Identification of immune-related genes from the tobacco hornworm, Manduca sexta, and characterization of two immune-inducible proteins, serpin-3 and leureptin

Insect immune processes are mediated by programs of differential gene expression. To understand the molecular regulation of the immune response in the tobacco hornworm, Manduca sexta, the relevant subset of differentially expressed genes of interest must be identified, cloned and studied in detail. In this study, I performed suppression subtractive hybridization (SSH), a PCR-based method for cDNA subtraction, to identify mRNAs from fat body of immunized larvae that are not present (or present at a low level) in control larvae. A subtracted cDNA library enriched in immune-inducible genes was constructed. Sequence analysis of 238 expressed sequence tags (ESTs) revealed that 120 ESTs, representing 54 distinct genes or gene families, had sequences identical or similar to previously characterized genes. 112 ESTs showed no significant similarity to any known genes. I carried out further studies on two of the ESTs by using them as probes to obtain full length cDNAs. One of these belonged to serpin superfamily and was named serpin-3. The other clone contained leucine-rich repeat motifs and was named leureptin.; Serpin-3 was expressed in fat body and secreted into hemolymph. The expression level of serpin-3 mRNA and protein was increased after immune challenge. Serpin-3 blocked the activation of prophenoloxidase by specifically inhibiting two M. sexta prophenoloxidase activating proteinases, PAP-1 and PAP-3. Both in vitro and in vivo experiments showed that serpin-3 formed stable complexes with activated PAP-1 and PAP-3. The association rate constants for inhibition of PAP-1 and PAP-3 by serpin-3, together with serpin-3 concentration in hemolymph, indicated that serpin-3 could be an efficient regulator of PAP activity under physiological conditions.; Leureptin contained twelve leucine-rich repeats. Circular dichroism spectrum suggested that leureptin assumed a three-dimensional structure predominated by beta-structure. Leureptin mRNA level was induced after immune challenge, while protein level in hemolymph decreased. Four isoforms with identical amino-terminal sequences were purified from hemolymph. Leureptin was able to bind to bacterial lipopolysaccharide and associate with hemocyte membranes. Leureptin may function as a recognition molecule for detection and response to bacterial infection in M. sexta.