Expression and characterization of recombinant Manduca sexta serpin- 1B and site-directed mutants that change its inhibitory selectivity

• Published in: Insect biochemistry and molecular biology 1995-12, Vol.25 (10), p.1093-1100
• Main Authors: Jiang, H., Mulnix, A.B., Kanost, M.R.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Base Sequence; DNA Primers; Elastase inhibitor; Escherichia coli; Genes, Insect; Hemolymph protein; Manduca - enzymology; Manduca - genetics; Molecular Sequence Data; Mutagenesis, Site-Directed; Proteinase inhibitor; Recombinant Fusion Proteins - genetics; Recombinant protein; Serine Proteinase Inhibitors - chemistry; Serine Proteinase Inhibitors - genetics; Serine Proteinase Inhibitors - metabolism; Serpin; Serpins - chemistry; Serpins - genetics; Serpins - metabolism; Structure-Activity Relationship; Tobacco hornworm
• ISSN: 0965-1748; 1879-0240
• DOI: 10.1016/0965-1748(95)00042-9

Hemolymph of Manduca sexta contains a number of serine proteinase inhibitors from the serpin superfamily. During formation of a stable complex between a serpin and a serine proteinase, the enzyme cleaves a specific peptide bond in an exposed loop (the reactive-site region) at the surface of the serpin. The amino acid residue on the amino-terminal side of this scissile bond, the P 1 residue, is important in defining the selectivity of a serpin for inhibiting different types of serine proteinases. M. sexta serpin-1B, with alanine at the position predicted from sequence alignments to be the P 1 residue, was previously named alaserpin. This alanyl residue was changed by site-directed mutagenesis to lysine (A343K) and phenylalanine (A343F). The serpin-1B cDNA and its mutants were inserted into an expression vector, H6pQE-60, and the serpin proteins were expressed in Escherichia coli. Affinity-purified recombinant serpins selectively inhibited mammalian serine proteinases: serpin-1B inhibited elastase; serpin-1B(A343K) inhibited trypsin, plasmin, and thrombin; serpin-1B(A343F) inhibited chymotrypsin as well as trypsin. All three serpins inhibited human cathepsin G. This insect serpin and its site-directed mutants associated with mammalian serine proteinases at rates similar to those reported for mammalian serpins. Serpin-1B and its mutants formed SDS-stable complexes with the enzymes they 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 1 alanine residue defines the primary selectivity of serpin-1B for elastase-like enzymes, and that this selectivity can be altered by mutations at this position.