Structure of a novel glycopeptidolipid antigen containing a O‐methylated serine isolated from Mycobacterium xenopi

Abstract

GPL X-1, a novel glycopeptidolipid (GPL) isolated from Mycobacterium xenopi ( C q 140 35004), has recently been found to typify a new class of mycobacterial glycopeptidolipids devoid of C-mycoside core structure, the so-called serine-containing glycopeptidolipid [Rivikre, M. & Puzo, G. (1991) J. Biol. Chem. 266,9057-90631. Here we report the purification and characterization of a novel serine-containing GPL termed GPL X-IIb, isolated from the M. xenopi strain NCTC 10042.On thin-layer chromatography, this GPL was found to be present in some other M. xenopi strains isolated from patients with pulmonary infections. The sugar and amino-acid compositions of this GPL were elucidated from the native form using a combination of two-dimensional homonuclear and heteronuclear scalar coupling NMR. The peptide and sugar sequences, as well as the methoxyl group locations on the C-3 of the 6-deoxy-a-~-talopyranoside (6dTalp) and on a Ser, were unambiguously determined by heteronuclear multiple-bond correlation experiments. GPL X-1% was found to be composed of a lipotetrapeptide of the following structure C,,-Ser-OMe-Ser-Phe-a%-OMe (aThr = allothreonine). The sugar part is made up of 30Me-a-~-6dTalp and the following disaccharide : a-~-Rhap-(1+3)-2-O-Lau-a-~-Rhap (Rhap = rhanmopyranose). Unlike GPL X-I, the sugar attachment sites on the tetrapeptide were successfully determined from heteronuclear three-bond coupling correlation observed in the heteronuclear multiple bond correlation spectrum between the anomeric carbon resonances and the protons of aThr-OMe and Ser. It was established that the 30Me-6dTalp glycosylates the Ser while the disaccharide is linked to the aThr-OMe. Thus both GPL X-I and GPL X-IIb share a common lipotetrapeptide core [with the exception of Ser(0Me)l but drastically differ in their oligosaccharide appendage. Thus, by analogy with the M. avium complex, the present report suggests that M. xenopi species can be divided in various serovars characterized by the unique structure of their C-mycoside GPL oligosaccharide appendage, enhancing the interest for this new type of serine-containing glycopeptidolipid.Research into mycobacterial infection has intensifying as a result of a resurgence in the prevalence of human tuberculosis. In developed countries, the epidemiology of the disease is changing, due mainly to the emergence of drug-resistant Mycobacterium tuberculosis strains (Weiss, 1992), but also from an increase in infections with nontuberculous mycobacteria such as Mycobacterium avium, Mycobacterium kansasii Correspondence to M.

A Unique Phenylalanine‐Containing Lipopeptide Isolated from a Rough‐Colony Variant of Mycobacterium Avium

“…This last point was confirmed by the 'H-13C HMQC spectrum (Fig. 5 ) revealing the presence of eight correlation plots the positions of which were in agreement with amino acid Ca chemical shifts [27]. Thus, from this and FAB-MS data, we can suspect that the last unidentified compound does not bear any amide proton.…”

“…5). Moreover this carbon exhibits correlations in the HMBC spectrum with the complex signal at 7.15 ppm attributed to Phe aromatic protons [27] (data not shown). Analysis of the two following spin systems arising from the amide proton signals at 9.031 ppm and 8.81 ppm revealed a similar pattern to the Ha s at 5.086 pprn and 5.23 ppm and the a methyl proton signals at 1 .I16 ppm and 1.498 ppm, respectively.…”

“…In the same way, the next amide proton resonance at 6 8.29 ppm was attributed to a second Phe which Ha resonates at 5.553 ppm and the Hp, H F at 3.271 ppm and 3.18 ppm. The two remaining proton spin systems appear less characteristic and were so attributed mainly on the basis of the HMQC, HMQC-TOCSY and HMBC experiments [27]. First, the one starting from the amide proton signal at 8.22 ppm exhibited a first cross-peak with the Ha at 5.366 ppm.…”

“…Finally, the last amide proton showing a correlation with Ha signal at 7.694 ppm and Hj? 2.345 ppm was attributed to the last Phe residue on the basis of its /?$' protons chemical shift [27], and heteronuclear correlations observed in the HMQC and HMBC spectra (Table 2). Due to the absence of any amide proton signal, analysis of the remaining spin system started from the putative a proton at 5.06 pprn which presents in the COSY spectrum a correlation with a signal at 2.345 ppm attributed to the corresponding Hp.…”

“…To confirm the sequence established by FAB-MS, the 'H-IT-HMBC spectrum was further analyzed in the aim of assigning the various magne- Lipopeptide-I amino acid proton chemical shifts. Referenced by setting the residual acetone methyl proton (CD,H-CO-CD3) tization transfers involving the amino acid carbonyl resonances [27]. First, these were attributed on the basis of the observed cross-peaks with their corresponding Ca and eventually other carbons of the same spin system.…”