hornworm, Manduca sexta, encodes, throu gh alternative exon usage, 12 reactive site variants (Jiang, H., Wang, Y. and Kanost, M. R., (1994) J. Blot. Chem. 269, 55-58; Jiang, H., Wa ng, Y., Huang, Y., Mulnix, A. B., Kadel, J., Cole, K., and Kanost, M. R. (1996) J. Biol. Chem. 271, 28017-28023). These 43-kDa proteins diff er from each other only in their COOH-terminal 39-46 residues, which i nclude the reactive site. To test the hypothesis that these proteins a re proteinase inhibitors of diverse selectivities and to begin to eluc idate their physiological functions, we expressed the 12 serpin-1 vari ants in Escherichia coli. Seven of the variants inhibited mammalian se rine proteinases, with association rate constants comparable with thos e of human serpins. Serpin-1A, with a P-1 Arg residue, inhibited both trypsin and plasmin. Serpin-1B (P-1 Ala) and serpin-1F (P-1 Val) inhib ited porcine pancreatic elastase and human neutrophil elastase. Serpin -1H, -1K, and -1Z, all with a Tyr residue at the P-1 position, inhibit ed chymotrypsin and cathepsin G. Serpin-1I (P-1 Leu) inhibited both el astase and chymotrypsin. Nine of the serpin variants were active as in hibitors of microbial serine proteinases, including subtilisin Carlsbe rg, proteinase K, and two proteinases secreted by an entomopathogenic fungus, Metarhizium anisopliae. In addition, one of the serpin variant s, serpin-1J, strongly inhibited activation of M. sexta hemolymph phen oloxidase, a pathway involving a serine proteinase cascade. This pathw ay is a component of the defensive response of insects to microbial in fection. These results suggest that the products of M. sexta serpin ge ne-1 may be important in regulating both exogenous and endogenous seri ne proteinases in hemolymph.