Interactions between sulfonamide homologues and glycosyltransferase induced metabolic disorders in rice (Oryza sativa L.).

Sulfadiazine and its derivatives (sulfonamides, SAs) could induce distinct biotoxic, metabolic and physiological abnormalities, potentially due to their subtle structural differences. This study conducted an in-depth investigation on the interactions between SA homologues, i.e. sulfadiazine (SD), sulfamerazine (SD1), and sulfamethazine (SD2), and the key metabolic enzyme (glycosyltransferase, GT) in rice (Oryza sativa L.). Untargeted screening of SA metabolites revealed that GT-catalyzed glycosylation was the primary transformation pathway of SAs in rice. Molecular docking identified that the binding sites of SAs on GT (D0TZD6) were responsible for transferring sugar moiety to synthesize polysaccharides and detoxify SAs. Specifically, amino acids in the GT-binding cavity (e.g., GLY487 and CYS486) formed stable hydrogen bonds with SAs (e.g., the sulfonamide group of SD). Molecular dynamics simulations revealed that SAs induced conformational changes in GT ligand binding domain, which was supported by the significantly decreased GT activity and gene expression level. As evidenced by proteomics and metabolomics, SAs inhibited the transfer and synthesis of sugar but stimulated sugar decomposition in rice leaves, leading to the accumulation of mono- and disaccharides in rice leaves. While the differences in the increased sugar content by SD (24.3%, compared with control), SD1 (11.1%), and SD2 (6.24%) can be attributed to their number of methyl groups (0, 1, 2, respectively), which determined the steric hindrance and hydrogen bonds formation with GT. This study suggested that the disturbances on crop sugar metabolism by homologues contaminants are determined by the interaction between the contaminants and the target enzyme, and are greatly dependent on the steric hindrance effects contributed by their side chains. The results are of importance to identify priority pollutants and ensure crop quality in contaminated fields.

Contribution of UDP-glycosyltransferases to chlorpyrifos resistance in Nilaparvata lugens.

As a multigene superfamily of Phase II detoxification enzymes, uridine diphosphate (UDP)-glycosyltransferases (UGTs) play important roles in the metabolism of xenobiotics including insecticides. In this study, 5-nitrouracil, an inhibitor of UGT enzyme activity, effectively increased the toxicity of chlorpyrifos to the chlorpyrifos-resistant strain of Nilaparvata lugens, one of the most resistant rice pests. The enzyme content of UGT in the resistant strain was significantly higher than that in the susceptible strain. Among 20 identified UGT genes, UGT386H2, UGT386J2, UGT386N2 and UGT386P1 were found significantly overexpressed in the resistant strain and can be effectively induced by chlorpyrifos. These four UGT genes were most highly expressed in the midgut and/or fat body, two main insect detoxification tissues. Amino acid sequence alignments revealed that these four UGTs contained a variable N-terminal substrate-binding domain and a conserved C-terminal sugar donor-binding domain. Furthermore, homology modeling and molecular docking analyses showed that these UGTs could stably bind to chlorpyrifos and chlorpyrifos oxon, with the binding free energies from -19.4 to -110.62 kcal mol-1. Knockdown of UGT386H2 or UGT386P1 by RNA interference dramatically increased the susceptibility of the resistant strain to chlorpyrifos. These findings suggest that overexpression of these two UGT genes contributes to chlorpyrifos resistance in N. lugens.

Plant species compositions alleviate toxicological effects of bisphenol A by enhancing growth, antioxidant defense system, and detoxification.

Bisphenol A (BPA), a broadly disseminated endocrine disturbing chemicals in environment, is harmful to creatures and plants. Plants can uptake and metabolize BPA, but a single plant species ability is limited. Undeniably, plant species compositions have a more vital ability to remove pollutants than a single plant species. However, the mechanisms of plant species compositions alleviating toxicological effects of bisphenol A are poorly understood. Here, we administered plant species compositions, which based on a full-factorial design of Phragmites australis (A), Typha latifolia (B), and Arundo donax (C), to unveil their role in BPA exposure. The results illustrated that the root activity, biomass, and photosynthetic pigment contents of the mixed hydroponic group (e.g., sp(ABC)) were significantly increased under concentration of BPA(1.5, 5, and 10 mg L-1), which showed that the root activity, fresh weight, dry weight, chlorophyll a, and total chlorophyll contents of shoots were increased. While mixed-hydroponic culture groups (e.g., sp(AB), sp(ABC)) significantly increased antioxidant enzyme activity and antioxidant substances under concentration of BPA(5 and 10 mg L-1), it astoundingly diminished responsive oxygen species (ROS) and malondialdehyde (MDA) substance, proposing that mixed-hydroponic culture groups calmed oxidative stress. Further analysis revealed that mixed-hydroponic culture groups (e.g., sp(AB), sp(AC), sp(ABC)) of 1.5, 5, and 10 mg L-1 BPA exposure significantly increased detoxification enzyme activity of NADPH-cytochrome P450 reductase (CPR), glutathione S-transferase (GST), and glycosyltransferase (GT). Moreover, mixed-hydroponic culture groups (e.g., sp(AB), sp(AC), sp(ABC)) decreased the BPA substance in leaves, proposing that mixed-hydroponic culture groups advanced BPA metabolism by improving CPR, GST, and GT enzyme activities. These results demonstrated that a mixed-hydroponic culture strategy can alleviate BPA phytotoxicity and possibly offer natural and potential phytoremediation methods for BPA.

Short-term treatment of golden retriever muscular dystrophy (GRMD) dogs with rAAVrh74.MHCK7.GALGT2 induces muscle glycosylation and utrophin expression but has no significant effect on muscle strength.

We have examined the effects of intravenous (IV) delivery of rAAVrh74.MHCK7.GALGT2 in the golden retriever muscular dystrophy (GRMD) model of Duchenne Muscular Dystrophy (DMD). After baseline testing, GRMD dogs were treated at 3 months of age and reassessed at 6 months. This 3-6 month age range is a period of rapid disease progression, thus offering a relatively short window to establish treatment efficacy. Measures analyzed included muscle AAV transduction, GALGT2 transgene expression, GALGT2-induced glycosylation, muscle pathology, and muscle function. A total of five dogs were treated, 4 at 2x1014vg/kg and one at 6x1014vgkg. The 2x1014vg/kg dose led to transduction of regions of the heart with 1-3 vector genomes (vg) per nucleus, while most skeletal muscles were transduced with 0.25-0.5vg/nucleus. GALGT2-induced glycosylation paralleled levels of myofiber vg transduction, with about 90% of cardiomyocytes having increased glycosylation versus 20-35% of all myofibers across the skeletal muscles tested. Conclusions from phenotypic testing were limited by the small number of dogs. Treated dogs had less pronounced fibrosis and overall lesion severity when compared to control groups, but surprisingly no significant changes in limb muscle function measures. GALGT2-treated skeletal muscle and heart had elevated levels of utrophin protein expression and GALGT2-induced expression of glycosylated α dystroglycan, providing further evidence of a treatment effect. Serum chemistry, hematology, and cardiac function measures were largely unchanged by treatment. Cumulatively, these data show that short-term intravenous treatment of GRMD dogs with rAAVrh74.MHCK7.GALGT2 at high doses can induce muscle glycosylation and utrophin expression and may be safe over a short 3-month interval, but that such treatments had only modest effects on muscle pathology and did not significantly improve muscle strength.

Extracellular transglycosylase and glyceraldehyde-3-phosphate dehydrogenase attributed to the anti-staphylococcal activity of Lactobacillus plantarum USM8613.

Increasing levels of antibiotic resistance in pathogens, including Staphylococcus aureus, remains a serious problem for public health, leading to the need for better alternative antimicrobial strategies. The antimicrobial proteins produced by Lactobacillus plantarum USM8613 attributed to its anti-staphylococcal activity were identified as extracellular transglycosylase and glyceraldehyde-3-phosphate dehydrogenase (GADPH), both with different mechanisms of action. Extracellular transglycosylase, which contains a LysM domain, exerts a cell wall-mediated killing mechanism, while GADPH penetrates into S. aureus cells and subsequently induces the overexpression of autolysis regulators, resulting in S. aureus autolysis. Both extracellular transglycosylase and GADPH exert anti-inflammatory effects in S. aureus-infected HaCaT cells by reducing the expression and production of TLR-2, hBDs and various pro-inflammatory cytokines (IL-1α, IL-1ß, IL-6, TNF-α, and IL-8). Taken together, extracellular transglycosylase and GADPH produced by L. plantarum USM8613 could potentially be applied as an alternative therapeutic agent to treat S. aureus skin infections and promote skin health.

Effect of the glycosyltransferases on the capsular polysaccharide synthesis of Streptococcus suis serotype 2.

Streptococcus suis serotype 2 (S. suis 2) is a serious zoonotic pathogen causing septicemia and meningitis in piglets and humans. The capsular polysaccharide (CPS) is an essential virulence factor for S. suis 2 to infect the host. The synthesis of CPS repeating units involves multiple glycosyltransferases. In this study, four genes (cps2E, cps2G, cps2J and cps2L) encoding different glycosyltransferases involved in CPS synthesis were researched in S. suis 2. Four deletion mutants (Δcps2E, Δcps2G, Δcps2J and Δcps2L) with their CPS incomplete and their sialic acid content significantly decreased were constructed in S. suis 2 SC19. All these four mutant strains showed enhanced adhesion to Hep-2 cells and increased sensitivity to phagocytosis. Flow cytometric analysis also revealed that these four mutants were more susceptible to the attack by the complement system. In a mouse model of infection, the mutant strains were rapidly cleared by the immune system, compared with the wild-type strain. In summary, this study characterized four genes (cps2E, cps2G, cps2J and cps2L) involved in CPS synthesis of S. suis 2 SC19 and it revealed that these genes were all crucial for SC19 to invade and survive in the host.

Tyrosine glycosylation of Rho by Yersinia toxin impairs blastomere cell behaviour in zebrafish embryos.

Yersinia species cause zoonotic infections, including enterocolitis and plague. Here we studied Yersinia ruckeri antifeeding prophage 18 (Afp18), the toxin component of the phage tail-derived protein translocation system Afp, which causes enteric redmouth disease in salmonid fish species. Here we show that microinjection of the glycosyltransferase domain Afp18(G) into zebrafish embryos blocks cytokinesis, actin-dependent motility and cell blebbing, eventually abrogating gastrulation. In zebrafish ZF4 cells, Afp18(G) depolymerizes actin stress fibres by mono-O-GlcNAcylation of RhoA at tyrosine-34; thereby Afp18(G) inhibits RhoA activation by guanine nucleotide exchange factors, and blocks RhoA, but not Rac and Cdc42 downstream signalling. The crystal structure of tyrosine-GlcNAcylated RhoA reveals an open conformation of the effector loop distinct from recently described structures of GDP- or GTP-bound RhoA. Unravelling of the molecular mechanism of the toxin component Afp18 as glycosyltransferase opens new perspectives in studies of phage tail-derived protein translocation systems, which are preserved from archaea to human pathogenic prokaryotes.

A conserved Hpa2 protein has lytic activity against the bacterial cell wall in phytopathogenic Xanthomonas oryzae.

The type III secretion system (TTSS) proteins form a needle-like structure injecting effector proteins into eukaryotic target cells. Although the TTSS forms an important pathway for bacterium-host interaction, its assembly process in vivo is poorly understood. The process is thought to include the opening of a pore before TTSS proteins are inserted into the bacterial cell wall. The proteins that break the bacterial cell wall have not yet been identified. We hypothesize that a hypersensitive response and pathogenicity (hrp) gene functions to digest the bacterial cell wall because it contains a conserved protein sequence similar to lytic transglycosylase. In this study, we cloned hrp-associated 2 (hpa2) genes from the bacteria Xanthomonas oryzae pathovars. We show in vitro that expressed Hpa2 protein has a lytic activity against bacterial cell walls. The analysis of a loss-of-function mutant of the hpa2 gene suggests that the hpa2 affects bacterial proliferation in host plants and a hypersensitive response in nonhost plants. As this is the first of such enzyme activity identified in the Hrp protein family, we speculate that the Hpa2 contributes to the assembly of the TTSS by enlarging gaps in the peptidoglycan meshwork of bacterial cell walls.

Xyloglucan endotransglucosylase activity loosens a plant cell wall.

BACKGROUND AND AIMS: Plant cells undergo cell expansion when a temporary imbalance between the hydraulic pressure of the vacuole and the extensibility of the cell wall makes the cell volume increase dramatically. The primary cell walls of most seed plants consist of cellulose microfibrils tethered mainly by xyloglucans and embedded in a highly hydrated pectin matrix. During cell expansion the wall stress is decreased by the highly controlled rearrangement of the load-bearing tethers in the wall so that the microfibrils can move relative to each other. Here the effect was studied of a purified recombinant xyloglucan endotransglucosylase/hydrolase (XTH) on the extension of isolated cell walls. METHODS: The epidermis of growing onion (Allium cepa) bulb scales is a one-cell-thick model tissue that is structurally and mechanically highly anisotropic. In constant load experiments, the effect of purified recombinant XTH proteins of Selaginella kraussiana on the extension of isolated onion epidermis was recorded. KEY RESULTS: Fluorescent xyloglucan endotransglucosylase (XET) assays demonstrate that exogeneous XTH can act on isolated onion epidermis cell walls. Furthermore, cell wall extension was significantly increased upon addition of XTH to the isolated epidermis, but only transverse to the net orientation of cellulose microfibrils. CONCLUSIONS: The results provide evidence that XTHs can act as cell wall-loosening enzymes.

ABO glycosyltransferases as potential source of minor histocompatibility antigens in allogeneic peripheral blood progenitor cell transplantation.

BACKGROUND: Most studies indicate that the incidence of graft-versus-host disease (GVHD) is not increased in ABO-mismatched allogeneic peripheral blood progenitor cell transplantation. These studies exclusively looked at ABO phenotypes without considering the fact that different genotypes hide behind identical phenotypes that encode for different sets of glycosyltransferases, thus providing a source for minor histocompatibility antigens (mHags). STUDY DESIGN AND METHODS: Therefore, whether peptides derived from ABO glycosyltransferases are capable of stimulating peptide-specific T cells was investigated. T-cell responses were identified by measuring intracellular interleukin-2 expression. RESULTS: Individuals with ABO genotypes encoding glycosyltransferases lacking the peptide sequences used for stimulation showed T-cell responses, whereas those expressing glycosyltransferases containing the respective peptide sequences proved to be tolerant, indicating that ABO peptides are allogeneic and may act as mHags. Interestingly, even ABO*O individuals were tolerant to O glycosyltransferase-derived peptides, which strongly suggests that truncated O transferases are expressed. CONCLUSION: Considering allelic ABO sequences, at least 15 percent of all phenotypically ABO-matched transplant pairs can be expected to have genotype constellations relevant to GVHD. Therefore, the genotype behind the ABO blood group phenotype should be considered to answer the question of whether ABO mismatch is a risk factor of GVHD.

Metabolism of [14C]-2,4,6-trinitrotoluene in tobacco cell suspension cultures.

The metabolism of 2,4,6-trinitrotoluene (TNT) was investigated in tobacco cell suspension cultures amended with [14C]-TNT. Five metabolites were purified and characterized. Temporal evolution of metabolites was monitored during a 120 h incubation period. Metabolites structure was identified by acid and enzymatic hydrolysis, followed by electrospray ionization mass spectrometry and 1H and 13C NMR spectroscopy analyses. The majority of metabolites were conjugates formed by glycose conjugation on the hydroxylamine group of either 2-hydroxylamino-4,6-dinitrotoluene (2-HADNT) or 4-hydroxylamino-2,6-dinitrotoluene (4-HADNT), which led to monoglycoside then to diglycoside. Various diglycosides were observed with gentiobioside or sophoroside formation. Bound residues represented a small fraction (<10% of initial 14C) irrespective of the interval after TNT amendment. Free ADNT was detected only in the medium. This study highlights the central role played by HADNT in the TNT metabolic pathway in tobacco cell suspension culture, and the key role of these compounds and of glycosyltransferases in TNT phytoremediation processes.

Human fibrinopeptide A mediates allergic reaction in mice in the acute phase.

We detected a human humoral peptide that deglycosylates mouse antibody IgE, and found that this peptide had the amino acid sequence ADSGEGDFLAEGGGV. The peptide synthesized according to the sequence also deglycosylated the antibody IgE. The deglycosylated IgE did not induce mouse systemic anaphylaxis. Human fibrinopeptide A has the amino acid sequence indicated above, and a polypeptide extracted from human urine showed an antiallergic effect on humans and mice, which strongly suggests that fibrinopeptide A mediates allergic reaction via antibody IgE-deglycosylation, and is excreted as a polypeptide in urine.

Biochemical characterization of a microbial glucosyltransferase that converts sucrose to palatinose

Foi isolada de fruta deteriorada uma linhagem de klebsiella sp que transforma sacarose em palatinose. Foi verificado que a linhagem produz uma glicosiltransferase intracelular que transforma sacarose para palatinose. A enzima de Klebsiella sp foi purificada por fracionamento com sulfato de amônio e cromatografia em colunas de DEAE-Sephadex A-50 e CM-celulose. A enzima purificada apresentou ótima atividade a 35ºC e na faixa de pH 6,0 a 6,5. A enzima foi inibida por Hg2+ e Ag+, mas näo foi inibida por p-cloromercuribenzoato. Os valores de km e Vmax da enzima purificada foram respectivamente 120mM e 110µg palatinose formada por minuto por ml de enzima para o substrato sacarose. O peso molecular da enzima foi estimado em 74.000 Da. A enzima converteu 4 por cento (p/w) de sacarose para isomaltulose com uma eficiência de 86 por cento a 25ºC, pH 6,5