Mannosyl Transferase Research Articles

Composition and Functional Capacity of Gut Microbes are Associated with Arterial Stiffness: A Prospective Study

Objective: Arterial stiffness is an important predictor of cardiovascular disease. Microbial diversity in the gut has been shown to be associated inversely with arterial stiffness in Caucasian populations. However, due to the different profiles of the gut microbiota among ethnicities, the relationship between gut-microbiota dysbiosis and the progression of arterial stiffness merits further investigation. This study aimed to investigate the association between the composition and functional capacity of the gut microbiota and the progression of arterial stiffness. Methods: “Shotgun” metagenomics sequencing were undertaken in 96 individuals from a hypertension-associated gut-microbiota study in the Kailuan cohort, who measured brachial-ankle pulse wave velocity (baPWV) and provided fecal samples between September 2014 and February 2015 at Kailuan General Hospital and 11 affiliated hospitals. The different composition and functional capacity of the gut microbiota were compared between individuals without arterial stiffness (normal arterial stiffness group, baPWV <1,400 cm/s, n = 27) and participants with arterial stiffness (increased arterial stiffness group, baPWV ≥1,400 cm/s, n = 69) at baseline. These participants were followed up prospectively for a mean duration of 2.6 years, and 50 underwent a repeat baPWV measurement. Associations between the gut microbiota and severity and progression of arterial stiffness were assessed using MaAsLin2 software after adjustment for age, sex, and mean arterial blood pressure and correction for multiple testing. Gene “catalogs” were aligned to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database to obtain information for potential functional capacities of the gut microbiota. Results: In this study, 14 genera and 50 species of bacteria were identified to be abundant in participants with normal arterial stiffness compared with those with increased arterial stiffness. Of 14 genera, the prevalence of beneficial bacteria of the genera Leadbetterella and Cytophaga was correlated inversely with baPWV (P < 0.05). Analyses of functional capacity revealed gut-microbial dysfunctions in the synthetic processes of “threonine dehydratase” “hypothetical protein” “mannosyl transferase” and “type-IV secretion-system proteins” in individuals suffering from arterial stiffness. During follow-up, bacteria of the proinflammatory genera Escherichia, Shigella, and Ruegeria were enriched in individuals with increased baPWV. Functional analyses showed that 26 KEGG orthologs of gut microbes were associated with an increase in baPWV and involved in “carbohydrate metabolism” “amino acid metabolism” and “protein families related to genetic information processing.” Conclusions: The composition and functional capacity of the microbial community in the gut of people suffering from arterial stiffness differed from those in individuals not suffering from arterial stiffness. Our data provide a new direction for the causality of the host-gut microbiota in arterial stiffness.

S16 and T18 mannosylation sites of LppX are not essential for its activity in phthiocerol dimycocerosates localization at the surface of Mycobacterium tuberculosis

LppX is an important virulence factor essential for surface localization of phthiocerol dimycocerosates (DIM) in Mycobacterium tuberculosis. Based on Concanavalin A recognition, M. tuberculosis LppX (LppX-tb) was initially proposed to be glycosylated in M. tuberculosis and more recently this glycosylation was characterized by mass spectrometry analysis on LppX-tb expressed and purified from Corynebacterium glutamicum. Here, using this model organism and Mycobacterium smegmatis, we show that S16 and T18 residues of LppX-tb are indeed glycosylated with several hexoses units. Interestingly this glycosylation is strictly dependent on the mannosyl transferase PMT which, in M. tuberculosis, has been reported to be crucial for virulence. Using a site directed mutagenesis approach, we were able to show that the absence of S16 and T18 glycosylation does not alter phthiocerol dimycocerosates (DIM) localization in M. tuberculosis.

Candida albicans β-1,2 mannosyl transferase Bmt3: Preparation and evaluation of a β (1,2), α (1,2)-tetramannosyl fluorescent substrate

We describe for the first time the chemical synthesis of a tetramannoside, containing both α (1→2) and β (1→2) linkages. Dodecylthio (lauryl) glycosides were prepared from odorless dodecyl thiol and used as donors for the glycosylation steps. This tetramannoside, was coupled to a mantyl group, and revealed to be a perfect substrate of β-mannosyltransferase Bmt3, confirming the proposed specificity and allowing the preparation of a pentamannoside sequence (β Man (1,2) β Man (1,2) α Man (1,2) α Man (1,2) α Man) usable as a novel substrate for further elongation studies.

VCP/Cdc48 rescues the growth defect of a GPI10 mutant in yeast

We identified a yeast mutant with temperature-sensitive growth defects that were rescued by VCP expression. The mutation occurred in GPI10, which encodes a mannosyl transferase for glycosylphosphatidylinositol anchor formation in the endoplasmic reticulum, and caused a Gly469Glu substitution in Gpi10. The mutant exhibited increased unfolded protein response, which was partially rescued by VCP or Cdc48, and showed sensitivity against cell-wall stressors, which were not rescued by VCP. These results suggest a potential link between VCP/Cdc48 and Gpi10 functions in the control of cell growth.

Altered N-glycosylation modulates TgrB1- and TgrC1-mediated development but not allorecognition in Dictyostelium

Cell surface adhesion receptors play diverse functions in multicellular development. In Dictyostelium, two immunoglobulin-like adhesion proteins, TgrB1 and TgrC1, are essential components with dual roles in morphogenesis and allorecognition during development. TgrB1 and TgrC1 form a heterophilic adhesion complex during cell contact and mediate intercellular communication. The underlying signaling pathways, however, have not been characterized. Here, we report on a mutation that suppresses the tgrB-tgrC1-defective developmental arrest. The mutated gene alg9 encodes a putative mannosyl transferase that participates in N-linked protein glycosylation. We show that alteration in N-linked glycosylation, caused by an alg9 mutation with a plasmid insertion (alg9(ins)) or tunicamycin treatment, can partially suppress the developmental phenotypes caused by tgrC1 deletion or replacement with an incompatible allele. The alg9(ins) mutation also preferentially primed cells toward a stalk-cell fate. Despite its effect on development, we found that altered N-linked glycosylation had no discernable effect on TgrB1-TgrC1-mediated allorecognition. Our results show that N-linked protein glycosylation can modulate developmental processes without disturbing cell-cell recognition, suggesting that tgrB1 and tgrC1 have distinct effects in the two processes.

Inactivation of the lpcC gene alters surface-related properties and symbiotic capability of Bradyrhizobium japonicum

We investigated the role of the Bradyrhizobium japonicum lpcC gene, encoding a mannosyl transferase, involved in the lipopolysaccharide (LPS) biosynthesis. The inactivation of the lpcC gene considerably altered the LPS structure and the cell surface properties. LPS analysis showed that the lpcC mutant JS715 had an abnormal LPS structure deficient in O-antigen. The cell surface hydrophobicity increased approximately threefold in JS715 compared to the wild type. The increased cell surface hydrophobicity is likely to be related with cell aggregation in the mutant culture. For the growth comparison, JS715 showed slower growth rate than the wild type. The motility of JS715 decreased in soft agar plates, but it showed enhanced biofilm-forming ability. Interestingly, JS715 was not able to nodulate the host legume soybean (Glycine max). This study shows not only that lpcC is involved in the biosynthesis of O-antigen in the B.japonicum LPS, but also that inactivation of the lpcC gene affects symbiotic capability of B.japonicum and surface-related properties such as cell hydrophobicity, biofilm formation and motility. This study demonstrates the role of the B.japonicum lpcC in nodulation with soybean and importance of cell surface hydrophobicity. The results also highlight that intact LPS is required for successful symbiosis between B.japonicum and soybeans. Our findings not only support previous studies emphasizing the necessity of LPS on the interaction between the two symbiotic partners, but also contribute to a better understanding of the symbiotic mechanisms.

LipoproteinN-acyl transferase (Lnt1) is dispensable for proteinO-mannosylation byStreptomyces coelicolor

A protein glycosylation system related to that for protein mannosylation in yeast is present in many actinomycetes. This system involves polyprenyl phosphate mannose synthase (Ppm), protein mannosyl transferase (Pmt), and lipoprotein N-acyl transferase (Lnt). In this study, we obtained a series of mutants in the ppm (sco1423), lnt1 (sco1014), and pmt (sco3154) genes of Streptomyces coelicolor, which encode Ppm, Lnt1, and Pmt, to analyze their requirement for glycosylation of the heterologously expressed Apa glycoprotein of Mycobacterium tuberculosis. The results show that both Ppm and Pmt were required for Apa glycosylation, but that Lnt1 was dispensable for both Apa and the bacteriophage φC31 receptor glycosylation. A bacterial two-hybrid assay revealed that contrary to M.tuberculosis, Lnt1 of S.coelicolor does not interact with Ppm. The D2 catalytic domain of M.tuberculosisPpm was sufficient for complementation of an S.coelicolor double mutant lacking Lnt1 and Ppm, both for Apa glycosylation and for glycosylation of φC31 receptor. On the other hand, M.tuberculosisPmt was not active in S.coelicolor, even when correctly localized to the cytoplasmic membrane, showing fundamental differences in the requirements for Pmt activity in these two species.

Bacterial protein-O-mannosylating enzyme is crucial for virulence ofMycobacterium tuberculosis

A posttranslational protein O-mannosylation process resembling that found in fungi and animals has been reported in the major human pathogen Mycobacterium tuberculosis (Mtb) and related actinobacteria. However, the role and incidence of this process, which is essential in eukaryotes, have never been explored in Mtb. We thus analyzed the impact of interrupting O-mannosylation in the nonpathogenic saprophyte Mycobacterium smegmatis and in the human pathogen Mtb by inactivating the respective putative protein mannosyl transferase genes Msmeg_5447 and Rv1002c. Loss of protein O-mannosylation in both mutant strains was unambiguously demonstrated by efficient mass spectrometry-based glycoproteomics analysis. Unexpectedly, although the M. smegmatis phenotype was unaffected by the lack of manno-proteins, the Mtb mutant had severely impacted growth in vitro and in cellulo associated with a strong attenuation of its pathogenicity in immunocompromised mice. These data are unique in providing evidence of the biological significance of protein O-mannosylation in mycobacteria and demonstrate the crucial contribution of this protein posttranslational modification to Mtb virulence in the host.

Mantyl tagged oligo α (1 → 2) mannosides as Candida albicans β-mannosyl transferases substrates: a comparison between synthetic strategies

Fluorescent oligomannosides are important tools for the evaluation of mannosyl transferase activities and selectivities. In a project dealing with Candida albicans β-mannosyl transferases, three mantyl tagged α (1 → 2) oligomannosides were prepared by different ways: using all ester strategy compatible with the presence of an azido group suitable for direct click chemistry; and alternatively using the more classic benzyl protecting groups. Although more elegant, the all ester strategy has shown important limitations: reduced reactivity of mannosyl donors, and 3 → 2 ester migration. Preliminary enzymatic studies have shown that the synthetic oligomannosides are efficient substrate of β-mannosyl transferases.

Gene expression during activation of Paracoccidioides brasiliensis conidia

This study focuses on gene expression during crucial biological phenomena of the dimorphic fungal human pathogen Paracoccidioides brasiliensis, the conidia-to-yeast (C-Y) transition and the conidia-to-mycelia (C-M) germination. We studied 10 genes involved in different cellular functions: oxidative stress response (alternative oxidase (AOX), superoxide dismutase (SOD), flavodoxin, conserved hypothetical protein (Y20)); cell metabolism (glyceraldehyde-3-phosphate dehydrogenase (GADPH), cholestenol Delta-isomerase (ChDI), glycine dehydrogenase (GDh)) and heat shock response (Heat shock protein 90 (HSP90)), and cell synthesis and wall structure (glucan synthase-1 (GS-1), α-1,3-glucan synthase (αGS), and mannosyltransferase (MT)). Gene expression was measured during the first 72 h and 96 h of C-Y and C-M, respectively, previously shown to be a fundamental time frame for the consolidation of these cellular processes. The gene expression of AOX, GAPDH, HSP90, MT, αGS, and GDh was significantly increased during the C-Y transition, while SOD, ChDI, GAPDH, MT, GDh, and GS-1 were increased during C-M germination. Additionally, some were highly expressed in each process: AOX, HSP90, and αGS during C-Y; SOD, ChDI, and GS-1 during C-M. Altogether, these data add new information regarding gene expression during the C-Y and C-M processes. Future research will be targeted to further characterize the true relevance of the studied genes during the morphological transition, either during adaptation to the environment or to the infected host.

Topography of Lipid A Assembly and Modification in the Inner Membrane of Francisella

Francisella tularensis is Gram‐negative bacteria that cause tularemia in mammals. The outer leaflet of its outer membrane is formed by lipid A, the hydrophobic membrane anchor of lipopolysaccharide (LPS). However, about two thirds of the lipid A molecules in Francisella do not have a core oligosaccharide. We have discovered that LPS and free lipid A are not fully separated from each other during purification. We took advantage of a mutation in a mannosyl transferase (LpcC), which adds the proximal mannose residue to Kdo2‐lipid A in the first step of outer core assembly. The LpcC mutant produces a truncated LPS with Kdo residues as the only remaining core sugars. The Kdo modified lipid A molecules lack of both the 1 and 4′‐phosphate groups, whereas free lipid A only lacks the 4′‐phosphate residue. This difference is attributed to the selectivity of the 1‐phosphatase LpxE, which located on the outer surface of the inner membrane and is dependent upon the presence of the Kdo disaccharide for activity. Likewise, LpcC, which faces the cytoplasm, is selective in vivo and in vitro for acceptor substrates with two Kdo units instead of one. Our findings have implications for the order and topography of lipid A modification reactions in the Francisella inner membrane. Supported by NIH grant GM‐51796 to C.R.H.R.

Viable mouse gene ablations that robustly alter brain Aβ levels are rare

BackgroundAccumulation of amyloid-β (Aβ) peptide in the brain is thought to play a key pathological role in Alzheimer's disease. Many pharmacological targets have therefore been proposed based upon the biochemistry of Aβ, but not all are equally tractable for drug discovery.ResultsTo search for novel targets that affect brain Aβ without causing toxicity, we screened mouse brain samples from 1930 novel gene knock-out (KO) strains, representing 1926 genes, using Aβ ELISA assays. Although robust Aβ lowering was readily apparent in brains from a BACE1 KO strain, none of the novel strains exhibited robust decreases in brain Aβ, including a GPR3 KO strain, which had previously been proposed as an Aβ target. However, significantly increased Aβ was observed in brain samples from two KO strains, corresponding to genes encoding the glycosylphosphatidylinositol mannosyl transferase PIGZ and quinolinate phosphoribosyltransferase (QPRT).ConclusionsThus, gene ablations that are permissive for mouse survival and that also have a robust effect on Aβ levels in the brain are rare.

A Multifunctional Mannosyltransferase Family in Candida albicans Determines Cell Wall Mannan Structure and Host-Fungus Interactions

The cell wall proteins of fungi are modified by N- and O-linked mannosylation and phosphomannosylation, resulting in changes to the physical and immunological properties of the cell. Glycosylation of cell wall proteins involves the activities of families of endoplasmic reticulum and Golgi-located glycosyl transferases whose activities are difficult to infer through bioinformatics. The Candida albicans MNT1/KRE2 mannosyl transferase family is represented by five members. We showed previously that Mnt1 and Mnt2 are involved in O-linked mannosylation and are required for virulence. Here, the role of C. albicans MNT3, MNT4, and MNT5 was determined by generating single and multiple MnTΔ null mutants and by functional complementation experiments in Saccharomyces cerevisiae. CaMnt3, CaMnt4, and CaMnt5 did not participate in O-linked mannosylation, but CaMnt3 and CaMnt5 had redundant activities in phosphomannosylation and were responsible for attachment of approximately half of the phosphomannan attached to N-linked mannans. CaMnt4 and CaMnt5 participated in N-mannan branching. Deletion of CaMNT3, CaMNT4, and CaMNT5 affected the growth rate and virulence of C. albicans, affected the recognition of the yeast by human monocytes and cytokine stimulation, and led to increased cell wall chitin content and exposure of β-glucan at the cell wall surface. Therefore, the MNT1/KRE2 gene family participates in three types of protein mannosylation in C. albicans, and these modifications play vital roles in fungal cell wall structure and cell surface recognition by the innate immune system.

Babesia bovis contains an abundant parasite-specific protein-free glycerophosphatidylinositol and the genes predicted for its assembly

Autonomous glycosylphosphatidylinositol (GPI) molecules (also protein-free GPIs or free GPIs) have been reported to be particularly abundant in some parasitic protozoa and mediate strong immunomodulatory effects on the host immune system. In the work at hand we have investigated the existence of free GPIs in Babesia bovis. Comparative thin layer chromatographic analysis of the protein-free glycolipid fraction of in vitro cultured B. bovis merozoites and erythrocyte membranes demonstrated the presence of an abundant parasite-specific band. Its chemical analysis revealed a GPI species containing a chain of two mannose residues, N-glucosamine and non-acylated inositol. The lipid moiety linked to inositol was diacylglycerol. The total fatty acid composition showed predominantly long-carbon chain molecules (12% of C22:0 and 45% of C24:0). The potential of B. bovis to assemble the presented free GPI species was verified by the existence of seven genes in its genome that putatively encode the following GPI biosynthetic enzymes: PI N-acetyl-GlcN-transferase (PIG-A and GPI-1), N-acetyl-GlcN-PI-de-N-acetylase (PIG-L), acyltransferase (PIG-W), dolichyl-phosphate mannosyl transferase (DPM-1), GPI mannosyltransferase I (PIG-M), and GPI mannosyltransferase II (PIG-V). GPI biosynthesis is vital for the intraerythrocytic parasite stage as mannosamine, an inhibitor of GPI biosynthesis, impaired in vitro growth of B. bovis merozoites. Absence of the vast majority of N-glycan metabolism encoding genes in the B. bovis genome underscores that the growth inhibitory effect of mannosamine is attributable to its interference with GPI biosynthesis and not with assembly of N-linked oligosaccharides, as has been described for higher eukaryotes. Elucidation of the structure and biosynthesis of GPI may allow to facilitate the development of future immune interventions against bovine babesiosis.

The Schizosaccharomyces pombe Map4 adhesin is a glycoprotein that can be extracted from the cell wall with alkali but not with β‐glucanases and requires the C‐terminal DIPSY domain for function

In fungi, cell adhesion is required for flocculation, mating and virulence, and it is mediated by covalently bound cell wall proteins termed adhesins. Map4, an adhesin required for mating in Schizosaccharomyces pombe, is N-glycosylated and O-glycosylated, and is an endogenous substrate for the mannosyl transferase Oma4p. Map4 has a modular structure with an N-terminal signal peptide, a serine and threonine (S/T)-rich domain that includes nine repeats of 36 amino acids (rich in serine and threonine residues, but lacking glutamines), and a C-terminal DIPSY domain with no glycosylphosphatidyl inositol (GPI)-anchor signal. Map4 can be extracted from cell walls with SDS/mercaptoethanol sample buffer or with mild alkali solutions. After extensive extraction with hot sample buffer, no more protein can be released by beta-glucanases or alkali. Additionally, none of the cysteine residues of the protein is required for its retention at the cell wall. These results show that Map4 is not directly bound to beta-glucans and point to the existence of alkali- and SDS/mercaptoethanol-sensitive linkages between cell wall proteins. The N-terminal S/T-rich regions are required for cell wall attachment, but the C-terminal DIPSY domain is required for agglutination and mating in liquid and solid media.

Genome-wide expression profile of the mnn2Δ mutant of Saccharomyces cerevisiae

The MNN2 gene of S. cerevisiae encodes an alpha (1,2) mannosyl transferase required for branching the outer chain of N-linked oligosaccharides (Rayner J.C. and Munro S. 1998. J. Biol. Chem. 273: 26836-26843) and it also seems to have some effect on the transfer of mannosyl phosphate groups to the inner core (Olivero I. et al. 2000. FEBS Lett. 475: 111-116). In order to reveal possible interactions of MNN2 expression with other cellular pathways, we analyzed the transcriptome of the deletion mutant S. cerevisiae mnn2 Delta using cDNA microarrays. We found 151 genes that showed an altered expression level of > or =2-fold, 58 of them up-regulated and 93 down-regulated. Quite a high proportion of these genes (29%) encode unclassified proteins. In contrast to other defects affecting the integrity of the cell wall, deletion of MNN2 does not stimulate the expression of any of the genes included in the previously defined 'cell wall compensatory cluster' (Lagorce et al. 2003. J. Biol. Chem. 278: 20345-20357). We also found that 15% of the selected genes are related to central metabolic pathways. In addition, the mnn2 Delta strain seems to have a certain level of stimulation of DNA processing reactions while some genes involved in intracellular transport pathways are under-regulated.

Biosynthesis of Glycoproteins in the Human Pathogenic Fungus Sporothrix Schenckii: Synthesis of Dolichol Phosphate Mannose and Mannoproteins by Membrane-Bound and Solubilized Mannosyl Transferases

A membrane fraction obtained from the filamentous form of Sporothrix schenckii was able to transfer mannose from GDP-Mannose into dolichol phosphate mannose and from this inTermediate into mannoproteins in coupled reactions catalyzed by dolichol phosphate mannose synthase and protein mannosyl transferase(s), respectively. Although the transfer reaction depended on exogenous dolichol monophosphate, membranes failed to use exogenous dolichol phosphate mannose for protein mannosylation to a substantial extent. Over 95% of the sugar was transferred to proteins via dolichol phosphate mannose and the reaction was stimulated several fold by Mg2+ and Mn2+. Incubation of membranes with detergents such as Brij 35 and Lubrol PX released soluble fractions that transferred the sugar from GDP-Mannose mostly into mannoproteins, which were separated by affinity chromatography on Concanavilin A-Sepharose 4B into lectin-reacting and non-reacting fractions. All proteins mannosylated in vitro eluted with the lectin-reacting proteins and analytical electrophoresis of this fraction revealed the presence of at least nine putative mannoproteins with molecular masses in the range of 26-112 kDa. The experimental approach described here can be used to identify and isolate specific glycoproteins mannosylated in vitro in studies of O-glycosylation.

Rhodanine‐3‐acetic Acid Derivatives as Inhibitors of Fungal Protein Mannosyl Transferase 1 (PMT1).

AbstractFor Abstract see ChemInform Abstract in Full Text.

Rhodanine-3-acetic acid derivatives as inhibitors of fungal protein mannosyl transferase 1 (PMT1)

The first inhibitors of fungal protein: mannosyl transferase 1 (PMT1) are described. They are based upon rhodanine-3-acetic acid and several compounds have been identified, for example, 5-[[3-(1-phenylethoxy)-4-(2-phenylethoxy)phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineacetic acid ( 5a), which inhibit Candida albicans PMT1 with IC 50s in the range 0.2–0.5 μM. Members of the series are effective in inducing changes in morphology of C. albicans in vitro that have previously been associated with loss of the transferase activity. These compounds could serve as useful tools for studying the effects of protein O-mannosylation and its relevance in the search for novel antifungal agents.

Partial purification and characterization of a mannosyl transferase involved in O -linked mannosylation of glycoproteins in Candida albicans.

Incubation of a mixed membrane fraction of C. albicans with the nonionic detergents Nonidet P-40 or Lubrol solubilized a fraction that catalyzed the transfer of mannose either from endogenously generated or exogenously added dolichol-P-[14C]Man onto endogenous protein acceptors. The protein mannosyl transferase solubilized with Nonidet P-40 was partially purified by a single step of preparative nondenaturing electrophoresis and some of its properties were investigated. Although transfer activity occurred in the absence of exogenous mannose acceptors and thus depended on acceptor proteins isolated along with the enzyme, addition of the protein fraction obtained after chemical de-mannosylation of glycoproteins synthesized in vitro stimulated mannoprotein labeling in a concentration-dependent manner. Other de-mannosylated glycoproteins, such as yeast invertase or glycoproteins extracted from C. albicans, failed to increase the amount of labeled mannoproteins. Mannosyl transfer activity was not influenced by common metal ions such as Mg(2+), Mn(2+) and Ca(2+), but it was stimulated up to 3-fold by EDTA. Common phosphoglycerides such as phosphatidylglycerol and, to a lower extent, phosphatidylinositol and phosphatidylcholine enhanced transfer activity. Interestingly, coupled transfer activity between dolichol phosphate mannose synthase, i.e., the enzyme responsible for Dol-P-Man synthesis, and protein mannosyl transferase could be reconstituted in vitro from the partially purified transferases, indicating that this process can occur in the absence of cell membranes.