Abstract
Lipooligosaccharides (LOSs) have recently been proposed as markers of mycobacterial avirulence. They have been characterized in Mycobacterium kansasii cell wall and were investigated in Mycobacterium gastri since the distinction between the two mycobacterial species remains in some question. A set of unknown LOSs was isolated from M. gastri W471. The highly antigenic lipooligosaccharide, LOS-III, was purified and appeared in all the M. gastri strains investigated regardless of their morphology. Moreover, by enzyme-linked immunosorbent assay and chromatographic approaches, it was found that LOS-III unambiguously distinguished M. gastri from the opportunistic pathogen M. kansasii. The LOS-III structure was established from its native form using NMR spectroscopy. This strategy revealed the presence of a supplementary monosaccharide (X) which was not characterized by routine carbohydrate analysis. Its core structure, 3,6-dideoxy-alpha-hexopyranose, was established from the complete assignment of the 1H and 13C spectra by two-dimensional homonuclear (COSY, HOHAHA) and heteronuclear 1H-13C heteronuclear multiple quantum correlation spectroscopy (HMQC) and HMQC-HOHAHA spectroscopy. Due to the absence of a proton at C4, the key data of the C4 side chain structure came from the heteronuclear multiple bond correlation spectroscopy (HMBC) spectrum. It was revealed to be a C-alkyl chain of partial structure 1,3-dimethoxypropyl. From the HMBC spectrum, this novel C-branched monosaccharide was located at the nonreducing end of the LOS, while the putative reducing end was found to consist of a 2',4,6-triacylated alpha-alpha-trehalose. The following structure, based on the evidence presented in this paper, is proposed for LOS-III: Xp alpha(1-->3)[L-Xylp beta(1-->4)](6)3-O-Me-Rhap alpha(1-->3)D- Galp beta(1-->3)-D-Glcp beta(1-->4)2-O-acyl-D-Glcp-alpha(1<==>1)alpha 4,6-di-O- acyl-D-Glcp.
Highlights
Lipooligosaccharides (LOSs)have recently been pro- UnitedStates,due in parttothe rise of HIV-1’-related posed as markers of mycobacterial avirulence
This is notthe case of thePhe GI which appearas complete assignment of the ‘H and 13C spectra by two- permanentmarkers of the M. kansasii strains.,an dimensional homonuclear (COSY, HOHAHA) and het- interesting correlation has beenproposed between virulence eronuclear ‘H-13C heteronuclearmultiplequantum andLOScontent:LOSsare considered to be markers of correlation spectroscopy (HMQC) and HMQC-HO- avirulence (4).rough variants of M. kansasii cause a HAHA spectroscopy
Trehalose containing lipooligosaccharides (LOSs) have been described as glycosylated extensions of asometimes methylated trehalose invariably tri-0-acylated at C3, C4,and suggests an organization inwhich the lipid moiety is involved in the mycocerosic layer, while the glycosidic moiety is exposed at thecell wall surface
Summary
0 1993by The American Society for Biochemistry and Molecular Biology, Inc. Vol 268, No 5, Issue of February 15, pp. 3168-3179. 1993 Prmted in U.S.A. Gen M. kansasii.The LOS-I11structure was established M. kansasii cell wall is endowed with two distinct classes of from its native form using NMR spectroscopy This surface glycolipid antigens: the trehalose-containing lipoolistrategyrevealedthepresence of a supplementary gosaccharides (LOSs) and thephenolic glycolipids (Phe Gls). Glycosylation of the LOSs compared to thatof the PheGls, it Fromthe HMBC spectrum,this novelC-branched has been suggested(4) that in smooth strainLsOSs may mask monosaccharide was located at thenonreducing endof some important cell wall glycolipids that areotherwise effecthe LOS, while the putative reducing end was found titvoe. These substances may be the phenolic glycolipids deconsist of a 2’,4,6-triacylated a-a-trehalose. The suspension was centrifuged at 4 "C for 20 min (3000 X g), and the supernatanwt as evaporated toyield 10 g
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