Cloning and characterization of a putative inhibitor of melanization from Anopheles gambiae

Abstract

Phenoloxidases, including tyrosinases and laccases, are enzymes involved in the synthesis of melanin, a process that can be elicited during insect immune responses, cuticle maturation, wound healing and egg chorion development. We cloned a putative inhibitor of melanization (POI) from Anopheles gambiae on the basis of homology with a functionally characterized peptide from Musca domestica (Daquinag et al ., Proc Natl Acad Sci USA 1995; 92: 2964 -2968). The 335 amino acid protein predicted from the A. gambiae cDNA consists of five tandemly arranged inhibitor motifs. The A. gambiae POI gene was expressed in all mosquito stages from egg to adult. POI transcript levels were high in the fat body and were measurable but comparatively reduced in the midgut. The POI transcript level increased after wounding or Sephadex bead injection. Gene knockdown did not result in faster or more extensive bead melanization but did result in more extensive melanization of wound sites following a thoracic bead injection.

Regulation of phenoloxidase activity by high- and low-molecular-weight inhibitors from the larval hemolymph of Manduca sexta

“…This is consistent with the finding that POI knockdown did not lead to a faster or more extensive melanization of Sephadex beads injected to the mosquitoes (Shi et al, 2006). To test if a posttranslational modification (e.g.…”

Regulation and function of the melanization reaction in Drosophila

“…46 POI homologues have been found in M. sexta, where its PO inhibitory activity was demonstrated by in vitro assays, 47 and in the mosquito Anopheles gambiae, where melanization is compromised when POI is knocked down by RNAi. 48 In T. molitor, a novel 43-kD melanization-inhibiting protein (MIP) was found to specifically inhibit melanin synthesis. The proposed function of MIP is to negatively regulate PO activation and thus to prevent possible tissue damage.…”

A Novel Protein Acts as a Negative Regulator of Prophenoloxidase Activation and Melanization in the Freshwater Crayfish Pacifastacus leniusculus

“…Although Tm MIP so far does not show any significant similarity with any known protein, Pl MIP in contrast contains a FReD, most similar to the recognition sites of vertebrate ficolins (14). Lack of the collagenous domain and the molecular size (43 kDa) of Pl MIP is similar to that of TL5s in horseshoe crabs (21), but Pl MIP did not show any hemagglutinating activity and is not likely to have lectin activity as the TL5s.…”

“…Other proteins affecting melanization are known as proteinase inhibitors that will inhibit one or several of the proteinases that are components of the proPO system, and therefore they will inhibit proPO activation. Among these latter inhibitors is a large molecular weight inhibitor, pacifastin, from P. leniusculus acting as a potent inhibitor of the proPO-activating protease, and Drosophila Spn27, a small serpin supposed to inhibit the proPO-activating enzyme (14,26). In contrast, Pl MIP and Tm MIP do not affect PO activity in itself but instead interfere with the melanization reaction from quinone compounds to melanin.…”

“…In addition to inhibitors of the proPO system, activation of a number of endogenous factors acting as competitive inhibitors of PO activity has been described (5). For example the so-called phenoloxidase inhibitors have been found in Musca domestica, and homologous basic lysine-rich peptides were also detected in mosquitoes and M. sexta (14,15).…”