Characterization and Functional Analysis of 12 Naturally Occurring Reactive Site Variants of Serpin-1 from Manduca sexta

Abstract

To test the hypothesis that these proteins are proteinase inhibitors of diverse selectivities and to begin to elucidate their physiological functions, we expressed the 12 serpin-1 variants in Escherichia coli. Seven of the variants inhibited mammalian serine proteinases, with association rate constants comparable with those 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) inhibited porcine pancreatic elastase and human neutrophil elastase. Serpin-1H, -1K, and -1Z, all with a Tyr residue at the P 1 position, inhibited chymotrypsin and cathepsin G. Serpin-1I (P 1 Leu) inhibited both elastase and chymotrypsin. Nine of the serpin variants were active as inhibitors of microbial serine proteinases, including subtilisin Carlsberg, proteinase K, and two proteinases secreted by an entomopathogenic fungus, Metarhizium anisopliae. In addition, one of the serpin variants, serpin-1J, strongly inhibited activation of M. sexta hemolymph phenoloxidase, a pathway involving a serine proteinase cascade. This pathway is a component of the defensive response of insects to microbial infection. These results suggest that the products of M. sexta serpin gene-1 may be important in regulating both exogenous and endogenous serine proteinases in hemolymph.The serpin 1 superfamily is composed of a large number of proteins, most of which function as serine proteinase inhibitors (1). Serpins have been identified in animals, plants, and viruses (2) and therefore appear to have evolved from a common ancestor before divergence of the animal and plant kingdoms. Individual mammalian species have a large number of serpin genes, each encoding a protein with a unique reactive site sequence and physiological function (3). Members of the serpin superfamily are involved in the regulation of a wide variety of physiological processes, including blood clotting, complement activation, inflammatory responses, hormone transport, and tumor suppression (1).Serpins are typically 370 -390 amino acid residues long, with a reactive site loop near the carboxyl terminus. This loop, exposed on the surface of the molecule, is the site at which a serpin interacts with a target serine proteinase (1). During reaction with a proteinase, the P 1 residue (4) in the reactive site loop forms a bond with the active site serine residue in the enzyme. The amino acid sequence and conformation of the reactive site loop largely determine the selectivity of inhibition. Altering the loop sequence, especially at the P 1 position, can cause dramatic changes in the inhibitory selectivity of a serpin (3).Serpins have been found in the hemolymph of invertebrates, including three groups of arthropods: insects, crayfish, and horseshoe crabs (5). These invertebrate serpins have 12-30% amino acid sequence identity with various mammalian serpins. Functions of invertebrate serpins are largely unknown, although they may regulate proteinases released from blood cells during inflammation-like processes and may regul...

Analysis of Mutually Exclusive Alternatively Spliced Serpin-1 Isoforms and Identification of Serpin-1 Proteinase Complexes in Manduca sexta Hemolymph

“…These isoforms differ in their carboxyl-terminal 39 -46 residues, including the P1 residue, and inhibit serine proteinases with different specificities (Fig. 1) (27)(28)(29)(30)(31). Similar alternative splicing occurs in some serpin genes from other insect species, with 3-15 alternative exons encoding the reactive center loop present in genes studied so far (32)(33)(34).…”

“…Only two of the 12 serpin-1 isoforms have been found to form complexes with M. sexta serine proteases. Serpin-1J can inhibit activation of the phenoloxidase pathway and form a complex with prophenoloxidase activating proteinase-3 in vitro (27,38), whereas serpin-1I can complex with HP14 3 in vitro (39). 27 hemolymph proteinases are known in M. sexta (40,41) and some of these are likely endogenous proteinase targets of serpin-1 isoforms.…”

Manduca sexta Serpin-4 and Serpin-5 Inhibit the Prophenol Oxidase Activation Pathway

“…There are 35 clip-domain protease genes in the D. melanogaster genome and 41 such genes in the mosquito A. gambiae (46), and many of these are likely to be plasma proteins. Insect plasma also contains a complex mixture of serine protease inhibitors (14,16), including at least 16 serpins in M. sexta (serpins 3-6 and 12 variants of serpin-1) (25,28,29). Understanding the physiological functions of such a complex system will require further characterization of the hemolymph proteases and serpins and their roles in immunity, development, or other processes.…”

“…Four serpins from M. sexta have been characterized previously. The serpin-1 gene encodes 12 variants with different inhibitory selectivity, due to alternative splicing of exons that encode the reactive center loop (23)(24)(25). Serpin-1 mRNA is constitutively expressed in the larval fat body and at a lower level in hemocytes (26).…”

“…Serpin-1 mRNA is constitutively expressed in the larval fat body and at a lower level in hemocytes (26). One of the reactive site variants, serpin-1J, inhibits pro-PO activation in plasma by inhibiting PAPs (8,25). Serpin-2 is an intracellular protein (27), expressed in the cytoplasm of hemocytes after bacterial challenge.…”