Revised structure for the 6-O-methylglucose polysaccharide of Mycobacterium smegmatis.

Properties of the mycobacterial polymethylpolysaccharide-lipid complex have been investigated by fluorometric techniques. From the dissociation constant for the O-methyglucose polysaccharide-parinaric acid complex at 293 K, a Gibbs free energy (delta G degree) of -33.65 kJ/mol was obtained. The Kd decreased with increasing temperature, giving an enthalpy (delta H degree) of 15.4 kJ/mol. From these data, a molar entropy (delta S degree) of 167.4 J K-1 was obtained. Thus, the reaction is slightly endothermic, but the large positive entropy change leads to an overall negative free energy favoring complex formation. From fluorescence depolarization measurements, the methylglucose polysaccharide-parinaric acid complex appears to display isotropic rotation with a correlation time of 2.55 ns at 23 degrees C. This may be compared to a rotational correlation time of 6.17 ps for free parinaric acid in water at 23 degrees C calculated from the value determined in cyclohexanol at the same temperature, which demonstrates that the mobility of the fatty acid in the complex is restricted. Assuming the complex is spherical, it was calculated to have a diameter of 23-26 A, whereas a helical methyglucose polysaccharide molecule assembled from space-filling models has the dimensions of a cylinder of 18 X 24 A. The polysaccharide and fatty acid chain-length dependence of the interaction shows a discontinuity for helical polysaccharide segments shorter than 12 sugars and for fatty acids shorter than palmitate.

Section: Resultsmentioning

confidence: 65%

Thermodynamic parameters and shape of the mycobacterial polymethylpolysaccharide-fatty acid complex

Kiho¹,

Ballou²

1988

“…The relatively large amount of material obtained in this way has allowed a study of the polysaccharide-lipid interactions at millimolar concentrations.The anomeric protons for all of the al-*4-linked hexose units in the mycobacterial methylglucose polysaccharide occur in an envelope centered at 5.40, and, on titration with hexadecyltrimethylammonium bromide, the majority of these resonances move upfield to about 5.15. This shift is consistent with a change in the polysaccharide from a less ordered chain to one that has a significant proportion of helical conformation, and it is probable that the alkyl chain is included IN^Iycobacteria synthesize two methylated polysaccharides, one composed of 3-0-methylmannose and the other of 6-0methylglucose (Ballou, 1981;Yamada & Ballou, 1979;Forsberg et al, 1982). These substances are found in the cytoplasm of the cell where they appear to regulate fatty acid metabolism (Bloch, 1977) as a consequence of their ability to form tight complexes with long-chain acyl coenzyme A derivatives (Machida & Bloch, 1973;Yabusaki & Ballou, 1978;Yabusaki et al, 1979).…”

mentioning

confidence: 59%

Affinity purification of mycobacterial polymethyl polysaccharides and a study of polysaccharide-lipid interactions by proton NMR

Hindsgaul¹,

Ballou²

1984

Mycobacterial polymethyl polysaccharides, which bind long-chain fatty acids tightly [Ballou, C.E. (1981) Pure Appl. Chem. 53, 107-112], have been purified on a preparative scale by use of an affinity column packing consisting of (palmitoylamino)alkylsilyl silicate. The relatively large amount of material obtained in this way has allowed a study of the polysaccharide-lipid interactions at millimolar concentrations. The anomeric protons for all of the alpha 1----4-linked hexose units in the mycobacterial methylglucose polysaccharide occur in an envelope centered at delta 5.40, and, on titration with hexadecyltrimethylammonium bromide, the majority of these resonances move upfield to about delta 5.15. This shift is consistent with a change in the polysaccharide from a less ordered chain to one that has a significant proportion of helical conformation, and it is probable that the alkyl chain is included in the coiled portion of the polysaccharide in a manner analogous to the interaction of methylmannose polysaccharide with palmitic acid [Yabusaki, K. K., Cohen, R. E., & Ballou, C. E. (1979) J. Biol. Chem. 254, 7282-7286]. The native methylglucose lipopolysaccharide, which contains several short-chain acyl groups as well as an esterified octanoyl group, has an anomeric proton nuclear magnetic resonance spectrum similar to that of the methylglucose polysaccharide-hexadecyltrimethylammonium bromide complex. This suggests that the acylation stabilizes the polysaccharide chain in the same conformation it assumes when complexed to a long-chain lipid. Thus, acylation of the methylglucose polysaccharide could have an important role in regulating its shape and lipid-binding properties.

“…The fast-atom bombardment mass spectrometry technique has been applied to reducing oligosaccharides, small polysaccharides, and glycolipids (Forsberg et al, 1982;Dell & Ballou, 1983;. In the positive ion recording mode, underivatized reducing oligosaccharides usually show signals for the protonated, cationized (Na+, K+) species, together with fragment ions from these species (Bosso et al, 1984).…”

Section: Discussionmentioning

confidence: 99%

“…Interestingly, the trehalose-containing oligosaccharides, devoid as they are of a reducing terminus, conform to the principles observed with reducing oligosaccharides, as does their acquisition of the molecular anion in negative-mode recording. In addition, the trehalose-containing oligosaccharides behave in both modes as the methylglucose lipopolysaccharides (Forsberg et al, 1982;Dell & Ballou, 1983), the only other nonreducing oligosaccharides examined in detail, albeit nonreducing by virtue of a glycerate aglycon, but still VI figure 8: ELISA reactivity of monoclonal antibody to various LOS. Each well was coated with 8 ng of glycolipid, and an excess (ca.…”

Section: Discussionmentioning

confidence: 99%

Trehalose-containing lipooligosaccharides from mycobacteria: structures of the oligosaccharide segments and recognition of a unique N-acylkanosamine-containing epitope

Hunter¹,

Jardine²,

Yanagihara³

et al. 1985

The structures of the oligosaccharide segments of nine trehalose-containing lipooligosaccharides (LOS) of Mycobacterium kansasii have been established by positive and negative fast-atom bombardment mass spectrometry, acetolysis, partial acid hydrolysis, methylation analyses, and nuclear magnetic resonance. Upon acetolysis, all produce the alpha,alpha-trehalose-containing tetraglucose (Glc4) "core" -beta-D-Glcp(1----3)-beta-D-Glcp(1----4)-alpha-D-Glcp(1----1)-alpha-D-Gl cp. The simplest (LOS I') contains an additional alpha 1----3-linked 3-O-methyl-L-rhamnopyranose (3-O-Me-L-Rhap) unit; those of intermediate complexity (LOS I-III) contain an additional D-xylopyranose (D-Xylp) residue or xylobiose in beta 1----4 linkage; and those of ultimate complexity (LOS IV-VIII) contain further D-Xylp residues and the distal N-acylkanosamine- (KanNAcyl) and fucopyranosyl- (Fucp) containing disaccharide KanNAcyl(1----3)Fucp. Thus, the structure of the oligosaccharide from LOS VII is KanNAcyl(1----3)Fucp(1----4) [-beta-D-Xylp(1----4)]6-alpha-L-3-O-Me-Rhap(1----3)Glc4++ +. Polyclonal rabbit and murine monoclonal antibodies react only with the more complex KanNAcyl-Fucp-containing lipooligosaccharides, indicating that the KanNAcyl distal end, not the trehalose end, contains the antibody binding site unique to M. kansasii and is responsible for the serological distinctiveness of M. kansasii among mycobacterial species.