Prophenoloxidase-activating Proteinase-2 from Hemolymph ofManduca sexta

Abstract

Proteolytic activation of prophenoloxidase in insects is a component of the host defense system against invad- ing pathogens and parasites. We have purified from he- molymph of the tobacco hornworm, Manduca sexta,a new serine proteinase that cleaves prophenoloxidase. This enzyme, designated prophenoloxidase-activating proteinase-2 (PAP-2), differs from another PAP, previ- ously isolated from integuments of the same insect (PAP-1). PAP-2 contains two clip domains at its amino terminus and a catalytic domain at its carboxyl termi- nus, whereas PAP-1 has only one clip domain. Purified PAP-2 cleaved prophenoloxidase at Arg51 but yielded a product that has little phenoloxidase activity. However, in the presence of two serine proteinase homologs, ac- tive phenoloxidase was generated at a much higher level, and it formed covalently linked, high molecular weight oligomers. The serine proteinase homologs asso- ciate with a bacteria-binding lectin in M. sexta hemo- lymph, indicating that they may be important for ensur- ing that the activation of prophenoloxidase occurs only in the vicinity of invading microorganisms. PAP-2 mRNA was not detected in naive larval fat body or he- mocytes, but it became abundant in these tissues after the insects were injected with bacteria.