Structure Elucidation of Sphingolipids from the Mycopathogen Paracoccidioides brasiliensis:  An Immunodominant β-Galactofuranose Residue Is Carried by a Novel Glycosylinositol Phosphorylceramide Antigen^†

Abstract

Two major acidic glycolipid components (Pb-1 and Pb-2) have been extracted from the mycopathogen Paracoccidioides brasiliensis, a thermally dimorphic fungus endemic to rural areas of South and Central America. Sera of all patients exhibiting paracoccidioidomycosis were found to be reactive with Pb-1, but not with Pb-2; no reactivity was observed with sera of healthy patients or those with histoplasmosis [Toledo, M. S., Suzuki, S., Straus, A. H., and Takahashi, H. K. (1995) J. Med. Vet. Mycol. 33, 247-251]. We report here the complete structure elucidation of both P. brasiliensis glycolipids using monosaccharide, fatty acid, sphingosine, and inositol component analysis by GC-MS; 1H- and 31P NMR spectroscopy; ESI-MS and -MS/CID-MS, linkage analysis, and exoglycosidase digestion. The compounds were found to be glycosylinositol phosphorylceramides (GIPCs) with the following structures: Pb-2, Manpalpha1-->3Manpalpha1-->2Ins1-P-1Cer; and Pb-1, Manpalpha1-->3[Galfbeta1-->6]Manpalpha1-->2Ins1- P-1Cer. The serologically nonreactive Pb-2 appears to be a biosynthetic intermediate between mannosylinositol phosphorylceramide (MIPC), which is widely distributed among fungi, and the antigenic Pb-1. Pb-1 is a novel glycosphingolipid, similar to a triglycosyl IPC (Hc-VI) reported from Histoplasma capsulatum [Barr, K., Laine, R.A, and Lester, R. L. (1984) Biochemistry 23, 5589-5596], but differing in the anomeric configuration of the terminal Galf1-->6 residue, which is immunodominant. The significance of these structures as serological and taxonomic markers, as well as their potential utility as targets for immunodiagnostic agents, is discussed.

Analysis of glycosylinositol phosphorylceramides expressed by the opportunistic mycopathogen Aspergillus fumigatus

“…5A. Aside from the presence of minor impurities, it is essentially identical to a spectrum previously published for Mana3Mana2IPC (Pb-2) from P. brasiliensis and for which most of the resonances were assigned by 2-D homonuclear COSY and TOCSY experiments (30) (22). The minor anomeric resonances at 5.030 and 4.905 ppm are consistent with a small contamination by a triglycosyl-IPC component (see below) incompletely separated from Af-2 in this fraction.…”

“…2 These compare the reactivity profiles of GIPCs from A. fumigatus strains 9197 and 237 (cultured at 37jC; lanes 1 and 2, respectively) with those from P. brasiliensis strain 18 (lane 3) and A. nidulans strain A28 (lane 4). The characterizations of the components from P. brasiliensis and A. nidulans have been described previously (30,40), except that the component formerly named Pb-1 is here named Pb-3.…”

“…Among other observations, these studies revealed the presence of GIPCs containing an antigenic branched 6RYManp(a1Y3)[Galf(b1Y6)]Manp(a1Y2) Ins structural motif. Although one GIPC antigen isolated from A. fumigatus, Manp(a1Y3)[Galf(b1Y6)]Manp(a1Y2) InsPCer (Af-3b), was characterized previously as a major GIPC component of the dimorphic mycopathogen Paracoccidioides brasiliensis (30), other GIPCs characterized here, including structural isomers and derivatives of Af-3b, have not been identified previously. A third major triglycosyl-IPC component of A. fumigatus was characterized as Manp(a1Y3) Manp(a1Y6)GlcpN(a1Y2)Ins1-P-1Cer (Af-3c); in addition, a minor component with an N-acetylated GlcN residue was identified (Af-3c*).…”

“…Myo -inositol (Ins) was detected in the monosaccharide analysis as its per-O -trimethylsilyl derivative, although optimal conditions for its release (20) were not used. All derivatives were prepared according to protocols described previously (30). Linkage analysis was performed by the method of partially methylated alditol acetates.…”

Unusual Galactofuranose Modification of a Capsule Polysaccharide in the Pathogenic Yeast Cryptococcus neoformans

“…The monosaccharide components of glycans primarily occur in cyclic forms with aldohexoses forming either five-or six-membered rings (1). Galactose, for example, occurs as either a six-membered galactopyranose (Galp) 6 or five-membered galactofuranose (Galf) ring. Galp occurs ubiquitously across all domains of life, but Galf is curiously restricted to simpler organisms that include prokaryotes (2-4), protozoa (5), and fungi (6 -8) as well as certain algae (9), Archaea (10), and invertebrates (11).…”