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Structural basis for dolichylphosphate mannose biosynthesis

Studies of the biosynthesis of the mannose 6-phosphate receptor in receptor-positive and -deficient cell lines

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Pathway Commons::GDP-mannose biosynthesis

Vitamin C (L-ascorbic acid; AsA) acts as a potent antioxidant and cellular reductant in plants and animals. AsA has long been known to have many critical physiological roles in plants, yet its biosynthesis is only currently being defined. A pathway for AsA biosynthesis that features GDP- mannose and L-galactose has recently been proposed for plants. We have isolated a collection of AsA-deficient mutants of Arabidopsis thaliana that are valuable tools for testing of an AsA biosynthetic pathway. The best- characterized of these mutants (vtc1) contains ≃25% of wild-type AsA and is defective in AsA biosynthesis. By using a combination of biochemical, molecular, and genetic techniques, we have demonstrated that the VTC1 locus encodes a GDP-mannose pyrophosphorylase (mannose-1-P guanyltransferase). This enzyme provides GDP-mannose, which is used for cell wall carbohydrate biosynthesis and protein glycosylation as well as for AsA biosynthesis. In addition to genetically defining the first locus involved in AsA biosynthesis, this work highlights the power of using traditional mutagenesis techniques coupled with the Arabidopsis Genome Initiative to rapidly clone physiologically important genes.

The de novo pathway for GDP-l-fucose biosynthesis starts from GDP-l-mannose.

The enzymatic function of C. burnetii CBU0294 was evaluated by complementation of an algC mutant of P. aeruginosa PAO1 serotype O5. PMM function of AlgC catalyzes the formation of mannose-1-phosphate, which is a metabolic precursor for synthesis of GDP-D-mannose . The latter is converted to GDP-D-rhamnose, the sugar residue composing the O5 A-band homopolymer . It has been shown that PGM function of P. aeruginosa AlgC is required for formation of D-glucose-1-phosphate, which is necessary for biosynthesis of UDP-D-glucose, a component of the core heterooligosaccharide , . Therefore both, PMM and PGM functions of AlgC are required to visualize the P. aeruginosa PAO A-band. C. burnetii CBU0294 was cloned into the P. aeruginosa shuttle vector pUCP20 and the resulting plasmid, pCN620, used for transformation of P. aeruginosa PAO1 algC::tet. LPS samples prepared from transformed PAO1 algC::tet strains were separated by SDS-PAGE and visualized by silver staining (). Both, the parental and complemented mutant strains produced a typical LPS banding pattern, while PA01 algC::tet alone as well as the vector control did not produce A-band LPS.

Biosynthesis and processing of the mannose receptor …

Characterization of the GDP-D-Mannose Biosynthesis …

Phosphomannosyl residues present on lysosomal enzymes are specifically recognized by the mannose 6-phosphate receptor protein. This interaction results in the selective targeting of lysosomal enzymes to lysosomes. While this pathway is operative in many cell types, we have found four cultured cell lines that are deficient in the ability to bind lysosomal enzymes containing phosphomannosyl residues to their intracellular or surface membranes (Gabel, C., D. Goldberg, and S. Kornfeld, 1983, Proc. Natl. Acad. Sci. USA, 80:775-779). These cells appear to segregate lysosomal enzymes by an alternate intracellular pathway. To determine the basis for the lack of mannose 6-phosphate receptor activity in these cell lines, we studied the biosynthesis of the receptor in receptor-positive (BW5147) and receptor-deficient (P388D1 and MOPC 315) cells. The cells were labeled with [2-3H]mannose or [35S]methionine and the receptor was immunoprecipitated with an antireceptor antiserum. BW5147 cells synthesize a receptor protein whose size increases after translation/glycosylation. MOPC 315 cells produce an apparently normal receptor and degrade it rapidly. P388D1 cells fail to synthesize any detectable receptor. The receptor from BW5147 and MOPC 315 cells is a glycoprotein with both high mannose and complex asparagine-linked oligosaccharides. The complex-type units become fully sialylated and remain so during long periods of chase.

Coxiella burnetii, the etiologic agent of human Q fever, is a Gram-negative and naturally obligate intracellular bacterium. The O-specific polysaccharide chain (O-PS) of the lipopolysaccharide (LPS) of C. burnetii is considered a heteropolymer of the two unusual sugars β-D-virenose and dihydrohydroxystreptose and mannose. We hypothesize that GDP-D-mannose is a metabolic intermediate to GDP-β-D-virenose. GDP-D-mannose is synthesized from fructose-6-phosphate in 3 successive reactions; Isomerization to mannose-6-phosphate catalyzed by a phosphomannose isomerase (PMI), followed by conversion to mannose-1-phosphate mediated by a phosphomannomutase (PMM) and addition of GDP by a GDP-mannose pyrophosphorylase (GMP). GDP-D-mannose is then likely converted to GDP-6-deoxy-D-lyxo-hex-4-ulopyranose (GDP-Sug), a virenose intermediate, by a GDP-mannose-4,6-dehydratase (GMD). To test the validity of this pathway in C. burnetii, three open reading frames (CBU0671, CBU0294 and CBU0689) annotated as bifunctional type II PMI, as PMM or GMD were functionally characterized by complementation of corresponding E. coli mutant strains and in enzymatic assays. CBU0671, failed to complement an Escherichia coli manA (PMM) mutant strain. However, complementation of an E. coli manC (GMP) mutant strain restored capsular polysaccharide biosynthesis. CBU0294 complemented a Pseudomonas aeruginosa algC (GMP) mutant strain and showed phosphoglucomutase activity (PGM) in a pgm E. coli mutant strain. Despite the inability to complement a manA mutant, recombinant C. burnetii PMI protein showed PMM enzymatic activity in biochemical assays. CBU0689 showed dehydratase activity and determined kinetic parameters were consistent with previously reported data from other organisms. These results show the biological function of three C. burnetii LPS biosynthesis enzymes required for the formation of GDP-D-mannose and GDP-Sug. A fundamental understanding of C. burnetii genes that encode PMI, PMM and GMP is critical to fully understand the biosynthesic pathway of GDP-β-D-virenose and LPS structure in C. burnetii.

A biosynthetic pathway via GDP-mannose, ..

The properties of this compound were in accord with it being dolichyl-mannosyl-phosphate, a possible lipid intermediate required for the biosynthesis of some glycoproteins.

Structural and compositional studies revealed several unique characteristics of the LPS molecule of C. burnetii LPS , , , , , , , , . The lipid A moiety contains a typical 1 and 4′ phosphorylated, β-(1?6)-linked D-glucosamine (GlcN) disaccharide backbone, but is tetraacylated . The inner core oligosaccharide is composed of D-mannose (D-Man), D-glycero-D-manno-heptose (D,D-Hep) and 3-deoxy-α-D-manno-oct-2-ulopyranoside (Kdo), in the molar ratio 223, comparable to the enterobacterial inner core region . However, composition and structure of the O-PS chain is not entirely resolved. Two unique branched sugar residues, β-D-virenose (6-deoxy-3-C-methyl-D-gulose) and L-dihydrohydroxystreptose (3-C-(hydroxymethyl)-L-lyxose), were detected in heteropolysaccharide fractions of isolated LPS , . To our knowledge, virenose is not found on the surface structures of any other microorganism except C. burnetii LPS. Subsequent studies resolved the structure of virenose, while linkage and chemical compositional analysis indicated that C. burnetii O-PS is likely a heteropolymer of 1?4 linked β-D-virenose, dihydrohydroxystreptose and mannose , . These findings are consistent with the observation that ABC transporter encoding genes wzm (CBU0703) and wzt (CBU0704) are located in a genomic region associated with O-PS synthesis . ABC transporters are usually involved in biosynthesis of homopolymeric or small repeating units containing herteropolymeric O-PS .

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  • Glycogen, Starch and Inulin are storage polysaccharides

    Studies of the biosynthesis of the mannose 6-phosphate receptor in receptor-positive and -deficient cell lines.

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01/03/1998 · Abstract

AB - Vitamin C (L-ascorbic acid; AsA) acts as a potent antioxidant and cellular reductant in plants and animals. AsA has long been known to have many critical physiological roles in plants, yet its biosynthesis is only currently being defined. A pathway for AsA biosynthesis that features GDP- mannose and L-galactose has recently been proposed for plants. We have isolated a collection of AsA-deficient mutants of Arabidopsis thaliana that are valuable tools for testing of an AsA biosynthetic pathway. The best- characterized of these mutants (vtc1) contains ≃25% of wild-type AsA and is defective in AsA biosynthesis. By using a combination of biochemical, molecular, and genetic techniques, we have demonstrated that the VTC1 locus encodes a GDP-mannose pyrophosphorylase (mannose-1-P guanyltransferase). This enzyme provides GDP-mannose, which is used for cell wall carbohydrate biosynthesis and protein glycosylation as well as for AsA biosynthesis. In addition to genetically defining the first locus involved in AsA biosynthesis, this work highlights the power of using traditional mutagenesis techniques coupled with the Arabidopsis Genome Initiative to rapidly clone physiologically important genes.

Fructose and mannose metabolism - Reference pathway …

N2 - Vitamin C (L-ascorbic acid; AsA) acts as a potent antioxidant and cellular reductant in plants and animals. AsA has long been known to have many critical physiological roles in plants, yet its biosynthesis is only currently being defined. A pathway for AsA biosynthesis that features GDP- mannose and L-galactose has recently been proposed for plants. We have isolated a collection of AsA-deficient mutants of Arabidopsis thaliana that are valuable tools for testing of an AsA biosynthetic pathway. The best- characterized of these mutants (vtc1) contains ≃25% of wild-type AsA and is defective in AsA biosynthesis. By using a combination of biochemical, molecular, and genetic techniques, we have demonstrated that the VTC1 locus encodes a GDP-mannose pyrophosphorylase (mannose-1-P guanyltransferase). This enzyme provides GDP-mannose, which is used for cell wall carbohydrate biosynthesis and protein glycosylation as well as for AsA biosynthesis. In addition to genetically defining the first locus involved in AsA biosynthesis, this work highlights the power of using traditional mutagenesis techniques coupled with the Arabidopsis Genome Initiative to rapidly clone physiologically important genes.

Glucosyldiphosphoundecaprenol, the Mannose Acceptor in …

Phase variation in C. burnetii is accompanied by the deletion of a large chromosomal fragment which contains glycosyl transferases and sugar processing genes required to complete β-D-virenose biosynthesis, O-PS chain elongation and inner membrane transport , . This deletion is likely the O-PS operon and is responsible for the loss of O-PS in the C. burnetii Nine Mile strain RSA439 . Based on the structure of β-D-virenose and the genes located within the deleted region of the C. burnetii phase II variant, the in presented GDP-β-D-virenose biosynthesis pathway is proposed. The aim of this study was to demonstrate the biological significance of three C. burnetii enzymes for the biosynthesis of GDP-D-mannose and examine the initial steps of GDP-β-D-virenose biosynthesis. The presented data provide fundamental knowledge necessary to further characterize the formation of GDP-β-D-virenose, a novel saccharide, and may help develop potential vaccine candidates such as in vivo and in vitro generated glycoconjugates.

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