Organization of Serpin Gene-1 from Manduca sexta

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Mutually exclusive alternative splicing produces transcripts for 12 serpin-1 isoforms in Manduca sexta that differ only in the region encoding the carboxyl-terminal 36 -40-amino acid residues. This variable region includes the reactive center loop, which determines the inhibitory selectivity of the serpin. We investigated mRNA levels of individual serpin-1 isoforms by quantitative PCR. The 12 isoforms were expressed at similar levels in hemocytes, but in fat body isoform B mRNA was present at significantly higher levels than isoforms C, D, E, F, G, J, K, and Z. To investigate the presence of individual serpin-1 isoforms in plasma we used immunoaffinity purification of serpin-1 isoforms from M. sexta plasma, followed by two-dimensional PAGE and identification of protein spots by digestion with a series of proteinases and analysis of the resulting peptides by MALDI-TOF/TOF. We identified nine of the 12 serpin-1 isoforms and, through analysis of putative serpin-1-proteinase complexes, identified three endogenous M. sexta proteinase targets of serpin-1. Our results suggest that M. sexta serpin-1 isoforms A, E, and J can inhibit hemolymph proteinase 8, which activates the cytokine spätzle. At least one isoform of serpin-1 can inhibit hemocyte proteinase 1, another M. sexta blood proteinase. In addition, a complex of serpin-1K in a complex with M. sexta midgut chymotrypsin was identified, suggesting serpin-1 isoforms may also function to protect insect tissues from digestive proteinases that may leak into the hemocoel.Serpins are a superfamily of proteins that are named for being serine proteinase inhibitors, a function that many serpins perform. Serpins contain an exposed reactive center loop, formed from a region near the carboxyl-terminal end of the protein. A proteinase that begins to cleave a serpin in the reactive center loop typically becomes covalently bound to the serpin and inactivated. The proteinase active site serine forms an ester bond with the acyl group of the serpin P1 residue at the amino-terminal side of the scissile bond, but cannot complete the hydrolysis because the serpin reactive center loop rapidly inserts into the A ␤-sheet, moving the proteinase ϳ70 Å and deforming the proteinase catalytic site (1, 2). Extracelluar serpins exert control over serine proteinase cascades in vertebrate blood, including complement activation, blood clotting, and fibrinolysis (3, 4). In insects, serine proteinase cascades initiated by microbial infection elicit antimicrobial peptide production and lead to activation of prophenoloxidase, which catalyzes the melanization response (5-13 In insects, serpin genes have evolved alternative exon splicing, which produces variation in the sequence of much of the reactive center loop, producing multiple functional serpins from a single gene. This was first described in M. sexta serpin-1, which has 12 different copies of exon 9 that undergo mutually exclusive alternative splicing to produce 12 putative protein isoforms. These isoforms differ in their carboxyl-terminal 39 -46 ...

Section: Discussionmentioning

confidence: 95%

mentioning

confidence: 85%

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

Ragan

Yang

et al. 2010

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“…Thus, these motifs can be used to identify additional RSL-containing exons that could have been overlooked in the previous searches. This is an important consideration, since some serpin genes in Drosophila (19) and Manduca sexta (20) contain alternatively spliced 3Ј exons with variable RSL sequences. We rescreened the C. elegans sequence data base (available on the World Wide Web at genome.wustl.edu/blast/ client.pl) using the RSL-containing exon of serpin C05E4.1.…”

Section: Identification Of Novel Serpin Genes In C Elegans-mentioning

confidence: 99%

SRP-2 Is a Cross-class Inhibitor That Participates in Postembryonic Development of the Nematode Caenorhabditis elegans

Pak

Kumar

Tsu

et al. 2004

High molecular weight serpins are members of a large superfamily of structurally conserved proteins that inactivate target proteinases by a suicide substrate-like mechanism. In vertebrates, different clades of serpins distribute predominantly to either the intracellular or extracellular space. Although much is known about the function, structure, and inhibitory mechanism of circulating serpins such as ␣ 1 -antitrypsin (SERPINA1) and antithrombin III (SERPINC1), relatively little is known about the function of the vertebrate intracellular (clade B) serpins. To gain a better understanding of the biology of the intracellular serpins, we initiated a comparative genomics study using Caenorhabditis elegans as a model system. A screen of the C. elegans genomic and cDNA databases revealed nine serpin genes, tandemly arrayed on chromosome V. Although the C. elegans serpins represent a unique clade (L), they share significant functional homology with members of the clade B group of intracellular serpins, since they lack typical N-terminal signal peptides and reside intracellularly. To determine whether nematode serpins function as proteinase inhibitors, one family member, srp-2, was chosen for further characterization. Biochemical analysis of recombinant SRP-2 protein revealed SRP-2 to be a dual cross-class inhibitor of the apoptosis-related serine proteinase, granzyme B, and the lysosomal cysteine proteinases, cathepsins K, L, S, and V. Analysis of temporal and spatial expression indicated that SRP-2 was present during early embryonic development and highly expressed in the intestine and hypoderm of larval and adult worms. Transgenic animals engineered to overexpress SRP-2 were slow growing and/or arrested at the first, second, or third larval stages. These data suggest that perturbations of serpin-proteinase balance are critical for correct postembryonic development in C. elegans.Serpins are a unique class of proteinase inhibitors that irreversibly neutralize target proteinases by a mechanism involving the conformational distortion of the proteinase. Serpins have been identified in animals, plants, insects, and certain viruses (1, 2). More recently, serpins have been detected in prokaryotes (3). A search of genome data bases provides evidence for Ͼ500 serpins that are grouped into 17 clades (plus Ͼ10 unclassified orphans) based upon phylogenetic relationships (4). In humans, ϳ35 serpins have been identified. These serpins are distributed among nine clades (A-I), and most are secreted and function in the circulation or extracellular spaces. These serpins regulate proteinases involved in blood coagulation, fibrinolysis, complement activation, inflammation, and extracellular matrix remodeling. In contrast, serpins belonging to clade B reside predominantly intracellularly and have been implicated in regulating apoptosis, tumor progression, and metastasis (5). However, their biological functions in terms of an intact organism have not been well defined. To date, no naturally occurring mutations with an identifiable phenotype...

“…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).…”

mentioning

confidence: 99%

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

Tong

Kanost

2005

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108

Infection stimulates the innate immune responses of insects, including activation of prophenol oxidase (pro-PO) in plasma as the last step of a serine protease cascade. To investigate the roles of protease inhibitors in regulating this pathway, we cloned cDNAs for two new serpins (serpin-4 and serpin-5) from the tobacco hornworm, Manduca sexta. Serpin-4 and serpin-5 mRNAs are constitutively expressed at a low level in larval hemocytes and fat body and increased dramatically upon bacterial challenge. These serpins are present in larval plasma at ϳ3 (serpin-4) and ϳ1 g/ml (serpin-5) and increased 3-8-fold by 24 h after injection of bacteria or fungi. Recombinant serpin-4 and serpin-5 decreased pro-PO activation when added to plasma, but they did not directly inhibit the pro-PO-activating proteases. Instead, they apparently regulate the pathway by inhibiting one or more target proteases upstream of the pro-PO-activating proteases.Prophenol oxidase (pro- PO) 1 activation is an innate immune response in arthropods. Upon injury or infection, a pro-PO zymogen in plasma is activated by a specific protease. Phenol oxidase (PO) hydroxylates monophenols to o-diphenols and then oxidizes o-diphenols to quinones, which can polymerize to form melanin at the injury site or around invading organisms (1, 2). Quinones may also be involved in the production of cytotoxic molecules such as superoxides and hydroxyl radicals that could help kill the invading organisms (1, 3).A serine protease cascade functions in pro-PO activation (2, 4, 5). The final protease in the pathway, pro-PO-activating protease (PAP) (also called pro-PO-activating enzyme or factor) has been identified from several insects, including the tobacco hornworm, Manduca sexta (6 -8), the silkworm, Bombyx mori (9), a beetle, Holotrichia diomphalia (10, 11), and from a crustacean, the crayfish Pacifastacus leniusculus (12). These PAPs contain one or two clip domains at their amino terminus and a serine protease domain at their carboxyl terminus (13). PAPs are synthesized as zymogens, which are activated by a specific proteolytic cleavage between the clip domain and the protease domain. However, protease(s) that activate PAPs have not yet been characterized.Serpins present in plasma regulate the pro-PO activation pathway (4,5,(13)(14)(15)(16). Serpins are serine protease inhibitors, about 400 amino acid residues long (17-20), with an exposed reactive center loop near their carboxyl terminus (21,22). The P1 residue located in this loop determines the primary specificity of inhibition. 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 (23-25). 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 ...