Here we report identification of a novel member of the thiol protease superfamily in the yellow fever mosquito, Aedes aegypti. It is synthesized and secreted as a latent proenzyme in a sex-, stage-, and tissue-specific manner by the fat body, an insect metabolic tissue, of female mosquitoes during vitellogenesis in response to blood feeding. The secreted, hemolymph form of the enzyme is a large molecule, likely a hexamer, consisting of 44-kDa subunits. The deduced amino acid sequence of this 44-kDa precursor shares high similarity with cathepsin B but not with other mammalian cathepsins. We have named this mosquito enzyme vitellogenic cathepsin B (VCB). VCB decreases to 42 kDa after internalization by oocytes. In mature yolk bodies, VCB is located in the matrix surrounding the crystalline yolk protein, vitellin. At the onset of embryogenesis, VCB is further processed to 33 kDa. The embryo extract containing the 33-kDa VCB is active toward benzoyloxycarbonyl-ArgArg-para-nitroanilide, a cathepsin B-specific substrate, and degrades vitellogenin, the vitellin precursor. Both of these enzymatic activities are prevented by transepoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64), a thiol protease inhibitor. Furthermore, addition of the anti-VCB antibody to the embryonic extract prevented cleavage of vitellogenin, strongly indicating that the activated VCB is involved in embryonic degradation of vitellin.Cathepsin B is a thiol (cysteine) protease with both endopeptidase and peptidyldipeptidase activities. Due to its broad specificity, cathepsin B plays a key role in intracellular protein catabolism in the lysosomal system (1). Cathepsin B has been well characterized both enzymatically and molecularly (2-10). The mammalian cathepsin B has been implicated in tumor invasion, progression, and metastasis (11-15). Tumor-specific cathepsin B is secreted by malignant cells as a latent high molecular weight precursor, presumably activated at cell contacts (16,17).In addition, cathepsins B, as well as the related cathepsins L, have been identified in numerous parasitic protozoa and helminthes, including prevalent pathogens of human and domestic animals (18 -26). In the blood-sucking bug, Rhodnius prolixus, cathepsin B is the major gut proteolytic enzyme (27). In these organisms, cathepsins B and L are presumably involved in the degradation of host hemoglobin.In insects and other arthropods, cathepsins B and L also participate in key developmental processes. In the flesh fly, Sarcophaga peregrina, hemocytes produce the extracellular form of a cathepsin B-like enzyme that participates in decomposition of the larval fat body during metamorphosis (28 -30). Moreover, cathepsins B and L have been implicated in degradation of yolk proteins during embryonic development (31-41).The elucidation of developmental mechanisms in the mosquito is important because this insect transmits the most devastating of vector-borne human diseases, including malaria, lymphatic filariasis, Dengue fever, and many others. Little is known, however, about t...

Microsporidia are obligatory intracellular parasites, most species of which live in the host cell cytosol. They synthesize and then transport secretory proteins from the endoplasmic reticulum to the plasma membrane for formation of the spore wall and the polar tube for cell invasion. However, microsporidia do not have a typical Golgi complex. Here, using quick-freezing cryosubstitution and chemical fixation, we demonstrate that the Golgi analogs of the microsporidia Paranosema (Antonospora) grylli and Paranosema locustae appear as 300-nm networks of thin (25- to 40-nm diameter), branching or varicose tubules that display histochemical features of a Golgi, but that do not have vesicles. Vesicles are not formed even if membrane fusion is inhibited. These tubular networks are connected to the endoplasmic reticulum, the plasma membrane and the forming polar tube, and are positive for Sec13, γCOP and analogs of giantin and GM130. The spore-wall and polar-tube proteins are transported from the endoplasmic reticulum to the target membranes through these tubular networks, within which they undergo concentration and glycosylation. We suggest that the intracellular transport of secreted proteins in microsporidia occurs by a progression mechanism that does not involve the participation of vesicles generated by coat proteins I and II.

The vitellogenic female fat body of the mosquito Aedes aegypti produces three yolk protein precursors that are deposited in the yolk bodies of developing oocytes: vitellogenin, vitellogenic carboxypeptidase (VCP), and 44-kDa protein (44KP). We have used gold immunocytochemistry to investigate the pathways of their secretion in fat body trophocytes and their internalization by oocytes. In fat body trophocytes, all three yolk protein precursors are co-localized in the Golgi complex and secretory granules, indicating that they proceed simultaneously through the secretory pathway. The lysosomal system plays an important role in the termination of vitellogenesis in mosquito trophocytes, by degrading biosynthetic organelles and secretory granules. At this time, VCP and 44KP are found together with vitellogenin in trophocyte autophagolysosomes, suggesting that all three yolk protein precusors are redirected from the secretory to the lysosomal degradative pathway. Localization of VCP and 44KP in developing mosquito oocytes clearly shows that the internalization of these yolk protein precursors by oocytes occurs via the same endocytotic route as vitellogenin: all three yolk protein precursors are found on the oocyte microvillus membrane, in coated vesicles, and early endosomes. They are observed intermixed with one another in the late endosomes or in transitional yolk bodies. In mature yolk bodies, however, 44KP and VCP are segregated from vitellin, the crystallized storage form of vitellogenin; 44KP and VCP reside in the non-crystalline cortex, surrounding the vitellin core in nature yolk bodies.

The major yolk protein precursor in mosquito oocytes, vitellogenin (Vg), is internalized by a 205-kDa membrane-bound receptor (VgR). Recently, VgR has been isolated permitting the production of polyclonal anti-VgR antibodies. To elucidate the pathway of VgR internalization and recycling in mosquito oocytes during Vg uptake, we carried out an immunogold electron-microscopic study, labeling both Vg and VgR in ultrathin frozen sections of ovarian tissue. VgR immunolabeling demonstrated that the oocyte plasma membrane was subdivided into microdomains, with VgR being located between and at the lower portions of the oocyte microvilli. During the early stages of internalization, Vg and VgR were observed together in coated pits, coated vesicles, and early endosomes. Fusion of early endosomes created transitional yolk bodies (TYB) in which Vg and VgR became segregated. VgR label was present in the numerous tubular compartments that protruded from the TYBs. These tubular organelles extended to and fused with the plasma membrane, suggesting that they represented the vehicle for VgR recycling. Vg label was not observed in the tubular compartments. Instead, Vg accumulated in the core of the TYB, a region free of VgR label. Mature yolk bodies (MYB) were heavily labeled for Vg, but completely lacked any VgR label, indicating that MYB are storage compartments that do not participate in receptor recycling. Thus, our immunocytochemical data clearly visualize the steps in Vg/VgR internalization, dissociation, sorting, and recycling of the receptor to the plasma membrane.

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