THE PLASMA-PROTEINS OF BIOMPHALARIA-GLABRATA IN THE PRESENCE AND ABSENCE OF SCHISTOSOMA-MANSONI

Snail plasma serves as both a sink for metabolites and a source of nutrients for parasites developing within their intermediate hosts. It also contains molecules involved in immunological events like non-self recognition, phagocytosis and encapsulation. In this study we present improved protocols for the separation and partial characterization of plasma proteins of schistosome-susceptible and resistant strains of Biomphalaria glabrata. Within each strain, the plasma of snails 12, 24, 48 and 72 h post-exposure to Schistosoma mansoni and of non-exposed snails was compared. Protein concentrations in hemolymph of all snail strains, non-exposed or parasite-exposed, were about 29 mg/mL and were not found to differ significantly. The dominant plasma molecule (80-85%) is extracellular hemoglobin (Hb) with a native mass of >1 M Da, and subunits of 190 kDa. It is the only protein bearing heme as shown after separation by native-PAGE and LDS-PAGE. The relatively large amounts of Hb and its large size cause problems if native plasma components are to be separated in PAGE. To obtain satisfactory separation, we used short-term ultracentrifugation to deplete Hb from plasma without qualitative loss of other proteins. Using this methodology, we have examined proteins by native polyacrylamide gel electrophoresis, in the presence of SDS or LDS only or SDS and mercaptoethanol, and by isoelectric focusing. Proteins have been detected in gels by silver stains and staining for heme groups, and, after transfer to membranes, by means of lectins and neoglycoproteins. Molecular weights of plasma proteins range between 10 and >450 kDa, and isoelectric points are from pH 4 to 9.4. All strains show similar protein patterns, although minor inter- and intrastrain differences occur. These differences are quantitative rather than qualitative, not consistent, and cannot be correlated with the snail's ability to effectively attack and kill S. mansoni sporocysts. In all snail strains, plasma proteins remained qualitatively stable during 3 days after exposure to S. mansoni. New proteins were not evident, and none was lost as a consequence of exposure to parasites. Our new Hb-depletion technique is an excellent approach to separate and examine Biomphalaria plasma proteins in their native state. The use of lectins to probe for the presence of carbohydrates showed that the majority of plasma proteins is glycosylated. Mannose, galactose, and N-acetylgalactosamine are their major carbohydrate components; fucose was not detected. Several lectins apparently in the molecular mass range of 330-500 and 56-135 kDa with major carbohydrate - specificities for N-acetylgalactosamine, N-acetylglucosamine, mannose, glucose, galactose and fucose were detected in the plasma of both resistant and susceptible snails by using neoglycoproteins as probes.