A novel 43-kDa protein as a negative regulatory component of phenoloxidase-induced melanin synthesis.

The melanization reaction induced by activated phenoloxidase in arthropods is important in the multiple host defense innate immune reactions, leading to the sequestration and killing of invading microorganisms. This reaction ought to be tightly controlled because excessive formation of quinones and systemic hypermelanization are deleterious to the hosts, suggesting that a negative regulator(s) of melanin synthesis may exist in hemolymph. Here, we report the purification and cloning of a cDNA of a novel 43-kDa protein, from the meal-worm Tenebrio molitor, which functions as a melanization-inhibiting protein (MIP). The deduced amino acid sequence of 352 residues has no homology to known sequences in protein data bases. When the concentration of the 43-kDa protein was examined by Western blot analysis in a melanin-induced hemolymph prepared by injection of Candida albicans into T. molitor larvae, the 43-kDa protein specifically decreased in the melanin-induced hemolymph compared with control hemolymph. Recombinant MIP expressed in a baculovirus system had an inhibitory effect on melanin synthesis in vitro. RNA interference using a synthetic 445-mer double-stranded RNA of MIP injected into Tenebrio larvae showed that melanin synthesis was markedly induced. These results suggest that this 43-kDa MIP inhibits the formation of melanin and thus is a modulator of the melanization reaction to prevent the insect from excessive melanin synthesis in places where it should be inappropriate.

Human mannose-binding lectin and L-ficolin function as specific pattern recognition proteins in the lectin activation pathway of complement.

The innate immune response in vertebrates and invertebrates requires the presence of pattern recognition receptors or proteins that recognize microbial cell components including lipopolysaccharide, bacterial peptidoglycan (PGN), and fungal 1,3-beta-D-glucan. We reported previously that PGN and 1,3-beta-D-glucan recognition proteins from insect hemolymph were able to induce the activation of the prophenoloxidase-activating system, one of the major invertebrate innate immune reactions. The goal of this study was to characterize the biochemical properties and effects of the human counterparts of these molecules. Soluble pattern recognition proteins were purified from human serum and identified as human mannose-binding lectin (MBL) and L-ficolin. The use of specific microbial cell component-coupled columns demonstrated that MBL and L-ficolin bind to PGN and 1,3-beta-D-glucan, respectively. Purified MBL and L-ficolin were associated with MBL-associated serine proteases-1 and -2 (MASPs) and small MBL-associated protein as determined by Western blot analysis. Finally, the binding of purified MBL/MASP and L-ficolin/MASP complexes to PGN and 1,3-beta-D-glucan, respectively, resulted in the activation of the lectin-complement pathway. These results indicate that human PGN and 1,3-beta-D-glucan recognition proteins function as complement-activating lectins.

Characterization and properties of a 1,3-beta-D-glucan pattern recognition protein of Tenebrio molitor larvae that is specifically degraded by serine protease during prophenoloxidase activation.

Although many different pattern recognition receptors recognizing peptidoglycan and 1,3-beta-D-glucan have been identified in vertebrates and insects, the molecular mechanism of these molecules in the pattern recognition and subsequent signaling is largely unknown. To gain insights into the action mechanism of 1,3-beta-D-glucan pattern recognition protein in the insect prophenoloxidase (proPO) activation system, we purified a 53-kDa 1,3-beta-D-glucan recognition protein (Tm-GRP) to homogeneity from the hemolymph of the mealworm, Tenebrio molitor, by using a 1,3-beta-d-glucan affinity column. The purified protein specifically bound to 1,3-beta-D-glucan but not to peptidoglycan. Subsequent molecular cloning revealed that Tm-GRP contains a region with close sequence similarity to bacterial glucanases. Strikingly, two catalytically important residues in glucanases are replaced with other nonhomologous amino acids in Tm-GRP. The finding suggests that Tm-GRP has evolved from an ancestral gene of glucanases but retained only the ability to recognize 1,3-beta-D-glucan. A Western blot analysis of the protein level of endogenous Tm-GRP showed that the protein was specifically degraded following the activation of proPO with 1,3-beta-D-glucan and calcium ion. The degradation was significantly retarded by the addition of serine protease inhibitors but not by cysteine or acidic protease inhibitor. These results suggest that 1,3-beta-D-glucan pattern recognition protein is specifically degraded by serine protease(s) during proPO activation, and we propose that this degradation is an important regulatory mechanism of the activation of the proPO system.