Structural characterization of the galactoxylomannan of Cryptococcus neoformans Cap67

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Abstract

The galactoxylomannan (GalXM) obtained from the culture supernatant of an acapsular mutant of Cryptococcus neoformans Cap67 was purified by Concanavalin A affinity, ion-exchange, and gel-filtration chromatographies. The structure of GalXM was determined by methylation analysis and by 1D and 2D NMR spectroscopic studies of the intact polysaccharide and of the oligosaccharide fragments generated by Smith degradation and by acetolysis. GalXM is a complex polysaccharide with an α-(1→6)-galactan backbone. The polysaccharide is branched at C-3 of alternate Gal units of the backbone. C-3 is the point of attachment of the oligosaccharide side chains comprised of α-d-Man-(1→3)-α-d-Man-(1→4)-β-d-Gal-substituted with zero to three terminal β-Xyl residues as shown in the following structure:

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Introduction

Cryptococcus neoformans is an encapsulated pathogenic yeast that is responsible for meningoencephalitis in individuals whose immune system is debilitated by chemotherapy or by illness. Cryptococcosis has emerged as a common life-threatening disease in patients with AIDS 1, 2.

The cell envelope of C. neoformans is composed of cell-wall glucans, a major capsular glucuronoxylomannan (GXM), and two minor polysaccharides, galactoxylomannan (GalXM), and mannoprotein (MP) 3, 4, 5. All three antigens are present in cryptococcal culture supernatants. GXM determines the serotype of the yeast, and it is considered to be a major virulence factor [6].

In an early study, Xyl, Man, Gal, and GlcA were identified by paper chromatography as components of the soluble antigen of C. neoformans serotypes A, B, and C [7]. A serotype A capsular polysaccharide was precipitated with hexadecyltrimethylammonium bromide (CTAB) and shown to be composed of Xyl, Man, and Gal. The polysaccharide cross-reacted with Streptococcus pneumoniae type XIV antiserum [8]. The specificity of this antiserum depends on terminal, (1→3)-, (1→6)-, and (1→3,6)-linked galactosyl residues [8]. The Gal content of the immunoprecipitated serotype A polysaccharide increased because of the reaction with the type XIV antiserum [8]. The results suggested that the capsular polysaccharide was comprised of a mixture of heteroglycans. In later studies, the capsular polysaccharide of C. neoformans serotype B was partially purified from the culture supernate by precipitation with EtOH [9]. The precipitate was comprised of Xyl, Man, Gal, and GlcA in the molar ratios of 2:3:0.5:1, respectively. In recent studies, the capsular polysaccharides from several serotypes of C. neoformans were isolated and purified 10, 11, 12, 13. The purified polysaccharides were comprised of Xyl, Man, and GlcA; Gal was not present in any of the purified GXMs. This suggested that Gal was present in the C. neoformans capsule as a separate glycan. The high viscosity of solutions containing GXM impeded the resolution of the mixture of polysaccharides into individual components in earlier studies.

GalXM was first isolated as a distinct entity from the culture supernate of C. neoformans serotype A [3]. It is believed to be loosely associated with the cell-wall rather than covalently linked to it [14]. A monoclonal antibody specific for GalXM was used in conjunction with immunocytochemical analysis to localize GalXM on the surface of the acapsular mutant C. neoformans Cap67 [15]. Immunogold transmission electron microscopy and micro agglutination assay indicate that the epitopes recognized by the monoclonal antibody are located within the cytoplasm and the cell-wall of C. neoformans [A. van de Moer, R. Cherniak, S.L. Salhi, N. Schnoy, S. Jouvert, M. Bastide, and J.M. Bastide, unpublished results].

Purified GalXM was obtained from three different strains (serotype A, C and Cap67) of C. neoformans[16]. GalXM from all three strains were comprised of Gal, Man, and Xyl in similar, but not identical, molar ratios. Methylation analysis of all the isolated GalXM indicated that some of the glycosyl residues are branched and that all of the Xyl and Galf are present as nonreducing terminal residues.

Herein, we report the structural characterization of the GalXM of C. neoformans Cap67. The isolation of GalXM and MP is complicated by the presence of GXM. Therefore, an acapsular mutant, C. neoformans Cap67, was used for the analysis of the primary structure of GalXM.

Section snippets

Analytical methods

The neutral carbohydrates were analyzed by the phenol–sulfuric acid method [17]. Protein was determined by the bicinchoninic acid (BCA) (Pierce, Rockford, IL) analysis method using BSA as a standard [18]. The 2-amino-2-deoxyhexoses were analyzed by the method of Smith and Gilkerson [19]. The constituent monosaccharides of GalXM were analyzed as their per-O-acetylated aldononitrile derivatives by GLC 16, 20. Underivatized monosaccharides were determined by HPAEC with pulsed amperometric

Antigen purification

The 10 K retentate obtained by ultrafiltration was used for the isolation of GalXM and MP (Fig. 1). Con A affinity chromatography yielded three fractions: (1) GalXM-A, the effluent that did not bind to the Con A column (38%); (2) 0.2 MP, a glycoprotein that was eluted with 0.2 M methyl α-d-mannopyranoside (Manp) (24%); and (3) 0.4 MP, a glycoprotein that was eluted with 0.4 M Manp (2%). The fractions eluted with Manp were reserved for future analysis. GalXM-A was resolved by anion-exchange

Discussion

Methylation analysis of GalXM-I indicated that 44% of the Man and Gal residues were 3-O-linked, and that 31% of the Gal were 6-O-linked. Based on these data, two degradation procedures were selected: (1) Smith degradation [to take advantage of the resistance of (1→3)-linked residues to oxidation by sodium periodate], and (2) acetolysis [to take advantage of the selective cleavage of (1→6)-linked Gal residues]. GalXM-A was used as starting material for the fragmentation experiments since the

Acknowledgements

This research was supported by Public Health Service grant NIH AI31769. V.V.V. was supported by a GAAN graduate fellowship in chemistry (Department of Education). The Varian UnityPlus 500 MHz NMR spectrometer was purchased with the help of grant BIR-9214443 from the National Science Foundation. We acknowledge Dr. Shuqun Sheng for her assistance with some of the NMR experiments and Eric O'Neill for his technical assistance.

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