Recombinant expression and analysis of hemolymph proteinase 16 from an insect, Manduca sexta

 

 

Hemolymph of the caterpillar, Manduca sexta, contains at least twenty-five serine proteinases, some of which function in aspects of innate immune responses. These hemolymph proteinases are synthesized as zymogens and are activated by specific proteolytic cleavage. Hemolymph proteinase 16 (HP16) is 47 kDa protein with predicted chymotrypsin/elastase-like specificity. HP16 lacks a clip domain, but contains an amino terminal 170 residue region that has no significant match to any sequence in current databases. The catalytic domain of HP16 is most similar to Drosophila gastrulation defective. The goal of this project was to investigate the proteolytic activity and biological function of HP16. Immunoaffinity chromatography was used to isolate a serpin-proteinase complex from the hemolymph of fifth instar larvae that were immune challenged by injection of Micrococcus luteus. Immunoblot analysis revealed that serpin-1 and serpin-3 form a complex with HP16, and MALDI-TOF analysis confirmed the presence of serpin-3. Using a baculovirus-based expression system, we expressed and purified recombinant HP16 and a HP16 mutant in which the activation site can be cleaved by bovine factor Xa. We are using the recombinant proteins to determine target proteins of HP16 during an immune response, the ability of HP16 to induce the synthesis of antimicrobial peptides, the role of HP16 in the phenoloxidase pathway, and potential interactions between HP16 and microbial elicitors. Supported by NIH grant GM41247.