Patchwork‐structure serpins from silkworm (<i>Bombyx mori</i>) larval hemolymph

Sasaki, Takuji

doi:10.1111/j.1432-1033.1991.tb16370.x

Cited by 51 publications

(26 citation statements)

References 31 publications

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“…Homologs of M. sexta serpin-1 are also found in other insects, including the Lepidopterans Bombyx mori, in which there are three alternative exons in serpin-1 and four in serpin-28 (32,33), Mamestra configurata, which has nine isoforms (51), and Choristoneura fumiferana, which has at least four isoforms (34). Also homologous is serpin-1 from the flea Ctenophalides felis, which consists of 15 isoforms (35).…”

Section: Discussionmentioning

confidence: 99%

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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

Journal of Biological Chemistry

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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 ...

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Section: Discussionmentioning

confidence: 99%

“…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). M. sexta serpin-1 is expressed in fat body and, less strongly, in hemocytes (36,37).…”

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confidence: 98%

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

Journal of Biological Chemistry

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“…(Feng and Doolittle, 1987). SWAC, silkworm antichymotrypsin (this study); SWAC2, silkworm antichymotrypsin I1 (Sasaki, 1991); SWAT, silkworm antitrypsin ; ASMS, Munduca sextu alaserpin (Kanost et al, 1989); AlAC, human a-1-antichymotrypsin (Chandra et al, 1983a); AlAT, human a-1-antitrypsin (Long et al, 1984). (a), Well-conserved amino acids in serpins, also observed in SWAC (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…They are thought to be important to maintain the tertiary structure common to the serpins. The sw-Achy is a new member of the serpin superfamily of proteins and the fourth serpin found in insects after alaserpin in Munducu sextu (Kanost et al, 1989), sw-AT ) and sw-Achy11 (Sasaki, 1991).…”

Section: Discussionmentioning

confidence: 99%

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Molecular cloning of silkworm (Bombyx mori) antichymotrypsin

Narumi

Hishida

Sasaki

et al. 1993

European Journal of Biochemistry

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The cDNA of silkworm (Bornbyx rnori) antichymotrypsin (sw-Achy) was cloned from larval fat body and its nucleotide sequence was determined. The deduced amino acid sequence of mature swAchy begins with Phel and ends with Phe384, with a preceding 16-amino-acid signal peptide. The amino-acid sequence similarities of sw-Achy with the serine-proteinase inhibitors (serpins) silkworm antitrypsin, tobacco hornworm alaserpin, human a-1-antitrypsin and human a-1 -antichymotrypsin were 29.6%, 30.3%, 26.1%, and 25.0%, respectively. The highly conserved amino acids in other serpins are also conserved in sw-Achy. sw-Achy is thought to be a new member of the serpin family. Multiple alignment of sw-Achy with 23 other kinds of serpin by the progressive method produced a phylogenetic tree in which all four insect serpins are grouped separately within one branch. The reactive site of sw-Achy with a-chymotrypsin was identified as Thr343-Ser344 by direct aminoacid sequence analysis of cleaved and purified protein.

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Isolation, characterization, and recombinant expression of multiple serpins from the cat flea, Ctenocephalides felis

Brandt

Silver

et al. 2004

Arch Insect Biochem Physiol

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Several clones encoding serine protease inhibitors were isolated from larval and adult flea cDNA expression libraries by immunoscreening and PCR amplification. Each cDNA contained an open reading frame encoding a protein of approximately 45 kDa, which had significant sequence similarity with the serpin family of serine protease inhibitors. The thirteen cDNA clones isolated to date encode serpin proteins, which share a primary structure that includes a nearly identical constant region of about 360 amino acids, followed by a C-terminal variable region of about 40-60 amino acids. The variable C-terminal sequences encode most of the reactive site loop (RSL) and are generated by mutually exclusive alternative exon splicing, which may confer unique protease selectivity to each serpin. Utilization of an alternative exon splicing mechanism has been verified by sequence analysis of a flea serpin genomic clone and adjacent genomic sequences. RNA expression patterns of the cloned genes have been examined by Northern blot analysis using variable region-specific probes. Several putative serpins have been overexpressed using the cDNA clones in Escherichia coli and baculovirus expression systems. Two purified baculovirus-expressed recombinant proteins have N-terminal amino acid sequences identical to the respective purified native mature flea serpins indicating that appropriate N-terminal processing occurred in the virus-infected insect cells.

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Patchwork‐structure serpins from silkworm (Bombyx mori) larval hemolymph

Cited by 51 publications

References 31 publications

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

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

Molecular cloning of silkworm (Bombyx mori) antichymotrypsin

Isolation, characterization, and recombinant expression of multiple serpins from the cat flea, Ctenocephalides felis

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