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

Abstract

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 ...

A Venom Serpin Splicing Isoform of the Endoparasitoid Wasp Pteromalus puparum Suppresses Host Prophenoloxidase Cascade by Forming Complexes with Host Hemolymph Proteinases

“…Pulldown results indicate that possible targets of PpS1V are hemolymph proteinase PrHP8 and PrPAP1. This is similar to serpin-1J in M. sexta (40,47,54,55,68). Manduca serpin-1J regulates PPO activation through inhibition of M. sexta PAP3 (MsPAP3) (40,54) and the Toll pathway through inhibition of MsHP8 (47).…”

Changes in the Plasma Proteome of Manduca sexta Larvae in Relation to the Transcriptome Variations after an Immune Challenge: Evidence for High Molecular Weight Immune Complex Formation

“…Fortunately, proteolytic activation of the precursors of SPs (including HPs, PAPs), SPHs, POs, PSP, and spä tzle-1 has been well documented in M. sexta (2,16,32,43). It is also known that serpins and other protease inhibitors are involved in the control of immune SPs (19,(33)(34)(35)44). While the current analysis lacks the resolution needed to reveal components of the serpin-protease complexes, it did provide consistent results and new leads (e.g.…”

“…Likewise, each hemolymph protease may be detected as a precursor, N-and C-terminal fragments of the active SP, and a 75 kDa complex with the serpin fragment. Among five of the M. sexta serpin-1 splicing variants, serpin-1E forms SDSstable complexes with HP1 and HP8 (33). Serpin-3 through -6 are also known to form complex with HPs, including PAP1, PAP3, HP1, HP6, HP8, and HP21 (34,35).…”

Manduca sexta proprophenoloxidase activating proteinase-3 (PAP3) stimulates melanization by activating proPAP3, proSPHs, and proPOs

“…We now know that HP1, HP6, HP8, HP14, HP17, HP21, PAPs, SPH1, and SPH2 are probably involved in proPO and pro-spätzle activation. Serpin-1 variants and serpins 3 through 7 form covalent complexes with HPs (Ragan et al, 2010; Zhu et al, 2003; Christen et al, 2012; Tong et al, 2005; Zou et al, 2005; Suwanchaichinda et al, 2013). In this paper, we report the isolation of a major activating proteinase of proSPH1 and proSPH2 from hemolymph of pharate pupae and changes in the cell-free hemolymph from naïve and bacteria-injected larvae.…”