Vitellin Research Articles

Characterization of yolk protein and its receptor on the oocyte membrane in Rhodnius prolixus

Vitellogenim (VG) and vitellin (VN) purified from hemolymph and oocytes respectively of Rhodnius prolixus have the same molecular weight of 385 kDa and each consists of the same four subunits (155, 145, 43 and 38 kDa). They show the same antigenicity and both are phospho-lipo-glycoproteins. VG and VN differ in the absolute contents of their non-protein components: VN contains more lipid, especially phospholipids and triacylglycerols, and more carbohydrate than VG. Preparations of oocyte membranes bind both VG and VN in a specific and saturable manner. Non-specific binding of VN is increased and specific binding is very markedly reduced in the absence of Ca 2+. It appears that the uptake of VG in Rhodnius is a receptor-mediated process requiring the presence of Ca 2+. Binding of VN is enhanced by juvenile hormone I, which appears to increase the number of receptors.

Vitellin processing and degradation during embryogenesis in Rhodnius prolixus

The fate of vitellin (VT), the major yolk protein in Rhodnius prolixus, was studied during embryogenesis by electrophoretic and immunological techniques. Analysis of the protein content of the eggs by polyacrylamide gel electrophoresis (PAGE) under non-denaturing conditions shows a continuous alteration in the VT molecule following oviposition. Two distinct phases of VT processing can be observed: (1) when limited proteolysis is dominant; and (2) when an extensive degradation of VT occurs inside the digestive system of the embryo. In the first phase (days 0–10 after oviposition), as embryogenesis proceeds, the two principal bands of VT give rise to several bands that migrate progressively further into the gel. Analysis of the same samples by SDS-PAGE reveals a continuous proteolysis of the subunits of the VT molecule. When the VT is analysed by rocket immunoelectrophoresis very little alteration in the total VT content per egg is observed from day 0 to day 10, suggesting that the antibodies used also recognize polypeptides produced by partial proteolysis. Western blot analysis demonstrated that some of the new polypeptides (112, 103, 64 and 56 kDa) generated during embryonic development are in fact derived from VT by limited proteolysis. After cleavage, the polypeptides remain in the same lipoprotein macromolecular complex. In the second phase of VT degradation, an extensive degradation of VT takes place, such that by day 14 after oviposition (hatching), VT content has declined to about half of the initial value. The VT remaining in the first instar is located entirely in the crop and disappears within 5 days after hatching.

Selective endocytosis of vitellogenin by oocytes of the mosquito, Aedes aegypti: An in vitro study

An in vitro assay was used to study the uptake of vitellogenin (VG) by oocytes of the mosquito, Aedes aegypti. The VG, obtained from the culture of fat bodies in vitro, was metabolically labeled to a high specific activity ( 3–6 × 10 5 cpm/μg) using [ 35S]methionine. Purification of both VG and vitellin (VN) from ovaries was achieved with ion-exchange chromatography on DEAE-Sepharose CL-6B. In an optimized in vitro system, mosquito oocytes in intact ovaries maintained stable endocytotic activity for at least 4 h. The rate of VG uptake was maximal at pH 7.5. It declined at acid pH and ceased abruptly at pH 6.3. Accumulation of VG by mosquito oocytes demonstrated features of receptor-mediated endocytosis; i.e. temperature dependence, saturability, selectivity and tissue specificity. The uptake of VG was inhibited at 4°C with only 6% of uptake achieved at 27°C. At 24 h post-blood meal, uptake of VG by oocytes at 27°C, neared saturation with a VG concentration in the medium of 8 μg/μl. Saturation kinetics generated for VG endocytosis by these oocytes produced a V max = 3.2 μg/μl/h and an apparent K uptake = 8.4 × 10 −6 M. Ovaries accumulated 10 times the amount of VG compared to mouse IgG, while uptake of VN was 71.6% of that for VG, indicating that oocytes from Aedes are able to distinguish these proteins. Non-ovarian tissues, fat body and Malpighian tubules, accumulated both VG and IgG at equally low levels. During the vitellogenic cycle, the rate of VG uptake by oocytes showed a steep and linear increase between 6 and 24 h post-blood meal. The peak of VG uptake occurred between 24 and 30 h post-blood meal, followed by a precipitous decline and cessation of uptake by 36 h.

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

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.

Structure and embryonic degradation of two native vitellins in the cockroach, Periplaneta americana

Multiple vitellogenins (VG) are found in species from all major families of cockroaches. Proof that observed multiple VGs are actually distinct proteins and not artifacts of differential processing requires careful examination. In Periplaneta americana two immunologically and electrophoretically distinct vitellins (VTs) of similar native size exist in the egg. VT1 is composed of four major peptides of molecular weights 170, 105, 92 and 78 K. VT2 is composed of three major peptides of molecular weights 105, 101 and 60 K. A peptide with molecular weight of about 105 K is found in both VG1 and VG2 and a similar sized peptide is also conserved in the VGs of all other Blatinae subfamily members examined despite the distant immunological relation of these proteins. The 170 K peptide of VT1 is likely to be a processing intermediate of the 92 K and the 78 K peptides. Each VT from a freshly ovulated egg is immunologically identical to and has essentially the same peptide substructure as its serum precursor. The cumulative M r of the constituent peptides of each VG is approx. 260 K, consistent with 17S native dimeric molecules of approx. 520 K M r . A specific and limited cleavage of VT major peptides occurs during early embryogenesis (at 30°C, 9 days after ovulation) resulting in a partial loss of immunological determinants. During subsequent yolk utilization the VTs undergo gradual degradation.

Purification and properties of vitellogenin and vitellin from Locusta migratoria

The vitellogenin (VG) and vitellin (VN) of Locusta migratoria have been purified from ovariectomized female haemolymph and from oocytes, respectively, by a new procedure. The products are free of lipoprotein I, which was present as a contaminant in previous preparations. The isoelectric point of VG and VN is pH 6.3. The amino acid compositions of VG and VN do not differ significantly. The lipid moiety (8.2% by weight of VG and 6.7% by weight of VN) includes diacylglycerol, a trace of triacylglycerol, cholesterol, phosphatidylcholine and phosphatidylethanolamine. The content of diacylglycerol is much lower in VN than in VG, which suggests the release of lipid within the oocyte.