Recognition Signal for C-Mannosylation of Trp-7 in RNase 2 Consists of Sequence Trp-x-x-Trp

C²-α-Mannosyltryptophan was discovered in human RNase 2, an enzyme that occurs in eosinophils and is involved in host defense. It represents a novel way of attaching carbohydrate to a protein in addition to the well-known N- and O-glycosylations. The reaction is specific, as in RNase 2 Trp-7, but never Trp-10, which is modified. In this article, we address which structural features provide the specificity of the reaction. Expression of chimeras of RNase 2 and nonglycosylated RNase 4 and deletion mutants in HEK293 cells identified residues 1–13 to be sufficient for C-mannosylation. Site-directed mutagenesis revealed the sequence Trp-x-x-Trp, in which the first Trp becomes mannosylated, as the specificity determinant. The Trp residue at position +3 can be replaced by Phe, which reduces the efficiency of the reaction threefold. Interpretation of the data in the context of the three-dimensional structure of RNase 2 strongly suggests that the primary, rather than the tertiary, structure forms the determinant. The sequence motif occurs in 336 mammalian proteins currently present in protein databases. Two of these proteins were analyzed protein chemically, which showed partial C-glycosylation of recombinant human interleukin 12. The frequent occurrence of the protein recognition motif suggests that C-glycosides could be part of the structure of more proteins than assumed so far.     

Overexpression of GDP-mannose pyrophosphorylase in Saccharomyces cerevisiae corrects defects in dolichol-linked saccharide formation and protein glycosylation

Thermosensitive mutants of Saccharomyces cerevisiae, affected in the endoplasmic reticulum (ER) located glycosylation, i.e. in Dol-P-Man synthase (dpm1), in beta-1,4 mannosyl transferase (alg1) and in alpha-1,3 mannosyltransferase (alg2), were used to assess the role of GDP-Man availability for the synthesis of dolichol-linked saccharides. The mutants were transformed with the yeast gene MPG1 (PSA1/VIG9) encoding GDP-Man pyrophosphorylase catalyzing the final step of GDP-Man formation. We found that overexpression of MPG1 allows growth at non-permissive temperature and leads to an increase in the cellular content of GDP-Man. In the alg1 and alg2 mutants, complemented with MPG1 gene, N-glycosylation of invertase was in part restored, to a degree comparable to that of the wild-type control. In the dpm1 mutant, the glycosylation reactions that depend on the formation of Dol-P-Man, i.e. elongation of Man(5)GlcNAc(2)-PP-Dol, O-mannosylation of chitinase and synthesis of GPI anchor were normal when MPG1 was overexpressed.Our data indicate that an increased level of GDP-Man is able to correct defects in mannosylation reactions ascribed to the ER and to the Golgi.     

O-mannosylation precedes and potentially controls the N-glycosylation of a yeast cell wall glycoprotein

Secretory proteins in yeast are N- and O-glycosylated while they enter the endoplasmic reticulum. N-glycosylation is initiated by the oligosaccharyl transferase complex and O-mannosylation is initiated by distinct O-mannosyltransferase complexes of the protein mannosyl transferase Pmt1/Pmt2 and Pmt4 families. Using covalently linked cell-wall protein 5 (Ccw5) as a model, we show that the Pmt4 and Pmt1/Pmt2 mannosyltransferases glycosylate different domains of the Ccw5 protein, thereby mannosylating several consecutive serine and threonine residues. In addition, it is shown that O-mannosylation by Pmt4 prevents N-glycosylation by blocking the hydroxy amino acid of the single N-glycosylation site present in Ccw5. These data prove that the O- and N-glycosylation machineries compete for Ccw5; therefore O-mannosylation by Pmt4 precedes N-glycosylation.     

An interaction effect between glucokinase gene variation and carbohydrate intakes modulates the plasma triglyceride response to a fish oil supplementation

A large inter-individual variability in the plasma triglyceride (TG) response to fish oil consumption has been observed. The objective was to investigate the gene–diet interaction effects between single-nucleotide polymorphisms (SNPs) within glucokinase (GCK) gene and dietary carbohydrate intakes (CHO) on the plasma TG response to a fish oil supplementation. Two hundred and eight participants were recruited in the greater Quebec City area. The participants completed a 6-week fish oil supplementation (5 g fish oil/day: 1.9–2.2 g EPA and 1.1 g DHA). Thirteen SNPs within GCK gene were genotyped using TAQMAN methodology. A gene–diet interaction effect on the plasma TG response was observed with rs741038 and CHO adjusted for age, sex and BMI (p = 0.008). In order to compare the plasma TG response between genotypes according to CHO, participants were divided according to median CHO. Homozygotes of the minor C allele of rs741038 with high CHO >48.59 % had a greater decrease in their plasma TG concentrations following the intake of fish oil (p < 0.05) than C/C homozygotes with low CHO and also than the other genotypes either with high or low CHO. The plasma TG response to a fish oil supplementation may be modulated by gene–diet interaction effects involving GCK gene and CHO.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-014-0395-5) contains supplementary material, which is available to authorized users.     

Identification of DPY19L3 as the C-mannosyltransferase of R-spondin1 in human cells

R-spondin1 (Rspo1) is a secreted protein that enhances Wnt signaling, which has crucial functions in embryonic development and several cancers. C-mannosylation is a rare type of glycosylation and might regulate secretion, protein–protein interactions, and enzymatic activity. Although human Rspo1 contains 2 predicted C-mannosylation sites, C-mannosylation of Rspo1 has not been reported, nor have its functional effects on this protein. In this study, we demonstrate by mass spectrometry that Rspo1 is C-mannosylated at W¹⁵³ and W¹⁵⁶. Using Lec15.2 cells, which lack dolichol-phosphate-mannose synthesis activity, and mutant Rspo1-expressing cells that replace W¹⁵³ and W¹⁵⁶ by alanine residues, we observed that C-mannosylation of Rspo1 is required for its secretion. Further, the enhancement of canonical Wnt signaling by Rspo1 is regulated by C-mannosylation. Recently DPY19 was reported to be a C-mannosyltransferase in Caenorhabditis elegans, but no C-mannosyltransferases have been identified in any other organism. In gain- and loss-of-function experiments, human DPY19L3 selectively modified Rspo1 at W¹⁵⁶ but not W¹⁵³ based on mass spectrometry. Moreover, knockdown of DPY19L3 inhibited the secretion of Rspo1. In conclusion, we identified DPY19L3 as the C-mannosyltransferase of Rspo1 at W¹⁵⁶ and found that DPY19L3-mediated C-mannosylation of Rspo1 at W¹⁵⁶ is required for its secretion.     

Design of antisense oligonucleotides stabilized by locked nucleic acids

The design of antisense oligonucleotides containing locked nucleic acids (LNA) was optimized and compared to intensively studied DNA oligonucleotides, phosphorothioates and 2′-O-methyl gapmers. In contradiction to the literature, a stretch of seven or eight DNA monomers in the center of a chimeric DNA/LNA oligonucleotide is necessary for full activation of RNase H to cleave the target RNA. For 2′-O-methyl gapmers a stretch of six DNA monomers is sufficient to recruit RNase H. Compared to the 18mer DNA the oligonucleotides containing LNA have an increased melting temperature of 1.5–4°C per LNA depending on the positions of the modified residues. 2′-O-methyl nucleotides increase the T_m by only <1°C per modification and the T_m of the phosphorothioate is reduced. The efficiency of an oligonucleotide in supporting RNase H cleavage correlates with its affinity for the target RNA, i.e. LNA > 2′-O-methyl > DNA > phosphorothioate. Three LNAs at each end of the oligonucleotide are sufficient to stabilize the oligonucleotide in human serum 10-fold compared to an unmodified oligodeoxynucleotide (from t_1/2 = ~1.5 h to t_1/2 = ~15 h). These chimeric LNA/DNA oligonucleotides are more stable than isosequential phosphorothioates and 2′-O-methyl gapmers, which have half-lives of 10 and 12 h, respectively.     

Identification and characterization of UDP-mannose in human cell lines and mouse organs: Differential distribution across brain regions and organs

Mannosylation in the endoplasmic reticulum is a key process for synthesizing various glycans. Guanosine diphosphate mannose (GDP-Man) and dolichol phosphate-mannose serve as donor substrates for mannosylation in mammals and are used in N-glycosylation, O-mannosylation, C-mannosylation, and the synthesis of glycosylphosphatidylinositol-anchor (GPI-anchor). Here, we report for the first time that low-abundant uridine diphosphate-mannose (UDP-Man), which can serve as potential donor substrate, exists in mammals. Liquid chromatography-mass spectrometry (LC-MS) analyses showed that mouse brain, especially hypothalamus and neocortex, contains higher concentrations of UDP-Man compared to other organs. In cultured human cell lines, addition of mannose in media increased UDP-Man concentrations in a dose-dependent manner. These findings indicate that in mammals the minor nucleotide sugar UDP-Man regulates glycosylation, especially mannosylation in specific organs or conditions.     

The extraction and hydrolysis of steroid monoglucuronides

1. A method in use for the extraction of urinary steroid conjugates has been applied to study the recovery of synthetic steroid monoglucuronides from aqueous solution. 2. In the presence of dissolved ammonium sulphate (50g./100ml.), ether–ethanol (3:1, v/v, 3×0·5vol.) extracted the monoglucuronides of steroids of the C₁₈, C₁₉ and C₂₁ series, quantitatively at values pH2–9. 3. The hydrolysis of the synthetic steroid monoglucuronides by β-glucuronidase (Patella vulgata) has been examined with reference to the pH value of the medium, enzyme concentration and substrate concentration. 4. The rate of hydrolysis of steroid monoglucuronides was dependent upon steroid structure and upon site of conjugation. 5. The rate of hydrolysis of the monoglucuronides decreased in the order C-3 (phenolic) >C-3β>C-17β>C-3α.     

A Novel Mechanism for Ribonuclease Regulation: TRANSFER-MESSENGER RNA (tmRNA) AND ITS ASSOCIATED PROTEIN SmpB REGULATE THE STABILITY OF RNase R*

The amount of RNase R, an important degradative exoribonuclease, increases 3–10-fold under a variety of stress conditions. This elevation is due to posttranslational regulation in which the highly unstable RNase R protein becomes stabilized during stress. Here we identify two components of the trans-translation machinery, transfer-messenger RNA (tmRNA) and SmpB, that are responsible for the short half-life of RNase R in exponential phase cells. The absence of either lengthens the half-life of RNase R in vivo >6-fold. SmpB directly interacts with RNase R in vitro and is stimulated by tmRNA. The C-terminal region of RNase R, encompassing its basic region and adjacent S1 domain are required for the interaction; their removal eliminates binding and stabilizes RNase R in vivo. However, the binding of SmpB and tmRNA does not alter RNase R activity. These data define a previously unknown regulatory process in which the stability of an RNase is determined by its interaction with an RNA and an RNA-associated protein.     

Tumor Necrosis Factor (TNF) –308G>A,Nitric Oxide Synthase 3 (NOS3) +894G>T Polymorphisms and Migraine Risk: A Meta-Analysis

Background and Objective

Conflicting data have been reported on the association between tumor necrosis factor (TNF) –308G>A and nitric oxide synthase 3 (NOS3) +894G>T polymorphisms and migraine. We performed a meta-analysis of case-control studies to evaluate whether the TNF –308G>A and NOS3 +894G>T polymorphisms confer genetic susceptibility to migraine.

Method

We performed an updated meta-analysis for TNF –308G>A and a meta-analysis for NOS3 +894G>T based on studies published up to July 2014. We calculated study specific odds ratios (OR) and 95% confidence intervals (95% CI) assuming allele contrast, dominant model, recessive model, and co-dominant model as pooled effect estimates.

Results

Eleven studies in 6682 migraineurs and 22591 controls for TNF –308G>A and six studies in 1055 migraineurs and 877 controls for NOS3 +894G>T were included in the analysis. Neither indicated overall associations between gene polymorphisms and migraine risk. Subgroup analyses suggested that the “A” allele of the TNF –308G>A variant increases the risk of migraine among non-Caucasians (dominant model: pooled OR = 1.82; 95% CI 1.15 – 2.87). The risk of migraine with aura (MA) was increased among both Caucasians and non-Caucasians. Subgroup analyses suggested that the “T” allele of the NOS3 +894G>T variant increases the risk of migraine among non-Caucasians (co-dominant model: pooled OR = 2.10; 95% CI 1.14 – 3.88).

Conclusions

Our findings appear to support the hypothesis that the TNF –308G>A polymorphism may act as a genetic susceptibility factor for migraine among non-Caucasians and that the NOS3 +894G>T polymorphism may modulate the risk of migraine among non-Caucasians.     

Serology,infection, and clinical trachoma as tools in prevalence surveys for re-emergence of trachoma in a formerly hyperendemic district

BackgroundTo eliminate trachoma as a public health problem, countries must achieve a district-level prevalence of trachomatous inflammation—follicular (TF) <5% in children ages 1–9 years. Re-emergence of TF could trigger additional rounds of mass drug/antibiotic administration (MDA), so accurate tools for use in surveys assessing trachoma prevalence are essential.Methodology & principal findingsWe surveyed 2401 children ages 1–9 years from 50 villages in Kongwa, Tanzania, 2 years post-MDA and 1.5 years after an impact survey found TF <5% in the same villages. Our survey included multiple tools: clinical determination of TF, Cepheid testing for Chlamydia trachomatis infection, and testing for anti-pgp3 antibodies via multiplex bead array. Photographs of the upper tarsal conjunctiva were taken in a subset of children to corroborate the field grades.Overall TF prevalence in 1–9 year olds was 7.1% (95% CI: 5.6%-8.9%), which decreased with age (p = <0.0001). TF prevalence by village was heterogeneous, with 19 villages having TF <5% and 16 villages having TF >10%. There was a strong correlation between field and photo grading of TF (kappa = 0.69; 95% CI: 0.60–0.78) and between TF and infection, with 21.5% of TF-positive children also testing positive for infection, as compared to only 1.6% of TF-negative children (p = 0.0010). Overall seroprevalence was 18.2% (95% CI: 14.8%-22.1%), which increased with age (p = <0.0001). Notably, 1–2 year olds, who were born after the cessation of MDA and theoretically should not have had exposure to C. trachomatis in the absence of transmission, had an average seroprevalence of 6.7%.Conclusions & significanceField TF prevalence, supported by photographic review and infection data, suggested re-emergence of trachoma in Kongwa. Moreover, seropositivity in the children born after cessation of MDA indicated exposure to C. trachomatis despite a previous survey finding of TF <5%. Examining seropositivity in specific age groups expected to have limited exposure to C. trachomatis can be used to detect re-emergence.     

Reversal by Triton WR-1339 of ethynyloestradiol-induced hepatic cholesterol esterification

Rats treated with ethynyloestradiol have marked hypolipidaemia: serum cholesterol is decreased to 5%, triacylglycerol to 10% and phospholipid to 70% of control concentrations. Loss of serum cholesterol follows an exponential decay, with a half-life of 1.13±0.09 days. After 4 days of treatment, serum cholesterol concentrations remain relatively constant (ranging from 1 to 20mg/100ml) for at least 30 days. There is a concomitant 20-fold decrease in the d<1.21 fraction of serum proteins and a similar decrease in serum apolipoproteins as measured by sodium dodecyl sulphate/10%-polyacrylamide-gel electrophoresis. The activity of hepatic microsomal acyl-CoA–cholesterol O-acetyltransferase (EC 2.3.1.26) was significantly increased by ethynyloestradiol treatment (P<0.05). This activation caused hepatic cholesteryl esters containing mainly C_18:1 fatty acids to increase linearly as serum cholesterol concentrations decreased (r=0.9675, P<0.001). Triton WR-1339, a non-ionic detergent that inhibits lipoprotein catabolism, was used to estimate hepatic lipid secretion by measuring the increment in serum lipids after its administration. At 15h after Triton WR-1339 administration, serum cholesterol concentrations were increased equally in both control and ethynyloestradiol-treated rats. In contrast, the increment of serum triacylglycerol of treated rats was 40% of that found in control rats, indicating that ethynyloestradiol inhibits hepatic triacylglycerol secretion. Triton WR-1339 inhibited the oestrogen activation of hepatic microsomal acyl-CoA–cholesterol O-acyltransferase and restored hepatic cholesteryl ester concentrations to normal values. These data suggest that ethynyloestradiol and its pharmacological `antagonist' Triton WR-1339 alter hepatic triacylglycerol secretion via a mechanism associated with changes in hepatic cholesterol esterification.     

Association between XRCC1 and XRCC3 Polymorphisms with Lung Cancer Risk: A Meta-Analysis from Case-Control Studies

Many studies have reported the association of X-ray repair cross-complementing group 1 (XRCC1) Arg399Gln, Arg194Trp, Arg280His, −77T>C, and X-ray repair cross-complementing group 3 (XRCC3) T241M polymorphisms with lung cancer risk, but the results remained controversial. Hence, we performed a meta-analysis to investigate the association between lung cancer risk and XRCC1 Arg399Gln (14,156 cases and 16,667 controls from 41 studies), Arg194Trp (7,426 cases and 9,603 controls from 23 studies), Arg280His (6,211 cases and 6,763 controls from 16 studies), −77T>C (2,487 cases and 2,576 controls from 5 studies), and XRCC3 T241M (8,560 cases and 11,557 controls from 19 studies) in different inheritance models. We found that −77T>C polymorphism was associated with increased lung cancer risk (dominant model: odds ration [OR] = 1.45, 95% confidence interval [CI] = 1.27–1.66, recessive model: OR = 1.73, 95% CI = 1.14–2.62, additive model: OR = 1.91, 95% CI = 1.24–1.94) when all the eligible studies were pooled into the meta-analysis. In the stratified and sensitive analyses, significantly decreased lung cancer risk was observed in overall analysis (dominant model: OR = 0.83, 95% CI = 0.78–0.89; recessive model: OR = 0.90, 95% CI = 0.81–1.00; additive model: OR = 0.82, 95% CI = 0.74–0.92), Caucasians (dominant model: OR = 0.82, 95% CI = 0.76–0.87; recessive model: OR = 0.89, 95% CI = 0.80–0.99; additive model: OR = 0.81, 95% CI = 0.73–0.91), and hospital-based controls (dominant model: OR = 0.81, 95% CI = 0.76–0.88; recessive model: OR = 0.89, 95% CI = 0.79–1.00; additive model: OR = 0.80, 95% CI = 0.71–0.90) for XRCC3 T241M. In conclusion, this meta-analysis indicates that XRCC1 −77T>C shows an increased lung cancer risk and XRCC3 T241M polymorphism is associated with decreased lung cancer risk, especially in Caucasians.     

Plant mitochondria use two pathways for the biogenesis of tRNAHis

All tRNA^His possess an essential extra G_–1 guanosine residue at their 5′ end. In eukaryotes after standard processing by RNase P, G_–1 is added by a tRNA^His guanylyl transferase. In prokaryotes, G_–1 is genome-encoded and retained during maturation. In plant mitochondria, although trnH genes possess a G_–1 we find here that both maturation pathways can be used. Indeed, tRNA^His with or without a G_–1 are found in a plant mitochondrial tRNA fraction. Furthermore, a recombinant Arabidopsis mitochondrial RNase P can cleave tRNA^His precursors at both positions G₊₁ and G_–1. The G_–1 is essential for recognition by plant mitochondrial histidyl-tRNA synthetase. Whether, as shown in prokaryotes and eukaryotes, the presence of uncharged tRNA^His without G_–1 has a function or not in plant mitochondrial gene regulation is an open question. We find that when a mutated version of a plant mitochondrial trnH gene containing no encoded extra G is introduced and expressed into isolated potato mitochondria, mature tRNA^His with a G_–1 are recovered. This shows that a previously unreported tRNA^His guanylyltransferase activity is present in plant mitochondria.     

The proportion of mutants in bacterial cultures

The proportion of mutants in a growing culture of organisms will depend upon (a) the rate at which the wild cells produce them (with or without growth), (b) the back mutation rate, and (c) the growth rates of the wild and mutant cells. If the mutation rate without growth and the back mutation rate are neglected, the growth of a mutant is expressed by See PDF for Equation and the ratio of the mutant to wild by See PDF for Equation in which λ = mutation frequency rate constant, "mutation rate," A = growth rate constant of wild cells W, B = growth rate constant of mutant cells M. If the term [B – (1 – 2λ)A] is positive, the proportion of mutants increases continuously. If it is negative, the proportion of mutants reaches a constant value See PDF for Equation If mutation is assumed to occur without growth at the rate C, then the corresponding equations are (11), (12), and (14). See PDF for Equation If (B + C – A) is negative and t = ∞, See PDF for Equation If C << A, See PDF for Equation     

Differential effects of non-ionic detergents on microsomal and sarcolemmal adenylate cyclase in cardiac muscle

1. About 4 and 23% of the homogenate adenylate cyclase activity was recovered in the microsomal and sarcolemmal fractions isolated from guinea-pig heart ventricles. 2. Cardiac microsomal adenylate cyclase activity [basal as well as p[NH]ppG (guanyl-5′-yl imidodiphosphate)- and NaF-stimulated] was increased over 2-fold in the presence of Lubrol-PX (0.01–0.1%). 3. The sarcolemmal enzyme, however, showed concentration-dependent inhibition caused by the detergent under all assay conditions, except when p[NH]ppG was included in the assay. In the latter case, the detergent (0.01–0.02%) caused a modest increase (30–45%) in enzyme activity. 4. Another non-ionic detergent, Triton X-100, also stimulated the microsomal cyclase and inhibited the sarcolemmal enzyme. 5. With either membrane fraction, Lubrol-PX solubilized the enzyme when the detergent/membrane protein ratio was 2.5 (μmol of detergent/mg of protein). 6. The findings with homogenate and a washed particulate fraction resembled those obtained with sarcolemma, and those with isolated sarcoplasmic reticulum resembled those with microsomal preparations. 7. p[NH]ppG, and to some extent NaF, protected the detergent-induced inactivation of the enzyme observed at higher detergent concentrations (0.5% Lubrol-PX and 0.05–0.5% Triton X-100). 8. In the absence of detergents, p[NH]ppG increased the basal enzyme activity about 2-fold in microsomal fractions, but did not appreciably stimulate the sarcolemmal enzyme. Isoproterenol, on the other hand, increased the sarcolemmal enzyme activity (>2-fold) in the presence of p[NH]ppG and caused only moderate stimulation (31%) of the microsomal enzyme under these conditions. 9. These findings support the view that, although the bulk of adenylate cyclase resides in heart sarcolemma (plasma membrane), the microsomal activity cannot be accounted for solely by contamination of the microsomal fraction with sarcolemma, as has been suggested by others [Besch, Jones & Watanabe (1976) Circ. Res. 39, 586–595; Engelhard, Plut & Storm (1976) Biochim. Biophys. Acta 451, 48–61]. Further, the results of this study show that cardiac sarcoplasmic-reticulum membranes possess this enzyme.     

Association of fibrinogen and plasmin inhibitor,but not coagulation factor XIII gene polymorphisms with coronary artery disease

BackgroundIn the final phase of clot formation, fibrinogen constitutes frame, whereas factor XIII (FXIII) active form is responsible for the covalent cross-linking of fibrin fibres and plasmin inhibitor (PI), thus contributing to clot stability. It could be expected that any change of coagulation factors'' structure affects the clot formation and modulates the atherothrombotic risk. The aim was to determine the frequency of four single nucleotide polymorphisms: (i) A > G in codon 312 of the fibrinogen α-chain gene (rs6050, Thr312AlaFGA), (ii) C > T at position 10034 of the 3 - untranslated region in the fibrinogen γ-chain gene (rs2066865, 10034C > T FGG), (iii) C > T in codon 564 of the FXIII-A subunit gene (rs5982, Pro564LeuFXIII-A), and (iv) C > T in codon 6 of the plasmin inhibitor gene (rs2070863, Arg6TrpPI) in Croatian patients and their association with coronary artery disease (CAD).MethodsWe performed the unrelated case-control association study on the consecutive sample of patients 18 years old, who had undergone coronary angiography for investigation of chest pain and suspected CAD. The cases were patients with confirmed CAD (N=201), and the controls were the subjects with no CAD (N=119). Samples were genotyped using PCR-RFLP analysis.ResultsObserved frequencies of the rare alleles of Thr312Ala FGA, 10034C > T FGG, Leu564Pro FXIII-A and Arg6Trp PI polymorphisms were 21%, 17%, 14%, 20%, respectively. Patients with 10034C > T FGG CC genotype had 3.5 times (95% CI 1.02-12.03) higher adjusted odds for CAD than patients with 10034C > T FGG TT genotype. Patients with Arg6Trp PI CC genotype had 3.86 times (95% CI 1.23-12.12) higher odds for CAD than patients with Arg6Trp PI TT genotype. It seems that those genotype-related higher odds are also male-gender related. No difference was observed regarding any other investigated polymorphism.ConclusionsOur finding suggests that 10034C > T FGG and Arg6Trp PI are associated with CAD.     

The fibrinogen C-terminal domain is seldom C-mannosylated but its C-mannosylation is important for the secretion of microfibril-associated glycoprotein 4

BackgroundC-mannosylation is the one of glycosylations. Microfibril-associated glycoprotein 4 (MFAP4), an important protein for tissue homeostasis and cell adhesion, contains a consensus sequence of C-mannosylation in its fibrinogen C-terminal domain. In this study, we sought to demonstrate that fibrinogen C-terminal domain is a new substrate domain for C-mannosylation.MethodsWe established an MFAP4-overexpresssing HT1080 cell line and purified recombinant MFAP4 protein from the conditioned medium for LC-MS/MS analysis. Subcellular localization of MFAP4 was observed under confocal fluorescence microscope.ResultsWe found that MFAP4 is C-mannosylated at Trp²³⁵ in the fibrinogen C-terminal domain by LC-MS/MS. To determine the functions of the C-mannosylation of MFAP4, we established a C-mannosylation-defective mutant MFAP4-overexpresssing HT1080 cell line and measured its secretion of MFAP4. The secretion of MFAP4 decreased significantly in the C-mannosylation-defective mutant MFAP4-overexpresssing cell line versus wild-type cells. Moreover, co-transfection experiments indicated that C-mannosylated MFAP4 accelerated its secretion.ConclusionsOur results demonstrate that the fibrinogen C-terminal domain is a novel C-mannosylation domain and that the C-mannosylation of MFAP4 is important for its secretion.General significanceThese results suggest that C-mannosylation has a role for dominant effect for MFAP4 secretion.     

Maternal dietary intake of folate,vitamin B12 and MTHFR 677C>T genotype: their impact on newborn’s anthropometric parameters

In this study, we evaluated the effects of dietary intake of vitamin B₁₂ and folate during pregnancy and their interactions with maternal polymorphism of MTHFR (677C>T; 1298A>C) on intrauterine development. Anthropometric parameters were obtained from 231 newborns that belong to a prospective birth cohort in Morelos, Mexico. Maternal dietary intake of vitamin B₁₂ and folate was assessed using a semi-quantitative questionnaire administered during the first and third trimesters of the pregnancy. Maternal MTHFR 677C>T and 1298 A>C genotypes were determined by PCR–RFLP. The associations between deficient dietary intake of vitamin B₁₂ (<2.0 μg/d) and folate (<400 μg/d) in the first and third trimesters and maternal polymorphisms of MTHFR on anthropometric parameters at birth were estimated using a multivariate linear regression model. During pregnancy, the deficient dietary intake was roughly 60 % for folate and 19 % for vitamin B₁₂. Allelic frequencies of 677T and 1298C were 59 and 10 %, respectively. After adjusting for confounders, deficiency in maternal dietary intake of vitamin B₁₂ (<2.0 μg/d) was associated with a significant reduction in length (β ~ −2.4; 95 % CI −4.3; −0.6) and length-for-age at birth (β ~ −1.2; 95 % CI −2.3; −0.1) among infants whose mothers were carriers of the 677TT genotype (p for interaction = 0.02). In contrast, no association was observed between deficiency in maternal dietary intake of folate (<400 μg/d) and any anthropometric parameter of newborns. These results suggest that supplementation with vitamin B₁₂ during pregnancy could have a favorable impact on intrauterine fetal development mainly in populations that are genetically susceptible.     

Impact of the CYP3A5, CYP3A4, COMT,IL-10 and POR Genetic Polymorphisms on Tacrolimus Metabolism in Chinese Renal Transplant Recipients

Tacrolimus is a widely used immunosuppressive drug for preventing the rejection of solid organ transplants. The efficacy of tacrolimus shows considerable variability, which might be related to genetic variation among recipients. We conducted a retrospective study of 240 Chinese renal transplant recipients receiving tacrolimus as immunosuppressive drug. The retrospective data of all patients were collected for 40 days after transplantation. Seventeen SNPs of CYP3A5, CYP3A4, COMT, IL-10 and POR were identified by the SNaPshot assay. Tacrolimus blood concentrations were obtained on days 1–3, days 6–8 and days 12–14 after transplantation, as well as during the period of the predefined therapeutic concentration range. Kruskal–Wallis test was used to examine the effect of genetic variation on the tacrolimus concentration/dose ratio (C ₀/D) at different time points. Chi-square test was used to compare the proportions of patients who achieved the target C ₀ range in the different genotypic groups at weeks 1, 2, 3 and 4 after transplantation. After correction for multiple testing, there was a significant association of C ₀/D with CYP3A5*3, CYP3A4*1G and CYP3A4 rs4646437 T>C at different time points after transplantation. The proportion of patients in the IL-10 rs1800871-TT group who achieved the target C ₀ range was greater (p = 0.004) compared to the IL-10 rs1800871-CT and IL-10 rs1800871-CC groups at week 3 after transplantation. CYP3A5*3, CYP3A4 *1G, CYP3A4 rs4646437 T>C and IL-10 rs1800871 C>T might be potential polymorphisms affecting the interindividual variability in tacrolimus metabolism among Chinese renal transplant recipients.