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1.  Diverse allosteric and catalytic functions of tetrameric d-lactate dehydrogenases from three Gram-negative bacteria 
AMB Express  2014;4:76.
NAD-dependent d-lactate dehydrogenases (d-LDHs) reduce pyruvate into d-lactate with oxidation of NADH into NAD+. Although non-allosteric d-LDHs from Lactobacilli have been extensively studied, the catalytic properties of allosteric d-LDHs from Gram-negative bacteria except for Escherichia coli remain unknown. We characterized the catalytic properties of d-LDHs from three Gram-negative bacteria, Fusobacterium nucleatum (FNLDH), Pseudomonas aeruginosa (PALDH), and E. coli (ECLDH) to gain an insight into allosteric mechanism of d-LDHs. While PALDH and ECLDH exhibited narrow substrate specificities toward pyruvate like usual d-LDHs, FNLDH exhibited a broad substrate specificity toward hydrophobic 2-ketoacids such as 2-ketobutyrate and 2-ketovalerate, the former of which gave a 2-fold higher kcat/S0.5 value than pyruvate. Whereas the three enzymes consistently showed hyperbolic shaped pyruvate saturation curves below pH 6.5, FNLDH and ECLDH, and PALDH showed marked positive and negative cooperativity, respectively, in the pyruvate saturation curves above pH 7.5. Oxamate inhibited the catalytic reactions of FNLDH competitively with pyruvate, and the PALDH reaction in a mixed manner at pH 7.0, but markedly enhanced the reactions of the two enzymes at low concentration through canceling of the apparent homotropic cooperativity at pH 8.0, although it constantly inhibited the ECLDH reaction. Fructose 1,6-bisphosphate and certain divalent metal ions such as Mg2+ also markedly enhanced the reactions of FNLDH and PALDH, but none of them enhanced the reaction of ECLDH. Thus, our study demonstrates that bacterial d-LDHs have highly divergent allosteric and catalytic properties.
PMCID: PMC4230899  PMID: 25401076
Allosteric regulation; NAD-dependent d-lactate dehydrogenase; Gram-negative bacteria; Escherichia coli; Fusobacterium nucleatum; Pseudomonas aeruginosa
2.  1,2-β-Oligoglucan Phosphorylase from Listeria innocua 
PLoS ONE  2014;9(3):e92353.
We characterized recombinant Lin1839 protein (Lin1839r) belonging to glycoside hydrolase family 94 from Listeria innocua. Lin1839r catalyzed the synthesis of a series of 1,2-β-oligoglucans (Sopn: n denotes degree of polymerization) using sophorose (Sop2) as the acceptor and α-d-glucose 1-phosphate (Glc1P) as the donor. Lin1839r recognized glucose as a very weak acceptor substrate to form polymeric 1,2-β-glucan. The degree of polymerization of the 1,2-β-glucan gradually decreased with long-term incubation to generate a series of Sopns. Kinetic analysis of the phosphorolytic reaction towards sophorotriose revealed that Lin1839r followed a sequential Bi Bi mechanism. The kinetic parameters of the phosphorolysis of sophorotetraose and sophoropentaose were similar to those of sophorotriose, although the enzyme did not exhibit significant phosphorolytic activity on Sop2. These results indicate that the Lin1839 protein is a novel inverting phosphorylase that catalyzes reversible phosphorolysis of 1,2-β-glucan with a degree of polymerization of ≥3. We propose 1,2-β-oligoglucan: phosphate α-glucosyltransferase as the systematic name and 1,2-β-oligoglucan phosphorylase as the short name for this Lin1839 protein.
PMCID: PMC3960220  PMID: 24647662
3.  FOXC2 Mutations in Familial and Sporadic Spinal Extradural Arachnoid Cyst 
PLoS ONE  2013;8(11):e80548.
Spinal extradural arachnoid cyst (SEDAC) is a cyst in the spinal canal that protrudes into the epidural space from a defect in the dura mater. Most cases are sporadic; however, three familial SEDAC cases have been reported, suggesting genetic etiological factors. All familial cases are associated with lymphedema-distichiasis syndrome (LDS), whose causal gene is FOXC2. However, FOXC2 mutation analysis has been performed in only 1 family, and no mutation analysis has been performed on sporadic (non-familial) SEDACs. We recruited 17 SEDAC subjects consisting of 2 familial and 7 sporadic cases and examined FOXC2 mutations by Sanger sequencing and structural abnormalities by TaqMan copy number assay. We identified 2 novel FOXC2 mutations in 2 familial cases. Incomplete LDS penetrance was noted in both families. Four subjects presented with SEDACs only. Thus, SEDAC caused by the heterozygous FOXC2 loss-of-function mutation should be considered a feature of LDS, although it often manifests as the sole symptom. Seven sporadic SEDAC subjects had no FOXC2 mutations, no symptoms of LDS, and showed differing clinical characteristics from those who had FOXC2 mutations, suggesting that other gene(s) besides FOXC2 are likely to be involved in SEDAC.
PMCID: PMC3838418  PMID: 24278289
4.  Meta-analysis of genome-wide association studies confirms a susceptibility locus for knee osteoarthritis on chromosome 7q22 
Evangelou, Evangelos | Valdes, Ana M. | Kerkhof, Hanneke J.M | Styrkarsdottir, Unnur | Zhu, YanYan | Meulenbelt, Ingrid | Lories, Rik J. | Karassa, Fotini B. | Tylzanowski, Przemko | Bos, Steffan D. | Akune, Toru | Arden, Nigel K. | Carr, Andrew | Chapman, Kay | Cupples, L. Adrienne | Dai, Jin | Deloukas, Panos | Doherty, Michael | Doherty, Sally | Engstrom, Gunnar | Gonzalez, Antonio | Halldorsson, Bjarni V. | Hammond, Christina L. | Hart, Deborah J. | Helgadottir, Hafdis | Hofman, Albert | Ikegawa, Shiro | Ingvarsson, Thorvaldur | Jiang, Qing | Jonsson, Helgi | Kaprio, Jaakko | Kawaguchi, Hiroshi | Kisand, Kalle | Kloppenburg, Margreet | Kujala, Urho M. | Lohmander, L. Stefan | Loughlin, John | Luyten, Frank P. | Mabuchi, Akihiko | McCaskie, Andrew | Nakajima, Masahiro | Nilsson, Peter M. | Nishida, Nao | Ollier, William E.R. | Panoutsopoulou, Kalliope | van de Putte, Tom | Ralston, Stuart H. | Rivadeneira, Fernado | Saarela, Janna | Schulte-Merker, Stefan | Slagboom, P. Eline | Sudo, Akihiro | Tamm, Agu | Tamm, Ann | Thorleifsson, Gudmar | Thorsteinsdottir, Unnur | Tsezou, Aspasia | Wallis, Gillian A. | Wilkinson, J. Mark | Yoshimura, Noriko | Zeggini, Eleftheria | Zhai, Guangju | Zhang, Feng | Jonsdottir, Ingileif | Uitterlinden, Andre G. | Felson, David T | van Meurs, Joyce B. | Stefansson, Kari | Ioannidis, John P.A. | Spector, Timothy D.
Annals of the rheumatic diseases  2010;70(2):349-355.
Osteoarthritis (OA) is the most prevalent form of arthritis and accounts for substantial morbidity and disability, particularly in the elderly. It is characterized by changes in joint structure including degeneration of the articular cartilage and its etiology is multifactorial with a strong postulated genetic component. We performed a meta-analysis of four genome-wide association (GWA) studies of 2,371 knee OA cases and 35,909 controls in Caucasian populations. Replication of the top hits was attempted with data from additional ten replication datasets. With a cumulative sample size of 6,709 cases and 44,439 controls, we identified one genome-wide significant locus on chromosome 7q22 for knee OA (rs4730250, p-value=9.2×10−9), thereby confirming its role as a susceptibility locus for OA. The associated signal is located within a large (500kb) linkage disequilibrium (LD) block that contains six genes; PRKAR2B (protein kinase, cAMP-dependent, regulatory, type II, beta), HPB1 (HMG-box transcription factor 1), COG5 (component of oligomeric golgi complex 5), GPR22 (G protein-coupled receptor 22), DUS4L (dihydrouridine synthase 4-like), and BCAP29 (the B-cell receptor-associated protein 29). Gene expression analyses of the (six) genes in primary cells derived from different joint tissues confirmed expression of all the genes in the joint environment.
PMCID: PMC3615180  PMID: 21068099
5.  A Novel Glycosylphosphatidylinositol-Anchored Glycoside Hydrolase from Ustilago esculenta Functions in β-1,3-Glucan Degradation 
Applied and Environmental Microbiology  2012;78(16):5682-5689.
A glycoside hydrolase responsible for laminarin degradation was partially purified to homogeneity from a Ustilago esculenta culture filtrate by weak-cation-exchange, strong-cation-exchange, and size-exclusion chromatography. Three proteins in enzymatically active fractions were digested with chymotrypsin followed by liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis, resulting in the identification of three peptide sequences that shared significant similarity to a putative β-1,3-glucanase, a member of glucoside hydrolase family 16 (GH16) from Sporisorium reilianum SRZ2. A gene encoding a laminarin-degrading enzyme from U. esculenta, lam16A, was isolated by PCR using degenerate primers designed based on the S. reilianum SRZ2 β-1,3-glucanase gene. Lam16A possesses a GH16 catalytic domain with an N-terminal signal peptide and a C-terminal glycosylphosphatidylinositol (GPI) anchor peptide. Recombinant Lam16A fused to an N-terminal FLAG peptide (Lam16A-FLAG) overexpressed in Aspergillus oryzae exhibited hydrolytic activity toward β-1,3-glucan specifically and was localized both in the extracellular and in the membrane fractions but not in the cell wall fraction. Lam16A without a GPI anchor signal peptide was secreted extracellularly and was not detected in the membrane fraction. Membrane-anchored Lam16A-FLAG was released completely by treatment with phosphatidylinositol-specific phospholipase C. These results suggest that Lam16A is anchored in the plasma membrane in order to modify β-1,3-glucan associated with the inner cell wall and that Lam16A is also used for the catabolism of β-1,3-glucan after its release in the extracellular medium.
PMCID: PMC3406117  PMID: 22685137
6.  Characterization of β-N-acetylhexosaminidase (LeHex20A), a member of glycoside hydrolase family 20, from Lentinula edodes (shiitake mushroom) 
AMB Express  2012;2:29.
We purified and cloned a β-N-acetylhexosaminidase, LeHex20A, with a molecular mass of 79 kDa from the fruiting body of Lentinula edodes (shiitake mushroom). The gene lehex20a gene had 1,659 nucleotides, encoding 553 amino acid residues. Sequence analysis indicated that LeHex20A belongs to glycoside hydrolase (GH) family 20, and homologues of lehex20a are broadly represented in the genomes of basidiomycetes. Purified LeHex20A hydrolyzed the terminal monosaccharide residues of β-N-acetylgalactosaminides and β-N-acetylglucosaminides, indicating that LeHex20A is a β-N-acetylhexosaminidase classified into EC The maximum LeHex20A activity was observed at pH 4.0 and 50°C. The kinetic constants were estimated using chitooligosaccharides with degree of polymerization 2-6. GH20 β-N-acetylhexosaminidases generally prefer chitobiose among natural substrates. However, LeHex20A had the highest catalytic efficiency (kcat/Km) for chitotetraose, and the Km values for GlcNAc6 were 3.9-fold lower than for chitobiose. Furthermore, the enzyme partially hydrolyzed amorphous chitin polymers. These results indicate that LeHex20A can produce N-acetylglucosamine from long-chain chitomaterials.
PMCID: PMC3430601  PMID: 22656067
β-N-acetylglucosaminide; Chitin; Fungal cell wall; Glycoside hydrolase family 20; Basidiomycete
7.  Recommendations for standardization and phenotype definitions in genetic studies of osteoarthritis: the TREAT-OA consortium 
To address the need for standardization of osteoarthritis (OA) phenotypes by examining the effect of heterogeneity among symptomatic (SOA) and radiographic osteoarthritis (ROA) phenotypes.
Descriptions of OA phenotypes of the 28 studies involved in the TREAT-OA consortium were collected. To investigate whether different OA definitions result in different association results, we created hip OA definitions used within the consortium in the Rotterdam Study-I and tested the association of hip OA with gender, age and BMI using one-way ANOVA. For radiographic OA, we standardized the hip, knee and hand ROA definitions and calculated prevalence's of ROA before and after standardization in 9 cohort studies. This procedure could only be performed in cohort studies and standardization of SOA definitions was not feasible at this moment.
In this consortium, all studies with symptomatic OA phenotypes (knee, hip and hand) used a different definition and/or assessment of OA status. For knee, hip and hand radiographic OA 5, 4 and 7 different definitions were used, respectively. Different hip OA definitions do lead to different association results. For example, we showed in the Rotterdam Study-I that hip OA defined as “at least definite JSN and one definite osteophyte” was not associated with gender (p=0.22), but defined as “at least one definite osteophyte” was significantly associated with gender (p=3×10−9). Therefore, a standardization process was undertaken for radiographic OA definitions. Before standardization a wide range of ROA prevalence's was observed in the 9 cohorts studied. After standardization the range in prevalence of knee and hip ROA was small. Standardization of SOA phenotypes was not possible due to the case-control design of the studies.
Phenotype definitions influence the prevalence of OA and association with clinical variables. ROA phenotypes within the TREAT-OA consortium were standardized to reduce heterogeneity and improve power in future genetics studies.
PMCID: PMC3236091  PMID: 21059398
8.  Prediction model for knee osteoarthritis based on genetic and clinical information 
Arthritis Research & Therapy  2010;12(5):R187.
Osteoarthritis (OA) is the most common bone and joint disease influenced by genetic and environmental factors. Recent association studies have uncovered the genetic factors behind OA, its susceptibility genes, which would enable us to predict disease occurrence based on genotype information. However, most previous studies have evaluated the effects of only a single susceptibility gene, and hence prediction based on such information is not as reliable. Here, we constructed OA-prediction models based on genotype information from a case-control association study and tested their predictability.
We genotyped risk alleles of the three susceptibility genes, asporin (ASPN), growth differentiation factor 5 (GDF5), and double von Willebrand factor A domains (DVWA) for a total of 2,158 Japanese subjects (933 OA and 1,225 controls) and statistically analyzed their effects. After that, we constructed prediction models by using the logistic regression analysis.
When the effects of each allele were assumed to be the same and multiplicative, each additional risk allele increased the odds ratio (OR) by a factor of 1.23 (95% confidence interval (CI), 1.12 to 1.34). Individuals with five or six risk alleles showed significantly higher susceptibility when compared with those with zero or one, with an OR of 2.67 (95% CI, 1.46 to 4.87; P = 0.0020). Statistical evaluation of the prediction power of models showed that a model using only genotyping data had poor predictability. We obtained a model with good predictability by incorporating clinical data, which was further improved by rigorous age adjustment.
Our results showed that consideration of adjusted clinical information, as well as increases in the number of risk alleles to be integrated, is critical for OA prediction by using data from case-control studies. To the authors' knowledge, this is the first report of the OA-prediction model combining both genetic and clinical information.
PMCID: PMC2991022  PMID: 20939878
9.  New Sequence Variants in HLA Class II/III Region Associated with Susceptibility to Knee Osteoarthritis Identified by Genome-Wide Association Study 
PLoS ONE  2010;5(3):e9723.
Osteoarthritis (OA) is a common disease that has a definite genetic component. Only a few OA susceptibility genes that have definite functional evidence and replication of association have been reported, however. Through a genome-wide association study and a replication using a total of ∼4,800 Japanese subjects, we identified two single nucleotide polymorphisms (SNPs) (rs7775228 and rs10947262) associated with susceptibility to knee OA. The two SNPs were in a region containing HLA class II/III genes and their association reached genome-wide significance (combined P = 2.43×10−8 for rs7775228 and 6.73×10−8 for rs10947262). Our results suggest that immunologic mechanism is implicated in the etiology of OA.
PMCID: PMC2841168  PMID: 20305777
10.  Identification of Lacto-N-Biose I Phosphorylase from Vibrio vulnificus CMCP6▿  
Applied and Environmental Microbiology  2008;74(20):6333-6337.
A β-1,3-galactosyl-N-acetylhexosamine phosphorylase (GalGlyNAcP) homolog gene was cloned from Vibrio vulnificus CMCP6. In synthetic reactions, the recombinant enzyme acted only with GlcNAc and GalNAc as acceptors in the presence of α-d-galactose-1-phosphate as a donor to form lacto-N-biose I (LNB) (Galβ1 → 3GlcNAc) and galacto-N-biose (GNB) (Galβ1 → 3GalNAc), respectively. GlcNAc was a much better acceptor than GalNAc. The enzyme also phosphorolysed LNB faster than it phosphorolysed GNB, and the kcat/Km for LNB was approximately 60 times higher than the kcat/Km for GNB. This result indicated that the enzyme was remarkably different from GalGlyNAcP from Bifidobacterium longum, which has similar activities with LNB and GNB, and GalGlyNAcP from Clostridium perfringens, which is a GNB-specific enzyme. The enzyme is the first LNB-specific enzyme that has been found and was designated lacto-N-biose I phosphorylase. The discovery of an LNB-specific GalGlyNAcP resulted in recategorization of bifidobacterial GalGlyNAcPs as galacto-N-biose/lacto-N-biose I phosphorylases.
PMCID: PMC2570299  PMID: 18723650
11.  Association of single-nucleotide polymorphisms in RHOB and TXNDC3 with knee osteoarthritis susceptibility: two case-control studies in East Asian populations and a meta-analysis 
Conflicting findings on the association of single nucleotide polymorphisms (SNPs) in RHOB and TXNDC3 with susceptibility to knee osteoarthritis (OA) have been reported in European Caucasians. To examine the associations of these SNPs with OA in East Asian populations and to evaluate their global significance, we conducted two case-control studies in 955 Chinese and 750 Japanese patients.
We genotyped the previously implicated SNPs rs585017 (in RHOB) and rs4720262 (in TXNDC3) in patients with primary symptomatic knee OA with radiographic confirmation and in matched control individuals, and analyzed their associations. We further conducted a meta-analysis of the study findings together with those of previously reported European studies using the DerSimonian-Laird procedure.
A significant association of RHOB with knee OA was observed in male Chinese patients (P = 0.02). No significant associations were found for RHOB in any other comparisons in the East Asian populations. The association of TXNDC3 was replicated in Chinese female (P = 0.04) and Japanese (P = 0.03) patients, although none of these associations persisted after Bonferroni correction. Significant association (P = 0.02 for the allelic frequency) with nonsignificant heterogeneity was found in the East Asian replication study. No significant association was found in any comparison in the meta-analysis for all studies.
Our study replicates the association, previously reported in European Caucasians, of TXNDC3 with knee OA susceptibility in an East Asian population.
PMCID: PMC2483443  PMID: 18471322
12.  Crystallization and preliminary X-ray analysis of cytosolic α-mannosidase from Thermotoga maritima  
Cytosolic class II α-mannosidase from T. maritima (TM1851), a family 38 glycoside hydrolase, was crystallized. A diffraction data set was collected to 2.9 Å resolution.
Class II α-mannosidase cleaves off α-1,2-, α-1,3- and α-1,6-mannose residues. In this paper, the crystallization and preliminary X-ray analysis of cytosolic class II α-mannosidase from Thermotoga maritima (TM1851), a family 38 glycoside hydrolase, is described. The crystal of recombinant TM1851 belongs to the C-­centred monoclinic space group C2, with unit-cell parameters a = 244.7, b = 87.4, c = 166.6 Å, β = 124.7°. X-ray diffraction data were collected to a resolution of 2.9 Å.
PMCID: PMC2150957  PMID: 16511275
class II α-mannosidase; glycoside hydrolase family 38; Thermotoga maritima; TM1851

Results 1-12 (12)