Insect Biochem. Vol. 20, No. 2, pp. 141-147, 1990 0020-1790/90$3.00 + 0.00 Printed in Great Britain. All rights reserved Copyright© 1990PergamonPress pie ISOLATION AND CHARACTERIZATION OF FOUR SERINE PROTEINASE INHIBITORS (SERPINS) FROM HEMOLYMPH OF MANDUCA SEXTA MICHAELR. KANOST Department of Biochemistry, Bioscienees West, University of Arizona, Tucson, AZ 85721, U.S.A. (Received 5 May 1989; revised and accepted 18 September 1989) Al~tract--Four serine proteinase inhibitors have been isolated from hemolymph of fifth instar larvae of Manduca sexta. One of these, an inhibitor specific for elastase, has been previously shown to be a member of the serpin family of serine proteinase inhibitors. Of the three remaining inhibitors, two are specific for chymotrypsin and one for trypsin. The four inhibitors have molecular weights of approx. 47,000 and isoelectric points between 4.4 and 4.8. The four proteins have very similar amino acid compositions, and NH2-terminal sequence analysis suggests that they represent members of a gene family. Key Word lndex: serine proteinase inhibitor, serpin, hemolymph proteins, Manduca sexta, tobacco hornworm INTRODUCTION The serpin (serine proteinase inhibitor) gene super- family (Carrdi-and Travis, 198~-Carreil and Boswell, 1986) is made up of proteins of 35-100 kDa with related amino acid sequences but diverse functions. The range in size of the proteins results from un- related NH2-terminal sequences of different lengths in some serpins as well as from varying degrees of glycosylation. Ten serpin proteins have now been identified in human serum. Most serpins so far discovered regulate endogenous serine proteinases. These include proteinase inhibitors which regulate blood clotting (antithrombin, antiplasmin, plasmino- gen activator inhibitor, protein C inhibitor, heparin cofaetor II), complement activation (C1 inhibitor) and proteinases released from neutrophils (~ l-anti- trypsin, ct 1-antichymotrypsin) (Boswell and Carrell, 1988). Other serpins have no detectable proteinase inhibitor activity and function as hormone carriers (thyroxine binding globulin, corticosteroid binding globulin) (Flink et al., 1986; Hammond et aL, 1987), and one serpin serves as a hormone precursor (angio- tensinogen) (Doolittle, 1983). Serpins also appear to have roles in reproduction (chicken ovalbumin, sheep uterus milk proteins) (Hunt and Dayhoff, 1980; Ing and Roberts, 1989) and development of the nervous system (glia derived nexin) (Monard, 1988). Although serine proteinase inhibitors are also known to be present in insects, their properties and functions are not as well understood. Several low molecular weight (7-10 kDa) serine proteinase inhibitors have been isolated from insect hemolymph (Sasaki, 1978; Kang and Fuchs, 1980; Ramesh et al., 1988), and inhibitors from Bombyx mori and Mand- uca sexta have been sequenced and found to be homologous with vertebrate Kunitz type inhibitors (Sasaki, 1984; Ramesh et al., 1988). Higher molecular weight inhibitors (40-50 kDa) have also been isolated from hemolymph of B. mori and M. sexta. A trypsin inhibitor (42kDa) and a chymotrypsin inhibitor (43 kDa) from B. mori (Sasaki and Kobayashi, 1984; Eguchi and Shomoto, 1985) have amino acid compositions similar to that of human ct l-antitrypsin and, like the vertebrate serpins, inhibit serine proteinases by forming stable complexes with the enzymes after cleavage of a reactive site near the COOH-terminal of the inhibitor (Sasaki, 1985; Sasaki et al., 1987). We have recently isolated a serpin clone from a M. sexta fat body eDNA library (Kanost et al., 1989). From its deduced amino acid sequence, it was predicted that the protein would inhibit elastase due to the presence of alanine at the Pj position of the reactive site. The protein corresponding to the eDNA, an alaserpin according to the convention of naming serpins based on their Pl residue, was subse- quently isolated from hemolymph of M. sexta and shown to inhibit elastase and chymotrypsin (Kanost et al., 1989). Manduca sexta alaserpin is a 47 kDa glycoprotein with 25-30% amino acid sequence identity with most members of the serpin superfamily. Its highest similarity (31.7% identical residues) is with human plasminogen activator inhibitor. During the course of isolating the alaserpin, we observed that several other less abundant proteinase inhibitors are present in M. sexta hemolymph with properties similar to alaserpin but with different proteinase specificities. I report here the isolation and characterization of two chymotrypsin inhibitors and one trypsin inhibitor that also appear to be members of the set-pin family of proteinase inhibitors. MATERIALSAND METHODS Insects M. sexta eggs were supplied by Dr J. Buckner, United States Department of Agriculture, Fargo, N.D. Larvae were reared as described by Prasad et al. (1986). 141