EXPRESSION AND CHARACTERIZATION OF RECOMBINANT MANDUCA-SEXTA SERPIN-1B AND SITE-DIRECTED MUTANTS THAT CHANGE ITS INHIBITORY SELECTIVITY

Hemolymph of Manduca sexta contains a number of serine proteinase inhi bitors from the serpin superfamily. During formation of a stable compl ex between a serpin and a serine proteinase, the enzyme cleaves a spec ific peptide bond in an exposed loop (the reactive-site region) at the surface of the serpin, The amino acid residue on the amino-terminal s ide of this scissile bond, the P-1 residue, is important in defining t he selectivity of a serpin for inhibiting different types of serine pr oteinases. M, sexta serpin-1B, with alanine at the position predicted from sequence alignments to be the P, residue, was previously named al aserpin, This alanyl residue was changed by site-directed mutagenesis to lysine (A343K) and phenylalanine (A343F). The serpin-1B cDNA and it s mutants were inserted into an expression vector, H6pQE-60, and the s erpin proteins were expressed in Escherichia coli, Affinity-purified r ecombinant serpins selectively inhibited mammalian serine proteinases: serpin-1B inhibited elastase; serpin1B(A343K) inhibited trypsin, plas min, and thrombin; serpin-1B(A343F) inhibited chymotrypsin as well as trypsin, All three serpins inhibited human cathepsin G, This insect se rpin and its site-directed mutants associated with mammalian serine pr oteinases at rates similar to those reported for mammalian serpins. Se rpin-1B and its mutants formed SDS-stable complexes with the enzymes t hey inhibited, The scissile bond was determined to be between residues 343 and 344 in wild-type serpin-1B and in serpin-1B with mutations at residue 343, These results demonstrate that the P, alanine residue de fines the primary selectivity of serpin1B for elastase-like enzymes, a nd that this selectivity can be altered by mutations at this position.