Shoji Watabe

Purification and Characterization of Fumarase from Corynebacterium glutamicum

Fumarase (EC 4.2.1.2) from Corynebacterium glutamicum (Brevibacterium flavum) ATCC 14067 was purified to homogeneity. Its amino-terminal sequence (residues 1 to 30) corresponded to the sequence (residues 6 to 35) of the deduced product of the fumarase gene of C. glutamicum (GenBank accession no. BAB98403). The molecular mass of the native enzyme was 200 kDa. The protein was a homotetramer, with a 50-kDa subunit molecular mass. The homotetrameric and stable properties indicated that the enzyme belongs to a family of Class II fumarase. Equilibrium constants (K eq) for the enzyme reaction were determined at pH 6.0, 7.0, and 8.0, resulting in K eq=6.4, 6.1, and 4.6 respectively in phosphate buffer and in 16, 19, and 17 in non-phosphate buffers. Among the amino acids and nucleotides tested, ATP inhibited the enzyme competitively, or in mixed-type, depending on the buffer. Substrate analogs, meso-tartrate, D-tartrate, and pyromellitate, inhibited the enzyme competitively, and D-malate in mixed-type.

PURIFICATION AND CHARACTERIZATION OF BOMBYX CYSTEINE PROTEINASE SPECIFIC INHIBITORS FROM THE HEMOLYMPH OF BOMBYX MORI

Protein inhibitors capable of inhibiting BCP (Bombyx cysteine proteinase) were found in the larval-pupal hemolymph of Bombyx mori. Two forms of the inhibitors, named BCPI (BCP inhibitor) alpha and BCPI beta, were purified from the pupal hemolymph by heat treatment and column chromatographies on CM-cellulose, Toyopearl HW-50, Phenyl-Sepharose, and Mono Q. Purified BCPI beta gave a single protein band with a molecular mass of 10,500 daltons on SDS-PAGE. BCPI alpha is mostly composed of the same molecular mass protein as BCPI beta. Both forms were inhibitory towards other cysteine proteinases such as cathepsins L,B and papain but had no effects on trypsin and pepsin. Both forms inhibited the processing of the enzymatically inactive proform of BCP (pro-BCP) to the activated mature BCP. BCPI alpha and BCPI beta shared many other features such as molecular mass determined by gel filtration, antigenicity, and HPLC profiles. NH(2)-terminal amino acid sequencing of the purified inhibitors revealed that three amino acid residues were different in the BCPI alpha and BCPI beta sequences, all others being identical. The hemolymph BCP inhibitor increased activity approximately four- to fivefold at the time of spinning and maintained this level of activity during pupation.

In vivo activation of pro-form Bombyx cysteine protease (BCP) in silkmoth eggs : localization of yolk proteins and BCP, and acidification of yolk granules

The present study was designed to investigate the process of acidification of yolk granules during embryogenesis. In oocytes of mature Bombyx mori silkmoth, yolk proteins and a cysteine protease (pro-form BCP) were found in yolk granules. BCP was localized in small sized yolk granules (SYG, 3-6 microm in diameter) and yolk proteins in large sized granules (LYG, 6-11 microm in diameter), which might result in a spatial separation of protease and its substrates to avoid unnecessary hydrolysis. The granules were isolated on Percoll density gradient centrifugation. Although separation of LYG and SYG was incomplete, the granules sedimented in different fractions when using unfertilized egg extract, in which LYG was recovered from heavier fractions and BCP from lighter fractions. Acid phosphatase, as well as other lysosomal marker enzymes tested, was recovered from LYG-containing fractions. When extracts were prepared from developing eggs (day 3), some BCP-containing granules co-sedimented with LYG. The inactive pro-form BCP was activated in vivo, in parallel with yolk protein degradation, and as demonstrated previously in vitro under acidic conditions (). These results suggest that acidification occurs in yolk granules during embryogenesis. This was also confirmed using acridine orange fluorescent dye. In early development, most yolk granules were neutral, but became acidic during embryonic development. SYG were progressively recovered in heavier density fractions, displaying acidic interior. In this fraction, BCP-containing granules seem to be associated with larger granules (6-11 microm in size). In addition, SYG (BCP containing granules) were likely to be acidified earlier than LYG. Our results suggest that acidification initiates yolk degradation through activation of pro-form BCP.

A novel inhibitor protein for Bombyx cysteine proteinase is homologous to propeptide regions of cysteine proteinases

A cDNA clone for an inhibitor of Bombyx cysteine proteinase was isolated and sequenced. Active inhibitor proteins were expressed in Escherichia coli using the cDNA. The open reading frame of the cDNA encodes a 105 residues protein with 19 residues of a signal sequence. The inhibitor has amino acid sequences homologous to several cysteine proteinases, but only to their propeptide sequences. The results suggest that some cysteine proteinase proregions may have evolved as autonomous modules and become inhibitor proteins for cysteine proteinases.

PROREGION OF BOMBYX MORI CYSTEINE PROTEINASE FUNCTIONS AS AN INTRAMOLECULAR CHAPERONE TO PROMOTE PROPER FOLDING OF THE MATURE ENZYME

A cDNA encoding the proform of Bombyx cysteine proteinase (BCP) was expressed at a high level in Escherichia coli using the T7 polymerase expression system. The insoluble recombinant zymogen was solubilized and renatured by modifying a method applied to human pro-cathepsin L. Like the natural BCP precursor, the recombinant proenzyme was spontaneously converted to an active proteinase at pH 3.75. A deletion in the central region of the propeptide resulted in much loss of the activity, suggesting that the propeptide is essential for proper folding during renaturation. In contrast, the renatured mature form of recombinant BCP was not active but regained activity by including the propeptide in the renaturing buffer, suggesting that the propeptide, acting as an intramolecular chaperone, promotes refolding of the associated proteinase domain into an active conformation. The mature form of natural BCP rapidly lost its activity at neutral pH, whereas its proform was stable. The mature enzyme retained some activity in the presence of the propeptide. Arch.

Bombyx acid cysteine proteinase

Summary Three kinds of yolk proteins (vitellin, egg-specific protein and 30 k-proteins) are found in silkmoth eggs and have been well characterized. Essentially these proteins are considered to be amino acid reserves for developing embryos. Since at an early stage of egg development the cysteine proteinase accounts for the majority of the total proteinase activity, it may be involved in the degradation of yolk proteins. The enzyme is stored in the eggs as an inactive pro-form, indicating that the activation of the enzyme might be one of the key steps in yolk protein degradation. To investigate at the molecular level how yolk proteins degradation takes place, we have studied Bombyx acid cysteine proteinase (BCP) during an early period of embryonic development. We summarize how proteinases are regulated and are involved in the degradation of Bombyx yolk proteins during embryogenesis. These will be discussed mainly in light of recent results obtained from eggs of the silkmoth, Bombyx mori.

Drosophila CTLA-2-like Protein (D/CTLA-2) Inhibits Cysteine Proteinase 1 (CP1), a Cathepsin L-like Enzyme

Abstract In this study, we present a propeptide-like cysteine proteinase inhibitor, Drosophila CTLA-2-like protein (D/CTLA-2), a CG10460 (crammer) gene product, with an amino acid sequence significantly similar to the proregion of Drosophila cysteine proteinase 1 (CP1). Recombinant D/CTLA-2, expressed in E. coli, strongly inhibited Bombyx cysteine proteinase (BCP) with a Ki value of 4.7 nM. It also inhibited cathepsins L and H with Ki values of 3.9 (human liver) and 0.43 (rabbit liver) nM, and 7.8 nM (human liver), respectively. Recombinant D/CTLA-2 exhibited low but significant inhibitory activities to cathepsin B with Ki values of 15 nM (human liver) and 110 nM (rat liver), but hardly inhibited papain. We attempted to purify cysteine proteinases inhibited by D/CTLA-2 from total bodies of adult Drosophila. Recombinant D/CTLA-2 significantly inhibited CP1 with a Ki value of 12 nM, indicating that CP1, a cognate enzyme of D/CTLA-2, is a target enzyme of the inhibitor in Drosophila cells. These results indicate that D/CTLA-2 is a selective inhibitor of cathepsin L-like cysteine pro-teinases similar to other propeptide-like cysteine proteinase inhibitors such as Bombyx cysteine proteinase inhibitors (BCPI) and cytotoxic T-lymphocyte antigen-2 (CTLA-2). D/CTLA-2 was expressed over the whole life cycle of Drosophila. Strong expression was observed in the garland cells and prothoracic gland in the late stages of embryonic development. These results suggest that D/CTLA-2, implicated in intra- and extra-cellular digestive processes, functions in these tissues by suppressing uncontrolled enzymatic activities of CP1.

R352Q mutation of the DHCR7 gene is common among Japanese Smith-Lemli-Opitz syndrome patients.

AbstractSmith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive malformation syndrome characterized by microcephaly, syndactyly of toes, ambiguous genitalia, and mental retardation. The underlying DHCR7 gene has been identified and a wide variety of distinct mutations were reported in USA and European SLOS patients. A significant difference has been suggested in the frequency of SLOS among different ethnic populations. Here, we report mutational analysis of seven Japanese SLOS patients. Five mutations, R352Q, R242H, G303R, X476Q, and S192F, were identified, and R352Q appeared most frequent, since nine out of the 13 mutations of Japanese origin were the same R352Q. These results suggest that R352Q is a predominant founder mutation in Japanese SLOS patients.

Bombyx acid cysteine protease (BCP): hormonal regulation of biosynthesis and accumulation in the ovary.

We previously reported purification of the cysteine protease from Bombyx eggs (BCP) and the occurrence of the enzyme in various tissues of this insect. In the present paper, we present a detailed analysis of stage-specific changes in activity of BCP between the fourth larval instar and pupal-adult development. A synthetic fluorescent peptide, carbobenzoxy-L-Phenylalanyl-L-Arginine4-methylcoumaryl-7-amide (Z-Phe-Arg-MCA), was used to assay proteolytic activity. When tissue extracts were treated with anti-BCP serum before assay of enzyme activity, most activity towards Z-Phe-Arg-MCA was removed from the extracts. Therefore proteolytic activity in the present experiments is due mainly to BCP. We used Western blot and Northern blot analyses to determine tissue and stage specific expression of the enzyme. In the 5th larval fat body and hemolymph, BCP activity dramatically increased at the time of spinning, returning to the basal level before ecdysis. Northern blot analysis showed that a 1.5 kilobase mRNA which hybridizes to BCPcDNA suddenly appears during this period. Similar results were obtained in 4th instar fat body. In pupal hemolymph and fat body, low basal activity of BCP was detected early (day 0 to day 3 after pupal ecdysis), followed by a pronounced increase to a maximum six days after ecdysis, before returning to the basal level. In ovariectomized female pupae, a significant amount of proteolytic activity accumulated in hemolymph, suggesting that the enzyme is synthesized in the fat body and transferred into the ovary along with vitellogenin. BCP activity increased three days after injection of 20-hydroxyecdysone into ligated pupae. Furthermore, putative BCPmRNA appeared in the fat body within 24 hours after injection. This increase was completely blocked by the administration of cycloheximide. The results suggest that, BCP is synthesized in extraovarian tissues such as fat body and ovarian follicle cells and accumulates in the ovary, thus representing a new class of yolk protein.

Autolytic activation mechanism of Bombyx acid cysteine protease (BCP)

Acid cysteine proteinase in the eggs of the silkmoth, Bombyx mori, exists as an inactive proenzyme. This 47‐kDa pro‐BCP1 zymogen molecule can be processed in vitro into an enzymatically active 39‐kDa BCP molecule (11, 12). In this current study, the maximum rate of processing in vitro was achieved at approximately pH 4.0, at a temperature of 37 °C under reducing conditions. The rate of conversion was not affected by increasing concentrations of pro‐BCP. We prepared immobilized BCP bound to AH‐Sepharose and examined the activation. Immobilized pro‐BCP was autolysed, although the rate of processing was slow, indicating that the reaction might be an intramolecular one. Kinetic experiments suggest that the mechanism is likely to involve a stepwise reaction, in which pro‐BCP is converted to an active enzyme through intermediate forms releasing small peptides stepwise.