A Pattern Recognition Serine Proteinase Triggers the Prophenoloxidase Activation Cascade in the Tobacco Hornworm, Manduca sexta

Abstract

A serine proteinase cascade in insect hemolymph mediates prophenoloxidase activation, a defense mechanism against pathogen or parasite infection. Little is known regarding its initiating proteinase or how this enzyme is activated in response to invading microorganisms. We have isolated from the tobacco hornworm, Manduca sexta, a cDNA encoding a modular protein designated hemolymph proteinase 14 (HP14). It contains five low density lipoprotein receptor class A repeats, a Sushi domain, a unique Cys-rich region, and a proteinase-catalytic domain. The HP14 mRNA exists in fat body and hemocytes of the naive larvae, and its level increases significantly at 24 h after a bacterial challenge. We expressed proHP14 with a carboxyl-terminal hexahistidine tag in a baculovirus/insect cell system and detected the recombinant protein in two forms. The 87-kDa protein was primarily intracellular, whereas the 75-kDa form was present in the medium. Interaction with peptidoglycan resulted in proteolytic processing of the purified zymogen and generation of an amidase activity. Supplementation of hemolymph with proHP14 greatly enhanced prophenoloxidase activation in response to Micrococcus luteus. These data suggest that proHP14 is a pattern recognition protein that binds to bacteria and autoactivates and triggers the prophenoloxidase activation system in the hemolymph of M. sexta.Similar to other invertebrates, insects lack an adaptive immune system and rely solely on their innate immune mechanisms to fight against invading microorganisms (1-3). These defense mechanisms are mediated by hemocytes (e.g. phagocytosis and encapsulation), fat body (e.g. induced synthesis of antimicrobial peptides), and plasma factors (e.g. hemolymph coagulation and melanization). Accumulating evidence indicates that a complex serine proteinase network in insect hemolymph coordinates some of these responses (4). The prophenoloxidase (proPO) 1 activation cascade is probably composed of several serine proteinases that are sequentially activated in response to microbial infection and lead to the proteolytic activation of proPO to phenoloxidase (PO). PO catalyzes the formation of quinones that are reactive intermediates for melanin synthesis (5, 6). Quinones are also involved in cuticle sclerotization, wound healing, and sequestration of parasites or pathogens.Although the physiological importance of the proPO activation in insect immune system has been appreciated for many years, the molecular characterization of the cascade components was reported only recently. We isolated three proPOactivating proteinases from cuticular extract or hemolymph of M. sexta prepupae, and they all required a protein cofactor for proPO activation (7-10). Similar results were obtained from the beetles Holotrichia diomphalia and Tenebrio molitor (11-13). In contrast, the silkworm proPO-activating enzyme does not appear to need any auxiliary factor for proPO activation (14). Limited by substrate availability, current biochemical research is focused on the components at the end of th...

Reconstitution of a branch of the Manduca sexta prophenoloxidase activation cascade in vitro: Snake-like hemolymph proteinase 21 (HP21) cleaved by HP14 activates prophenoloxidase-activating proteinase-2 precursor

“…Based on these data, we concluded that: 1) the amino-terminal light chain of HP21 (Ala 1 to Leu 152 ) began at the predicted starting site of mature proHP21 (Fig. 1); 2) HP14, which cut itself immediately after Leu 387 and hydrolyzed Ala-AlaPro-Leu-p-nitroanilide (Ji et al, 2004), cleaved proHP21 at the predicted activation site between Leu 152 and Ile 153 .…”

“…We reported the molecular cloning and functional analysis of M. sexta HP14, a modular serine proteinase containing seven disulfide knotted structures and one catalytic domain (Ji et al, 2004). The recombinant HP14 precursor binds to peptidoglycan, autoactivates, and triggers the proPO activation cascade.…”

Manduca sexta Hemolymph Proteinase 21 Activates Prophenoloxidase-activating Proteinase 3 in an Insect Innate Immune Response Proteinase Cascade

“…Upon binding to these microbial polysaccharides, HP14 undergoes autoactivation, presumably via an initial conformational change that enables one molecule of HP14 to cleave a second molecule at its activation site (GTEL*VLGG). Key residues in the putative substrate binding pocket of HP14 (Gly 585 , Ala 616 , and Thr 630 ) are consistent with cleavage after a bulky, hydrophobic residue such as leucine (3). The cleaved form of HP14 can initiate activation of proPO in hemolymph (3,4).…”

“…Until now, only proteinases in the first and final positions of the melanization pathways have been conclusively identified. Just one initiating proteinase has been discovered: Manduca sexta hemolymph proteinase 14 (HP14), which autoactivates in the presence of microbial surface components (3,4). In contrast, several proPO activativing proteinases (PAPs, also named PPAF and PPAE) have been identified through biochemical studies of M. sexta, Holotrichia diomphalia, and Bombyx mori (5)(6)(7)(8)(9).…”

“…HP14 is a complex, multidomain serine proteinase that associates with peptidoglycan or ␤-1,3-glucan found on the surface of bacteria and fungi, respectively (3,4). At least in some cases, the association is mediated by a pathogen recognition protein; for example, HP14 combines with a ␤-1,3-glucan recognition protein (␤GRP2) to bind to ␤-1,3-glucan (4).…”