Trehalose-based oligosaccharides isolated from the cytoplasm of Mycobacterium smegmatis Relation to trehalose-based oligosaccharides attached to lipid Masaya Ohta 1 , Y. T. Pan 2 , Roger A. Laine 3 and Alan D. Elbein 2 1 Department of Biochemistry, Fukuyuma University, Japan; 2 Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA; 3 Departments of Biological Sciences and Chemistry, Louisiana State University, Baton Rouge, LA, USA A series of trehalose-based oligosaccharides were isolated from the cytoplasmic fraction of Mycobacterium smegmatis and purified by gel-filtration and paper chromatography and TLC. Their structures were determined by HPLC and GLC to determine sugar composition and ratios, MALDI-TOF MS to measure molecular mass, methylation analysis to determine linkages, 1 H-NMR to obtain anomeric configu- rations of glycosidic linkages, and exoglycosidase digestions followed by TLC to determine sequences and anomeric configurations of the monosaccharides. Six different oligo- saccharides were identified all with trehalose as the basic structure and additional glucose or galactose residues attached in various linkages. One of these oligosaccharides is the disaccharide trehalose (Glca1–1aGlc), which is present in substantial amounts in these cells and also in other mycobacteria. Two other oligosaccharides, the tetrasaccha- rides Glca1–4Glca1–1aGlc6–1aGal and Gala1–6Gala1– 6Glca1–1aGlc, have not previously been isolated from natural sources or synthesized chemically. The fourth oligosaccharide, Glcb1–6Glcb1–6Glca1–1aGlc, has been isolated from corynebacteria, but not reported in other organisms. Two other oligosaccharides, Glca1–4Glca1– 1aGlc, which has been synthesized chemically and isolated from insects but not previously reported in mycobacteria, and Glcb1–6Glca1–1aGlc, which was previously isolated from Mycobacterium fortuitum and yeast, were also charac- terized. Another trisaccharide found in the cytosol has been partially characterized as arabinosyl-1–4trehalose, but nei- ther the anomeric configuration nor the D or L configuration of the arabinose is known. In analogy with sucrose and its higher homologs, raffinose and stachyose, which may act as protective agents during maturation drying in plants, these trehalose homologs may also have a protective role in mycobacteria, perhaps during latency. Keywords: Mycobacteria, oligosaccharides; trehalose. The mycobacterial cell wall is an exceedingly complex network of interacting molecules and includes various polysaccharides such as mycolic acid–arabinogalactan and lipoarabinomannan, as well as a variety of complex glycoli- pids [1]. Among the interesting and important glycolipds are a number of trehalose-containing lipids, such as trehalose monomycolate and dimycolate, and other acylated trehalose compounds [2]. In addition to its role as a structural component, trehalose dimycolate has also been implicated as a donor of mycolic acids to the arabinogalactan [3,4]. Furthermore, in yeast, bacteria and various other organisms, free trehalose has been shown to have a protective role as a stabilizer of proteins and membranes during dehydration, dessication, heat shock and other adverse conditions [5,6]. Trehalose (Glca1–1aGlc) is not found in any vertebrates, nor is it synthesized in these organisms. Thus, the synthesis and acylation of trehalose represent excellent target sites for the design of new drugs against tuberculosis. Therefore, we have purified the trehalose phosphate synthase [7] and trehalose phosphate phosphatase (S. Klutts, I. Pastuszak, D. Carroll, Y. T. Pan & A. D. Elbein, unpublished results) from Mycobacterium smegmatis and examined cytosolic and lipid extracts of M. smegmatis for possible analogs of trehalose. In this paper, we report on the isolation from the cytoplasm and characterization of trehalose and five other trehalose-based oligosaccharides. Two of these oligosaccha- rides are newly described tetrasaccharides, so far only reported here in M. smegmatis. Another trehalose oligosac- charide has been synthesized chemically but not previously isolated from any living organisms, and two others have been demonstrated in other cells, but not in M. smegmatis. One other oligosaccharide, with an arabinose linked to trehalose, is also new but its complete structure is not known. At this point, we do not know the function of these trehalose oligosaccharides. However, in analogy with the plant disaccharide sucrose and its higher homologs raffinose and stachyose, these higher derivatives of trehalose may also stabilize M. smegmatis and other mycobacteria during stress and perhaps latency. In plants, sucrose, raffinose and stachyose have been implicated as protective agents during maturation drying [9]. Sucrose is a nonreducing disac- charide of glucose and fructose, whereas trehalose is a Correspondence to A. D. Elbein, Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA. Fax: 501 686 8169, Tel. 501 686 5176, E-mail: elbeinaland@uams.edu Abbreviation: MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight. (Received 11 January 2002, revised 27 March 2002, accepted 30 April 2002) Eur. J. Biochem. 269, 3142–3149 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02971.x nonreducing disaccharide of two glucoses. Thus, the sucrose and the trehalose series may have analogous functions. EXPERIMENTAL PROCEDURES Materials M. smegmatis was obtained from the American Type Culture Collection. Trypticase Soy broth was from Fisher Chemical Co. a-Glucosidase and a-galactosidase were purchased from Sigma Chemical Co. Whatman 3 MM paper was from Whatman Co., and silica gel thin-layer plates were from Analtech, Inc. All other chemicals were from reliable chemical companies and were of the best grade available. Growth of M. smegmatis and isolation of oligosaccharides M. smegmatis was grown in trypticase soy broth for 24–48 h at 37 °C. A 125-mL Erlenmeyer flask containing 25 mL medium was inoculated from a slant of the organism, and grown overnight. This culture was then used as the starter to inoculate a number of large culture flasks (2-L conical flasks containing 1 litre of medium). Cells were grown for 36–48 h, harvested by centrifugation, and stored as a paste in aluminium foil at )20 °C until used. M. smegmatis cells were suspended in water (100 g cell paste in 200 mL water) and disrupted by sonication. The sonicate was centrifuged at  37 000 g for 30 min, and the supernatant, representing the cytosolic fraction of the cell, was removed and saved. To this ice-cold supernatant, was added trichloroacetic acid to a final concentration of 5% to precipitate the protein. After removal of the protein by centrifugation, the trichloroacetic acid was removed by extraction with diethyl ether, and then alcohol was added to the supernatant to a final concentration of 70% to precipitate glycogen and any other trichloroacetate-soluble polymers present in the cytoplasm. The alcohol mixture was allowed to stand overnight in the cold, and any precipitate was removed by centrifugation. The supernatant from this centrifugation was concentrated to a small volume and deionized by treatment with mixed- bed ion-exchange resin [equal mixture of Dowex-1 (CO 3 2– ) and Dowex-50 (H + )]. The deionized solution was applied to a Bio-Gel P-2 column (1.5 · 200 cm) and eluted with 1% acetic acid. Fractions were collected, and an aliquot of each was analyzed with the anthrone reagent [10] for the presence of hexose. A number of anthrone-positive peaks were identified which corresponded to monosaccharide, disac- charide, trisaccharide and tetrasaccharide (Fig. 1). A large anthrone-positive peak emerged early in the elution and may represent polymeric material. Each of these peaks was pooled and subjected to additional gel filtrations for further purification. In some cases, the peaks from the Bio-Gel column were also subjected to paper chromatography in various solvents to separate the oligosaccharides further. Sugars and oligosaccharides were visualized with silver nitrate reagent [11]. Oligosaccharides were also separated by TLC on both analytical plates and, in some cases, prepar- ative plates and by preparative HPLC. As trehalose is a nonreducing sugar, each of the oligosaccharides was tested with the reducing sugar test [12] to determine whether it was also a nonreducing sugar. Paper chromatography and TLC Oligosaccharides were streaked on 9 inch sheets of What- man 3 MM paper (22 inches long) and chromatographed by descending chromatography in the following solvent sys- tems: butan-1-ol/pyridine/0.1 M HCl (5:3:2, by vol.); propan-2-ol/butan-1-ol/water (140 : 20 : 40, by vol.); pro- panol/ethyl acetate/water (140 : 20 : 40, by vol.). TLC of sugars and oligosaccharides was performed on silica-gel plates in acetonitrile/water (4 : 1). Usually the thin-layer plates were subjected to multiple developments in the acetonitrile/water solvent with complete drying between each run. HPLC HPLC analysis was performed with a Shimadzu model LC-10A liquid chromatograph (Shimadzu Co.) equipped with a TSK-gel Amide-80 column (0.46 · 250 mm; TOSOHO) at 40 °C. Elution was isocratic with acetonitrile/ water (56 : 42, v/v) at a flow rate of 0.5 mLÆmin )1 .Elution of oligosaccharides was monitored with a refractive index detector [13]. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS characterization The molecular mass of each oligosaccharide was determined by MALDI-TOF MS (Voyager DE STR, Perseptive Biosystems) in a positive-ion reflector mode using 20 kV total accelerating voltage. Desorption/ionization was via a nitrogen laser operating at 337 mm. The matrix solution contained 10 mgÆmL )1 2,5-dihydroxybenzoic acid in 10% ethanol. One microlitre of the matrix solution and 1 lLof the peracetylated oligosaccharides in chloroform were mixed on the sample plate directly and dried to avoid Fig. 1. Separation of the cytosolic oligosaccharides on columns of Bio- Gel P-2. A1.5· 200 cm column of Bio-Gel P-2 was prepared and washed with 1% acetic acid. The samples were applied to the column and eluted with the same solution. Fractions were collected and an aliquot of each was removed and assayed for its hexose content by the anthrone reaction. Letters shown at the top indicate the elution posi- tions of the sugar standards: G ¼ glucose, T ¼ trehalose, R ¼ raffinose, S ¼ stachyose. Ó FEBS 2002 Cytoplasmic trehalose oligosaccharides (Eur. J. Biochem. 269) 3143 crystallization. The analyzed substances were detected as their pseudomolecular ions ([M + Na] + ). Oligosaccharides were peracetylated by suspending lyophilized samples in 50 lL acetic anhydride and incubating at 80 °C for 2 h. The reaction mixture was evaporated to dryness under a stream of N 2. Methylation analysis Oligosaccharides were permethylated by the method of Ciucanu & Kerek [14]. Methylated samples were hydro- lyzed in acid, reduced and acetylated as described by Liang et al. [15]. The sugar acetates were chromatographed on a silica-gel capillary column (DB-5MS; 0.25 mm · 30 m; J and W Scientific Co., Folsom, CA, USA) in a Finnigan GCQ (Trace GC 2000 coupled with Polaris MSD). The column was operated at 50 °C for 1 min, increased to 170 °Catarateof20° per min, and then increased to 260 °Cat3° per min. It was then held at this final temperature for 4 min. The column was calibrated with various methylated glucose standards. Exoglycosidase digestions To determine the sequence of sugars in these oligosaccha- rides, as well as the anomeric configurations of the linkages, various oligosaccharides were subjected to digestion by specific exoglycosidases that remove sugars from the nonreducing ends of the oligosaccharides. The following enzymes and incubation conditions were used: (a) 1 unit a-glucosidase (from Bacillus stearothermophilus)wasincu- bated with substrates in 0.1 M sodium phosphate buffer, pH 6.8; (b) 0.5 unit a-galactosidase (from Aspergillus niger) was incubated with substrates in 50 m M sodium acetate buffer, pH 4.0. All incubations were performed at 37 °C for 24 h, and were stopped by placing the incubation mixtures in a boiling water bath for 3 min. The mixtures were diluted in water and treated with mixed-bed ion-exchange resin to remove salt and other charged compounds. The digestion products were then analyzed by TLC. RESULTS Isolation of cytosolic oligosaccharides M. smegmatis cells were disrupted by sonication and the cytosolic fraction was isolated by high-speed centrifugation to remove cell walls and membranes. The supernatant was treated with trichloroacetic acid and ethanol to remove protein and glycogen as described, and after treatment with mixed-bed ion-exchange resin, it was applied to a column of Bio-Gel P-2 to roughly separate the various oligosaccha- rides into their different sizes, i.e. mono, di, tri, and tetra. Figure 1 shows a profile of the elution pattern of sugars (i.e. anthrone-positive material) from this column. A number of anthrone-positive peaks were eluted from the column, with the three major peaks corresponding to glucose (fractions 111–120), trehalose (fractions 101–110) and high-molecular-mass material (fractions 50–62). How- ever, a number of other peaks containing smaller amounts of anthrone-positive material were also detected and each of these peaks was designated as follows: CO7, CO6, CO5, CO4, CO3, CO2, CO1 (see Fig. 1 for positions of each peak). CO1 was the monosaccharide peak containing mostly glucose, and CO7 was composed of several higher- molecular-mass oligosaccharides, which were not further characterized because of limiting amounts of material (see Fig. 2 for TLC profiles). A large peak was eluted very early (fractions 50–65), which probably represents polysaccharide material. This peak has not yet been characterized. Each of the peaks labeled CO2 through CO6 were pooled and analyzed to determine the amount of oligosaccharide present. Several were also analyzed by the reducing sugar test to compare the amount of hexose present with the amount of reducing sugar. This was to determine whether nonreducing oligosaccharides of the trehalose class were present in these fractions. That is, for a reducing tetrasac- charide, one would expect one reducing sugar for every four hexoses, whereas for a trisaccharide there should be one reducing sugar for every three hexoses. Table 1 shows the results from the gel-filtration column, indicating the position of elution of each peak (fraction numbers), the amount of hexose based on anthrone determinations [10] and reducing sugar based on the Nelson method [12] compared with a glucose and maltose standard. These standards gave hexose/ reducing sugar values of 1 : 1 and 2 : 1 as expected. The ratio of total hexose to reducing sugar of several of the trehalose oligosaccharides are shown in Table 1. The amount of each oligosaccharide was as follows (total lmol glucose/100 g cell paste): CO1, 90; CO2, 150; CO3, 3; CO4, 14; CO5, 7; CO6, 2. It can be seen that these cells contained substantial amounts of trehalose (CO2) and free glucose (CO1), and considerably smaller amounts of the various higher oligosaccharides. However, most of these peaks appeared to be composed mostly of nonreducing oligosac- charides, presumably of the trehalose class, because the ratio of number of hexoses/reducing sugar was greater than 8 and as high as 50. Fig. 2. TLC of the various oligosaccharides after isolation by gel fil- tration and paper chromatography. Samples were applied to the plate and it was developed twice in acetonitrile/water (4 : 1, v/v). Carbo- hydrates were detected by spraying the dried plates with 50% con- centrated sulfuric acid in ethanol followed by heating at 110 °C for 5–10 min. Lane 1, various sugar standards (G ¼ glucose, T ¼ trehalose, R ¼ raffinose, S ¼ stachyose); lanes 2–8, the isolated oligosaccharides designated CO1 through CO7. 3144 M. Ohta et al.(Eur. J. Biochem. 269) Ó FEBS 2002 Each of the peaks shown in Table 1 was rerun on the Bio- Gel column for further purification, and then each peak from these columns was streaked on sheets of Whatman 3 MM paper and subjected to chromatography in one or more of the solvents described in Experimental Procedures. Areas corresponding to various oligosaccharides were cut from the papers, and the oligosaccharides eluted with water and rechromatographed until they appeared to be homo- geneous. Figure 2 shows a thin-layer chromatogram of the indi- vidual oligosaccharides characterized in this study. Oligo- saccharide CO3 was found to be composed of two compounds, on the basis of Amide-80 HPLC (Fig. 3), and these were separated and designated CO3-1 and CO3-2. Likewise, CO4 was separated into four fractions by HPLC (Fig. 3), and these were designated CO4-1, CO4-2, CO4-3 and CO4-4. Structures of the oligosaccharides Fractions CO1 and CO2. The mobility on TLC (Fig. 2), the methylation data (Table 2), and the 1 H-NMR analysis revealed that CO-1 was glucose and CO-2 (referred to as oligosaccharide CD2, see Table 4) was trehalose. Fraction CO3. CO3 was composed of two different oligosaccharides as shown by TLC (Fig. 2). These two components, designated as CO3-1 and CO3-2, were separated in sufficient amounts by HPLC (Fig. 3) to allow chemical characterization of each. CO3-1 was identified as trehalose by TLC as well as by MALDI-TOF MS analysis of the products obtained by permethylation and hydrolysis, andalsoby 1 H-NMR. Oligosaccharide CO3-2 appeared to be a trisaccharide, as MALDI-TOF MS analysis of the peracetylated oligosac- charide gave a pseudomolecular ion [M + Na] + at m/z 989, suggesting that it was composed of three hexoses. Methylation, followed by hydrolysis and reduction, of this oligosaccharide, as shown in Table 2, gave 1 mol 2,3,6- tri-O-methylglucitol acetate and 2 mol 2,3,4,6-tetra-O- methylglucitol acetate. These results establish that the oligosaccharide has the structure Glc1–4Glc1–1Glc. The 1 H-NMR analysis, presented in Fig. 4, shows three ano- meric proton signals at 5.21 p.p.m. (J 12 ¼ 4.0), 5.21 p.p.m. (J 12 ¼ 4.0), and 5.41 p.p.m. (J 12 ¼ 3.6), attributable to all a-glycosidic linkages. The overlapping a-linked signals at 5.21 p.p.m. were suggestive of an a,a¢-linked disaccharide, i.e. a,a-trehalose [16]. Moreover, these values for the anomeric proton signals are the same as seen for bemisiose isolated from Bemisia honeydew [17]. On the basis of these results, oligosaccharide CO3-2 is identified as Glca1– 4Glca1–1aGlc and is termed CT3A (see Table 4). Fraction CO4. The migration of CO4 on TLC plates, as seen in Fig. 2, indicated that it was composed of several poorly separated components. Subsequent fractionation by Amide-80 HPLC (Fig. 3) resulted in separation of four peaks, designated CO4-1, CO4-2, CO4-3 and CO4-4, in the proportions 11%, 45%, 23%, and 21%, respectively. Each oligosaccharide was subjected to structural characterization by methylation analysis, MALDI-TOF MS and 1 H-NMR. CO4-1, when peracetylated and subjected to MALDI- TOF MS, gave a single peak at m/z 917, indicating that this oligosaccharide was a trisaccharide consisting of 1 mol pentose and 2 mol hexose. Further investigation of the permethylated alditol acetates from this oligosaccharide gave Fig. 3. Amide-HPLC separataion of trehalose oligosaccharide fractions CO3 and CO4. An Amide89 column, 4.6 · 250 mm, was run with isocratic 58% acetonitrile in water at a flow rate of 0.5 mLÆmin )1 . Oligosaccharides were detected by refractive index. Standard sugars wererunonthiscolumnandemergedinpositionsshownatthetopof the profile, i.e. T ¼ trehalose, R ¼ raffinose, S ¼ stachyose. Table 1. Amount of oligosaccharides isolated from 100g cells. Peak designation Fractions from P-2 column lmol Glucose a Ratio hexose/reducing sugar Polymer 51–60 164 – CO7 61–70 5 – CO6 73–79 2 8.3 (5 : 0.6) CO5 77–85 7 69 (6.9 : 0.1) CO4 85–92 14 – CO3 97–103 3 8 (3 : 0.4) CO2 100–105 150 CO1 111–114 90 a Based on anthrone assay. Ó FEBS 2002 Cytoplasmic trehalose oligosaccharides (Eur. J. Biochem. 269) 3145 three peaks corresponding to terminal arabinose, terminal glucose, and 4-linked glucose (Table 2). These results suggested that the arabinose was attached to position 4 of one of the glucoses of trehalose. Because of limited amounts of this oligosaccharide, further analysis was not possible. This peak is most likely Ara-1,4Glca1–1aGlc, but the anomeric configuration and the D or L form of the arabi- nose have not been determined. No arabinose-containing oligosaccharides of trehalose have previously been reported. The most prominent peak, CO4-2, gave the same results as CO3-2 when subjected to MALDI-TOF MS, methyla- tion analysis and 1 H-NMR. Thus CO4-2 appears to be the trisaccharide, Glca1–4Glca1–1aGlc, and was referred to as CT3A (Table 4). In oligosaccharide CO4-3, a major pseudomolecular ion [M + Na] + signal at m/z 989 was observed corresponding to a trisaccharide composed of three hexoses. A minor pseudomolecular ion [M + Na] + at m/z 1073 was also detected, which probably corresponds to a compound composed of two hexoses and one hexitol. Table 2 shows that methylation of CO4-3 gave two peaks, indicating the presence of terminal glucose and 6-linked glucose in the ratio 2 : 1. 1 H-NMR of the CO4-3 fraction is presented in Fig. 4 and showed the presence of three anomeric proton signals at 5.20 p.p.m. (J 12 ¼ 4.0), 5.20 p.p.m. (J 12 ¼ 4.0), and 4.41 p.p.m. (J 12 ¼ 7.6). The signals at 5.20 were assigned to two a-linkages and the signal at 4.41 p.p.m. was assigned to a b-linkage. The b-linked signal (4.41 p.p.m) suggested that the terminal glucose residue was attached to C-6 of an internal glucose [18]. From these results, the structure of the major oligosaccharide in the CO4-3 peak is Glcb1–6Glca1–1aGlc (termed CT3b, Table 4). MALDI-TOF MS analysis of the peracetylated oligo- saccharide in CO4-4 gave a pseudomolecular ion [M + Na] + at m/z 1277, suggesting a tetrasaccharide structure composed of four hexoses. Methylation analysis, as seen in Table 2, gave an almost equal amount of 2,3,4,6- tetramethylglucitol acetate and 2,3,4-trimethylglucitol acet- ate, indicating that the structure of this tetrasaccharide was either Glc1–6Glc1–1Glc6–1Glc or Glc1–6Glc1–6Glc1– 1Glc. The 1 H-NMR spectrum showed four anomeric proton signals at 5.21 p.p.m. (J 12 ¼ 4.0), 5.19 p.p.m. (J 12 ¼ 4.0), 4.41 p.p.m. (J 12 ¼ 7.6) and 4.51 p.p.m. (J 12 ¼ 8.0), attributable to two a-glycosidic and two b-glycosidic linkages (Fig. 4). The two b-linked glucose signals at 4.41 p.p.m and 4.51 p.p.m. are consistent with a Fig. 4. 1 H-NMR spectra of the cytoplasmic oligosaccharides, CO3-2, CO4-3 and CO4-4. The methodology is described in Experimental Procedures. All oligosaccharides obtained in sufficient quantity were subjected to NMR as described here and gave the results described in the text. Table 2. Methylation analysis of cytoplasmic oligosaccharides from M. smegmatis. Residues Oligosaccharides (molar ratio) CO1 CO2 CO3–1 CO3–2 CO4-1 CO4–2 CO4-3 CO4-4 CO5 CO6 Arabinitol 2,3,5-Tri-O-methyl- – – – – 0.8 – – – – – Glucitol 2,3,4,6-Tetra-O-methyl- 1.0 a 2.0 b 2.0 b 2.0 b 1.0 a 2.0 b 2.0 b 2.0 b 1.0 a 0.7 2,3,6-Tri-O-methyl- – – 0.8 0.7 0.9 – – 1.2 – 2,3,4-Tri-O-methyl- – – – – – – 0.8 1.8 0.9 1.1 Galactitol 2,3,4,6-Tetra-O-methyl- – – – – – – – + c 0.9 1.0 d 2,3,4-Tri-O-methyl- – – – – – – – – – 1.1 a Values normalized to one residue of 2,3,4,6-tetra-O-methylglucitol. b Values normalized to two residues of 2,3,4,6-tetra-O-methylglucitol. c + means < 0.1. d Values normalized to one residue of 2,3,4,6-tetra-O-methylgalactiol. 3146 M. Ohta et al.(Eur. J. Biochem. 269) Ó FEBS 2002 Glcb1–6Glcb1–6 disaccharide linked to a trehalose core [19]. Therefore the most likely structure of this tetrasaccha- ride is Glcb1–6Glcb1–6Glca1–1aGlc. It was designated CT4AasindicatedinTable4. Fraction CO5. MALDI-TOF MS analysis of the peracet- ylated CO5 showed a pseudomolecular ion at 1277, characteristic of a tetrasaccharide of four hexoses. Table 2 presents the results of methylation analysis of this tetrasac- charide. This method gave the following four methylated sugars in almost equal amounts: 2,3,4,6-tetramethylglucitol acetate, 2,3,6-trimethylglucitol acetate, 2,3,4-trimethylgluc- itol acetate and 2,3,4,6-tetramethylgalactitol acetate, as seen in Fig. 5. The presence of 2,3,4-trimethylglucitol acetate and 2,3,6-trimethylglucitol acetate indicated that these two glucoses were linked together in a 1–1 linkage (i.e. as trehalose) with a terminal galactose linked to one of the glucoses of the trehalose in either a 1–6 or 1–4 linkage, and the nonreducing glucose linked to the other trehalose glucose in either a 1–6 or a 1–4 linkage. To determine the sugar sequence of this tetrasaccharide, it was subjected to exoglycosidase digestions with specific glycosidases, and the products of the digestions were isolated and characterized. When CO5 was incubated with purified a-galactosidase, it was converted into a trisaccha- ride that migrated on TLC plates with Glca1–4Glca1– 1aGlc as shown in Fig. 6. When the trisaccharide product was subjected to methylation analysis, 2 mol 2,3,4,6-tetra- methylglucitol acetate and 1 mol 2,3,6-trimethylglucitol acetate were found (see Table 3 and Fig. 5). This indicated that the galactose residue was linked in an a1–6 linkage to one of the glucoses of trehalose, and the glucose was linked to the other trehalose glucose in an a1–4 linkage. The trisaccharide resulting from the a-galactosidase digestion was susceptible to digestion by a-glucosidase with the release of glucose. When the original tetrasaccharide was treated with a-glucosidase, a trisaccharide product was obtained. As shown in Table 3, methylation of this trisac- charide gave 1 mol 2,3,4,6-tetramethylglucitol acetate and 1 mol 2,3,4-trimethylglucitol acetate and 1 mol 2,3,4,6- tetramethyl galactitol acetate. These results show that this tetrasaccharide has the structure Glca1–4Glca1–1aGlc6– 1aGal. This oligosaccharide is designated CT4b (Table 4). Fig. 5. Methylation analysis of the various oligosaccharides isolated as described. The methylation results of CO5 and CO6 are shown here before and after glycosidase treatment, but similar results and similar methodology was used with other oligosaccharides. Fig. 6. Determination of the sugar sequence and the anomeric confi- guration using specific exoglycosidases. Oligosaccharides were subjected to digestion with a-glucosidase, a-galactosidase, or both and the products determined by TLC in acetonitrile/water (4 : 1, v/v). Lane 1, sugar standards (G ¼ glucose, T ¼ trehalose, R ¼ raffinose, S ¼ stachyose); lane 2, a-galactosidase treatment of CO5; lane 3, a-galactosidase treatment of CO6; lane 4, a-glucosidase digestion of CO5; lane 5, a-glucosidase digestion of CO6. After chromatography and drying of the plates, carbohydrates were detected with sulfuric acid and heating. Ó FEBS 2002 Cytoplasmic trehalose oligosaccharides (Eur. J. Biochem. 269) 3147 Fraction CO6. The peracetylated CO6 gave only a single peak at m/z 1277 as a pseudomolecular ion [M + Na] + , indicating that this oligosaccharide was also a tetrasaccha- ride composed of four hexose residues. When this oligosac- charide was subjected to methylation analysis (see Table 2), four different methylated products, all present in about equal amounts, were identified as follows: 2,3,4-trimethyl- glucitol acetate, 2,3,4,6-tetramethylglucitol acetate, 2,3, 4-trimethylgalactitol acetate and 2,3,4,6-tetramethylgalacti- tol acetate. As the amount of CO6 available for character- ization was too low for NMR analysis, additional charac- terization was performed using various exoglycosidases to remove terminal sugars, followed by analysis of the products of these digestions. The tetrasaccharide was resistant to hydrolysis by a-glucosidase (Fig. 6, lane 5), but was susceptible to digestion by a-galactosidase (Fig. 6, lane 3). Treatment of CO6 with a-galactosidase resulted in the formation of trehalose, as identified by TLC (Fig. 6). The disaccharide resulting from treatment with a-galactosi- dase was further converted into free glucose by digestion with a-glucosidase (data not shown). In addition, methyla- tion of the disaccharide resulting from a-galactosidase digestion gave only 2,3,4,6-tetramethylglucitol acetate (Table 3). This product could only arise from a disaccharide linked in a 1–1-glycosidic bond. These results indicate that this tetrasaccharide has the structure Gala1–6Gala1– 6Glca1–1aGlc,andisreferredtoasCT4c(Table4). DISCUSSION The isolation and characterization of a number of trehalose- based oligosaccharides from the cytoplasmic fraction of M. smegmatis are described. Besides trehalose, five other nonreducing oligosaccharides were isolated and character- ized. Two of these, the tetrasaccharides CT4b (Glca1– 4Glca1–1aGlc6–1aGal) and CT4c (Gala1–6Gala1– 6Glca1–1aGlc) are newly described and have not been previously reported from any natural sources, nor are there any reports on their chemical synthesis. On the other hand, a number of trehalose-containing oligosaccharides have been synthesized chemically, some of which are related to those described here. For example, trehalose with a b-galactose linked to the 6-hydroxy group of each glucose of the trehalose (i.e. Galb1–6Glca1–1aGlc6–1bGal) has been synthesized chemically [20], as well as a trehalose with an a1,6-linked disaccharide of glucose linked to the 6 position of one of the trehalose glucoses (i.e. Glca1– 6Glca1–6Glca1–1aGlc) [21]. A number of other tetrasac- charides of trehalose have been chemically synthesized with either galactose or glucose linked in a or b linkages. The third tetrasaccharide isolated from M. smegmatis, CT4a (Glcb1–6Glcb1–6Glca1–1aGlc), has been reported in corynebacteria, but has not previously been described in mycobacteria [19]. However, a pentasaccharide, with the same structure as CT4a except for an additional glucose linked b to the 3-hydroxy group of the second glucose of trehalose, has been isolated from the cell wall lipids of Mycobacterium kansasii [1]. That study strongly suggests that some of the oligosaccharides described here, and isolated from the cytoplasm of M. smegmatis, will also be found as part of the cell wall components of this organism. All trehalose-containing structures reported from mycobac- terial glycolipids thus far contain b-linked additional sugars. The cytoplasm of M. smegmatis also contains at least two trisaccharides that have trehalose as the base. One of these compounds identified in both CO3-2 and CO4-2 is the trisaccharide, Glca1–4Glca1–1aGlc, i.e. CT3a. This com- pound has been synthesized chemically [22] and also isolated from insects and yeast. It is interesting to note that this trisaccharide is also related to the new tetrasaccharide reported here as CT4b and may be a precursor in the synthesis of that tetrasaccharide. It is important to deter- mine whether there are galactosyltransferases and glucosyl- transferases in these cells that can convert trehalose into these various oligosaccharides, because some of these enzymes could be target sites for chemotherapy. The other trisaccharide found in fraction CO4-3 has the structure Glcb1–6Glca1–1aGlc. This compound has been reported in Aspergillus sydowi [23], and it may also be a precursor of CT4a. Table 4. Proposed structures of cytoplasmic oligosaccharides from M. smegmatis. Oligosaccharide Structure Fraction CD2 G1ca1-1aG1c CO2, CO3-1 CT3a G1ca1- 4G1ca1-1aG1c CO3-2,CO4-2 CT3b G1cb1- 6G1ca1-1aG1c CO4-3 CT4a G1cb1-6 G1cb1-6G1ca1-1aG1c CO4-4 CT4b G1ca1-4G1ca1-1aG1c6-1aGal CO5 CT4c Ga1a1-6 Gala1-6G1ca1-1aG1c CO6 Table 3. Methylation analysis of exo-glycosidase digestion products. Residues CO5 CO6 Native a-Galactosidase digestion a-Glucosidase digestion Native a-Galactosidase digestion Glucitol 2,3,4,6-Tetra-O-methyl 1.0 2.1 2.2 a 0.7 1.9 2,3,6-Tri-O-methyl- 1.2 1.0 ___ 2,3,4-Tri-O-methyl- 0.9 _ 0.9 1.1 _ Galactitol 2,3,4,6-Tetra-O-methyl- 0.9 0.9 b 0.9 1.0 2.1 b 2,3,4-Tri-O-methyl- __ _ 1.1 _ a Values include the amount of the released glucose residue. b Values indicate the amount of the released galactose residue. 3148 M. Ohta et al.(Eur. J. Biochem. 269) Ó FEBS 2002 The origin of these cytosolic oligosaccharides is not known. They could be synthesized in the cytosol by glycosyltransferases which add glucosyl or galactosyl resi- dues to the free trehalose that arises from trehalose phosphate by the action of the specific trehalose phosphate phosphatase (S. Klutts, I. Pastuszak, D. Carroll, Y. T. Pan & A. D. Elbein, unpublished results). Thus the synthesis of the higher homologs of trehalose may be analogous to synthesis of raffinose and stachyose from sucrose [9]. In that case the donor of galactose to sucrose to form raffinose is galactinol, i.e. galactosyl myoinositol, rather than the expected UDP- galactose. As trehalose and its higher homologs may have key roles as protectants or stabilizers of mycobacteria and may also be important structural components, any of the reactions involved in their biosynthesis represent excellent potential target sites for chemotherapeutic intervention in tuberculosis. However, in mycobacteria there may be as many as three different pathways for the formation of trehalose. The well-established pathway involves forma- tion of trehalose 6-phosphate by transfer of glucose from UDP-glucose to glucose 6-phosphate by the trehalose phosphate synthase [7], and then removal of the phos- phate by a highly specific trehalose phosphate phospha- tase to give free trehalose (S. Klutts, I. Pastuszak, D. Carroll, Y. T. Pan & A. D. Elbein, unpublished results). Both of these enzymes have been purified and character- ized from M. smegmatis [7,8] and other mycobacteria. Two other pathways have been proposed on the basis of sequence homologies between established enzymes and the sequences obtained from the Mycobacterium tuberculosis genome [24]. One of these pathways involves the direct rearrangement of the a1,4-glucosidic linkage of maltose to the a,a1,1 linkage of trehalose, and this enzyme has been demonstrated in Pimelobacter species [25]. A third path- way involves the generation of trehalose from glycogen, and this pathway was reported in Arthrobacter species [26]. These alternative pathways have not been demon- strated in mycobacteria, but, if they do exist, they could represent different ways to make trehalose for different functions in the cell. 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They could be synthesized in the cytosol by glycosyltransferases which add glucosyl or