Purification, CDNA Cloning And Functional Characterization Of MAP-5, A Kind Of Melanization-associated Protein In Tenebrio Molitor

The pro-PO (prophenoloxidase) system is an important non-self recognition system in the insect innate immunity. Like the vertebrate complement system, it is a proteolytic cascade composed of pattern recognition proteins (PRPs), several serine proteases, their inhibitors and the terminal zymogen, pro-PO. The recognition of PRPs with microbial polysaccharides including LPS, PGN andβ-1, 3-glucan triggers the activation of serine proteases, which results in the transfer of pro-PO to the active form, PO. The melanization reaction induced by the activated PO leads to the sequestration and killing of invading microorganisms. It is also found to play a role in wound healing and blood coagulation of insects. Recently, our knowledge of pro-PO activation mechanism has increased dramatically. However, little is known about the controlling and regulatory mechanism of the ultimate step, PO-mediated melanin synthesis.To identify the proteins possibly associated with melanization, we firstly induced melanin synthesis of the hemolymph harvested from Tenebrio molitor larvae byβ-1,3-glucan and Ca²⁺ in vitro and analyzed the protein changes in the reaction by SDS-PAGE. Six protein bands clearly disappeared following the melanin synthesis in a time-dependent manner, and PTU, a kind of PO inhibitor, could prevent the disappearance of those bands, indicating those proteins might be involved in PO-induced melanin synthesis. We named them as melanization-associated proteins (MAPs)-1～6. The amino-terminal sequences were determined to further characterize these six bands. Both MAP-1 and 2 were T.molitor vitellogenin-like protein that was reported as a melanization-enhancing protein. The amino-terminal sequences of MAP-3 were blocked. MAP-4 was a novel protein whose sequences showed high homology to those of silkworm imaginal disc growth factor-like protein or human chitinase 3-like protein. MAP-5 was also a novel protein whose sequences showed no homology to those of known proteins in the database. The sequences of MAP-6 perfectly matched with the T.molitor desiccation protein whose function in the melanin synthesis is unknown. Among MAPs, we firstly purified denatured MAP-5 to homogeneity by electroelution. Four internal amino acid sequences of MAP-5 were determined. Based on them, a degenerate oligonucleotide probe was synthesized and used to screen a cDNA library of T. molitor larvae. The open reading frame of the obtained positive clone includes 1056 nucleotide bases and encodes MAP-5 consisting of 352 amino acid residues. Three potential attachment sites for the N-linked carbohydrate chains are found in the amino acid sequences of MAP-5. The deduced amino acid sequence of MAP-5 shows no significant homology to other proteins reported so far, but it contains an Asp-rich region, including 11 contiguous Asp residues in the central part of MAP-5, which shows high degree of homology to several unrelated proteins with different functions,To further investigate the potential function of MAP-5 in the melanin synthesis, we purified the native MAP-5 from T. molitor hemolymph by gel filtration and anion-exchange chromatography. Recombinant MAP-5 was constructed and expressed in Bac-to-Bac Baculovirus expression system and purified by Ni-NTA affinity resin and anion-exchange chromatography. Antibodies against MAP-5 were raised with the injection of denatured MAP-5 to an albino rabbit and affinity-purified by protein A sepharose CL-4B column.The activation of pro-PO cascade and PO-mediated melanization ought to be localized, target-specific and tightly controlled, since excessive formation of quinones and systematic hyper-melanization are deleterious to the hosts, suggesting that the endogenous negative regulators of the melanin synthesis may exist in hemolymph. In our study, both purified native MAP-5 and recombinant MAP-5 showed inhibitory effects on the melanin synthesis in a dose-dependent manner in vitro. RNA interference experiment also showed the melanin synthesis of hemolymph from T. molitor larvae injected with a synthetic 445mer double stranded-RNA of MAP-5 was markedly induced. All of these results support that MAP-5 functions as a negative modulator of the melanin synthesis. MAP-5 showed no effects on the serine protease activity and PO activity in the pro-PO system, which suggests the target step inhibited by MAP-5 in the activation of pro-PO cascade may be the ultimate step, PO-mediated melanin synthesis. Therefore, we named it melanization-inhibitory protein (MIP). MAP-5 existed in a complex with several hemolymph proteins including vitellogenin-like protein, the truncated PO fragment, MAP-3 and a 35kDa novel protein with homology to actin related protein 2/3 complex subunit 1A, suggesting the co-reaction of MAP-5 with the proteins in the complex might be the potential mechanism of it's inhibitory effects. The MAP-5 was detected to exist in the plasma of T. molitor larvae by western blot analysis.This is the first report of a novel 43 kDa protein exhibiting inhibitory effects on the down-stream part of the pro-PO system, PO-mediated melanin synthesis. The ongoing and further investigation of the mechanism underlying MAP-5 inhibitory effects and the relationship among MAPs will eventually offer clues how the pro-PO cascade and melanogenesis function as an important innate immune reaction in arthropods.