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Yoshihisa Hagihara

Publications and source records attributed to Yoshihisa Hagihara.

18 recordsLinked to original sources
PubMed2006

Structure of the catalytic domain of the hyperthermophilic chitinase from Pyrococcus furiosus.

The crystal structure of the catalytic domain of a chitinase from the hyperthermophilic archaeon Pyrococcus furiosus (AD2(PF-ChiA)) has been determined at 1.5 A resolution. This is the first structure of the catalytic domain of an archaeal chitinase. The overall structure of AD2(PF-ChiA) is a TIM-barrel fold with a tunnel-like active site that is a common feature of family 18 chitinases. Although the catalytic residues (Asp522, Asp524 and Glu526) are conserved, comparison of the conserved residues and structures with those of other homologous chitinases indicates that the catalytic mechanism of PF-ChiA is different from that of family 18 chitinases.

Amino Acid Sequence↗
PubMed2006

Crystallization and X-ray diffraction analysis of a catalytic domain of hyperthermophilic chitinase from Pyrococcus furiosus.

The crystallization and preliminary X-ray diffraction analysis of a catalytic domain of chitinase (PF1233 gene) from the hyperthermophilic archaeon Pyrococcus furiosus is reported. The recombinant protein, prepared using an Escherichia coli expression system, was crystallized by the hanging-drop vapour-diffusion method. An X-ray diffraction data set was collected at the undulator beamline BL44XU at SPring-8 to a resolution of 1.50 angstroms. The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 90.0, b = 92.8, c = 107.2 angstroms.

Amino Acid Sequence↗
PubMed2005

Structural studies reveal that the diverse morphology of beta(2)-microglobulin aggregates is a reflection of different molecular architectures.

Amyloid deposition accompanies over 20 degenerative diseases in human, including Alzheimer's, Parkinson's, and prion diseases. Recent studies revealed the importance of other type of protein aggregates, e.g., non-specific aggregates, protofibrils, and small oligomers in the development of such diseases and proved their increased toxicity for living cells in comparison with mature amyloid fibrils. We carried out a comparative structural analysis of different monomeric and aggregated states of beta(2)-microglobulin, a protein responsible for hemodialysis-related amyloidosis. We investigated the structure of the native and acid-denatured states, as well as that of mature fibrils, immature fibrils, amorphous aggregates, and heat-induced filaments, prepared under various in vitro conditions. Infrared spectroscopy demonstrated that the beta-sheet compositions of immature fibrils, heat-induced filaments and amorphous aggregates are characteristic of antiparallel intermolecular beta-sheet structure while mature fibrils are different from all others suggesting a unique overall structure and assembly. Filamentous aggregates prepared by heat treatment are of importance in understanding the in vivo disease because of their stability under physiological conditions, where amyloid fibrils and protofibrils formed at acidic pH depolymerize. Atomic force microscopy of heat-induced filaments represented a morphology similar to that of the low pH immature fibrils. At a pH close to the pI of the protein, amorphous aggregates were formed readily with association of the molecules in native-like conformation, followed by formation of intermolecular beta-sheet structure in a longer time-scale. Extent of the core buried from the solvent in the various states was investigated by H/D exchange of the amide protons.

Amyloid↗
PubMed2005

Cellular quality control screening to identify amino acid pairs for substituting the disulfide bonds in immunoglobulin fold domains.

We are interested in determining which amino acid pairs can be substituted for the disulfide (S-S) bonds in proteins without disrupting their native structures under physiological conditions. In this study, we focused on the intradomain S-S bonds in Ig fold domains and aimed to determine a simple rule for replacement of their S-S bonds. The cysteines of four different Ig fold domains were mutated randomly, and the amino acid pairs substituted for the S-S bonds were screened by the method utilizing a cellular quality control system. Among the 36 selected mutants, 31 were natively folded without S-S bonds, as judged from the cooperativity of thermal unfolding. In addition, the selected mutant llama heavy chain antibodies retained antigen-binding affinity. At least two of the pairs Ala:Ala, Ala:Val, Val: Ala, and Val:Val were found in the selected mutants for all four different Ig fold domains, and they were stably folded at 30 degrees C. This suggests that examination of these four pairs could be enough to obtain natively folded Ig fold domains without S-S bonds.

Amino Acid Sequence↗
PubMed2005

Crystallization and preliminary X-ray diffraction analysis of a chitin-binding domain of hyperthermophilic chitinase from Pyrococcus furiosus.

The crystallization and preliminary X-ray diffraction analysis of the chitin-binding domain of chitinase from a hyperthermophilic archaeon, Pyrococcus furiosus, are reported. The recombinant protein was prepared using an Escherichia coli overexpression system and was crystallized by the hanging-drop vapour-diffusion method. An X-ray diffraction data set was collected to 1.70 A resolution. The crystal belonged to space group P4(3)2(1)2 or P4(1)2(1)2. The unit-cell parameters were determined to be a = b = 48.8, c = 85.0 A.

Chitin↗
PubMed2005

Crystallization and preliminary X-ray diffraction analysis of thioredoxin peroxidase from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1.

Thioredoxin peroxidase is a member of the peroxiredoxin family and plays a dominant role in a hydrogen peroxide metabolism. A recombinant form of the hyperthermostable thioredoxin peroxidase from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1, a polypeptide consisting of 250 amino acids, was purified. The C207S mutant protein was crystallized by the hanging-drop vapour-diffusion method using potassium sodium tartrate as the precipitant at 298 K. Diffraction data were collected and processed to 2.7 A resolution. The crystal belongs to space group P1, with unit-cell parameters a = 126.2, b = 126.3, c = 213.7 A, alpha = 80.4, beta = 80.3, gamma = 70.7 degrees. Calculation of the self-rotation function showed that the protein quaternary structure includes a fivefold axis and five twofold axes.

Aeropyrum↗
PubMed2005

Amyloid fibril formation by the CAD domain of caspase-activated DNase.

Caspase-activated DNase (CAD) is a key protein in the process of apoptosis that degrades DNA through the action of caspases. Its N-terminal region, the CAD domain (CAD-CD), is highly conserved among CAD family proteins and is responsible for the interaction with its inhibitor. We report here that CAD-CD spontaneously aggregates to form amyloid fibrils, without a lag time, under the conditions of low pH (below 4) and the presence of anions. Interestingly, the secondary structure of CAD-CD in the fibril state comprised not only beta-sheet but also alpha-helix, as found in CD, FTIR, and x-ray fiber diffraction experiments. Aromatic side chains have a defined orientation and are in the hydrophobic environment occurring with the CAD-CD fibrillogenesis. These findings provide new insights into the architecture of amyloid fibrils.

Amyloid↗
PubMed2005

Identification of the region responsible for fibril formation in the CAD domain of caspase-activated DNase.

Caspase-activated DNase (CAD) has a compact domain at its N-terminus (CAD domain, 87 amino acid residues), which comprises one alpha-helix and five beta-strands forming a single sheet. The CAD domain of CAD (CAD-CD) forms amyloid fibrils containing alpha-helix at low pH in the presence of salt. To obtain insights into the mechanism of amyloid fibril formation, we identified the peptide region essential for fibril formation of CAD-CD and the region responsible for the salt requirement. We searched for these regions by constructing a series of deletion and point mutants of CAD-CD. Fibril formation by these CAD-CD mutants was examined by fluorescence analysis of thioflavin T and transmission electron microscopy. C-Terminal deletion and point mutation studies revealed that an aromatic residue near the C-terminus (Trp81) is critical for fibril formation. In addition, the main chain conformation of the beta5 strand, which forms a hydrophobic core with Trp81, was found to be important for the fibril formation by CAD-CD. The N-terminal 30 amino acid region containing two beta-strands was not essential for fibril formation. Rather, the N-terminal region was found to be responsible for the requirement of salt for fibril formation.

Amyloid↗
PubMed2003

Amyloid fibril formation in the context of full-length protein: effects of proline mutations on the amyloid fibril formation of beta2-microglobulin.

Beta2-microglobulin (beta2-m), a typical immunoglobulin domain made of seven beta-strands, is a major component of amyloid fibrils formed in dialysis-related amyloidosis. To understand the mechanism of amyloid fibril formation in the context of full-length protein, we prepared various mutants in which proline (Pro) was introduced to each of the seven beta-strands of beta2-m. The mutations affected the amyloidogenic potential of beta2-m to various degrees. In particular, the L23P, H51P, and V82P mutations significantly retarded fibril extension at pH 2.5. Among these, only L23P is included in the known "minimal" peptide sequence, which can form amyloid fibrils when isolated as a short peptide. This indicates that the residues in regions other than the minimal sequence, such as H51P and V82P, determine the amyloidogenic potential in the full-length protein. To further clarify the mutational effects, we measured their stability against guanidine hydrochloride of the native state at pH 8.0 and the amyloid fibrils at pH 2.5. The amyloidogenicity of mutants showed a significant correlation with the stability of the amyloid fibrils, and little correlation was observed with that of the native state. It has been proposed that the stability of the native state and the unfolding rate to the amyloidogenic precursor as well as the conformational preference of the denatured state determine the amyloidogenicity of the proteins. The present results reveal that, in addition, stability of the amyloid fibrils is a key factor determining the amyloidogenic potential of the proteins.

Amyloid↗
PubMed2002

Screening for stable mutants with amino acid pairs substituted for the disulfide bond between residues 14 and 38 of bovine pancreatic trypsin inhibitor (BPTI).

We have developed a screening method to identify stable protein mutants from a large number of sequences using a cellular quality control system. This method was used to screen amino acid pairs substituted for the disulfide (S-S) bond between residues 14 and 38 of bovine pancreatic trypsin inhibitor. The mutants selected could be divided into two groups: one with mutation C14G and the other with mutation C38V. Although each mutation did not fully compensate for the destabilizing effect of removal of the S-S bond, these mutants have midpoint temperatures of thermal unfolding that are 12-17 degrees C higher than that of the C14A/C38A mutant. This fact indicates that these mutations are better substitutions for the S-S bond than C14A/C38A. The C14G mutants inhibited trypsin more strongly at 37 degrees C than did the C14A/C38A mutant, although bulky amino acids at position 14 largely diminished the inhibitory activity of the C38V mutants. Thermodynamic analysis indicated that the enthalpy of unfolding of the C14G and C38V mutant groups differed considerably, which suggests different stabilizing mechanisms in these two groups. Because renaturation of S-S bonds is often difficult in the large scale production of proteins, this method should provide a useful tool with which to increase the production of recombinant proteins by eliminating S-S bonds with minimum concomitant stability loss.

Amino Acid Sequence↗
PubMed2002

Toward development of a screen to identify randomly encoded, foldable sequences.

The ability to identify sequences in a randomly encoded polypeptide library that are capable of acquiring unique and stably folded structures would be valuable in the examination of protein-folding issues. The quality control system of the yeast secretory pathway prevents the release of incompletely folded polypeptides. Earlier work has shown that this feature can be used in a screen to identify mutations that increase the stability of a protein. We sought to extend this strategy for use with random sequence libraries by combining a quality-control system-based screen with generic tag-based immunodetection that can be applied to any sequence. To test this method, we screened a library encoding random mutations in a bovine pancreatic trypsin inhibitor variant containing a small generic tag. Initial on-plate screening resulted in a large number of false positives: sequences that were secreted but not foldable. These false positives were excluded successfully in additional screening steps that used a liquid-culture secretion screen and a gel electrophoresis assay. Three positive clones were obtained that showed midpoint thermal denaturation temperatures 10-16 degrees C higher than the original bovine pancreatic trypsin inhibitor variant. Thus, this multistep screening method may be useful for finding novel, foldable sequences.

Amino Acid Sequence↗
PubMed2002

Aggregation of beta(2)-glycoprotein I induced by sodium lauryl sulfate and lysophospholipids.

beta(2)-Glycoprotein I (beta(2)-GPI) is a plasma protein that binds to negatively charged substances such as DNA, heparin, and anionic phospholipids. The interaction of beta(2)-GPI with anionic phospholipids is intriguing in the context of the autoimmune disease antiphospholipid syndrome. To extend understanding of the binding mechanism to phospholipids, the interactions of beta(2)-GPI with amphiphiles, i.e., sodium lauryl sulfate and lysophospholipids, were examined. These amphiphiles induced the aggregation of beta(2)-GPI below the critical micelle concentration, indicating that the interaction of beta(2)-GPI with monodispersed amphiphiles is unstable, resulting in the formation of large aggregates. However, highly soluble monocaproylphosphatidic acid did not induce aggregation, suggesting that the hydrophobicity of the acyl chain is also an important factor for aggregate formation in addition to negative charges in the headgroup. A series of experiments using deletion mutants and a peptide showed that the fifth domain of beta(2)-GPI (domain V) is responsible for formation of aggregates observed for intact full-length beta(2)-GPI. In addition, the flexible loop (F307-C326) in the C-terminal of domain V, which consists of hydrophobic and positively charged residues, was identified as the important region for aggregation. These results indicate that beta(2)-GPI binds to the amphiphiles through the flexible loop of domain V, resulting in formation of large aggregates where both electrostatic and hydrophobic interactions are involved.

Amino Acid Sequence↗
PubMed2002

Mapping the core of the beta(2)-microglobulin amyloid fibril by H/D exchange.

Despite numerous efforts, the lack of detailed structural information on amyloid fibrils has hindered clarification of the mechanism of their formation. Here, we describe a novel procedure for characterizing the conformational flexibility of beta(2)-microglobulin amyloid fibrils at single-residue resolution that uses H/D exchange of amide protons combined with NMR analysis. The results indicate that most residues in the middle region of the molecule, including the loop regions in the native structure, form a rigid beta-sheet core, whereas the the N- and C-termini are excluded from this core. The extensively hydrogen-bonded beta-sheet core explains the remarkable rigidity and stability of amyloid fibrils. The present method could be used to obtain residue-specific conformational information of various amyloid fibrils, even though it does not provide a high resolution three-dimensional structure.

Amyloidosis↗
PubMed2002

The intrachain disulfide bond of beta(2)-microglobulin is not essential for the immunoglobulin fold at neutral pH, but is essential for amyloid fibril formation at acidic pH.

beta(2)-Microglobulin (beta2M), the light chain of the type I major histocompatibility complex, is a major component of dialysis-related amyloid fibrils. beta2M in the native state has a typical immunoglobulin fold with a buried intrachain disulfide bond. The conformation and stability of recombinant beta2M in which the intrachain disulfide bond was reduced were studied by CD, tryptophan fluorescence, and one-dimensional NMR. The conformation of the reduced beta2M in the absence of denaturant at pH 8.5 was similar to that of the intact protein unless the thiol groups were modified. However, reduction of the disulfide bond decreased the stability as measured by denaturation in guanidine hydrochloride. Intact beta2M formed amyloid fibrils at pH 2.5 by extension reaction using sonicated amyloid fibrils as seeds. Under the same conditions, reduced beta2M did not form typical amyloid fibrils, although it inhibited fibril extension competitively, suggesting that the conformation defined by the disulfide bond is important for amyloid fibril formation of beta2M.

Amyloid↗
PubMed2002

The role of disulfide bond in the amyloidogenic state of beta(2)-microglobulin studied by heteronuclear NMR.

beta(2)-Microglobulin (beta2-m) is a major component of dialysis-related amyloid fibrils. Although recombinant beta2-m forms needle-like fibrils by in vitro extension reaction at pH 2.5, reduced beta2-m, in which the intrachain disulfide bond is reduced, cannot form typical fibrils. Instead, thinner and flexible filaments are formed, as shown by atomic force microscopy images. To clarify the role of the disulfide bond in amyloid fibril formation, we characterized the conformations of the oxidized (intact) and reduced forms of beta2-m in the acid-denatured state at pH 2.5, as well as the native state at pH 6.5, by heteronuclear NMR. [(1)H]-(15)N NOE at the regions between the two cysteine residues (Cys25-Cys80) revealed a marked difference in the pico- and nanosecond time scale dynamics between that the acid-denatured oxidized and reduced states, with the former showing reduced mobility. Intriguingly, the secondary chemical shifts, DeltaCalpha, DeltaCO, and DeltaHalpha, and (3)J(HNHalpha) coupling constants indicated that both the oxidized and reduced beta2-m at pH 2.5 have marginal alpha-helical propensity at regions close to the C-terminal cysteine, although it is a beta-sheet protein in the native state. The results suggest that the reduced mobility of the denatured state is an important factor for the amylodogenic potential of beta2-m, and that the marginal helical propensity at the C-terminal regions might play a role in modifying this potential.

Amino Acid Sequence↗
PubMed2001

Investigation of a peptide responsible for amyloid fibril formation of beta 2-microglobulin by achromobacter protease I.

To obtain insight into the mechanism of amyloid fibril formation from beta(2)-microglobulin (beta2-m), we prepared a series of peptide fragments using a lysine-specific protease from Achromobacter lyticus and examined their ability to form amyloid fibrils at pH 2.5. Among the nine peptides prepared by the digestion, the peptide Ser(20)-Lys(41) (K3) spontaneously formed amyloid fibrils, confirmed by thioflavin T binding and electron microscopy. The fibrils composed of K3 peptide induced fibril formation of intact beta2-m with a lag phase, distinct from the extension reaction without a lag phase observed for intact beta2-m seeds. Fibril formation of K3 peptide with intact beta2-m seeds also exhibited a lag phase. On the other hand, the extension reaction of K3 peptide with the K3 seeds occurred without a lag phase. At neutral pH, the fibrils composed of either intact beta2-m or K3 peptide spontaneously depolymerized. Intriguingly, the depolymerization of K3 fibrils was faster than that of intact beta2-m fibrils. These results indicated that, although K3 peptide can form fibrils by itself more readily than intact beta2-m, the K3 fibrils are less stable than the intact beta2-m fibrils, suggesting a close relation between the free energy barrier of amyloid fibril formation and its stability.

Amino Acid Sequence↗