Dataset Information

Lipoarabinomannan biosynthesis in Corynebacterineae: the interplay of two ?(1?2)-mannopyranosyltransferases MptC and MptD in mannan branching.

ABSTRACT: Lipomannan (LM) and lipoarabinomannan (LAM) are key Corynebacterineae glycoconjugates that are integral components of the mycobacterial cell wall, and are potent immunomodulators during infection. LAM is a complex heteropolysaccharide synthesized by an array of essential glycosyltransferase family C (GT-C) members, which represent potential drug targets. Herein, we have identified and characterized two open reading frames from Corynebacterium glutamicum that encode for putative GT-Cs. Deletion of NCgl2100 and NCgl2097 in C. glutamicum demonstrated their role in the biosynthesis of the branching ?(1?2)-Manp residues found in LM and LAM. In addition, utilizing a chemically defined nonasaccharide acceptor, azidoethyl 6-O-benzyl-?-D-mannopyranosyl-(1?6)-[?-D-mannopyranosyl-(1?6)](7) -D-mannopyranoside, and the glycosyl donor C(50) -polyprenol-phosphate-[(14) C]-mannose with membranes prepared from different C. glutamicum mutant strains, we have shown that both NCgl2100 and NCgl2097 encode for novel ?(1?2)-mannopyranosyltransferases, which we have termed MptC and MptD respectively. Complementation studies and in vitro assays also identified Rv2181 as a homologue of Cg-MptC in Mycobacterium tuberculosis. Finally, we investigated the ability of LM and LAM from C. glutamicum, and C. glutamicum?mptC and C. glutamicum?mptD mutants, to activate Toll-like receptor 2. Overall, our study enhances our understanding of complex lipoglycan biosynthesis in Corynebacterineae and sheds further light on the structural and functional relationship of these classes of polysaccharides.

SUBMITTER: Mishra AK

PROVIDER: S-EPMC3123699 | biostudies-literature | 2011 Jun

REPOSITORIES: biostudies-literature

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Lipoarabinomannan biosynthesis in Corynebacterineae: the interplay of two α(1→2)-mannopyranosyltransferases MptC and MptD in mannan branching.

Mishra Arun K AK Krumbach Karin K Rittmann Doris D Appelmelk Ben B Pathak Vibha V Pathak Ashish K AK Nigou Jerome J Geurtsen Jeroen J Eggeling Lothar L Besra Gurdyal S GS

Molecular microbiology 20110404 5

Lipomannan (LM) and lipoarabinomannan (LAM) are key Corynebacterineae glycoconjugates that are integral components of the mycobacterial cell wall, and are potent immunomodulators during infection. LAM is a complex heteropolysaccharide synthesized by an array of essential glycosyltransferase family C (GT-C) members, which represent potential drug targets. Herein, we have identified and characterized two open reading frames from Corynebacterium glutamicum that encode for putative GT-Cs. Deletion o ...[more]

PMID: 21435038

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Project description:Fungal-type galactomannan (FTGM) is a polysaccharide composed of α-(1 → 2)-/α-(1 → 6)-mannosyl and β-(1 → 5)-/β-(1 → 6)-galactofuranosyl residues located at the outer cell wall of the human pathogenic fungus Aspergillus fumigatus. FTGM contains a linear α-mannan structure called core-mannan composed of 9 or 10 α-(1 → 2)-mannotetraose units jointed by α-(1 → 6)-linkages. However, the enzymes involved in core-mannan biosynthesis remain unknown. We speculated that two putative α-1,2-mannosyltransferase genes in A. fumigatus, Afu5g02740/AFUB_051270 (here termed core-mannan synthase A [CmsA]) and Afu5g12160/AFUB_059750 (CmsB) are involved in FTGM core-mannan biosynthesis. We constructed recombinant proteins for CmsA and detected robust mannosyltransferase activity using the chemically synthesized substrate p-nitrophenyl α-D-mannopyranoside as an acceptor. Analyses of CmsA enzymatic product revealed that CmsA possesses the capacity to transfer a mannopyranoside to the C-2 position of α-mannose. CmsA could also transfer a mannose residue to α-(1 → 2)-mannobiose and α-(1 → 6)-mannobiose and showed a 31-fold higher specific activity toward α-(1 → 6)-mannobiose than toward α-(1 → 2)-mannobiose. Proton nuclear magnetic resonance (1H-NMR) spectroscopy and gel filtration chromatography of isolated FTGM revealed that core-mannan structures were drastically altered and shortened in disruptant A. fumigatus strains ∆cmsA, ∆cmsB, and ∆cmsA∆cmsB. Disruption of cmsA or cmsB resulted in severely repressed hyphal extension, abnormal branching hyphae, formation of a balloon structure in hyphae, and decreased conidia formation. The normal wild type core-mannan structure and developmental phenotype were restored by the complementation of cmsA and cmsB in the corresponding disruptant strains. These findings indicate that both CmsA, an α-1,2-mannosyltransferase, and CmsB, a putative mannosyltransferase, are involved in FTGM biosynthesis.

Project description:BackgroundDendrite morphogenesis plays an essential role in establishing the connectivity and receptive fields of neurons during the development of the nervous system. To generate the diverse morphologies of branched dendrites, neurons use external cues and cell surface receptors to coordinate intracellular cytoskeletal organization; however, the molecular mechanisms of how this signaling forms branched dendrites are not fully understood.MethodsWe performed in vivo time-lapse imaging of the PVD neuron in C. elegans in several mutants of actin regulatory proteins, such as the WAVE Regulatory Complex (WRC) and UNC-34 (homolog of Enabled/Vasodilator-stimulated phosphoprotein (Ena/VASP)). We examined the direct interaction between the WRC and UNC-34 and analyzed the localization of UNC-34 in vivo using transgenic worms expressing UNC-34 fused to GFP.ResultsWe identify a stereotyped sequence of morphological events during dendrite outgrowth in the PVD neuron in C. elegans. Specifically, local increases in width ("swellings") give rise to filopodia to facilitate a "rapid growth and pause" mode of growth. In unc-34 mutants, filopodia fail to form but swellings are intact. In WRC mutants, dendrite growth is largely absent, resulting from a lack of both swelling and filopodia formation. We also found that UNC-34 can directly bind to the WRC. Disrupting this binding by deleting the UNC-34 EVH1 domain prevented UNC-34 from localizing to swellings and dendrite tips, resulting in a stunted dendritic arbor and reduced filopodia outgrowth.ConclusionsWe propose that regulators of branched and linear F-actin cooperate to establish dendritic branches. By combining our work with existing literature, we propose that the dendrite guidance receptor DMA-1 recruits the WRC, which polymerizes branched F-actin to generate "swellings" on a mother dendrite. Then, WRC recruits the actin elongation factor UNC-34/Ena/VASP to initiate growth of a new dendritic branch from the swelling, with the help of the actin-binding protein UNC-115/abLIM. Extension of existing dendrites also proceeds via swelling formation at the dendrite tip followed by UNC-34-mediated outgrowth. Following dendrite initiation and extension, the stabilization of branches by guidance receptors further recruits WRC, resulting in an iterative process to build a complex dendritic arbor.

Dataset Information

Lipoarabinomannan biosynthesis in Corynebacterineae: the interplay of two ?(1?2)-mannopyranosyltransferases MptC and MptD in mannan branching.

Publications

Lipoarabinomannan biosynthesis in Corynebacterineae: the interplay of two α(1→2)-mannopyranosyltransferases MptC and MptD in mannan branching.

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