Kohji Yamamoto

Genes Encoding Small Heat Shock Proteins of the Silkworm, Bombyx mori

Small heat shock protein (sHSP) is a family of ubiquitous polypeptides involved in a variety of physiological phenomena. From the silkworm, Bombyx mori, we isolated and sequenced the following cDNAs encoding sHSPs: shsp19.9, shsp20.1, shsp20.4, shsp20.8, shsp21.4, and shsp23.7. shsp21.4 was nearly twice as large in size as other shsps. The deduced amino acid sequence of sHSP21.4 was similar to that of Drosophila melanogaster CG14207-PA. Other sHSPs were highly similar to each other and, in a phylogenetic tree, formed a cluster including Plodia interpunctella αCP25. It was speculated that shsp21.4 has at least one intron in genome while other shsps do not. The transcripts of all shsps were subtle, but were constitutively detected in various tissues. Heat shock triggered a substantial increase in the transcripts other than shsp21.4. The B. mori sHSPs are perhaps classified into at least two groups: sHSP21.4 and others.

Expression and Characterization of a Sigma-Class Glutathione S-Transferase of the Fall Webworm, Hyphantria cunea

A cDNA encoding glutathione S-transferase (GST) of the fall webworm, Hyphantria cunea, was cloned by reverse transcriptase-polymerase chain reaction. The resulting clone (hcGST) was sequenced and deduced for amino acid sequence, which revealed 87, 59, and 42% identities to Sigma-class GSTs from Bombyx mori, Manduca sexta, and Blattella germanica respectively. A recombinant hcGST protein (rhcGST) was functionally overexpressed in Escherichia coli cells in a soluble form and purified to homogeneity. rhcGST retained more than 75% of its original GST activity after incubation at pHs 6 to 11. Incubation for 30 min at temperatures below 50 °C scarcely affected the activity. rhcGST was able to catalyze the reaction of glutathione with 1-chloro-2,4-dinitrobenzene, a universal substrate for GST, as well as with 4-hydroxynonenal, a product of lipid peroxidation. We also found that as compared to B. mori Sigma-class GST, rhcGST had a higher affinity for fenitrothion, an organophosphorus insecticide.

Identification of a sigma‐class glutathione‐S‐transferase from the silkworm, Bombyx mori

Abstract:  A cDNA that encodes a glutathione‐S‐transferase belonging to sigma class (GSTS1) from the silkworm, Bombyx mori was cloned by reverse transcriptase‐polymerase chain reaction and sequenced. The deduced amino acid sequence revealed 66%, 48% and 41% identity to sigma‐class GSTs from Manduca sexta, Blattella germanica and Anopheles gambiae, respectively. The GSTS1 was also estimated to be close to those GSTs in a phylogenetic tree. GSTS1 mRNA was widely distributed in various tissues. The recombinant GST (rGSTS1) was functionally overexpressed in Escherichia coli in a soluble form, purified to homogeneity and characterized. The pH‐optimum of rGSTS1 was around pH 8. The rGSTS1 retained more than 75% of its original activity after incubation at pH 4–9. Incubation for 30 min at temperatures below 40°C did not affect its activity. rGSTS1 was able to catalyse the reaction of glutathione with 1‐chloro‐2,4‐dinitrobenzene, the universal substrate for GST, as well as with 4‐hydroxynonenal, the product of lipid peroxidation.

Biochemical properties of an omega-class glutathione S-transferase of the silkmoth, Bombyx mori

A cDNA encoding an omega-class glutathione S-transferase of the silkmoth, Bombyx mori (bmGSTO), was cloned by reverse transcriptase-polymerase chain reaction. The resulting clone was sequenced and deduced for amino acid sequence, which revealed 40, 40, and 39% identities to omega-class GSTs from human, pig, and mouse, respectively. A recombinant protein (rbmGSTO) was functionally overexpressed in Escherichia coli cells in a soluble form and purified to homogeneity. rbmGSTO was able to catalyze the biotranslation of glutathione with 1-chloro-2,4-dinitrobenzene, a model substrate for GST, as well as with 4-hydroxynonenal, a product of lipid peroxidation. This enzyme was shown to have high affinity for organophosphorus insecticide and was present abundantly in silkmoth strain exhibiting fenitrothion resistance. These results indicate that bmGSTO could be involved in the increase in level of insecticide resistance for lepidopteran insects.

The intrinsic ATPase activity of Mycobacterium tuberculosis DnaA promotes rapid oligomerization of DnaA on oriC.

Oligomerization of the initiator protein, DnaA, on the origin of replication (oriC) is crucial for initiation of DNA replication. Studies in Escherichia coli (Gram‐negative) have revealed that binding of DnaA to ATP, but not hydrolysis of ATP, is sufficient to promote DnaA binding, oligomerization and DNA strand separation. To begin understanding the initial events involved in the initiation of DNA replication in Mycobacterium tuberculosis (Gram‐positive), we investigated interactions of M. tuberculosis DnaA (DnaATB) with oriC using surface plasmon resonance in the presence of ATP and ADP. We provide evidence that, in contrast to what is observed in E. coli, ATPase activity of DnaATB promoted rapid oligomerization on oriC. In support, we found that a recombinant mutant DnaATB proficient in binding to ATP, but deficient in ATPase activity, did not oligomerize as rapidly. The corresponding mutation in the dnaA gene of M. tuberculosis resulted in non‐viability, presumably due to a defect in oriC–DnaA interactions. Dimethy sulphate (DMS) footprinting experiments revealed that DnaATB bound to DnaA boxes similarly with ATP or ADP. DnaATB binding to individual DnaA boxes revealed that rapid oligomerization on oriC is triggered only after the initial interaction of DnaA with individual DnaA boxes. We propose that ATPase activity enables the DnaA protomers on oriC to rapidly form oligomeric complexes competent for replication initiation.

Characterization of an omega-class glutathione S-transferase in the stress response of the silkmoth

The glutathione S‐transferase (GST) superfamily is involved in detoxification of various xenobiotics. Using real‐time PCR, mRNA encoding an omega‐class GST of Bombyx mori (bmGSTO) was shown to be induced after exposure to various environmental stresses. A soluble form of recombinant protein (rbmGSTO) was functionally overexpressed in Escherichia coli cells and purified to homogeneity. Cys 38 and Pro 39 were found to be highly conserved in omega‐class GSTs, and their roles were investigated by site‐directed mutagenesis/kinetic analysis. Mutations of Cys 38 and Pro 39 residues affected the catalytic efficiency of enzymes, indicating that the presence of Cys 38 and Pro 39 residues is important for bmGSTO activity. Thus, bmGSTO could contribute to increasing the environmental stress resistance of lepidopteran insects.

Molecular characterization of an insecticide-induced novel glutathione transferase in silkworm

BACKGROUND The glutathione transferase (GST) superfamily is involved in the detoxification of various xenobiotics. We have identified a GST mRNA that was induced in the fat bodies of a silkworm strain exhibiting diazinon resistance and have investigated the enzyme properties of this GST. METHODS A soluble recombinant protein was overexpressed in Escherichia coli. Amino acid residues of interest were changed to alanine by site-directed mutagenesis. RESULTS AND CONCLUSIONS Phylogenetic analysis of the deduced amino acid sequence indicates that this GST belongs to an unclassified group previously reported in mosquitoes. This enzyme, named bmGSTu, has highly conserved amino acid residues, including Tyr7, Ser12 and Asn50. A recombinant bmGSTu was able to catalyze the biotranslation of glutathione with 1-chloro-2,4-dinitrobenzene, a synthetic substrate of GST. Kinetic analysis of bmGSTu mutants indicated that Tyr7, Ser12 and Asn50 are involved in enzyme function. GENERAL SIGNIFICANCE These results support the hypothesis that bmGSTu may play a role in insecticide resistance in Bombyx mori.

Superoxide dismutase from the silkworm, Bombyx mori: sequence, distribution, and overexpression.

Superoxide dismutase (SOD) is an enzyme facilitating the removal of superoxide anions from living organisms. This study focused on SOD from the silkworm, Bombyx mori (bmSOD). cDNA encoding bmSOD was amplified by reverse transcriptase–polymerase chain reaction. The deduced amino acid sequence of bmSOD indicated that the residues forming the Cu/Zn binding site are conserved and that the sequence is in 60% identity to that of the Drosophila melanogaster. B. mori SOD was also close to the D. melanogaster SOD in a phylogenetic tree. The bmSOD mRNA and the enzyme activity were widely distributed in diverse tissues. bmSOD functionally overexpressed in Escherichia coli in a soluble form was purified, and its stability was examined. bmSOD at 4 °C retained almost all of its original activity after incubation at pH 4–11 for 24 h. Incubation (pH 7) for 30 min at temperatures below 40 °C also affected activity insignificantly.

Proteomic studies of lipopolysaccharide-induced polypeptides in the silkworm, Bombyx mori

Silkworm larvae at the 5th instar were injected with lipopolysaccharide from Escherichia coli and inducible polypeptides were examined within a pI range of 3–10 and a size range of 14–97 kDa by proteomics, including peptide mass fingerprinting. No polypeptides were induced in the midgut. FB1 and H1–4 polypeptides were significantly induced in fat body and hemolymph, respectively. FB1 and H1 were estimated to be antitrypsin and serpin-2 proteinase inhibitors respectively. H2 and H3 were novel polypeptides. H4 was estimated to be attacin antibacterial polypeptide with high coverage of sequence. The amounts of all the induced polypeptides decreased at 48 h after the injection.

Modulation of mycobacterium tuberculosis DnaA protein-adenine-nucleotide interactions by acidic phospholipids

The biochemical aspects of the initiation of DNA replication in Mycobacterium tuberculosis are unknown. To understand this process, we overproduced, purified and characterized the recombinant M. tuberculosis DnaA protein. The M. tuberculosis DnaA protein binds the origin of replication (oriC), ATP and ADP, and exhibited weak ATPase activity. ADP, after hydrolysis of ATP, remained strongly associated with DnaA and the exchange of ATP for bound ADP was weak. Vesicles prepared from acidic phospholipids, such as phosphatidylinositol, cardiolipin and phosphatidylglycerol, promoted dissociation of both ADP and ATP, whereas the neutral phospholipid phosphatidylethanolamine did not. The phospholipid-mediated dissociation of ATP was decreased in the presence of the M. tuberculosis oriC, whereas dissociation of ADP was stimulated in the presence of oriC. Acidic phospholipids in micelles, however, were not efficient in dissociating bound nucleotides from DnaA. Together, these results suggest that both polar head groups and membrane bilayer structure play an important role in M. tuberculosis DnaA-adenine-nucleotide interactions. We suggest that initiation of M. tuberculosis oriC involves intimate interactions between DnaA, adenine nucleotides and membrane phospholipids, and the latter helps to ensure that only the ATP form of the DnaA protein interacts continuously with oriC.