C-Terminal Domain of Hemocyanin, a Major Antimicrobial Protein from Litopenaeus vannamei: Structural Homology with Immunoglobulins and Molecular Diversity.

Invertebrates rely heavily on immune-like molecules with highly diversified variability so as to counteract infections. However, the mechanisms and the relationship between this variability and functionalities are not well understood. Here, we showed that the C-terminal domain of hemocyanin (HMC) from shrimp Litopenaeus vannamei contained an evolutionary conserved domain with highly variable genetic sequence, which is structurally homologous to immunoglobulin (Ig). This domain is responsible for recognizing and binding to bacteria or red blood cells, initiating agglutination and hemolysis. Furthermore, when HMC is separated into three fractions using anti-human IgM, IgG, or IgA, the subpopulation, which reacted with anti-human IgM (HMC-M), showed the most significant antimicrobial activity. The high potency of HMC-M is a consequence of glycosylation, as it contains high abundance of α-d-mannose relative to α-d-glucose and N-acetyl-d-galactosamine. Thus, the removal of these glycans abolished the antimicrobial activity of HMC-M. Our results present a comprehensive investigation of the role of HMC in fighting against infections through genetic variability and epigenetic modification.

Characterization of a novel hemolytic activity of human IgG fractions arising from diversity in protein and oligosaccharide components.

Human IgG is a well-established multifunctional antigen specific immunoglobulin molecule of the adaptive immune system. However, an antigen nonspecific immunological function of human IgG has never been reported. In this study, human IgG was isolated using ammonium sulfate fractional precipitation and diethylaminoethanol (DEAE) cellulose 52 ion exchange chromatography, from which h-IgG and hs-IgG fractions were purified on the basis of their differential binding to rabbit anti-shrimp hemocyanin antibody (h) and rabbit anti-shrimp hemocyanin's small subunit antibody (hs), respectively. We found that h-IgG had a higher hemolytic activity than hs-IgG against erythrocytes from humans, rabbits, mice and chickens, whereas the control IgG showed negligible activity. h-IgG could interact directly with erythrocyte membranes, and this interaction was suppressed by high molecular weight osmoprotectants, showing that it may follow a colloid-osmotic mechanism. In comparative proteomics and glycomics studies, h-IgG and hs-IgG yielded 20 and 5 significantly altered protein spots, respectively, on a 2-D gel. The mean carbohydrate content of h-IgG and hs-IgG was approximately 3.6- and 2-fold higher than that of IgG, respectively, and the α-d-mannose/α-d-glucose content was in the order of h-IgG>hs-IgG>IgG. In this study, a novel antigen nonspecific immune property of human IgG was investigated, and the diversity in the protein constituents and glycosylation levels may have functional signficance.

Hemocyanin from shrimp Litopenaeus vannamei shows hemolytic activity.

Hemocyanin is an extracellular copper-containing protein present in the hemolymph of both mollusks and arthropods. The traditionally recognized function of hemocyanin is for oxygen transport. Lately, it was demonstrated that hemocyanin is a multifunctional protein, especially participating in multiple roles of immune defense. For better understanding its actions in immune defense, the hemolytic activity of hemocyanin from shrimp Litopenaeus vannamei and the mechanism were investigated in this study. The results showed that shrimp hemocyanin exhibited hemolytic activity against vertebrate erythrocytes. The hemolysis displayed dependencies on hemocyanin concentration, pH, temperature and divalent cations. The highest activity occurred at a concentration of 0.125 mg ml(-1), and pH 6.0, 40 degrees C in the presence of calcium. Moreover, from the incubation products of erythrocytes with hemocyanin, besides two subunits of hemocyanin, two molecules around 150 and 230 kD were isolated and speculated as oligomers of hemocyanin. Further evidence revealed that the hemolysis could be inhibited to different degrees by osmoprotectants with high molecular masses, suggesting that it follows a colloid-osmotic mechanism. These results indicate that L. vannamei hemocyanin has a novel function with hemolytic activity, partly related to a colloid-osmotic mechanism mediated by its oligomers.