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• W2033352215 abstract "The cell wall of Mycobacterium spp. consists predominately of arabinogalactan chains linked at the reducing ends to peptidoglycan via a P-GlcNAc-(α1–3)-Rha linkage unit (LU) and esterified to a variety of mycolic acids at the nonreducing ends. Several aspects of the biosynthesis of this complex have been defined, including the initial formation of the LU on a polyprenyl phosphate (Pol-P) molecule followed by the sequential addition of galactofuranosyl (Galf) units to generate Pol-P-P-LU-(Galf)1,2,3, etc. and Pol-P-P-LU-galactan, catalyzed by a bifunctional galactosyltransferase (Rv3808c) capable of adding alternating 5- and 6-linked Galf units. By applying cell-free extracts of Mycobacterium smegmatis, containing cell wall and membrane fragments, and differential labeling with UDP-[14C]Galp and recombinant UDP-Galp mutase as the source of [14C]Galf for galactan biosynthesis and 5-P-[14C]ribosyl-P-P as a donor of [14C]Araf for arabinan synthesis, we now demonstrate sequential synthesis of the simpler Pol-P-P-LU-(Galf)n glycolipid intermediates followed by the Pol-P-P-LU-arabinogalactan and, finally, ligation of the P-LU-arabinogalactan to peptidoglycan. This first time demonstration of in vitro ligation of newly synthesized P-LU-arabinogalactan to newly synthesized peptidoglycan is a necessary forerunner to defining the genetics and enzymology of cell wall polymer-peptidoglycan ligation in Mycobacterium spp. and examining this step as a target for new antibacterial drugs. The cell wall of Mycobacterium spp. consists predominately of arabinogalactan chains linked at the reducing ends to peptidoglycan via a P-GlcNAc-(α1–3)-Rha linkage unit (LU) and esterified to a variety of mycolic acids at the nonreducing ends. Several aspects of the biosynthesis of this complex have been defined, including the initial formation of the LU on a polyprenyl phosphate (Pol-P) molecule followed by the sequential addition of galactofuranosyl (Galf) units to generate Pol-P-P-LU-(Galf)1,2,3, etc. and Pol-P-P-LU-galactan, catalyzed by a bifunctional galactosyltransferase (Rv3808c) capable of adding alternating 5- and 6-linked Galf units. By applying cell-free extracts of Mycobacterium smegmatis, containing cell wall and membrane fragments, and differential labeling with UDP-[14C]Galp and recombinant UDP-Galp mutase as the source of [14C]Galf for galactan biosynthesis and 5-P-[14C]ribosyl-P-P as a donor of [14C]Araf for arabinan synthesis, we now demonstrate sequential synthesis of the simpler Pol-P-P-LU-(Galf)n glycolipid intermediates followed by the Pol-P-P-LU-arabinogalactan and, finally, ligation of the P-LU-arabinogalactan to peptidoglycan. This first time demonstration of in vitro ligation of newly synthesized P-LU-arabinogalactan to newly synthesized peptidoglycan is a necessary forerunner to defining the genetics and enzymology of cell wall polymer-peptidoglycan ligation in Mycobacterium spp. and examining this step as a target for new antibacterial drugs. The cell envelope of Mycobacterium tuberculosis is composed of a conventional plasma membrane and a cell wall proper unique to some genera within the Actinomycetales order, consisting of a core of arabinogalactan (AG), 1The abbreviations used are: AG, arabinogalactan; Galf, galactofuranose; Araf, arabinofuranose; Galp, galactopyranose; GL, glycolipid; LU, linker unit; MOPS, 4-morpholinepropanesulfonic acid; PG, peptidoglycan; PRPP, 5-phosphoribose-pyrophosphate; Rha, rhamnose; UDP-MurNAc-pentapeptide, uridine diphosphoryl-N-acetylmuramate-l-Ala-d-Glu-meso-DAP-d-Ala-d-Ala; Tricine, N-[2-hydroxy-1,1-bis(hydoxymethyl)ethyl]glycine; MAPc, mycolate-arabinogalactan-peptidoglycan-complex; Pol-P, polyprenyl phosphate; MurNAc, N-acetylmuramic acid; TAPS, 3-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}-1-propanesulfonic acid; DAP, 2,6-diaminopimelic acid. peptidoglycan (PG), and mycolic acids interspersed with a variety of free lipids, lipoglycans, and proteins (1Brennan P.J. Nikaido H. Annu. Rev. Biochem. 1995; 64: 29-63Crossref PubMed Scopus (1572) Google Scholar); there is also evidence for polysaccharides on the outer face of the cell wall (2Crick D.C. Mahapatra S. Brennan P.J. Glycobiology. 2001; 11: 107R-118RCrossref PubMed Scopus (201) Google Scholar). The mycolic acids are attached to the nonreducing ends of the arabinogalactan, whereas the reducing ends are covalently attached to the cross-linked peptidoglycan via phosphoryl-N-acetylglucosaminosyl-rhamnosyl linkage units (P-GlcNAc-Rha). This massive structure, the mycolate-arabinogalactan-peptidoglycan-complex (MAPc), is the basis of many of the physiological and pathogenic features of M. tuberculosis and the site of susceptibility and resistance to many of the anti-tuberculosis drugs (3Crick D.C. Brennan P.J. Curr. Opin. Anti-inf. Invest. Drugs. 2000; 2: 154-163Google Scholar). Biosynthesis of this complex commences with attachment of the residues of the linkage unit, GlcNAc-1-P and Rha, donated by UDP-GlcNAc and dTDP-Rha, respectively, to a polyprenyl phosphate (Pol-P) carrier lipid (4Mikušová K. Mikuš M. Besra G.S. Hancock I. Brennan P.J. J. Biol. Chem. 1996; 271: 7820-7828Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Formation of the linkage unit is followed by the sequential addition of galactofuranosyl (Galf) units donated by UDP-Galf, to provide simple Pol-P-P-linked AG intermediates (4Mikušová K. Mikuš M. Besra G.S. Hancock I. Brennan P.J. J. Biol. Chem. 1996; 271: 7820-7828Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). The bulk, if not all, of galactan biosynthesis is catalyzed by a membrane-associated bifunctional galactosyltransferase capable of adding the alternating 5- and 6-linked Galf units (5Mikušová K. Yagi T. Stern R. McNeil M.R. Besra G.S. Crick D.C. Brennan P.J. J. Biol. Chem. 2000; 275: 33890-33897Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 6Kremer L. Dover L.G. Morehouse C. Hitchin P. Everett M. Morris H.R. Dell A. Brennan P.J. McNeil M.R. Flaherty C. Duncan K. Besra G.S. J. Biol. Chem. 2001; 276: 26430-26440Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). The demonstration that the direct donor of the arabinofuranosyl (Araf) units of the cell wall core is decaprenyl-P-Araf (7Wolucka B.A. McNeil M.R. de Hoffmann E. Chojnacki T. Brennan P.J. J. Biol. Chem. 1994; 269: 23328-23335Abstract Full Text PDF PubMed Google Scholar) and that 5-P-ribosyl-PP (PRPP) is a precursor of decaprenyl-P-Araf (8Scherman M.S. Kalbe-Bournonville L. Bush D. Xin Y. Deng L. McNeil M. J. Biol. Chem. 1996; 271: 29652-29658Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar) now provides us with the means to characterize the subsequent polymerization steps in AG biosynthesis and the final ligation of the AG lipid-linked intermediates to PG to generate the fully formed cell wall core. Preparation of UDP-Galp Mutase, dTDP-Rha, P[14C]RPP, and UDP-MurNAc-l-Ala-d-Glu-meso-DAP-d-Ala-d-Ala (UDP-MurNAc-pentapeptide)—Escherichia coli BL21 (DE3) (Stratagene, Cedar Creek, TX) was transformed with plasmid pORF6 containing Rv3809c as described (5Mikušová K. Yagi T. Stern R. McNeil M.R. Besra G.S. Crick D.C. Brennan P.J. J. Biol. Chem. 2000; 275: 33890-33897Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). The recombinant UDP-Galp mutase was prepared and assayed as described (5Mikušová K. Yagi T. Stern R. McNeil M.R. Besra G.S. Crick D.C. Brennan P.J. J. Biol. Chem. 2000; 275: 33890-33897Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar); the concentration of protein in the final preparation was 2.0 mg per 100 μl. dTDP-Rha and P[14C]RPP were prepared from dTDP-Glc (9Lee R. Monsey D. Weston A. Duncan K. Rithner C. McNeil M. Anal. Biochem. 1996; 242: 1-7Crossref PubMed Scopus (68) Google Scholar) and d-[U-14C]glucose (8Scherman M.S. Kalbe-Bournonville L. Bush D. Xin Y. Deng L. McNeil M. J. Biol. Chem. 1996; 271: 29652-29658Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), respectively, and were generous gifts from Dr. M. R. McNeil (Colorado State University). For the synthesis of UDP-MurNAc-pentapeptide, UDP-MurNAc was first prepared by a two-step coupled enzymatic conversion of UDP-GlcNAc to UDP-MurNAc (10Mahapatra S. Crick D.C. Brennan P.J. J. Bacteriol. 2000; 182: 6827-6830Crossref PubMed Scopus (55) Google Scholar) and identified through negative ion fast atom bombardment mass spectrometry as follows. The recombinant E. coli MurC, MurD, MurE, and MurF were overexpressed and purified from E. coli ER2566 as described (11Reddy S.G. Waddell S.T. Kuo D.W. Wong K.K. Pompliano D.L. J. Am. Chem. Soc. 1999; 121: 1175-1178Crossref Scopus (41) Google Scholar) but using the Impact CN system (New England Biolabs) following the manufacturer's instructions. The purified enzymes were dialyzed extensively against 50 mm Tris-HCl (pH 8.0) containing 10 mm MgCl2 and 10% glycerol (v/v), and aliquots were stored at –80 °C. Reaction mixtures (30 ml) containing UDP-MurNAc (250 μm), l-Ala, d-Glu, DAP, d-Ala-d-Ala (1 mm each), TAPS (50 mm, pH 8), MgCl2 (5 mm), ATP (2.5 mm), and MurC, MurD, MurE, and MurF (75 μg/ml each) were incubated at 30 °C overnight and deproteinated by ultrafiltration, and the filtrate was loaded on a 10-ml Q-Sepharose (Amersham Biosciences) column equilibrated with 20 mm ammonium acetate. The bound material was eluted with a 20–1000 mm gradient of ammonium acetate. Fractions were monitored at A 262 for the presence of UDP-containing compounds. Fractions containing UDP-MurNAc-pentapeptide were identified by TLC on silica gel plates in 2-butyric acid/1 m NH4OH (5:3), utilizing UV absorption and ninhydrin for detection. These fractions were pooled and lyophilized to remove buffer, and the final product, UDP-MurNAc-pentapeptide, was analyzed by mass spectrometry as described (11Reddy S.G. Waddell S.T. Kuo D.W. Wong K.K. Pompliano D.L. J. Am. Chem. Soc. 1999; 121: 1175-1178Crossref Scopus (41) Google Scholar, 12Hitchcock S.A. Eid C.N. Aikins J.A. Zia-Ebrahimi M. Blaszczak L.C. J. Am. Chem. Soc. 1998; 120: 1916-1917Crossref Scopus (54) Google Scholar). In most syntheses, the rate of conversion of UDP-MurNAc to UDP-MurNAc-pentapeptide was about 80%. Preparation of Chalaropsis Muramidase—Chalaropsis sp. ATCC 16003 (American Type Culture Collection, Manassas, VA) was grown at 25 °C in medium consisting of glucose at 40 g/liter and peptone at 10 g/liter for 5 days (13Hash J.H. Arch. Biochem. Biophys. 1963; 102: 379-388Crossref PubMed Scopus (27) Google Scholar, 14Hash J.H. Rothlauf M.V. J. Biol. Chem. 1967; 242: 5586-5590Abstract Full Text PDF PubMed Google Scholar). The secreted muramidase was adsorbed from crude culture filtrates with Amberlite CG-50-H+ (Sigma) buffered at pH 5.0; protein was eluted from the matrix with 0.5 m ammonium acetate and the muramidase was precipitated with ammonium sulfate at 70% saturation (13Hash J.H. Arch. Biochem. Biophys. 1963; 102: 379-388Crossref PubMed Scopus (27) Google Scholar, 14Hash J.H. Rothlauf M.V. J. Biol. Chem. 1967; 242: 5586-5590Abstract Full Text PDF PubMed Google Scholar). The precipitate was redissolved in 10 mm ammonium acetate (pH 6.5). After dialysis to remove residual ammonium sulfate, the sample was passed over a Sephadex G-75 column (Amersham Biosciences), and fractions containing muramidase activity (measured by the reduction in A 610 of Staphylococcus aureus whole cell suspension (13Hash J.H. Arch. Biochem. Biophys. 1963; 102: 379-388Crossref PubMed Scopus (27) Google Scholar, 14Hash J.H. Rothlauf M.V. J. Biol. Chem. 1967; 242: 5586-5590Abstract Full Text PDF PubMed Google Scholar)) were pooled. Purity was checked by SDS-PAGE, and the enzyme preparation showed a single band in SDS-PAGE gels stained with Coomassie Brilliant Blue R250. Yield from 10 liters of culture was about 200 mg of enzyme. One unit of enzyme was defined as the amount of enzyme that decreased the A 610 of a S. aureus cell suspension at a rate of 0.008 OD/min. Preparation of an Enzymatically Active Cell Envelope Fraction from M. smegmatis—M. smegmatis mc2155 cells were grown in nutrient broth to midlog phase (4Mikušová K. Mikuš M. Besra G.S. Hancock I. Brennan P.J. J. Biol. Chem. 1996; 271: 7820-7828Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar), harvested, and stored at –70 °C until required. Approximately 8 g of bacteria (wet weight) were washed with a buffer containing 50 mm MOPS (pH 8.0), 5 mm 2-mercaptoethanol, and 10 mm MgCl2 (buffer A), resuspended in 24 ml of buffer A at 4 °C, and subjected to probe sonication as described (4Mikušová K. Mikuš M. Besra G.S. Hancock I. Brennan P.J. J. Biol. Chem. 1996; 271: 7820-7828Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). The sonicate was centrifuged at 27,000 × g for 15 min at 4 °C, and the pellet, containing the cell envelope, was resuspended in buffer A to a final volume of 16 ml. Percoll was added to achieve a 60% suspension, and the mixture was centrifuged at 27,000 × g for 60 min at 4 °C. The particulate, upper band was collected, washed twice with buffer A, resuspended in buffer A to a protein concentration of 15–20 mg/ml, and used as the enzyme source in all experiments. Reaction Mixtures for [14C]Gal Labeling and Fractionation of Reaction Products—The basic reaction mixtures for assessing [14C]Gal incorporation into lipid-linked AG precursors were prepared as follows. UDP-[U-14C]Galp (1 μCi; 3.5 nmol; 289 mCi/mmol; PerkinElmer Life Sciences) was dried under a stream of N2, dissolved in 38 μl of buffer A, and incubated with 2 μl of the UDP-Galp mutase preparation (0.13 mg of protein) at 37 °C for 15 min. Other reagents and buffer A were added to yield a final volume of 320 μl containing a 10.8 μm mixture of UDP-[U-14C]Galp and UDP-[U-14C]Galf, 60 μm UDP-GlcNAc, 20 μm dTDP-Rha, 100 μm ATP, and the envelope enzyme fraction (2 mg of protein). The reaction mixtures were incubated at 37 °C for the indicated period of time. In some cases, 60 μm PRPP and/or 200 μm UDP-MurNAc-pentapeptide were also included in the reaction mixtures. In the case of the ligation assays, these [14C]Gal labeling reaction mixtures containing both PRPP and UDP-MurNAc-pentapeptide were incubated at 28 °C for appropriate periods. After incubation, reaction mixtures were extracted with CHCl3/CH3OH (2:1), the resultant pellet was washed thoroughly with 0.9% NaCl and extracted with CHCl3/CH3OH/H2O (10:10:3), followed by “E-soak” (water/ethanol/diethyl ether/pyridine/ammonium hydroxide; 15:15:5:1:0.017) (15Angus W.W. Lester R.L. Arch. Biochem. Biophys. 1972; 151: 483-495Crossref PubMed Scopus (69) Google Scholar) as described (16Besra G.S. Morehouse C.B. Rittner C.M. Waechter C.J. Brennan P.J. J. Biol. Chem. 1997; 272: 18460-18466Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). The CHCl3/CH3OH (2:1) extract was partitioned with water (17Folch J. Lees M. Sloane-Stanley G.H. J. Biol. Chem. 1957; 226: 479-509Abstract Full Text PDF Google Scholar). The backwashed lower (organic) phase was dried under a stream of N2, and the residue was dissolved in 200 μl of CHCl3/CH3OH/H2O/NH4OH (65:25:3.6:0.5) prior to liquid scintillation counting and analysis by TLC. In order to obtain a completely insoluble residue, rich in MAPc, the E-soak insoluble pellet was extracted three times with boiling 60% methanol containing 0.1% ammonium hydroxide. To examine product-precursor relationships between lipid-linked intermediates and the insoluble residue, [14C]Gal-labeled CHCl3/CH3OH/ H2O (10:10:3)-soluble lipid-linked polymers were synthesized using the basic reaction conditions for [14C]Gal labeling described above. These enzymatically synthesized [14C]Gal-labeled compounds (∼300,000 dpm/assay) were dried under N2, and resuspended in 100 μl of buffer A by bath sonication. Fresh enzyme (2 mg of protein), cold UDP-Galp preincubated with UDP-Galp mutase, UDP-GlcNAc, dTDP-Rha, ATP, PRPP, UDP-MurNAc-pentapeptide, and buffer A were added to achieve the same concentrations and volume used in the ligation assays. The resulting mixture was bath sonicated and incubated for periods of time up to 16 h. Reaction mixtures were extracted as described above. Labeling of the Arabinan Component of AG with P[14C]RPP—The basic reaction mixture contained 3.3 μm P[14C]RPP (∼600,000 dpm), 60 μm UDP-GlcNAc, 20 μm dTDP-Rha, 60 μm UDP-Galp preincubated with UDP-Galp mutase (as described above), 100 μm ATP, enzyme (2 mg of protein), and buffer A in a total volume of 320 μl. Reaction mixtures were incubated at 37 °C for 2 h, and fractionation of the reaction products was conducted as described above for [14C]Gal labeling. Analysis of the Insoluble Residue—The insoluble residue, enriched in MAPc, was subjected to base treatment with 2 ml of 0.5% KOH in methanol for 4 days at 37 °C with gentle stirring. After washing three times with methanol, the methyl esters of the mycolic acids were removed with two diethyl ether extractions. The residual pellet was dried under N2 and digested with 100 μg/ml of Proteinase K (Roche Applied Science) in 250 μl of 10 mm sodium acetate (pH 7.5) at 37 °C overnight. Radioactivity released into the supernatant from the insoluble pellet by Proteinase K treatment was quantitated by liquid scintillation counting and subjected to sugar analysis as described below. After washing with 10 mm sodium acetate buffer, the residual pellet was treated with 2.5 units of purified Chalaropsis muramidase in 250 μl of 10 mm sodium acetate (pH 5.0), 500 units/ml of lysozyme in 10 mm Tris-HCl buffer (pH 7.5), or Proteinase K at 37 °C overnight. Aliquots of radiolabeled materials solubilized by these treatments were subjected to liquid scintillation counting and sugar analysis. Analysis—In order to facilitate size exclusion chromatography of the polyprenyl-P-linked polymers, the CHCl3/CH3OH/H2O (10:10:3)-soluble, E-soak-soluble, and Chalaropsis muramidase-solubilized materials were hydrolyzed in mild acid as follows to selectively cleave the prenyl phosphate. Samples were suspended in 50 μl of 1-propanol by bath sonication, followed by 100 μl of 0.02 n HCl, and the resulting mixture was incubated for 30 min at 60 °C (18Lucas J.J. Waechter C.J. Lennarz W.J. J. Biol. Chem. 1975; 250: 1992-2002Abstract Full Text PDF PubMed Google Scholar, 19Turco S.J. Wilkerson M.A. Clawson D.R. J. Biol. Chem. 1984; 259: 3883-3889Abstract Full Text PDF PubMed Google Scholar). After neutralization with 10 μl of 0.2 n NaOH, the released water-soluble products were applied to a Biogel P-100 column (1 × 118 cm), equilibrated, and eluted with 100 mm ammonium acetate (pH 7.0). SDS-PAGE analysis of enzymatically radiolabeled products was done using Novex® 10–20% Tricine gels (Invitrogen) under conditions recommended by the manufacturer. After electrophoresis, samples were blotted to nitrocellulose membranes, which were dried at room temperature, and subjected to autoradiography. CHCl3/CH3OH (2:1)-soluble materials were analyzed on silica gel TLC plates developed in CHCl3/CH3OH/NH4OH/1 m ammonium acetate/H2O (180:140:9:9:23), which were then subjected to autoradiography. For [14C]sugar analysis, samples were subjected to acid hydrolysis in 2 m CF3COOH for1hat120 °C. Hydrolysates were analyzed on silica gel TLC plates (silica gel G60, aluminum-backed; EM Science, Gibbstown, NJ), developed in pyridine/ethyl acetate/glacial acetic acid/water (5:5:1:3), and autoradiography. Radioactive spots were identified by comparative chromatography with standard sugars. Protein concentrations were estimated using the BCA protein assay reagent (Pierce). Synthesis of Polyprenyl-P-linked Intermediates—We previously established a cell-free assay system using membranes from M. smegmatis for the synthesis of the simple Pol-P-P-GlcNAc, Pol-P-P-GlcNAc-Rha, and Pol-P-P-GlcNAc-Rha-(Galf)1–4 intermediates in AG biosynthesis (4Mikušová K. Mikuš M. Besra G.S. Hancock I. Brennan P.J. J. Biol. Chem. 1996; 271: 7820-7828Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). The evidence for the nature of these products was based on solubility in organic solvents, susceptibility to mild acid hydrolysis, the presence of the appropriate radiolabeled sugar, and pulse-chase experiments (5Mikušová K. Yagi T. Stern R. McNeil M.R. Besra G.S. Crick D.C. Brennan P.J. J. Biol. Chem. 2000; 275: 33890-33897Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). These experiments led to the identification of one of the galactosyltransferases involved in galactan synthesis, a bifunctional enzyme capable of adding the majority of the alternating 5- and 6-linked Galf units (5Mikušová K. Yagi T. Stern R. McNeil M.R. Besra G.S. Crick D.C. Brennan P.J. J. Biol. Chem. 2000; 275: 33890-33897Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 6Kremer L. Dover L.G. Morehouse C. Hitchin P. Everett M. Morris H.R. Dell A. Brennan P.J. McNeil M.R. Flaherty C. Duncan K. Besra G.S. J. Biol. Chem. 2001; 276: 26430-26440Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). In the present study, we modified this cell-free system in order to demonstrate the sequential synthesis of the simpler glycolipid intermediates, followed by polyprenyl-P-P-linked galactan and arabinan intermediates, and in vitro ligation of these lipid-linked arabinogalactan intermediates to PG. The cell wall-membrane fraction of M. smegmatis, the source of endogenous glycosyltransferases and polyprenyl-P, was supplemented with UDP-GlcNAc, dTDP-Rha, the precursors of LU, and UDP-[14C]Galp and UDP-Galp mutase as the source of the Galf units of galactan. Reaction products were extracted with the organic solvents, CHCl3/CH3OH (2:1), CHCl3/CH3OH/H2O (10:10:3), and “E-soak” (15Angus W.W. Lester R.L. Arch. Biochem. Biophys. 1972; 151: 483-495Crossref PubMed Scopus (69) Google Scholar), and finally with boiling 60% methanol containing 0.1% ammonium hydroxide to remove residual soluble material, providing the insoluble MAPc-containing cell wall core. The incorporation of [14C]Galf from UDP-[14C]Galp into these four fractions is shown in Table I. Analysis of the CHCl3/CH3OH (2:1)-, CHCl3/CH3OH/H2O (10:10:3)-, and E-soak-soluble materials for 14C-labeled sugars showed that [14C]Gal was the sole radioactive sugar component (data not shown). TLC analysis of CHCl3/CH3OH (2:1)-soluble materials revealed a hierarchical array of glycolipids previously identified (5Mikušová K. Yagi T. Stern R. McNeil M.R. Besra G.S. Crick D.C. Brennan P.J. J. Biol. Chem. 2000; 275: 33890-33897Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar) as polyprenyl-P-P-GlcNAc-Rha-Galf, polyprenyl-P-P-GlcNAc-Rha-(Galf)2, and polyprenyl-P-P-GlcNAc-Rha-(Galf)3,4 (a mixture of tri-Galf- and tetra-Galf-containing glycolipid intermediates) (Fig. 1). No further simple glycolipid intermediates were observed in the more polar CHCl3/CH3OH/H2O (10:10:3) extract; the bulk of its radioactivity remained at the origin of the TLC plate (results not shown), supporting the evidence that this fraction contained the polyprenyl-P-P-GlcNAc-Rha-AG intermediates (5Mikušová K. Yagi T. Stern R. McNeil M.R. Besra G.S. Crick D.C. Brennan P.J. J. Biol. Chem. 2000; 275: 33890-33897Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar) (see below). The addition of exogenous PRPP and UDP-MurNAc-pentapeptide, precursors of arabinan and peptidoglycan synthesis, respectively, stimulated incorporation of [14C]Gal into the MAPc-containing residue in an additive manner with a concomitant reduction of radioactivity in the CHCl3/CH3OH/H2O (10:10:3) and E-soak extracts (Table I), suggesting that the polyprenyl-P-P-GlcNAc-Rha-(Gal)1–4 and the polyprenyl-P-P-GlcNAc-Rha-AG intermediates in these extracts were precursors of the mature PG-bound AG. However, the increase in insoluble material is not fully matched by a concomitant decrease in the other fractions. There is a substantial loss of radioactivity from the CHCl3/CH3OH/H2O (10:10:3) and E-soak extracts in incubations containing the additional precursors UDP-MurNAc-pentapeptide and PRPP. This is presumably due to a shortage of endogenous lipid carrier, required for de novo PG and decaprenyl-P-Araf synthesis when the UDP-MurNAc-pentapeptide and PRPP precursors are added.Table IInfluence of UDP-MurNAc-pentapeptide and PRPP on the incorporation of [14C]Gal into organic solvent-soluble and insoluble fractionsReaction mixtureCHCl3/CH3OH (2:1)CHCl3/CH3OH/H2O (10:10:3)E-soakInsoluble residuedpm/fractiondpm/fractionBasic16,000400,000270,0002,200With UDP-MurNAc-pentapeptide14,000280,000110,0003,000 (36%)aValues shown in parentheses are percentage increases in radioactivity incorporated into the insoluble residue relative to that seen in the basic reaction. The basic reaction mixture consisted of 60 μM UDP-GlcNAc, 20 μM dTDP-Rha, 100 μM ATP, 1 μCi of UDP-[14C]Galp preincubated with UDP-Galp mutase for 20 min at 37 °C and enzyme (2 mg of protein) in 320 μl of buffer A. UDP-MurNAc-pentapeptide and PRPP were added at a final concentration of 200 and 60 μM, respectively. After a 2-h incubation at 37 °C, reaction mixtures were subjected to serial extractions with organic solvents to yield an insoluble residue as described under “Experimental Procedures.”With PRPP17,000240,000190,0003,700 (68%)With UDP-MurNAc-pentapeptide and PRPP13,000190,000140,0004,600 (109%)a Values shown in parentheses are percentage increases in radioactivity incorporated into the insoluble residue relative to that seen in the basic reaction. The basic reaction mixture consisted of 60 μM UDP-GlcNAc, 20 μM dTDP-Rha, 100 μM ATP, 1 μCi of UDP-[14C]Galp preincubated with UDP-Galp mutase for 20 min at 37 °C and enzyme (2 mg of protein) in 320 μl of buffer A. UDP-MurNAc-pentapeptide and PRPP were added at a final concentration of 200 and 60 μM, respectively. After a 2-h incubation at 37 °C, reaction mixtures were subjected to serial extractions with organic solvents to yield an insoluble residue as described under “Experimental Procedures.” Open table in a new tab Arabinan Polymerization Steps in AG Biosynthesis—To define the steps leading to the synthesis of the arabinan component of AG, cell-free reactions containing P[14C]RPP as the ultimate precursor of Araf were prepared in bulk and extracted with CHCl3/CH3OH (2:1), CHCl3/CH3OH/H2O (10:10:3), and E-soak. Parallel reactions containing UDP-[14C]Galp were run, and similar extracts were prepared. Complete acid hydrolysis and TLC analysis for radioactive sugar showed that all of the [14C]Gal remained as such, and the majority of the P[14C]RPP radiolabel was converted into [14C]Ara-containing material; a minority appeared as [14C]ribose, apparently from intermediates of an unidentified riban (8Scherman M.S. Kalbe-Bournonville L. Bush D. Xin Y. Deng L. McNeil M. J. Biol. Chem. 1996; 271: 29652-29658Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). TLC of the CHCl3/CH3OH (2:1)-soluble products showed a preponderance of polyprenyl (C50)-P-Araf in this fraction (7Wolucka B.A. McNeil M.R. de Hoffmann E. Chojnacki T. Brennan P.J. J. Biol. Chem. 1994; 269: 23328-23335Abstract Full Text PDF PubMed Google Scholar) (Fig. 1). Mild acid hydrolysis of the [14C]Ara-labeled CHCl3/CH3OH/H2O (10:10:3)-soluble and E-soak-soluble lipid polymers to remove the presumed polyprenyl-P and subsequent gel filtration showed considerable overlap but not complete coincidence in the profiles of these two sets of [14C]Ara-containing polymers (Fig. 2). Profiles were similar to those of the [14C]Gal-labeled polymers, labeled, released, and extracted under similar conditions (Fig. 2). In both cases, the E-soak-extractable material appeared to be slightly but reproducibly larger than that extractable with CHCl3/CH3OH/H2O (10:10:3), pointing to the presence of a population of polyprenyl-P-linked AG intermediates, partially resolvable by the two extractants. These lipid-linked intermediates were also analyzed by Tricine SDS-PAGE (Fig. 3). Overnight exposure of autoradiograms revealed a population of [14C]Gal-labeled CHCl3/CH3OH/H2O (10:10:3)- and E-soak-soluble lipid-linked polymers, whereas no images were seen in lanes containing [14C]Ara-labeled lipid-linked polymers, presumably due to lower labeling efficiency when using P[14C]RPP as the precursor. However, after 14 days of exposure, the [14C]Ara-containing CHCl3/CH3OH/H2O (10:10:3)- and E-soak-soluble lipid-linked polymers were also visible on the autoradiograms and showed a degree of heterogeneity similar to that of the [14C]Gal-labeled material. The E-soak soluble [14C]Ara- and [14C]Galf-containing lipid polymers again appeared to be larger than the CHCl3/CH3OH/H2O (10:10:3)-soluble material, supporting the trend seen in the size exclusion analysis. Analysis for radioactive sugar content in these extracts confirmed the sole presence of [14C]Ara and [14C]Gal in the respectively labeled polymers.Fig. 3Tricine SDS-PAGE analysis of radiolabeled, lipid-linked polymers. Autoradiograms were exposed overnight (A) or for 14 days (B). Lane 1, [14C]Gal-containing CHCl3/CH3OH/H2O (10:10:3)-soluble materials; lane 2, [14C]Ara-containing CHCl3/CH3OH/H2O (10:10:3)-soluble materials; lane 3, [14C]Gal-containing E-soak-soluble materials; lane 4, [14C]Ara-containing E-soak-soluble materials. [14C]Gal- or [14C]Ara-containing lipid-linked polymers were prepared as described in the legend to Fig. 1. Identical volumes of each [14C]Gal- or [14C]Ara-containing material were dried, 10 μl of Tricine SDS buffer was added, and the mixture was boiled for 3 min and applied to Novex® 10–20% Tricine gels. After electrophoresis, those materials were electroblotted to a nitrocellulose membrane and exposed to x-ray film at –70 °C for the indicated periods. The migration positions of protein molecular weight markers" @default.
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• W2033352215 title "Polymerization of Mycobacterial Arabinogalactan and Ligation to Peptidoglycan" @default.
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