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...

The eggs of most oviparous animals are provisioned with a class of protein called vitellogenin (Vg) which is stored as the major component of yolk. Until recently, deduced amino acid sequences were available only from vertebrate and nematode Vgs, which proved to be homologous. The sequences of several insect Vgs are now known, but early attempts at pairwise alignments with vertebrate and nematode Vgs have been problematic, leading to conflicting conclusions about how closely insect Vgs are related to the others. In this paper we demonstrate that insect Vg sequences can be confidently aligned with one another along their entire lengths and with multiple vertebrate and nematode Vg sequences along most of their spans. Although divergence is high, conservation among insect, vertebrate, and nematode Vg sequences is widespread with a preponderance of glycine, proline, and cysteine residues among strictly conserved amino acids, establishing conclusively that Vgs from the three phyla are homologous. Areas of least-certain alignment are primarily in and around insect and vertebrate polyserine domains which are not homologous. Phylogenetic reconstructions of Vgs based on sequence identities indicate that the insect lineage is the most diverged and that the mammalian serum protein, apolipoprotein B-100, arose from a Vg ancestor after the nematode/vertebrate divergence.

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