Studies On Phenoloxidase-like Activities And CDNA Cloning Of Hemoglobin From Blood Clam Scapharca Kagoshimensis

Blood clam, Scapharca kagoshimensis, is one of a marine invertebrates which has an important economic value and belongs to Mollusca, Lamellibranchia. It lacks antibodies and immune memory and has to resist parasites and diseases with non-specific immune system. Haemocytes and humoral immune factors play an important roles in immune responses of S.kagoshimensis to many kinds of pathogenic microorganism. Hemoglobin (Hb), responsible for oxygen transport, are a kind of iron-containing respiratory proteins found in the hemolymph of both vertebrates and some invertebrates. This study indicate that hemoglobin not only have oxygen-bearing function, but also have functions as follows: antimicrobial activity , adjusting blood pressure, transporting sulfide, adjusting acid-base balance, oxidase, peroxidase activity and so on. It is one of the most valuable respiratory proteins to worth researching. Many studies have indicated the hemocyanins can be functionally converted into a kind of phenoloxidase-like enzyme and display PO activities under certain conditions. In this study, phenoloxidase-like activities of the hemoglobin from blood clam S.kagoshimensis was found and it lay solid foundation in non-specific immune of invertebrates. In order to characterize the properties of PO-like activities of the hemoglobin from blood clam S.kagoshimensis, Hb from S.kagoshimensis was purified by biochemical and molecular biology methods, and characterized its biochemistry and enzymatic properties. To study the stimulating mechanisms of immunostimulates, the immunostimulating effects ofβ-glucan, lipopolysaccharide (LPS), inactivate Vibrio harveyi and Vibrio anguillarum on the PO-like activities of Hb from S. kagoshimensis were investigated and full-length cDNA sequence has been obtained. This study has not only great significance in understanding its roles in invertebrate non-specific immune system, but also helps us to fully cognize the multiple biological functions of Hb.In this study, Hb from hemolymph of S. kagoshimensis was purified by sephacryl S-100 gel-filtration and Q sepharose Fast Flow ion-exchange chromatography, and characterized its biochemistry and enzymatic properties by using L-dihydroxyphenylalanine (L-DOPA) as the specific substrate to study its PO-like activity. The result indicate that the purified Hb had a molecule weight of only 14.27kDa in sodium dodecyl sulfate polyacrylemide gel electrophoresis(SDS-PAGE), and it is testified by peptide mass fingerprint(PMF). The purified Hb can specific oxidate L-DOPA and dyed by L-DOPA after Native-PAGE .The result indicate that the Hb from S. kagoshimensis have the PO-like activities.The biochemical and enzymatic properties of Hb from S. kagoshimensis using L-dihydroxyphenylalanine (L-DOPA) as the specific substrate was studied and the result indicated that the optimum pH and temperature was 7.0 or so and 30℃and using L-dihydroxyphenylalanine (L-DOPA) as the specific substrate to study its PO-like activity. The Km values was 4.90 mmol/L against L-DOPA. The PO-like activities of Hb from S. kagoshimensis can be activated by isopropanol, and was extremely sensitive to some effective inhibitors of benzoic acid; sensitive to ascorbic acid,urea and sodium sulfite,citric acid; but not sensitive to cysteine and1-phenyl-2-thiourea. All these indicate that the PO-like activities of Hb from S. kagoshimensis in this study is a kind of tyrosinase. The PO-like activity of Hb from S. kagoshimensis was very sensitive to EDTA, Cu2+ and Zn2+, but not sensitive to DETC ,Ca2+ and Mg2+, and Fe2+ activate the PO activity effectively. Then it can be concluded that the Hb of S. kagoshimensis was a type of iron-containing protein.To study the stimulating mechanisms of immunostimulates, the immunostimulating effects ofβ-glucan, lipopolysaccharide (LPS), inactivated Vibrio harveyi and Vibrio anguillarum on the PO-like activities of Hb from S. kagoshimensis were investigated in this study. Under the stimulation ofβ-glucan, LPS, inactivated Vibrio Harvey and inactivated Vibrio anguillaru, the amount of Hb synthesized from S.kagoshimensis increased about 7.47%,6.17%,10.39% and 27.92%, respectively and PO-like activities increased about20.72%,4.03%,11.40% and 45.84%, but the unit activity of PO-like remains almost unchanged. The immunostimulation of Vibrio anguillaru is the most efficient on the amount of Hb synthesized and PO-like activity. Further more, the number of all hemocytes increased about 8.3%,6.3%,2.0% and 20.8%. It is concluded that under the stimulation ofβ-glucan, LPS, inactivated Vibrio Harvey and inactivated Vibrio anguillaru, the amount of Hb synthesized from S.kagoshimensis, PO-like activities and the number of all hemocytes are all increased, The result indicated that the hemocytes and Hb play an important roles in Scapharca kagoshimensis's immune responses.Under the infection of 1×107ind/mL Vibrio anguillaru, the amount of Hb synthesized from S.kagoshimensis increased about 19.78%, respectively and PO-like activities increased about5.29%, the number of all hemocytes reduced 37.71%. Under the infection of 1×108ind/mL Vibrio Harvey, the amount of Hb synthesized from S.kagoshimensis increased about 8.44%, respectively and PO-like activities increased about6.36%, the number of all hemocytes reduced 1.75%. Under the infection of 1×105ind/mL of Vibrio Harvey and Vibrio anguillaru, the amount of Hb synthesized from S.kagoshimensis reduced about 1.99% and 7.94%, respectively and PO-like activities reduced about1.15% and 5.59%, the number of all hemocytes reduced 35.63% and 16.67%. The result indicated that the infection results in reduction of the number of all hemocytes. Infection with high concentration pathogeny results in increase of the amount of Hb synthesized, PO-like activities and the number of all hemocytes. Otherwise, infection with low concentration pathogeny results in reduction of the amount of Hb synthesized, PO-like activities and the number of all hemocytes.Total RNA is isolated from the hemocytes of S.kagoshimensis and full-length cDNA sequence of HbI ,HbII-A and HbII-B has been obtained using reverse transcriptase(RT)-PCR and rapid amplification of cDNA end(RACE).The full-length cDNA of HbI from Scapharca kagoshimensis is 756 bp, encoding a 147-amino acid protein, the molecular weight is about 16.2 kDa, the identity with HbI from Scapharca inaequivalvis is 99.32%; HbII-A from Scapharca kagoshimensis is 755 bp, encoding a 150-amino acid protein, the molecular weight is about 16.5kDa, the identity with HbII-A from Scapharca inaequivalvis is 98.67%; HbII-B from Scapharca kagoshimensis is 745 bp, encoding a 152-amino acid protein, the molecular weight is about 16.7kDa, the identity with HbII-B from Scapharca inaequivalvis is 99.34%.In conclusion, the study of the PO-like activities of Hb from S. kagoshimensis has great significance in not only understanding the roles and mechanisms of Hb in invertebrate non-specific immune system, but also identification the molecular evolution status of Hb in the family of respiratory protein.