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 t
Trang 1Trehalose-based oligosaccharides isolated from the cytoplasm
Relation to trehalose-based oligosaccharides attached to lipid
Masaya Ohta1, Y T Pan2, Roger A Laine3and Alan D Elbein2
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;3Departments 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,1H-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 theDorLconfiguration
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 tetrasacchaoligosaccha-rides, 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)
Trang 2nonreducing disaccharide of two glucoses Thus, the sucrose
and the trehalose series may have analogous functions
E X P E R I M E N T A L P R O C E D U R E S
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 3MMpaper
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 ofM smegmatis and isolation
of oligosaccharides
M smegmatis was grown in trypticase soy broth for
24–48 h at 37C 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 smegmatiscells 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 (CO32–)
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 preparprepar-ative 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 3MMpaper (22 inches long) and chromatographed by descending chromatography in the following solvent sys-tems: butan-1-ol/pyridine/0.1M 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 40C 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)12,5-dihydroxybenzoic acid in 10% ethanol One microlitre of the matrix solution and 1 lL of 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 A 1.5 · 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.
Trang 3crystallization 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 80C for 2 h The
reaction mixture was evaporated to dryness under a stream
of N2.
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 50C for 1 min, increased to
170C at a rate of 20 per min, and then increased to
260C at 3 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) was
incu-bated with substrates in 0.1M sodium phosphate buffer,
pH 6.8; (b) 0.5 unit a-galactosidase (from Aspergillus niger)
was incubated with substrates in 50 mM sodium acetate
buffer, pH 4.0
All incubations were performed at 37C 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
R E S U L T S
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 TLCof 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.
Trang 4Each 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
3MM 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 the1H-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, and also by1H-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
1H-NMR analysis, presented in Fig 4, shows three ano-meric proton signals at 5.21 p.p.m (J12¼ 4.0), 5.21 p.p.m (J12¼ 4.0), and 5.41 p.p.m (J12¼ 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 and1H-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-HPLCseparataion 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
were run on this column and emerged in positions shown at the top of
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 Glucosea Ratio hexose/reducing sugar
a
Based on anthrone assay.
Trang 5three 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 theDorLform of the arabi-nose have not been determined No arabiarabi-nose-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 and1H-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 : 1.1H-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 (J12¼ 4.0), 5.20 p.p.m (J12¼ 4.0), and 4.41 p.p.m (J12¼ 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 1H-NMR spectrum showed four anomeric proton signals at 5.21 p.p.m (J12¼ 4.0), 5.19 p.p.m (J12¼ 4.0), 4.41 p.p.m (J12¼ 7.6) and 4.51 p.p.m (J12¼ 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 4.1H-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)
Arabinitol
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
Galactitol
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.dValues normalized to one residue of 2,3,4,6-tetra-O-methylgalactiol.
Trang 6Glcb1–6Glcb1–6 disaccharide linked to a trehalose core
[19] Therefore the most likely structure of this
tetrasaccha-ride is Glcb1–6Glcb1–6Glca1–1aGlc It was designated
CT4A as indicated in Table 4
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-tetramethylgalact2,3,4-trimethylgluc-itol 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-tri2,3,4,6-tetra-methylglucitol
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.
Trang 7Fraction 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-tetramethyl2,3,4-trimethyl-glucitol 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, and is referred to as CT4c (Table 4)
D I S C U S S I O N
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
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
Native
a-Galactosidase digestion
a-Glucosidase digestion Native
a-Galactosidase digestion Glucitol
Galactitol
a Values include the amount of the released glucose residue b Values indicate the amount of the released galactose residue.
Trang 8The 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 phosphos-phate
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 That is, one pathway could provide
trehalose for a structural role and another pathway could
produce it and its oligosaccharides to act as stabilizers
A C K N O W L E D G E M E N T S
This research was supported in part by NIH RO3 AI43292 to A.D.E.
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