Purification and characterization of a cysteine proteinase from silkworm eggs

Abstract

Eggs of the silkworm, Bombyx mori, contain a high level of a proteinase which is most active in acidic pH region. The proteinase was purified from an extract of eggs by a six-step procedure which included conventional chromatographic fractionations. The molecular mass of the proteinase was estimated to be 350 kDa by gel filtration and 47 kDa by electrophoresis on sodium dodecyl sulfate/polyacrylamide gels, suggesting an octameric structure. The amino acid composition was found to resemble that of mammalian lysosomal cysteine proteinases, in particular cathepsin L. The NH2-terminal 10-residue sequence is Val-Gln-Phe-Phe-Asp-Leu-Val-Lys-Glu-Glu-.The enzyme appears to be a member of the class of cysteine proteinases since it was strongly inhibited by sulfkydrylreactive compounds and N-[N-(1,3-trans-carboxyoxiran-2-carbonyl)-~-leucyl]-agmatine (E-64). The enzyme hydrolyzed various protein substrates, such as hemoglobin, vitellogenin, vitellin, and lipophorin, with maximal activity around pH 3 -3.5. The specificity of the cleavage sites in the oxidized B chain of insulin was rather well defined and there was high affinity for hydrophobic residues at the P2 and P3 positions. The cysteine proteinase is thought to be involved in protein degradation during embryonic development of silkworm eggs.Silkworm eggs contain high levels of yolk proteins such as vitellin and egg-specific protein [l]. Since these proteins are known to be utilized during embryogenesis, proteinases are thought to be involved in the catabolism of such proteins [2]. Proteinases from insect cells and tissues have been studied by various authors [3, 41. However, at least in part because of the difficulties in obtaining sufficient starting material, the information about these enzymes is much less extensive than that which has been published about mammalian proteinases. lntracellular proteinases, such as cathepsins B, H, L, and D, are present in mammalian lysosomes and play a significant role in the intracellular degradation of proteins [5]. The occurrence of these proteinases in insects is still unproven and remains to be clarified.We reported previously that silkworm eggs contain high levels of activity associated with cysteine proteinases [6]. In our efforts to learn more about the role of proteinases in the developing embryo, we have purified and characterized a cysteine proteinase from silkworm eggs. It is unusual in its high molecular mass and its maximum activity around pH 3.0. The amino acid composition and proteolytic specificity, however, are rather similar to those of other cysteine proteinases such as cathepsin L. EXPERIMENTAL PROCEDURES MaterialsEggs of the silkworm (Bombyx mori) were obtained by extraction from the fully matured ovaries of female moths. They were stored frozen at -20°C until use. Bovine hemoglobin was purchased from Worthington, synthetic chromogenic peptides, synthetic oligopeptides, microbial inhibitors were obtained from the Protein Research Foundation, Osaka; DEAE-cellulose (DE-32) from Whatman, DEAE-Toyopearl from Toso...

Molecular characterization of cathepsin L cDNA and its expression during oogenesis and embryogenesis in the oriental river prawn Macrobrachium nipponense (Palaemonidae)

“…The result showed that the MnCatL was possibly involved in hydrolyzing yolk protein. This deduction is supported by results reported for other arthropods that the cysteine enzyme purified from silkworm eggs can hydrolyze VTG, vitellin and lipophorin (Kageyama and Takahashi, 1990), and that CatL is stored in the yolk to degrade VTG in the soft tick (Fagotto, 1990a,b). The expression of MnCatL during oogenesis and embryogenesis in oriental river prawns implies that CatL can functionally degrade VTG and yolk proteins.…”

“…A confirmed function of CatL in the ovary is its involvement in VTG hydrolysis. The role of CatL in VTG hydrolysis has been widely studied in vertebrates (Carnevali et al, 1999(Carnevali et al, , 2006, while its research in arthropods is behind, although some progress has been made in the silkworm B. mori (Kageyama and Takahashi, 1990) and soft tick Orithodors moubata (Fagotto, 1990a,b).…”

A Boophilus microplus vitellin-degrading cysteine endopeptidase

“…Proteases of the cysteine endopeptidase subclass often participate in arthropod yolk degradation, as has been reported for B. germanica, Drosophila melanogaster, O. moubata and Bombyx mori (Liu et al 1996 ;Medina et al 1988 ;Fagotto, 1990 ;Kageyama & Takahashi, 1990).…”

Crystal structure of Bombyx mori arylphorins reveals a 3:3 heterohexamer with multiple papain cleavage sites

“…[14][15][16] Sequence blast of this cysteine protease and other insect papain genes against silkworm genome revealed 9 papain-like genes encoded by B. mori.…”