Properties of the mosquito yolk protein: A study using monoclonal antibodies

Abstract

The yolk protein (YP) of the mosquito Aedes aegypti was analyzed with radiolabeling experiments and monoclonal antibodies (mABs). Immunoprecipitation analyses with mAB demonstrated that the mature vitellogenin (VG) secreted by the fat body consisted of two subunits, 200 and 65 kDa. In the oocyte, vitellin (VT) was also composed of only these two subunits, and no additional processing of VT was detected after its internalization and crystallization. Immunoprecipitation experiments showed that the YP subunits were not associated covalently, since they behaved similarly during SDS-PAGE under reduced or non-reduced conditions. Labeling with radioactive carbohydrate precursors revealed that both VG subunits were glycosylated and retained their carbohydrate moieties after their internalization and processing into VT by the oocyte. They were composed of mannose and N- acetylglucosamine . The carbohydrate moieties of VG from the fat body were sensitive to endo-β-N- acetylglucosaminidase H (Endo H), and they continued to be Endo H-sensitive in crystalline VT in the oocyte. Therefore, the carbohydrate moieties of both YP subunits were high-mannose oligosaccharides. Treatment of [ 35S]methionine labeled VG and VT by Endo H demonstrated that carbohydrate moieties accounted for 10 kDa in a large subunit and for 15 kDa in a small subunit. Labeling with [ 32P]orthophosphate showed that both subunits of VG and VT were phosphorylated, and phosphorylated moieties were resistant to delipidation.