Investigations On The Immunological Functions And Their Mechanisms Of Hemocyanin From Crustacean

At present, crustaceans production of China is the largest in the world. However, the whole benefit of commercial crustaceans cultivation in our country is lower compared with the international shrimps and crabs farming, and the outlook of sustainable development is not optimistic. For the reasons related with disease and bio-security, most researchers consider that the effective strategies to prevent crustacean diseases and improve crustacean quality are breeding for stress tolerance species and establishing safe, ecotypic, protecting environmental and healthy culture model. It has been proved that one of the key to successfully resolve these problems is to reveal the self-defense mechanism of crustacean and search for the effective strategy to prevent crustacean diseases. Recently, hemocyanin of mollusks and arthropods was reported as an important non-specific immune protein present in the hemolymph with multifunction such as antiviral and antibacterial activities. In this paper, an attempt was made to investigation the immunological functions and their mechanisms of hemocyanins from Litopenaeus vannamei and Scylla serrata. The main findings are as follows:1. Hemocyanin from L. vannamei showing antibacterial activity was related to its glycosylation and OmpC was appeared to be one of the antibacterial targetFirstly, on the basis of our previous findings that two L. vannamei hemocyanins (named LHt and LH75) presenting different carbohydrate content and antibacterial activity were purified, in the current study, the methods of lectin-blotting, Tricine-SDS-PAGE and HPLC were used to analyse the variation of the two hemocyanins. The results indicated that the glycosylation level of?-D-mannose /?-D-glucose content of LH75 was higher than that of LHt. And molecular weights of main peptides digested by trypsin,of LH75 were also higher than those of LHt. In additional, 10 and 24 peaks of digested LH75 and LHt were observed in HPLC map, respectively. Furthermore, 7 and 6 protein spots were appeared in the 2-DE map of LHt and LH75, respectively, in which, the former contained 3 big subunit spots and 4 small subunit spots, while the later contained 4 big subunit spots and 2 small subunit spots. Thus, these results suggested two hemocyanins were probably discrepant with glycosylation.Secondly, more LH75 increased than that of LHt in serum after injecting Vibro parahaemolyticus. Combined to the previous results that antibacterial activity of LH75 was obvious higher than that of LHt, these results suggested that LH75 was a important component of hemocyanin with antibacterial activity, which was related to glycosylation.Finally, three V. parahaemolyticus outer membrane proteins (Omps) binding to LH75 was separated by affinity incubation, which named p1, p2, p3 respectively. The p2 was identified as OmpC by MALDI-TOF-TOF MS. Our previous research showed that antibacterial activity of LH75 with E. coli Omps gene-deleted mutants,?OmpC, decreased obviously in comparison with that of wild type. Thus, the results manifested that OmpC appeared to be one of the antibacterial target of LH75 and its other antibacterial target need further examination.2. S. serrata hemocyanin mediates agglutination by recognizing OmpA and OmpX of bacteria.First of all, we identified the S. serrata hemocyanin using affinity proteomics and the results showed that S. serrata hemocyanin consisted of five subunits with molecular weights of 70, 72, 75, 76 and 80 kDa, respectively. All five subunits showed high homology with hemocyanin subunits from other crab species including Carcinus aestuarii, Callinectes sapidus and Cancer magister, and three mainly subunit p75?p76 and p80 reacted with the rabbit anti-hemocyanin IgG. Together, these results suggested that S. serrata hemocyanin was purified to homogeneity and suitable for further investigation.Furthermore, seven bacterial species including V. parahaemolyticus, V. alginolyticus, V. harveyi,V. fluvialis, Aeromonas hydrophila, Staphylococcus aureus and Escherichia coli K12 were selected for agglutination and agglutination inhibition analysis. S. serrata hemocyanin presented agglutinative activities against the seven pathogenic bacteria ranging from 7.5-30 g/mL, which was inhibited by N-acetylneuraminic acid,?-D-glucose, D-galactose and D-xylose, respectively. These results suggested that S. serrata hemocyanin possessed agglutination properties.Additionaly, SDS-PAGE, Western-blotting, agglutination inhibition and gene knock-down analysis were performed. The results showed that the 76 kDa subunit of S. serrata hemocyanin was responsible for its agglutinative activities. Moreover, Omps of bacteria could completely stop the agglutination. The agglutinative activities of hemocyanin with?OmpA and?OmpX were obviously decreased. Together, the data suggest that the 76 kDa subunit of S. serrata hemocyanin mediates agglutination by recognizing OmpA and OmpX of bacteria.3. S. serrata hemocyanin mediated hemolysis by binding tightly to erythrocyte membranes and following a colloid-osmotic mechanism.Hemolysis assays was performed to investigate the hemolytic activity of S. serrata hemocyanin. The results showed that S. serrata hemocyanin exhibited Ca2+-dependent hemolytic activities against vertebrate erythrocytes including chicken, mouse, rabbit and human erythrocytes. The hemolysis assay of hemocyanin showed the“dose-response”positive relativity, and the hemolysis was sensitive to pH and temperature. The hemolytic activity of hemocyanin decreased with increasing pH from 5 to 8, and increased with the increasing temperature, the 100% hemolysis was occured at 37 oC. Further evidence revealed that the five subunits of hemocyanin were presented in the solubilized incubation products of erythrocytes with hemocyanin, and the hemolysis could be inhibited to different degrees by osmoprotectants with various molecular masses, indicating that S. serrata hemocyanin mediated hemolysis by binding tightly to erythrocyte membranes and following a colloid-osmotic mechanism. Thus, these finding suggested that S. serrata hemocyanin also can act as a hemolysin.In summary, our results indicated that hemocyain of crustacean (shrimps and crabs) showed agglutinative activity, antibacterial activity and hemolytic activity. And bacterial Omps were acted as the agglutinative and antibacterial targets of pathogen recognized by hemocyanin. Importantly, the mechanism of hemocyanin-induced hemilysis was colloid-osmotic. These findings lay the foundation for revealing the mechanism of immunological functions of hemocyanin and elucidating the pattern of recognizing pathogenic bacteria by hemocyanin. Also, they can provide evidence on the anti-diseases activitives of hemocyanin in crustacean, and will help us understand the immune status of hemocyanin in shellfish such as shrimps and crabs and develop the knowledge of shellfish's immune system and immune prevention.