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Hiroki Kaneko

Publications and source records attributed to Hiroki Kaneko.

7 recordsLinked to original sources
PubMed2006

Molecular dynamics calculations of wild type vs. mutant protein C: relationship between binding affinity to endothelial cell protein C receptor and hereditary disease.

Molecular dynamics simulations of the protein C gamma-carboxyglutamic acid (Gla) domain and endothelial cell protein C receptor (EPCR) complex were performed to determine the effect of a hereditary disease, which results in a mutation (Gla 25 --> Lys) in the protein C Gla domain. Our results suggest that the Gla 25 --> Lys mutation causes a significant reduction in the binding force between protein C Gla domain and EPCR due to destabilization of the helix structure of EPCR and displacement of a Ca2+ ion.

1-Carboxyglutamic Acid↗
PubMed2005

Rational design of thermostable lactate oxidase by analyzing quaternary structure and prevention of deamidation.

Our current knowledge of protein unfolding is overwhelmingly related to reversible denaturation. However, to engineer thermostable enzymes for industrial applications and medical diagnostics, it is necessary to consider irreversible denaturation processes and/or the entire quaternary structure. In this study we have used lactate oxidase (LOD), which is employed in lactic acid sensors, as a model example to design thermostable variants by rational design. Twelve mutant proteins were tested and one of them displayed a markedly greater thermostability than all the mutants we had previously obtained by random mutagenesis. This mutant was designed so as to strengthen the interaction between the subunits and stabilize the quaternary structure. Since LOD is difficult to crystallize, its three-dimensional structure remains unknown. This study shows that it is possible to carry out rational design to improve thermostability using a computer-aided quaternary structure model based on the known tertiary structure of a related protein. Critical factors required for increasing the thermal stability of proteins by rational design, where the 3-D structure is not available, are discussed.

Amides↗
PubMed2005

Use of random and saturation mutageneses to improve the properties of Thermus aquaticus amylomaltase for efficient production of cycloamyloses.

Amylomaltase from Thermus aquaticus catalyzes intramolecular transglycosylation of alpha-1,4 glucans to produce cyclic alpha-1,4 glucans (cycloamyloses) with degrees of polymerization of 22 and higher. Although the amylomaltase mainly catalyzes the transglycosylation reaction, it also has weak hydrolytic activity, which results in a reduction in the yield of the cycloamyloses. In order to obtain amylomaltase with less hydrolytic activity, random mutagenesis was perfromed for the enzyme gene. Tyr54 (Y54) was identified as the amino acid involved in the hydrolytic activity of the enzyme. When Y54 was replaced with all other amino acids by site-directed mutagenesis, the hydrolytic activities of the mutated enzymes were drastically altered. The hydrolytic activities of the Y54G, Y54P, Y54T, and Y54W mutated enzymes were remarkably reduced compared with that of the wild-type enzyme, while those of the Y54F and Y54K mutated enzymes were similar to that of the wild-type enzyme. Introducing an amino acid replacement at Y54 also significantly affected the cyclization activity of the amylomaltase. The Y54A, Y54L, Y54R, and Y54S mutated enzymes exhibited cyclization activity that was approximately twofold higher than that of the wild-type enzyme. When the Y54G mutated enzyme was employed for cycloamylose production, the yield of cycloamyloses was more than 90%, and there was no decrease until the end of the reaction.

Amino Acid Substitution↗
PubMed2005

Protein C Sapporo (protein C Glu 25 --> Lys): a heterozygous missense mutation in the Gla domain provides new insight into the interaction between protein C and endothelial protein C receptor.

Interaction of the gamma-carboxyglutamic acid (Gla) domain of protein C with endothelial protein C receptor (EPCR) is a critical step for efficient activation of protein C, though interactions by mutants in the Gla domain of protein C with EPCR have been rarely evaluated. We identified a 44-year-old Japanese woman with a history of recurrent thromboembolism as an inherited missense mutation, the first such case reported in Japan, which involved a protein C Gla 25 mutation. Total protein C antigen and Gla protein C antigen levels in the proband were normal. Protein C activity measured with an anticoagulant assay was reduced, whereas that measured with an amidolytic assay was normal. She was therefore phenotypically diagnosed as type IIb protein C deficiency. Direct sequencing of the PCR fragments revealed a heterozygous G to A transition at nucleotide position 1462 in exon 3, which predicted an amino acid substitution of Glu 25 by Lys. Her mother and one son were also heterozygous for this mutation. A molecular dynamics simulation of Gla 25-->Lys/EPCR complex in water suggested that the affinity between the molecules was decreased compared to the wild type Gla domain/EPCR complex. Since Gla 25 has been shown to play an important role in protein C function, not only in membrane phospholipid binding but also in binding to EPCR, our findings provide new insight into the mechanism by which the Glu 25-->Lys mutation induces type IIb protein C deficiency in individuals.

Adult↗
PubMed2003

Effect of mutations at Glu160 and Val198 on the thermostability of lactate oxidase.

We have obtained two types of thermostable mutant lactate oxidase - one that exhibited an E-to-G point mutation at position 160 (E160G) through error-prone PCR-based random mutagenesis, and another that exhibited an E-to-G mutation at position 160 and a V-to-I mutation at position 198 (E160G/V198I) through DNA shuffling-based random mutagenesis - both of which we have previously reported. Our molecular modeling of lactate oxidase suggests that the substitution of G for E at position 160 reduces the electrostatic repulsion between the negative charges of E160 and E130 in the (beta/alpha)8 barrel structure, but a thermal-inactivation experiment on the five kinds of single-mutant lactate oxidase at position 160 (E160A, E160Q, E160H, E160R, and E160K) showed that the side-chain volume of the amino acid at position 160 mainly contributes to the thermostability of lactate oxidase. We also produced V198I single-mutant lactate oxidase through site-directed mutagenesis, and analysed the thermostability of wild-type, V198I, E160G, and E160G/V198I lactate oxidase enzymes. The half-life of E160G/V198I lactate oxidase at 70 degrees C was about three times longer than that of E160G lactate oxidase, and was about 20 times longer than that of wild-type lactate oxidase. In contrast, the thermostability of the V198I lactate oxidase was almost identical to that of wild-type lactate oxidase. This indicates that the V198I mutation alone does not affect lactate oxidase thermostability, but does affect it when combined with the E160G mutation.

Amino Acid Sequence↗
PubMed2002

Calcium-binding analysis and molecular modeling reveal echis coagulation factor IX/factor X-binding protein has the Ca-binding properties and Ca ion-independent folding of other C-type lectin-like proteins.

Many biologically active heterodimeric proteins of snake venom consist of two C-type lectin-like subunits. One of these proteins, habu IX/X-bp, is a Gla domain-binding protein whose subunits both bind to a Ca2+ ion, with a total of two Ca2+-binding sites. The molecular modeling and Ca2+-binding analysis of echis IX/X-bp revealed that it lacks one of two Ca2+-binding sites, though the folding of this subunit is conserved. It is concluded that heterodimeric C-type lectin-like proteins function independent of Ca2+ and have essentially a similar folding to habu IX/X-bp.

Amino Acid Sequence↗