环糊精葡萄糖基转移酶182位点定点改造催化合成糖基化染料木素

染料木素及其单糖苷衍物在食品和医药领域具有重要作用,但难溶于水的特性极大地限制了其应用范围,研究表明糖基化反应可有效提高其水溶性.文中针对来源于软化芽孢杆菌的环糊精葡萄糖基转移酶,研究其对染料木素单糖苷衍生物槐角苷的糖基化反应.通过对D182位点的定点饱和突变,突变酶D182C较WT转化率提高了13.42％,主要糖基化...     

On the molecular nature of the Duarte variant of galactose-1-phosphate uridyl transferase (GALT)

Galactosemia is an inborn error of galactose metabolism secondary to deficiency of galactose-1-phosphate uridyl transferase (GALT). GALT is a polymorphic enzyme and Duarte (D) is the most common enzyme variant. This variant is characterized by faster electrophoretic mobility and reduced activity. Duarte/galactosemia compound heterozygotes (D/G) are commonly identified in galactosemia newborn screening programs. However, these patients do not generally require treatment. By using a candidate mutation approach to define the molecular basis of the Duarte variant of GALT, a close association between the previously reported N314D polymorphism and the Duarte variant of GALT was found. We suggest that N314D encodes the D variant of GALT and that molecular testing for N314D might be useful to confirm a biochemical diagnosis of Duarte variant of GALT.     

Selective expansion of the newly evolved genomic variants of retrotransposon 1731 in the Drosophila genomes

The structural variants of the regulatory and coding regions of the LTR-retrotransposon 1731 are described. Two classes of genomic copies of retrotransposon 1731, with and without frameshifting strategy to express Gag and Pol proteins, were earlier revealed in the D. melanogaster genome. Copies without frameshifting are shown to be evolved from an ancient variant with frameshifting and are widespread in the genomes of the melanogaster complex species. Position of a rare codon responsible for ribosome pausing and efficient frameshifting is identified. Two structural variants of 1731 LTRs were detected in the melanogaster complex species: the predominant structural variant A1A2 of 1731 LTR in the D. melanogaster, D. simulans, and D. sechellia genomes contains duplicated and diverged copies of 28 bp in the U3 region, whereas A1 variant lacking this duplication is expanded in the D. mauritiana genome. Selective expansion of the A1A2 variant was detected in the independently established D. melanogaster cell cultures. A1A2 variant is expressed in embryos, cell culture, and testes, whereas A1 is expressed only in testes of D. melanogaster. Relief of expression of the A1A2 but not A1 variant in the ovaries as a result of mutation in the RNA interference (RNAi) spn-E gene is shown. Thus, expansion of the recently evolved genomic variants of the LTR retrotransposon 1731 possessing a new translation strategy, duplication in the U3 region, and extended profile of expression is revealed.     

Frequency of single nucleotide polymorphisms of CYP2D6 in the Czech population

CYP2D6 is a member of cytochrome P450 enzymes that metabolise over 25% of commonly used drugs. Genetic polymorphisms can cause insufficient drug efficacy at usually administered doses or can be the cause of adverse drug reaction. CYP2D6 genotyping can be used to predict CYP2D6 phenotype and thereby explain some abnormalities in drug response and thus optimize pharmacotherapy. The aim of this study was to investigate the frequency of functionally important variant alleles of the CYP2D6 gene throughout the Czech population to predict the prevalence of ultra-rapid and poor metabolizer phenotypes. The DNA of 223 unrelated, healthy volunteers was analysed to detect the presence of CYP2D6*6, *5, *4, *3 and gene duplication. The variant allele frequencies in our population were 0.22%, 3.14%, 22.87%, 1.12% and 3.14% for CYP2D6*6, CYP2D6*5, CYP2D6*4, CYP2D6*3 and CYP2D6*MxN, respectively. Fifteen subjects carried two variant alleles leading to predicted poor type of metabolism, 84 subjects were heterozygous extensive metabolizers (het-EM). The full-text contains detailed comparison with European white populations. The distribution of variant alleles complies with the Hardy-Weinberg equilibrium. The frequencies of functional variant alleles of CYP2D6 in Czech population are in concordance with other Caucasian populations.     

The G473D mutation impairs dimerization and catalysis in human sulfite oxidase

Among the mutations identified in patients with isolated sulfite oxidase deficiency, the G473D variant is of particular interest since sedimentation analysis reveals that this variant is a monomer, and the importance of the wild-type dimeric state of mammalian sulfite oxidase is not yet well understood. Analysis of recombinant G473D sulfite oxidase indicated that it is severely impaired both in the ability to bind sulfite and in catalysis, with a second-order rate constant 5 orders of magnitude lower than that of the wild type. To elucidate the specific reasons for the severe effects seen in the G473D variant, several other variants were created, including G473A, G473W, and the double mutant R212A/G473D. Despite the inability to form a stable dimer, the G473W variant had 5-fold higher activity than G473D and nearly wild-type activity at pH 7.0 when ferricyanide was the electron acceptor. In contrast, the R212A/G473D variant demonstrated some ability to oligomerize but had undetectable activity. The G473A variant retained the ability to dimerize and had steady-state activity that was comparable to that of the wild type. Furthermore, stopped-flow analysis of the reductive half-reaction of this variant yielded a rate constant nearly 3 times higher than that of the wild type. Examination of the secondary structures of the variants by CD spectroscopy indicated significant random-coil formation in G473D, G473W, and R212A/G473D. These results demonstrate that both the charge and the large size of an Asp residue in this position contribute to the severe effects seen in a patient with the G473D mutation, by causing partial misfolding and monomerization of sulfite oxidase and attenuating both substrate binding and catalytic efficiency during the reaction cycle.     

Genetic variation of an odorant receptor OR7D4 and sensory perception of cooked meat containing androstenone

Although odour perception impacts food preferences, the effect of genotypic variation of odorant receptors (ORs) on the sensory perception of food is unclear. Human OR7D4 responds to androstenone, and genotypic variation in OR7D4 predicts variation in the perception of androstenone. Since androstenone is naturally present in meat derived from male pigs, we asked whether OR7D4 genotype correlates with either the ability to detect androstenone or the evaluation of cooked pork tainted with varying levels of androstenone within the naturally-occurring range. Consistent with previous findings, subjects with two copies of the functional OR7D4 RT variant were more sensitive to androstenone than subjects carrying a non-functional OR7D4 WM variant. When pork containing varying levels of androstenone was cooked and tested by sniffing and tasting, subjects with two copies of the RT variant tended to rate the androstenone-containing meat as less favourable than subjects carrying the WM variant. Our data is consistent with the idea that OR7D4 genotype predicts the sensory perception of meat containing androstenone and that genetic variation in an odorant receptor can alter food preferences.     

乙型肝炎表面抗原单克隆抗体的制备及其应用

目的：研制能够同时检测乙型肝炎表面抗原(HBsAg) 野生株和多种变异株的单克隆抗体（mAb）,将筛选出的mAb进行纯度、免疫学性状鉴定,并对其应用效果及质量做初步评价。方法：用中国乙型肝炎病毒感染者血清中分离的HBsAg免疫小鼠,利用杂交瘤细胞融合技术制备抗HBsAg野生株和多种变异株的mAb,将筛选出的mAb经饱和硫酸铵纯化后通过聚丙烯酰胺凝胶电泳、琼脂糖凝胶电泳、ELISA鉴定其纯度、特性,初步评价其应用效果及质量。结果：获得了一株能够同时检测HBsAg野生株和多种变异株的mAb,命名为D12。其纯度较高,特异性强,灵敏度好,Ig亚类测定结果为IgG1,识别位点存在于自然抗原上,其检测HBsAg野生株的能力优于现行3种国产试剂盒,检测HBsAg变异株的能力明显优于现行5种国产试剂盒。结论：成功研制了可以同时识别HBsAg野生株和大多数变异株的mAb,为进一步提高我国当前乙型肝炎病毒变异株的检出率以及加强预防和控制乙型肝炎病毒的传播奠定新的基础。     

The role of Glu39 in MnII binding and oxidation by manganese peroxidase from Phanerochaete chrysoporium

Manganese peroxidase (MnP) is a heme-containing enzyme produced by white-rot fungi and is part of the extracellular lignin degrading system in these organisms. MnP is unique among Mn binding enzymes in its ability to bind and oxidize Mn(II) and efficiently release Mn(III). Initial site-directed mutagenesis studies identified the residues E35, E39, and D179 as the Mn binding ligands. However, an E39D variant was recently reported to display wild-type Mn binding and rate of oxidation, calling into question the role of E39 as an Mn ligand. To investigate this hypothesis, we performed computer modeling studies which indicated metal-ligand bond distances in the E39D variant and in an E35D--E39D--D179E triple variant which might allow Mn binding and oxidation. To test the model, we reconstructed the E35D and E39D variants used in the previous study, as well as an E39A single variant and the E35D--E39D--D179E triple variant of MnP isozyme 1 from Phanerochaete chrysosporium. We find that all of the variant proteins are impaired for Mn(II) binding (K(m) increases 20--30-fold) and Mn(II) oxidation (k(cat) decreases 50--400-fold) in both the steady state and the transient state. In particular, mutation of the E39 residue in MnP decreases both Mn binding and oxidation. The catalytic efficiency of the E39A variants decreased approximately 10(4)-fold, while that of the E39D variant decreased approximately 10(3)-fold. Contrary to initial modeling results, the triple variant performed only as well as any of the single Mn ligand variants. Interestingly, the catalytic efficiency of the triple variant decreased only 10(4)-fold, which is approximately 10(2)-fold better than that reported for the E35Q--D179N double variant. These combined studies indicate that precise geometry of the Mn ligands within the Mn binding site of MnP is essential for the efficient binding, oxidation, and release of Mn by this enzyme. The results clearly indicate that E39 is a Mn ligand and that mutation of this ligand decreases both Mn binding and the rate of Mn oxidation.     

A functional variant in the 5′‐flanking region of the chicken serotonin transporter gene is associated with increased body weight and locomotor activity

In this study, we identified a polymorphism in the 5′‐flanking region of the chicken serotonin transporter (5‐HTT) gene. Sequencing analysis revealed that in comparison with the wild‐type variant (W), a deleted variant (D) is generated by deletion of four nucleotides (5′‐AATT‐3′) and a single nucleotide change (A→T). Using a polyacrylamide gel electrophoresis system, we found that the 360‐bp DNA fragment containing the W variant with the wild‐type sequence 5′‐AATTAATT‐3′ shows intrinsic DNA curvature while the 356‐bp fragment containing the D variant lacking the four base pairs AATT is not curved. Quantitative real‐time RT‐PCR and ELISA demonstrated that the expression of 5‐HTT in D/D chickens was higher than that in W/W and W/D chickens. In addition, transient transfection experiments with chloramphenicol acetyltransferase reporter gene constructs revealed increased 5‐HTT promoter activity mediated by the D variant and a silencer activity of the W variant. Interestingly, females and males with D/D genotype showed significant greater increase in body weight from 6 weeks and 16 weeks of age, respectively, and higher body mass index. Moreover, we found that D/D chickens of both genders were physically more active than W/W and W/D chickens.

     

SDS-PAGE Pattern Polymorphism of X-Chromosomal Glue Proteins in Natural Populations of Two Drosophila nasuta Subgroup Species

For the first time, the glue protein patternpolymorphism in natural populations of D. n.nasuta and D. s. neonasuta has beenanalyzed by SDS-PAGE. The study involving 200 and 185isofemale lines comprising 2028 and 1900 individuals of D. n.nasuta and D. s. neonasuta, respectively,revealed the occurrence of eight variant glue proteinphenotypes in D. n. nasuta and seven in D.s. neonasuta. The number and frequency of variant phenotypes in differentpopulations of both species were found to vary. Analysisof glue protein patterns in the F₁ progeny ofcrosses involving parents with variant glue proteinphenotypes revealed that the polymorphic fractions areproduced by co-dominant genes located on the Xchromosome. More than 87% of the naturally inseminatedadult females were found to produce polymorphic progeny. The heterozygous female larvae were found toexceed the homozygotes in the isofemale line progeny ofmost of the populations.     

Increased endosomal sorting of ligand to recycling enhances potency of an interleukin-2 analog

An interleukin-2 (IL-2) variant containing adjacent point mutations (L18M/L19S, termed 2D1) displaying binding affinity to the heterotrimeric IL-2 receptor similar to that of wild-type IL-2 (WT) had been previously found to surprisingly exhibit increased bioactivity in a peripheral blood lymphocyte proliferation assay. In order to provide an explanatory mechanism for this unexpected potency enhancement, we hypothesize that altered endocytic trafficking of the 2D1 variant might be responsible by increasing the number of ligand-receptor complexes. We demonstrate here that the internalization kinetics of 2D1 via the high affinity IL-2 receptor are equivalent to those of WT but that a significantly increased fraction of internalized 2D1 is sorted to recycling instead of to lysosomal degradation. We further find a reduced pH sensitivity of binding to IL-2 receptor alpha relative to IL-2 receptor beta compared with WT, which could be responsible for the altered sorting behavior of 2D1 in the acidic endosomal compartment. Accordingly, the 2D1 variant displays a half-life 36 h longer than that of IL-2 in T-lymphocyte culture at concentrations equal to the K(D) of the IL-2 receptor. The extended half-life of intact 2D1 provides enhanced mitogenesis as compared with IL-2. In addition, 2D1 stimulates natural killer cells to a lesser degree than IL-2 at equal concentrations. We conclude that this IL-2 variant provides increased mitogenic stimulation that could not be easily predicted from its cell surface receptor binding affinity while minimizing undesired stimulation of natural killer cells. This concept of altering trafficking dynamics may offer a generalizable approach to generating improvements in the pharmacological efficacy of therapeutic cytokines.     

Aspartate 313 in the Streptomyces plicatus hexosaminidase plays a critical role in substrate-assisted catalysis by orienting the 2-acetamido group and stabilizing the transition state

SpHex, a retaining family 20 glycosidase from Streptomyces plicatus, catalyzes the hydrolysis of N-acetyl-beta-hexosaminides. Accumulating evidence suggests that the hydrolytic mechanism involves substrate-assisted catalysis wherein the 2-acetamido substituent acts as a nucleophile to form an oxazolinium ion intermediate. The role of a conserved aspartate residue (D313) in the active site of SpHex was investigated through kinetic and structural analyses of two variant enzymes, D313A and D313N. Three-dimensional structures of the wild-type and variant enzymes in product complexes with N-acetyl-d-glucosamine revealed substantial differences. In the D313A variant the 2-acetamido group was found in two conformations of which only one is able to aid in catalysis through anchimeric assistance. The mutation D313N results in a steric clash in the active site between Asn-313 and the 2-acetamido group preventing the 2-acetamido group from providing anchimeric assistance, consistent with the large reduction in catalytic efficiency and the insensitivity of this variant to chemical rescue. By comparison, the D313A mutation results in a shift in a shift in the pH optimum and a modest decrease in activity that can be rescued by using azide as an exogenous nucleophile. These structural and kinetic data provide evidence that Asp-313 stabilizes the transition states flanking the oxazoline intermediate and also assists to correctly orient the 2-acetamido group for catalysis. Based on analogous conserved residues in the family 18 chitinases and family 56 hyaluronidases, the roles played by the Asp-313 residue is likely general for all hexosaminidases using a mechanism involving substrate-assisted catalysis.     

Colon cancer-associated DNA polymerase β variant induces genomic instability and cellular transformation

Rapidly advancing technology has resulted in the generation of the genomic sequences of several human tumors. We have identified several mutations of the DNA polymerase β (pol β) gene in human colorectal cancer. We have demonstrated that the expression of the pol β G231D variant increased chromosomal aberrations and induced cellular transformation. The transformed phenotype persisted in the cells even once the expression of G231D was extinguished, suggesting that it resulted as a consequence of genomic instability. Biochemical analysis revealed that its catalytic rate was 140-fold slower than WT pol β, and this was a result of the decreased binding affinity of nucleotides by G231D. Residue 231 of pol β lies in close proximity to the template strand of the DNA. Molecular modeling demonstrated that the change from a small and nonpolar glycine to a negatively charged aspartate resulted in a repulsion between the template and residue 231 leading to the distortion of the dNTP binding pocket. In addition, expression of G231D was insufficient to rescue pol β-deficient cells treated with chemotherapeutic agents suggesting that these agents may be effectively used to treat tumors harboring this mutation. More importantly, this suggests that the G231D variant has impaired base excision repair. Together, these data indicate that the G231D variant plays a role in driving cancer.     

Characterization of the structure and dynamics of amyloidogenic variants of human lysozyme by NMR spectroscopy

The structures and dynamics of the native states of two mutational variants of human lysozyme, I56T and D67H, both associated with non-neuropathic systemic amyloidosis, have been investigated by NMR spectroscopy. The (1)H and (15)N main-chain amide chemical shifts of the I56T variant are very similar to those of the wild-type protein, but those of the D67H variant are greatly altered for 28 residues in the beta-domain. This finding is consistent with the X-ray crystallographic analysis, which shows that the structure of this variant is significantly altered from that of the wild-type protein in this region. The (1)H-(15)N heteronuclear NOE values show that, with the exception of V121, every residue in the wild-type and I56T proteins is located in tightly packed structures characteristic of the native states of most proteins. In contrast, D67H has a region of substantially increased mobility as shown by a dramatic decrease in heteronuclear NOE values of residues near the site of mutation. Despite this unusual flexibility, the D67H variant has no greater propensity to form amyloid fibrils in vivo or in vitro than has I56T. This finding indicates that it is the increased ability of the variants to access partially folded conformations, rather than intrinsic changes in their native state properties, that is the origin of their amyloidogenicity.     

An electrophoretically cryptic alcohol dehydrogenase variant in Drosophila melanogaster. I. Activity ratios, thermostability, genetic localization and comparison with two other thermostable variants

The alcohol dehydrogenase (ADH) variant ADH-FCh.D. has a secondary alcohol/primary alcohol activity ratio characteristic of ADH-S although it has an electrophoretic mobility inseparable from ADH-F. ADH-FCh.D. is distinguished from these two common ADH variants by being much more thermostable. Genetic analysis suggests tht ADH-FCh.D. is specified by an allele at the Adh locus. Biochemical comparisons show that ADH-FCh.D. has the same electrophoretic mobility, activity ratio and thermostability as the two other heat-resistant variants which have been reported, ADH-F71K in Europe and ADH-Fr in North America. The geographically widespread distribution of a thermostable ADH variant within the ADH-F electrophoretic class indicates that it should be considered in attempts to explain the Adh polymorphism in natural populations.     

Biochemical and structural characterisation of cutinase mutants in the presence of the anionic surfactant AOT

The reactivity, stability and unfolding of wild-type (WT) Fusarium solani pisi cutinase and L153Q, S54D and T179C variants were studied in the absence and presence of the dioctyl sulfosuccinate sodium salt (AOT) surfactant. In the absence of surfactant the S54D variant catalytic activity is similar to that of the WT cutinase, whereas L153Q and T179C variants show a lower activity. AOT addition induces an activity reduction for WT cutinase and its variants, although for low AOT concentrations a small increase of activity was observed for S54D and T179C. The enzyme deactivation in the presence of 0.5 mM AOT is relatively slow for the S54D and T179C variants when compared to wild-type cutinase and L153Q variant. These results were correlated with secondary and tertiary structure changes assessed by the CD spectrum and fluorescence of the single tryptophan and the six tyrosine residues. The WT cutinase and S54D variant have similar secondary and tertiary structures that differ from those of T179C and L153Q variants. L153Q, S54D and T179C mutations prevent the formation of hydrophobic crevices responsible for the unfolding by anionic surfactants, with the consequent decrease of the AOT-cutinase interactions.