| Abstract |
The aim of the work presented here is the biochemical characterization of vitellogenin and vitellin from the land crab Potamon potamios, the identification of the molecular form of precursor vitellogenin and its transformation during its transport to developing oocytes, as well as the identification of the organ that synthesizes vitellogenin. Vitellogenins are proteins that have been found in all egg-laying organisms and are the precursor form of egg vitellin. They have been studied from egg-laying vertebrates (birds, amfibia, fish) and invertebrates (nematodes, annelids, arthropods). Vitellogenins are proteins with large molecular mass and carry in their molecules lipids, carbohydrates and in certain cases carotenoids. Vitellogenin synthesis is regulated on hormonal systems. Electrophoretic analysis of the hemolymph protein from Potamon potamios during the annual reproduction cycle, revealed that vitellogenesis did not occur in males or in females of a previtellogenic stage. The analysis of ovarian and egg extracts revealed the presence of vitelliin, which was identical to vitellogenin. Both proteins were lipo-glyco-carotenoproteins and were present in three different aggregational states that represented monomeric, dimeric and trimeric forms of the proteins. In both proteins the predominant form was the dimeric, which had a molecular mass of 551 kDa for vitellogenin and 510 kDa for vitellin. In spite of the difference in terms of native molecular mass, the two proteins were similar in their lipid, carotenoid and carbohydrate composition. Under denaturing conditions both proteins consisted of three polypeptides with molecular mass of 115, 105 and 85 kDa. However, vitellogenin of some animals contained a fourth polypeptide with a molecular mass of 181 kDa.This polypeptide was never identified in egg vitellin. The 181 kDa polypeptide of vitellogenin and the other three polypeptides of vitellogenin and vitellin (115, 105 and 85 kDa) were immunoreactive against antiserum prepared from the 85 kDa polypeptide of vitellogenin. Futhermore, proteolytic cleavage experiments confirmed that the 115 and 105 kDa vitellogenin polypeptides were derived from the 181 kDa polypeptide. From this result we concluded that it was possible for vitellogenin to be synthesized as a large and a small polypeptide subunit. Labeling studies using [35S]-methionine on normal vitellogenic animals showed that the radioactivity was first distributed among the 181 and 85 kDa polypeptides. SDS-PAGE analysis of purified hemolymph vitellogenin from eyestalk ablated females revealed, in most animals, two polypeptides with an apparent molecular mass of 181 and 85 kDa, respectively. These results, from the in vivo experiments, corroborated the view that the 115 kDa and 105 kDa vitellogenin and vitellin polypeptides are derived from the heaviest 181 kDa polypeptide. In addition, it was demonstrated that hepatopancreas and ovaries of female Potamon potamios incubated in vitro with [35S]-methionine synthesized five polypeptides with apparent molecular mass of 224, 181, 115, 105 and 85 kDa, while the hepatopancreas appeared to secrete the 181, 115, 105 and 85 kDa polypeptides. The major 115, 105 and 85 kDa polypeptides were found to be components of egg vitellin, while the 224 kDa polypeptide was found to be a minor component of vitellogenin and vitellin from hepatopancreas and ovaries extract, respectively. We infered that the 224 kDa polypeptide was the heavy intracellular form and the 181 kDa polypeptide the heavy transport form of vitellogenin and vitellin molecules.
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