Molecular identification of a bevy of serine proteinases in Manduca sexta hemolymph

Abstract

Extracellular serine proteinase pathways control immune and homeostatic processes in insects. Our current knowledge of their components is limited-prophenoloxidase-activating proteinases (PAPs) are among the few hemolymph proteinases (HPs) with known functions. To identify components of proteinase systems in the hemolymph of Manduca sexta, we amplified cDNAs from larval fat body or hemocytes using degenerate primers coding for two conserved regions in S1 family serine proteinases. PCR yielded fragments encoding seven known (HP1-HP4, PAP-1, PAP-2 and PAP-3) and 18 unknown (HP5-HP22) serine proteinases. We screened cDNA libraries and isolated clones for 17 of the newly discovered HPs (HP5-HP22 except for HP11) and prepared antibodies to 14 recombinant proteins (HP6, HP8-HP10, HP12, HP14-HP19, HP21 and HP22). Fourteen of the HPs contain regulatory clip domain(s) at their amino-terminus--HP1, HP2, HP6, HP8, HP13, HP17, HP18, HP21, HP22 and PAP-1 have one, whereas HP12, HP15, PAP-2 and PAP-3 have two clip domains. Multiple sequence alignment of catalytic domains in these and other arthropod serine proteinases provided useful clues for future functional analysis. Northern blot and reverse transcription PCR (RT-PCR) analyses showed increases in HP2, HP7, HP9, HP10, HP12-HP22 mRNA levels at 24h after a bacterial challenge, and immunoblot analysis confirmed elevated concentrations of HP12, HP14-HP19, HP21 and HP22 proteins in plasma in response to injected bacteria. Hemocytes express HP13 and HP18; fat body produces HP12, HP20-HP22; both tissues synthesize the other HPs. These results collectively indicate the existence of a complex serine proteinase network in M. sexta hemolymph, predicted to mediate rapid defense responses upon wounding and/or microbial infection.

Functions of Manduca sexta Hemolymph Proteinases HP6 and HP8 in Two Innate Immune Pathways

“…1A) (15). They are each most similar to clip domain proteinases known to function in activation of the Toll pathway (Fig.…”

“…HP6 may be an ortholog of Drosophila Persephone (35% identity), whereas HP8 has the highest identity to a group of proteinases whose substrate is the cytokine precursor prospätzle, including Drosophila Easter (42%) (42), SPE from Drosophila (39%) (18), and Tenebrio (45%) (11) and a predicted SPE named BAEEase from the silkworm, B. mori (70%) (18). The predicted proteolytic activation sites (2) are at 109 LDLH2ILGG in proHP6 and 111 NNDR2IVGG in proHP8 (15) (Fig. 1).…”

“…The sizes and positions of the molecular weight markers are indicated on the right. the carboxyl-terminal catalytic chain (our antisera to HP6 and HP8 do not recognize the light chain containing the clip domain (15)). The naturally occurring proHP6 zymogen band was detected in plasma, and this band disappeared after plasma was treated with bacteria or curdlan (Fig.…”

“…To investigate proPO activation and other immune pathways in M. sexta, we have isolated more than 20 serine proteinase cDNAs from fat body and hemocytes (15). Only a handful of these hemolymph proteinases have been studied functionally.…”

“…Recent research also has led to better characterization of the proPO activation pathway in Manduca sexta (7,15,16) and the Tollsignaling pathway in the Drosophila immune response (17, 18) and to both the proPO and Toll pathways in the beetle Tenebrio molitor (11,19).…”