Biomedical subjects

Shinzaburo Takamiya

Publications and source records attributed to Shinzaburo Takamiya.

5 recordsLinked to original sources

PubMed2006

Unique structure of Ascaris suum b5-type cytochrome: an additional alpha-helix and positively charged residues on the surface domain interact with redox partners.

Cytochrome b5 of the body wall of adult Ascaris suum, a porcine parasitic nematode, is a soluble protein that lacks a C-terminal membrane-anchoring domain, but possesses an N-terminal pre-sequence of 30 amino acids. During the maturation of cytochrome b5, the N-terminal pre-sequence is proteolytically cleaved to form the mature protein of 82 amino acid residues. A. suum cytochrome b5 is a basic protein containing more lysine residues and exhibiting a higher midpoint redox potential than its mammalian counterparts. We developed an expression system for the production of the recombinant nematode cytochrome b5, which is chemically and functionally identical with the native protein. Using this recombinant protein, we have determined the X-ray crystal structure of A. suum cytochrome b5 at 1.8 A (1 A=0.1 nm) resolution, and we have shown that this protein is involved in the reduction of nematode body-wall metmyoglobin. The crystal structure of A. suum cytochrome b5 consists of six alpha-helices and five beta-strands. It differs from its mammalian counterparts by having a head-to-tail disulphide bridge, as well as a four-residue insertion in the vicinity of the sixth ligating histidine, which forms an additional alpha-helix, alpha4A, between helices alpha4 and alpha5. A. suum cytochrome b5 exists predominantly as a haem-orientation B isomer. Furthermore, the haem plane is rotated approx. 80 degrees relative to the axis formed by haem-Fe and N atoms of the two histidine residues that are ligated to haem-Fe. The charge distribution around the haem crevice of A. suum cytochrome b5 is remarkably different from that of mammalian cytochrome b5 in that the nematode protein bears positively charged lysine residues surrounding the haem crevice. Using immunohistochemistry, we found that A. suum cytochrome b5 is present in the nematode hypodermis. Based on this histochemical and structural information, the physiological function of A. suum cytochrome b5 and its interaction with nematode metmyoglobin can be hypothesized.

Amino Acid Sequence↗

PubMed2005

Parasite mitochondria as a target of chemotherapy: inhibitory effect of licochalcone A on the Plasmodium falciparum respiratory chain.

Parasites have exploited unique energy metabolic pathways as adaptations to the natural host habitat. In fact, the respiratory systems of parasites typically show greater diversity in electron transfer pathways than do those of host animals. These unique aspects of parasite mitochondria and related enzymes may represent promising targets for chemotherapy. Natural products have been recognized as a source of the candidates of the specific inhibitors for such parasite respiratory chains. Chalcones was recently evaluated for its antimalarial activity in vitro and in vivo. However, its target is still unclear in malaria parasites. In this study, we investigated that licochalcone A inhibited the bc1 complex (ubiquinol-cytochrome c reductase) as well as complex II (succinate ubiquinone reductase, SQR) of Plasmodium falciparum mitochondria. In particular, licochalcone A inhibits bc1 complex activity at very low concentrations. Because the property of the P. falciparum bc1 complex is different from that of the mammalian host, chalcones would be a promising candidate for a new antimalarial drug.

Animals↗

PubMed2003

Heterologous expression of Ascaris suum cytochrome b5 precursor protein: a histidine-tagged full-length presequence is correctly processed to transport the mature protein to the periplasm of Escherichia coli.

The cytochrome b(5) of the body wall of adult Ascaris suum, a porcine parasitic nematode, is a novel type of cytochrome b(5). It is a soluble protein that lacks the COOH-terminal membrane-anchoring domain found in erythrocyte cytochrome b(5), but possesses an NH(2)-terminal extension (presequence) of 30 amino acids that are missing from the 82-residue protein purified from the nematode tissues [Yu, Y., Yamasaki, H., Kita, K., and Takamiya, S., 1996, Arch. Biochem. Biophys. 328, 165-172]. The nematode cytochrome b(5) is, therefore, probably synthesized as a precursor protein whose presequence is cleaved to form a mature protein, but the localization of the mature protein is still unknown. To investigate the processing of the putative precursor protein, the wild-type precursor of nematode cytochrome b(5) with a complete presequence (b5wt) and its NH(2) terminus-truncated derivatives, b5Delta18 and b5Delta28, with 18 and 28 residues deleted, respectively, were expressed using pET-28a(+) vector in Escherichia coli. As expected, all transformants, tb5wt, tb5Delta18, and tb5Delta28, produced recombinant proteins with a histidine-tagged NH(2)-terminal extension. However, only the recombinant protein with the full-length presequence, produced in tb5wt, was correctly processed and transported to the periplasm, from which the majority of the induced product was purified as a mature protein chemically and functionally identical to the native protein purified from the nematode body wall. These results clearly show that the nematode histidine-tagged presequence functions as a signal peptide in E. coli.

Amino Acid Sequence↗

PubMed2003

Isolation and characterization of the stage-specific cytochrome b small subunit (CybS) of Ascaris suum complex II from the aerobic respiratory chain of larval mitochondria.

We recently reported that Ascaris suum mitochondria express stage-specific isoforms of complex II: the flavoprotein subunit and the small subunit of cytochrome b (CybS) of the larval complex II differ from those of adult enzyme, while two complex IIs share a common iron-sulfur cluster subunit (Ip). In the present study, A. suum larval complex II was highly purified to characterize the larval cytochrome b subunits in more detail. Peptide mass fingerprinting and N-terminal amino acid sequencing showed that the larval and adult cytochrome b (CybL) proteins are identical. In contrast, cDNA sequences revealed that the small subunit of larval cytochrome b (CybS(L)) is distinct from the adult CybS (CybS(A)). Furthermore, Northern analysis and immunoblotting showed stage-specific expression of CybS(L) and CybS(A) in larval and adult mitochondria, respectively. Enzymatic assays revealed that the ratio of rhodoquinol-fumarate reductase (RQFR) to succinate-ubiquinone reductase (SQR) activities and the K(m) values for quinones are almost identical for the adult and larval complex IIs, but that the fumarate reductase (FRD) activity is higher for the adult form than for the larval form. These results indicate that the adult and larval A. suum complex IIs have different properties than the complex II of the mammalian host and that the larval complex II is able to function as a RQFR. Such RQFR activity of the larval complex II would be essential for rapid adaptation to the dramatic change of oxygen availability during infection of the host.

Aerobiosis↗

PubMed2002

Electron-transfer complexes in Ascaris mitochondria.

Parasites have developed a variety of physiological functions necessary for their survival within the specialized environment of the host. Using metabolic systems that are very different from those of the host, they can adapt to low oxygen tension present within the host animals. Most parasites do not use the oxygen available within the host to generate ATP, but rather employ anaerobic metabolic pathways. In addition, all parasites have a life cycle. In many cases, the parasite employs aerobic metabolism during its free-living stage outside the host. In such systems, parasite mitochondria play diverse roles. In particular, marked changes in the morphology and components of the mitochondria during the life cycle are very interesting elements of biological processes such as developmental control and environmental adaptation. Recent research on the respiratory chain of the parasitic helminth Ascaris suum has shown that the mitochondrial NADH-fumarate reductase system plays an important role in the anaerobic energy metabolism of adult parasites inhabiting hosts, as well as describing unique features of the developmental changes that occur during its life cycle.

Anaerobiosis↗