Tarlochan Singh Dhadialla

The mosquito ultraspiracle homologue, a partner of ecdysteroid receptor heterodimer: cloning and characterization of isoforms expressed during vitellogenesis.

We report the cloning and characterization of two isoforms of the Ultraspiracle homologue (AaUSP) from the mosquito, Aedes aegypti. The 2.33-kb AaUSPa cDNA has an open reading frame (ORF) of 484 amino acids encoding a polypeptide of 54 kDa, whereas the 2.14-kb AaUSPb ORF of 459 amino acids encodes a 51.3 kDa polypeptide. The AaUSPa and AaUSPb proteins differ only in the N-terminal portion of the variable A/B domain. The AaUSP DNA-binding domain shares 92% and 97% identities with the respective domains of the Drosophila (DmUSP) and Bombyx (BmUSP) Ultraspiracles. However, the AaUSP ligand-binding domain is only 57% and 52% identical to those of DmUSP and BmUSP, respectively. In spite of the relatively low level of sequence conservation, electrophoretic mobility shift assay (EMSA) and hormone-binding assay clearly demonstrated that the products of the AaUSPa and AaUSPb cDNAs are functional heterodimeric partners of the mosquito ecdysteroid receptor. In vitellogenic tissues, each of the two AaUSP isoforms is expressed differently: the AaUSPa is predominant in the fat body and the AaUSPb in the ovary. The kinetics of ovarian AaUSP mRNA coincide with those of the ecdysteroid receptor, being elevated during the previtellogenic period and shortly after the onset of vitellogenesis. In contrast, the level of the AaUSP in the fat body remains relatively constant throughout most of the vitellogenic cycle.

Characterization of the solubilized mosquito vitellogenin receptor

Abstract In this study we solubilized and characterized the receptor for the major egg yolk protein precursor, vitellogenin (Vg), from the yellow fever mosquito, Aedes aegypti . The receptor was solubilized from vitellogenic ovary membranes using octyl-β- d -glucoside (OG). Under equilibrium binding conditions, [ 35 S]Vg bound with high affinity ( K d = 2.8 × 10 −8 M) to a single class of binding sites in solubilized ovary extracts. The solubilized receptor was present in ovarian extracts and bound selectively A. aegypti Vg and its storage form, vitellin (Vn). The receptor preparation was heat and trypsin sensitive. Binding of Vg to its receptor could be inhibited as well dissociated with suramin. The receptor was visualized by ligand-blotting as a 205 kDa protein under non-reducing conditions. It did not share immunological cross-reactivity with antibodies to chicken and locust Vg receptors. Vitellogenin, Vn and its purified subunits competed for binding to the receptor in the order, Vg ≈ Vn > Vn large subunit > Vn small subunit. Binding of dephosphorylated Vg was significantly reduced. Deglycosylated Vg, on the other hand, formed high molecular weight aggregates resulting in artifactually high binding which indicates importance of glycosylation for the stability of Vg molecule. During egg maturation, the number of receptor binding sites in ovaries correlated with the rate of Vg uptake and peaked between 24–30 h after which it reduced to no binding by 48 h post blood meal.

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

Abstract 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 [ 35 S]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.

Purification and characterization of a lysosomal aspartic protease with cathepsin D activity from the mosquito

Abstract A lysosomal aspartic protease with cathepsin D activity, from the mosquito, Aedes aegypti , was purified and characterized. Its isolation involved ammonium sulfate (30–50%) and acid (pH 2.5) precipitations of protein extracts from whole previtellogenic mosquitoes followed by cation exchange chromatography. Purity of the enzyme was monitored by SDS-PAGE and silver staining of the gels. The native molecular weight of the purified enzyme as determined by polyacrylamide gel electrophoresis under nondenaturing conditions was 80,000. SDS-PAGE resolved the enzyme into a single polypeptide with M r = 40,000 suggesting that it exists as a homodimer in its non-denatured state. The pI of the purified enzyme was 5.4 as determined by isoelectric focusing gel electrophoresis. The purified enzyme exhibits properties characteristic of cathepsin D. It utilizes hemoglobin as a substrate and its activity is completely inhibited by pepstatin-A and 6M urea but not by 10 mM KCN. Optimal activity of the purified mosquito aspartic protease was obtained at pH 3.0 and 45°C. With hemoglobin as a substrate the enzyme had an apparent K m of 4.2 μ M. Polyclonal antibodies to the purified enzyme were raised in rabbits. The specificity of the antibodies to the enzyme was verified by immunoblot analysis of crude mosquito extracts and the enzyme separated by both non-denaturing and SDS-PAGE. Density gradient centrifugation of organelles followed by enzymatic and immunoblot analyses demonstrated the lysosomal nature of the purified enzyme. The N-terminal amino acid sequence of the purified mosquito lysosomal protease (19 amino acids) has 74% identity with N-terminal amino acid sequence of porcine and human cathepsins D.

Biosynthesis and Endocytosis of Yolk Proteins in the Mosquito

The maturation of insect eggs requires the fat body production of large amounts of a protein, vitellogenin (Vg), that is secreted into the hemolymph, selectively accumulated by the oocytes and stored in yolk bodies as crystalline vitellin (Vn). The coordination of activity in fat body and oocytes during vitellogenesis is achieved through a complex regulatory mechanism. The mosquito, Aedes aegypti, has proven to be a valuable organism in the study of vitellogenesis.