Abstract
A cDNA clone isolated from a fat body cDNA library from an insect, Manduca sexta, has been sequenced and shown to code for a member of the serpin family of proteinase inhibitors. The cDNA has an open reading frame which codes for a 392-residue polypeptide of Mr = 43,500 with a hydrophobic NH2-terminal sequence which appears to be a signal peptide. An alignment of this amino acid sequence with 11 members of the serpin superfamily reveals that the insect protein is 25-30% identical with most members of the superfamily. The alignment was used to construct an evolutionary tree of the serpin sequences analyzed, which indicates that the progenitor of the M. sexta serpin and the human serpins most closely related to it diverged from other serpin genes prior to the divergence of the vertebrates and invertebrates. The M. sexta serpin is predicted to inhibit elastase due to the presence of alanine at the P1 position of its reactive center and is classified as an alaserpin. A glycoprotein of Mr = 47,000 isolated from hemolymph of M. sexta larvae has an NH2-terminal sequence identical to that deduced from the alaserpin cDNA clone and inhibits porcine pancreatic elastase and bovine chymotrypsin.
Highlights
A cDNA clone isolated fromfaat body cDNAlibrary tebrates
The alanine at the PI position of its reactive center and is amino acid compositions of the silkwormproteinase inhibitors classified as analaserpin
The sequences of the first 18NH2-terminalamino has an NHz-terminal sequence identical to that dedaucicdesdof the two silkworm proteinase inhibitors have been from the alaserpin cDNA clone and inhibits porcine determined but reveal no similarity to the human serpins, pancreatic elastase and bovine chymotrypsin
Summary
Isolation of a Fat Body cDNA Clone and Hybrid Select Translation-Upon screening an M . sextu fat bodycDNA library with antiserumthought to be specific for apolipophorin-I1 (Shapiro et al, 1984),we isolated two clones which hybridized with a fatbody RNAof 1470nucleotides, too small to encode apolipophorin-I1(M,= 78,000). The 12 serpin sequences (Table I) were used to construct an 3’ evolutionary tree (Fig. 5) This treeyields a branching order similar to those proposed by Carrell and Boswell (1986) and. Four gene duplications occurred within a fairly short time, giving rise to: the cowpox virus serpin; the progenitor of al-antitrypsin, al-antichymotrypsin, and protein C inhibitor;the progenitor of antithrombin-111, M. sexta sites (CCA/GCC) (Kozak, 1984), including the highly conserved purine a t position -3. The NH2-terminaslequence of the polypeptide is quite hydrophobic and likely encodes a signal peptide, as serpin, plasminogen activatoirnhibitor, and glia-derived nexin; and the progenitor of ovalbumin and gene-Y These gene duplications must have occurred before the divergence of the vertebrates and invertebrates approximately 500 million years ago, twice as long ago as proposed by Bao et al. Et al, 1985).The significance of these similarities was evalu- Purification of a Hemolymph Elastase Inhibitor-Guided by
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