A β-1,2-Xylosyltransferase from Cryptococcus neoformans Defines a New Family of Glycosyltransferases
2007; Elsevier BV; Volume: 282; Issue: 24 Linguagem: Inglês
10.1074/jbc.m701941200
ISSN1083-351X
AutoresJ. Stacey Klutts, Steven B. Levery, Tamara L. Doering,
Tópico(s)Plant Pathogens and Fungal Diseases
ResumoCryptococcus neoformans is an opportunistic fungal pathogen characterized by a prominent polysaccharide capsule that envelops the cell. Although this capsule is dispensable for in vitro growth, its presence is essential for virulence. The capsule is primarily made of two xylose-containing polysaccharides, glucuronoxylomannan and galactoxylomannan. There are likely to be multiple xylosyltransferases (XTs) involved in capsule synthesis, and the activities of these enzymes are potentially important for cryptococcal virulence. A β-1,2-xylosyltransferase with specificity appropriate for capsule synthesis was purified ∼3000-fold from C. neoformans, and the corresponding gene was identified and cloned. This sequence conferred XT activity when expressed in Saccharomyces cerevisiae, which lacks endogenous XT activity. The gene, termed CXT1 for cryptococcal xylosyltransferase 1, encodes a 79-kDa type II membrane protein with an N-linked glycosylation site and two DXD motifs. These latter motifs are believed to coordinate divalent cation binding in the activity of glycosyltransferases. Site-directed mutagenesis of one DXD motif abolished Cxt1p activity, even though this activity does not depend on the addition of a divalent cation. This may indicate a novel catalytic mechanism for glycosyl transfer. Five homologs of Cxt1p were found in the genome sequence of C. neoformans and 34 within the sequences of other fungi, although none were found in other organisms. Many of the homologous proteins are similar in size to Cxt1p, and all are conserved with respect to the essential DXD motif. These proteins represent a new family of glycosyltransferases, found exclusively within the fungal kingdom. Cryptococcus neoformans is an opportunistic fungal pathogen characterized by a prominent polysaccharide capsule that envelops the cell. Although this capsule is dispensable for in vitro growth, its presence is essential for virulence. The capsule is primarily made of two xylose-containing polysaccharides, glucuronoxylomannan and galactoxylomannan. There are likely to be multiple xylosyltransferases (XTs) involved in capsule synthesis, and the activities of these enzymes are potentially important for cryptococcal virulence. A β-1,2-xylosyltransferase with specificity appropriate for capsule synthesis was purified ∼3000-fold from C. neoformans, and the corresponding gene was identified and cloned. This sequence conferred XT activity when expressed in Saccharomyces cerevisiae, which lacks endogenous XT activity. The gene, termed CXT1 for cryptococcal xylosyltransferase 1, encodes a 79-kDa type II membrane protein with an N-linked glycosylation site and two DXD motifs. These latter motifs are believed to coordinate divalent cation binding in the activity of glycosyltransferases. Site-directed mutagenesis of one DXD motif abolished Cxt1p activity, even though this activity does not depend on the addition of a divalent cation. This may indicate a novel catalytic mechanism for glycosyl transfer. Five homologs of Cxt1p were found in the genome sequence of C. neoformans and 34 within the sequences of other fungi, although none were found in other organisms. Many of the homologous proteins are similar in size to Cxt1p, and all are conserved with respect to the essential DXD motif. These proteins represent a new family of glycosyltransferases, found exclusively within the fungal kingdom. The basidomycetous fungus Cryptococcus neoformans causes a variety of maladies, including an often fatal meningoencephalitis (1Chayakulkeeree M. Perfect J.R. Infect. Dis. Clin. North Am. 2006; 20 (v-vi): 507-544Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar). Currently available chemotherapeutic agents are unable to completely clear the infection, necessitating long term treatment in those patients that survive (1Chayakulkeeree M. Perfect J.R. Infect. Dis. Clin. North Am. 2006; 20 (v-vi): 507-544Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar). The main virulence factor of this opportunistic pathogen is the large polysaccharide capsule that envelops the cell. Strains of C. neoformans that lack this capsule are avirulent in animal models (2Kwon-Chung K.J. Rhodes J.C. Infect. Immun. 1986; 51: 218-223Crossref PubMed Google Scholar), suggesting that the synthesis of this structure could be targeted therapeutically (3Bose I. Reese A.J. Ory J.J. Janbon G. Doering T.L. Eukaryot. Cell. 2003; 2: 655-663Crossref PubMed Scopus (187) Google Scholar). Two xylose-containing polysaccharides, termed glucuronoxylomannan (GXM) 2The abbreviations used are: GXM, glucuronoxylomannan; GalXM, galactoxylomannan; XT, xylosyltransferase; TOCSY, total correlation spectroscopy; PMAA, partially methylated alditol acetate; MS, mass spectrometry; HPLC, high pressure liquid chromatography. and galactoxylomannan (GalXM), make up ∼97% of the mass of the capsule, with the remaining mass contributed by mannoproteins (3Bose I. Reese A.J. Ory J.J. Janbon G. Doering T.L. Eukaryot. Cell. 2003; 2: 655-663Crossref PubMed Scopus (187) Google Scholar). The biochemical structures of both GXM and GalXM have been determined. GXM, with a molecular mass of 1-7 megadaltons (4McFadden D.C. De Jesus M. Casadevall A. J. Biol. Chem. 2006; 281: 1868-1875Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), is composed of a backbone of α-1,3-linked mannan substituted with glucuronic acid and xylose residues (5Cherniak R. Valafar H. Morris L.C. Valafar F. Clin. Diagn. Lab. Immunol. 1998; 5: 146-159Crossref PubMed Google Scholar) (Fig. 1). GalXM is a smaller polymer (100 kDa), consisting of an α-1,6-galactan backbone with galactomannan side chains that are decorated with xylose residues (6Vaishnav V.V. Bacon B.E. O'Neill M. Cherniak R. Carbohydr. Res. 1998; 306: 315-330Crossref PubMed Scopus (103) Google Scholar) (Fig. 2).FIGURE 2Repeating unit of GalXM from C. neoformans. Open circles, galactose; filled circles, mannose; stars, xylose.View Large Image Figure ViewerDownload Hi-res image Download (PPT) There are four serotypes of C. neoformans (A-D) (7Wilson D.E. Bennett J.E. Bailey J.W. Proc. Soc. Exp. Biol. Med. 1968; 127: 820-823Crossref PubMed Scopus (149) Google Scholar) that differ with respect to virulence and the amount of xylose present within GXM (Fig. 1, panels A-D). Serotype D GXM has the simplest xylosylation pattern, with only one β-1,2-linked xylose residue (Fig. 1, panel D), and is also the least virulent of the serotypes. Serotype A GXM has two β-1,2-linked residues (Fig. 1, panel A), and this serotype is more virulent than serotype D strains (8Barchiesi F. Cogliati M. Esposto M.C. Spreghini E. Schimizzi A.M. Wickes B.L. Scalise G. Viviani M.A. J. Infect. 2005; 51: 10-16Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). The GXM of serotypes B and C, which can cause disease in immunocompetent patients, contains additional β-1,4-linked xylose residues (Fig. 1, panels B and C). The structure of GalXM has only been studied in serotype D strains (6Vaishnav V.V. Bacon B.E. O'Neill M. Cherniak R. Carbohydr. Res. 1998; 306: 315-330Crossref PubMed Scopus (103) Google Scholar). GXM mediates a variety of detrimental effects on the host immune response that allow for the in vivo survival of C. neoformans (9Vecchiarelli A. Curr. Mol. Med. 2005; 5: 413-420Crossref PubMed Scopus (30) Google Scholar). In addition to the correlation between increased GXM xylosylation and virulence among the four serotypes described above, there is further evidence that capsular xylose participates in these immunological effects. A mutant strain of C. neoformans lacking UDP-xylose, the activated donor for xylose transfer, has a general defect in xylosylation (10Bar-Peled M. Griffith C.L. Doering T.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12003-12008Crossref PubMed Scopus (126) Google Scholar, 11Moyrand F. Klaproth B. Himmelreich U. Dromer F. Janbon G. Mol. Microbiol. 2002; 45: 837-849Crossref PubMed Scopus (84) Google Scholar). This strain lacks capsular xylose, has an aberrant capsule appearance, and is avirulent in mice (11Moyrand F. Klaproth B. Himmelreich U. Dromer F. Janbon G. Mol. Microbiol. 2002; 45: 837-849Crossref PubMed Scopus (84) Google Scholar, 12Griffith C.L. Klutts J.S. Zhang L. Levery S.B. Doering T.L. J. Biol. Chem. 2004; 279: 51669-51676Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). In addition, Fries and co-workers (13Goldman D.L. Fries B.C. Franzot S.P. Montella L. Casadevall A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14967-14972Crossref PubMed Scopus (91) Google Scholar, 14Jain N. Guerrero A. Fries B.C. FEMS Yeast Res. 2006; 6: 480-488Crossref PubMed Scopus (32) Google Scholar) have identified phenotypic switching in C. neoformans. Three phenotypic variants of one C. neoformans strain differ with respect to virulence and the arrangement of xylose moieties within GXM (15Fries B.C. Goldman D.L. Cherniak R. Ju R. Casadevall A. Infect. Immun. 1999; 67: 6076-6083Crossref PubMed Google Scholar), further implicating these residues in cryptococcal pathogenesis. Our current understanding of capsule biosynthesis is limited. By screening mutagenized C. neoformans cells for defects in capsule formation, Kwon-Chung, Janbon, and their co-workers (16Chang Y.C. Kwon-Chung K.J. Mol. Cell. Biol. 1994; 14: 4912-4919Crossref PubMed Scopus (399) Google Scholar, 17Chang Y.C. Kwon-Chung K.J. Infect. Immun. 1998; 66: 2230-2236Crossref PubMed Google Scholar, 18Chang Y.C. Kwon-Chung K.J. J. Bacteriol. 1999; 181: 5636-5643Crossref PubMed Google Scholar, 19Chang Y.C. Penoyer L.A. Kwon-Chung K.J. Infect. Immun. 1996; 64: 1977-1983Crossref PubMed Google Scholar, 20Janbon G. Himmelreich U. Moyrand F. Improvisi L. Dromer F. Mol. Microbiol. 2001; 42: 453-467Crossref PubMed Scopus (126) Google Scholar, 21Moyrand F. Chang Y.C. Himmelreich U. Kwon-Chung K.J. Janbon G. Eukaryot. Cell. 2004; 3: 1513-1524Crossref PubMed Scopus (51) Google Scholar) identified a number of genes that appear to be involved in capsule synthesis. Disruption of any one of the four CAP genes (CAP10, CAP59, CAP60, and CAP64) yields an acapsular phenotype, suggesting that these each play a central role in capsule synthesis (16Chang Y.C. Kwon-Chung K.J. Mol. Cell. Biol. 1994; 14: 4912-4919Crossref PubMed Scopus (399) Google Scholar, 17Chang Y.C. Kwon-Chung K.J. Infect. Immun. 1998; 66: 2230-2236Crossref PubMed Google Scholar, 18Chang Y.C. Kwon-Chung K.J. J. Bacteriol. 1999; 181: 5636-5643Crossref PubMed Google Scholar, 19Chang Y.C. Penoyer L.A. Kwon-Chung K.J. Infect. Immun. 1996; 64: 1977-1983Crossref PubMed Google Scholar). Five homologs of CAP10 (CAP1-5) have also been identified, but the biochemical functions of the CAP genes and these homologs have remained undefined (22Okabayashi K. Hasegawa A. Watanabe T. Mycopathologia. 2007; 163: 1-8Crossref PubMed Scopus (20) Google Scholar). Glycosyltransferases catalyze the specific transfer of a monosaccharide from an activated donor, such as a nucleotide disphosphosugar, to an acceptor. These enzymes are typically 30-50-kilodalton type II membrane proteins (23Klutts J.S. Yoneda A. Reilly M.C. Bose I. Doering T.L. FEMS Yeast Res. 2006; 6: 499-512Crossref PubMed Scopus (37) Google Scholar). Glycosyltransferases have been organized into families based on sequence similarity (24Coutinho P.M. Deleury E. Davies G.J. Henrissat B. J. Mol. Biol. 2003; 328: 307-317Crossref PubMed Scopus (931) Google Scholar) and can be broadly divided into two superfamiles (GT-A and GT-B) based on structural patterns. Most members of the GT-A group contain a DXD motif that is involved in the binding of a divalent cation cofactor important for catalytic activity (25Busch C. Hofmann F. Selzer J. Munro S. Jeckel D. Aktories K. J. Biol. Chem. 1998; 273: 19566-19572Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 26Hodson N. Griffiths G. Cook N. Pourhossein M. Gottfridson E. Lind T. Lidholt K. Roberts I.S. J. Biol. Chem. 2000; 275: 27311-27315Abstract Full Text Full Text PDF PubMed Google Scholar, 27Shibayama K. Ohsuka S. Sato K. Yokoyama K. Horii T. Ohta M. FEMS Microbiol. Lett. 1999; 174: 105-109Crossref PubMed Google Scholar, 28Unligil U.M. Rini J.M. Curr. Opin. Struct. Biol. 2000; 10: 510-517Crossref PubMed Scopus (346) Google Scholar, 29Unligil U.M. Zhou S. Yuwaraj S. Sarkar M. Schachter H. Rini J.M. EMBO J. 2000; 19: 5269-5280Crossref PubMed Scopus (239) Google Scholar, 30Wiggins C.A. Munro S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7945-7950Crossref PubMed Scopus (321) Google Scholar, 31Zhang Y. Malinovskii V.A. Fiedler T.J. Brew K. Glycobiology. 1999; 9: 815-822Crossref PubMed Scopus (19) Google Scholar). Although a few GT-A enzymes lack the DXD motif and cation requirement, this is more typical of the GT-B glycosyltransferases. The synthesis of large glycans such as GXM or GalXM generally requires the sequential action of several glycosyltransferases. Thus, it is likely that a number of these enzymes, including up to seven xylosyltransferases, are actively involved in capsule synthesis. It is also reasonable to suggest that some of the previously identified capsule synthesis associated genes may encode glycosyltransferases, especially as one of them, CAP59, encodes a homolog to a known mannosyltransferase (32Sommer U. Liu H. Doering T.L. J. Biol. Chem. 2003; 278: 47724-47730Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). However, no glycosyltransferase with a defined role in capsule synthesis has been identified. Capsule synthesis offers a potential target for antifungal chemotherapy. To understand this process, we must identify the glycosyltransferases involved in GXM and GalXM production. Because of the importance of xylose residues within these structures, we have focused on identifying xylosyltransferases (XTs) involved in capsule biosynthesis. As no homologs to known mammalian or plant XTs exist within the C. neoformans data base, we took a biochemical approach to this question. Here we describe the discovery, purification, characterization, and expression of Cxt1p, a β-1,2-XT from C. neoformans with activity appropriate for the synthesis of either GXM or GalXM. Cxt1p is a large, apparently cation-independent glycosyltransferase that defines a new family of glycosyltransferases. This family includes C. neoformans Cap10p and its homologs and is exclusive to fungi. Materials—Unless otherwise noted, all chemicals were from Sigma. Strains and Cell Growth—C. neoformans wild-type strain JEC21 (serotype D MATα) was provided by Joseph Heitman (Duke University). C. neoformans ags1Δ strain was generated in our laboratory (serotype D MATα ags1Δ) (33Reese A.J. Yoneda A. Breger J.A. Beauvais A. Liu H. Griffith C.L. Bose I. Kim M.J. Skau C. Yang S. Sefko J.A. Osumi M. Latge J.P. Mylonakis E. Doering T.L. Mol. Microbiol. 2007; 63: 1385-1398Crossref PubMed Scopus (122) Google Scholar), and Saccharomyces cerevisiae strain TDY172 (RSY620; MATa ade2-1 trp1-1 leu2-3,112 ura3-1 his3-11,15 pep4::TRP1) was from Randy Schekman (University of California, Berkeley). For activity analysis, 50 ml of minimal medium minus uracil (URA-) (34Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Albright L.M. Coen D.M. Varki A. Chanda V.B. Current Protocols in Molecular Biology. Vol. 2. John Wiley & Sons, Inc., New York2003: 13-14Google Scholar) was inoculated with a single colony of JEC21 or ags1Δ and incubated at 30 °C with continuous shaking (200 rpm) until the A600 was between 1 and 2. For purification, such a culture of ags1Δ was then inoculated into 3 liters of the same medium and grown at 30 °C with continuous shaking for 3-4 days until the A600 was between 4 and 5. TDY172 was grown at 30 °C with continuous shaking in 50 ml of minimal broth media supplemented with uracil to an A600 of 1-2. Xylosyltransferase (XT) Enzyme Assays—Activity was assayed by monitoring the transfer of [14C]xylose from UDP-[U-14C]xylose (264.4 mCi/mmol; PerkinElmer Life Sciences) to α-1,3-mannobiose (α-1,3-Man2; Carbohydrate Synthesis, Oxford, UK). Standard assay mixtures (50 μl) included 15 μl of protein sample, 8.5 mm α-1,3-Man2 (0.43 μmol), 57 nmol of UDP-[U-14C]xylose (15 nCi), and 100 mm Tris, pH 6.5. The reaction was incubated for 4 h at 20 °C and terminated by applying the assay mixture to a small disposable column containing 0.6 ml of AG2X-50 resin (Bio-Rad), followed by 30 μl of deionized water to ensure the sample was fully loaded into the resin. 600 μl of deionized water was then applied to the column, and the eluate collected and spun at 13,000 × g for 5 min to remove particulate materials, and the supernatant fraction removed to another tube. 14C-Labeled trisaccharide product was detected either by scintillation counting or TLC. For the latter, all or part of the supernatant was dried under N2 at 50 °C, resuspended in 15 μl of 40% n-propyl alcohol, and applied to a dried (70 °C for 30 min) 20 × 20-cm Silica Gel-60 TLC plate (EMD Chemicals, Gibbstown, NJ). The plate was developed in 5:4:2 n-propyl alcohol:acetone:water for 2 h, dried, and then developed for an additional 2 h in the same solvent. After drying, the standards were visualized by spraying with 0.2% orcinol in 75:15:10 ethanol:sulfuric acid:water and incubating for 5-10 min at 70 °C. The sample lanes were sprayed with Enhance surface autoradiography spray (PerkinElmer Life Sciences) and allowed to dry, and the radioactive products were visualized by autoradiography. Production and Purification of Trisaccharide Product for Analysis—To generate product for structural analysis, XT reactions were scaled up. Ten 70-μl reactions (20 μl of partially purified enzyme (post Sephacryl S-300, see below), 12.5 mm α-1,3-Man2 (0.88 μmol), 12.8 mm UDP-xylose (0.9 μmol) (Carbosource Services, Athens, GA), 57 nmol of UDP-[U-14C]xylose (0.015 μCi), and 147 mm Tris, pH 6.5) were incubated at 20 °C for 30 h and applied to AG2X-50 columns as above. Each column was eluted with 700 μl of deionized water, and the eluates were pooled, lyophilized, resuspended in 100 μl of 40% n-propyl alcohol, streaked onto two Silica Gel 60 TLC plates, and developed as above. The radioactive trisaccharide was localized using a TLC plate scanner (System 200A imaging scanner; Bioscan Inc., Washington, D.C.), and the corresponding area of silica was recovered. The product was eluted from the silica powder by vortexing for 1 min with 5 ml of water and allowing the mixture to remain for 1 h at room temperature. The silica was sedimented (10,000 × g, 10 min, room temperature), and the supernatant was lyophilized and resuspended in 1 ml of water. The product was then purified by solid phase extraction using a protocol adapted from Ref. 35Packer N.H. Lawson M.A. Jardine D.R. Redmond J.W. Glycoconj. J. 1998; 15: 737-747Crossref PubMed Scopus (417) Google Scholar. Briefly, the suspension was applied to a 0.25 g (1 ml) Envi-Carb solid phase extraction column (Supelco, Bellefonte, PA) that had been preconditioned with 3 ml of 80% (v/v) acetonitrile in 0.1% (v/v) trifluoroacetic acid followed by 2 ml of water. The loaded column was washed with 3 ml of water, and the product was eluted with 3 ml of 25% acetonitrile in water (v/v). The acetonitrile in the eluate was removed by evaporation under N2 at 50 °C, and the remainder of the sample was lyophilized. α-Mannosidase Treatment of the XT Product—XT product was partially purified by TLC as described above. The product was then incubated with jack bean α-mannosidase (Oxford Glycosystems, Oxford, UK) for 18 h at 37 °C in buffer supplied by the manufacturer. One-dimensional 1H and Two-dimensional 1H-1H Nuclear Magnetic Resonance Spectroscopy—Samples of the XT product and the α-1,3-Man2 substrate (∼0.3-1.0 mg) were deuterium-exchanged by repeated lyophilization from D2O and then dissolved in 0.5 ml of D2O for NMR analysis. One-dimensional 1H NMR, two-dimensional 1H-1H-gCOSY, two-dimensional 1H-1H-TOCSY, and one-dimensional 1H-1H nuclear Overhauser effect spectra were acquired at 25 °C on a Varian Unity Inova 500-MHz spectrometer (Department of Chemistry, University of New Hampshire), using standard acquisition software available in the Varian VNMR software package. Proton chemical shifts are referenced to internal acetone (δ = 2.225 ppm). Permethylation and Linkage Analysis—A portion of the XT product was permethylated using the method of Ciucanu and Kerek (36Ciucanu I. Kerek F. Carbohydr. Res. 1984; 131: 209-217Crossref Scopus (3215) Google Scholar) with the modification of Ciucanu and Costello (37Ciucanu I. Costello C.E. J. Am. Chem. Soc. 2003; 125: 16213-16219Crossref PubMed Scopus (218) Google Scholar). An aliquot of the permethylated product was further treated (by hydrolysis, reduction, and per-O-acetylation) for analysis of partially methylated alditol acetates (PMAAs), using the protocols described by Levery and Hakomori (38Levery S.B. Hakomori S. Methods Enzymol. 1987; 138: 13-25Crossref PubMed Scopus (90) Google Scholar). The PMAAs were analyzed on an Rtx-5MS-bonded phase-fused silica capillary column (30 m × 0.25 mm; 0.25 μm phase thickness; Restek Corp., Bellefonte, PA) in the splitless mode using a Trace GC ultra gas chromatograph interfaced to a Polaris Q ion trap mass spectrometer (Finnigan MAT, San Jose, CA), with MS operated in electron ionization mode, and gas chromatography programmed as described previously (38Levery S.B. Hakomori S. Methods Enzymol. 1987; 138: 13-25Crossref PubMed Scopus (90) Google Scholar). The derivatives were identified by retention times and characteristic electron impact mass spectra compared with standards (39Hellerqvist C.G. Methods Enzymol. 1990; 193: 554-573Crossref PubMed Scopus (55) Google Scholar, 40Hellerqvist C.G. Sweetman B. Suelter C.H. Watson D.T. Biomedical Applications of Mass Spectrometry. Vol. 34. Wiley-Interscience, New York1990: 91-143Google Scholar, 41Jansson P.-E. Kenne L. Liedgren H. Lindberg B. Lönngren J. Chem. Commun. Univ. Stockholm. 1976; 8: 1-75Google Scholar). Positive Ion Mode Electrospray-Ionization Mass Spectrometry—Mass spectrometry was performed in the positive ion mode on a linear ion trap (LTQ, ThermoFinnigan San Jose, CA), with sample introduction via direct infusion in 50% MeOH-H2O (for the native XT product) or 100% MeOH (for the permethylated XT product), and a sample concentration of ∼100 ng/μl. Xylosyltransferase Purification—All steps were carried out on ice or at 4 °C, unless otherwise indicated. Q-Sepharose, DEAE, and Sephacryl S-300 chromatography were carried out on an ÄKTAFPLC (GE Healthcare). Protein was measured with the Bio-Rad DC protein reagent (Bio-Rad) using IgG as the standard. All buffers used in the purification are listed in Table 1.TABLE 1Buffers used in xylosyltransferase purificationBufferA100 mm Tris 8.0, 0.1 mm EDTAB20 mm Tris 8.0, 0.1 mm EDTA, 0.05% Triton X-100, 500 mm NaClC20 mm Tris 8.0, 0.1 mm EDTA, 0.05% Triton X-100, 2 m NaClD20 mm Tris 8.0, 0.1 mm EDTA, 0.05% Triton X-100, 100 mm NaClE20 mm Tris 7.5, 0.05% Triton X-100, 400 mm NaCl, 1 mm CaCl2, 1 mm MnCl2F20 mm Tris 8.0, 0.05% Triton X-100, 400 mm NaCl, 0.01 mm EDTA, 200 mm αMMaαMM indicates α-methyl mannosideG20 mm Tris 8.0, 0.1 mm EDTA, 0.01% Triton X-100H20 mm Tris 8.0, 0.1 mm EDTA, 0.01% Triton X-100, 80 mm NaClI20 mm Tris 8.0, 0.1 mm EDTA, 0.01% Triton X-100, 225 mm NaClJ20 mm Tris 6.5, 0.1 mm EDTA, 0.01% Triton X-100K20 mm Tris 6.5, 0.1 mm EDTA, 0.01% Triton X-100, 20 mm NaClL20 mm Tris 6.5, 0.1 mm EDTA, 0.01% Triton X-100, 150 mm NaCla αMM indicates α-methyl mannoside Open table in a new tab Crude Membrane Preparation—ags1Δ cells grown in large scale as above were harvested by centrifugation (9,000 × g, 15 min) at 4 °C, resuspended in Buffer A, and recentrifuged. The cells were resuspended in 30 ml of Buffer A (total volume ≈ 75 ml), divided into three 50-ml conical tubes, and mixed with 12 ml of 0.5-mm glass beads. Cells were broken by vortex mixing six times for 2 min, with 2 min on ice between each mixing, and cell lysates were transferred to fresh 50-ml conical tubes. The beads were washed with 15 ml of Buffer A, and this wash was pooled with the lysate. To sediment broken cells and debris, each pool was centrifuged (1,200 × g, 20 min), and membranes were recovered from the supernatant fraction by ultracentrifugation (60,000 × g, 45 min). The supernatants were discarded, and each membrane pellet was suspended in 50 ml of Buffer A, resedimented (60,000 × g, 30 min), and resuspended in 2 ml of Buffer A. For smaller scale membrane preparations, glass bead lysis was performed, either in a Mini-Bead Beater 8 (Biospec Products, Bartlesville, OK) or by vortex mixing as in Ref. 42Doering T.L. J. Bacteriol. 1999; 181: 5482-5488Crossref PubMed Google Scholar. Solubilization—Pooled crude membranes were mixed with 20% Triton X-100 to a final concentration of 1% and incubated on ice for 30 min, with brief vortex mixing every 5 min. This solution was cleared by centrifugation (60,000 × g, 30 min), and the supernatant fraction (SolZ) was recovered. Q-Sepharose Chromatography—The SolZ fraction (2-3 ml) was filtered using 0.45-μm spin filters and promptly applied to a 20-ml HiPrep 16/10 Q-Sepharose Fast Flow column (GE Healthcare) that had been pre-equilibrated with Buffer B. The column was then washed with 200 ml of Buffer B, and activity was eluted with a 200-ml gradient from Buffer B to Buffer C. 4-ml fractions were assayed as above, and the activity peak was pooled and concentrated to ∼500 μl (SolQ) using a 15-ml Amicon Ultra 10-kDa molecular mass cutoff spin concentrator. Sephacryl S-300 Gel Filtration Chromatography—The Sol Q fraction was filtered and applied to a 316-ml HiPrep 26/60 Sephacryl S-300 gel filtration column (GE Healthcare) that was pre-equilibrated with Buffer D. The column was eluted with a 253-ml (0.8 column volume) isocratic gradient of Buffer D, with the first 95 ml going to waste (void volume). The remaining 158 ml were collected into 4-ml fractions and assayed, and the activity peak was concentrated to 1 ml (SolS). Concanavalin A Chromatography—The SolS fraction was diluted to 10 ml with Buffer E, reconcentrated to 1 ml, and diluted back to 2.5 ml with Buffer E. This was then applied to a 2.5-ml column of concanavalin A-Sepharose 4B (GE Healthcare) that had been pre-equilibrated with Buffer E. The column was capped and allowed to rock at 4 °C overnight. The flow-through was then collected and the column washed with 60 ml of Buffer E. 50 ml of Buffer F was then added to the column. After 10 ml of Buffer F had passed through and been collected, the column was capped and allowed to rock for 30 min at 4 °C. The remainder of the Buffer F was then allowed to elute, pooled with the first 10 ml, and concentrated to 1 ml (SolC). DEAE Chromatography—To desalt SolC, it was diluted to 10 ml with Buffer G, concentrated to 1 ml, rediluted to 10 ml, and concentrated to 0.8 ml. The final solution was filtered and applied to a 1-ml HiTrap DEAE-Fast Flow column (GE Healthcare). The column was washed with 15 ml of Buffer H and eluted with a 20-ml gradient from Buffer H to Buffer I. 1-ml fractions were assayed, and the activity peak was concentrated to 1 ml (SolD). α-1,3-Man2 Affinity Chromatography—The SolD fraction was diluted to 15 ml with Buffer G and extensively desalted with three successive concentrations and dilutions, to a final volume of 1 ml. This was mixed with 1 ml of Buffer J and loaded onto a column containing 5 ml of a custom-synthesized α-1,3-Man2-agarose resin (Carbohydrate Synthesis, Oxford, UK) that had been pre-equilibrated with Buffer J. The column was capped, allowed to rock at 4 °C for 30 min and then at 25 °C for 90 min, and then washed with 30 ml of Buffer K at 25 °C. The column was then moved to 4 °C and eluted with a 44-ml gradient from Buffer K to Buffer L, collecting 0.85-ml fractions. The activity peak was combined into two pools that were each concentrated to 25 μl; one pool that consisted of the shoulders of the peak and the other the middle of the peak. Protein Analysis—The two pools from the α-1,3-Man2 column were resolved by SDS-PAGE on a 12% gel, which was stained with SYPRO Ruby (Bio-Rad) as per the manufacturer's instructions and visualized on a Bio-Rad Molecular Imager FX. An ∼90-kDa band of interest was excised and submitted to the Protein and Nucleic Acid Chemistry Laboratory at Washington University School of Medicine for trypsin digestion followed by HPLC separation and mass spectrometry analysis of the resulting fragments. The mass spectra were compared with the C. neoformans protein data base, and the protein sequence corresponding to the band of interest was then analyzed using the Prosite domain prediction server and the TMpred transmembrane domain prediction server. Expression in S. cerevisiae—CXT1 was amplified from JEC21 cDNA by PCR using primers Exp-sense and Exp-antisense (Table 2) to incorporate a His6 tag at the C terminus, as well as HindIII and BamHI sites at the 5′ and 3′ ends, respectively. The product was cloned into a TOPO pCR2.1 vector (Invitrogen) and sequenced (Protein and Nucleic Acid Chemistry Laboratory, Washington University School of Medicine). A Topo-CXT1 clone with the correct sequence was then digested with HindIII and BamHI, and the released fragment was cloned into a2-μm yeast expression vector between the promoter and terminator of phosphoglycerate kinase (plasmid pPGK (43Kang Y.S. Kane J. Kurjan J. Stadel J.M. Tipper D.J. Mol. Cell. Biol. 1990; 10: 2582-2590Crossref PubMed Google Scholar); from K. Blumer, Washington University School of Medicine). The resulting plasmid and empty vector were transformed in
Referência(s)