Vol. 131, No. 1 Printed in U.S.A.
JOURNAL OF BACTERIOLOGY, JUlY 1977, P. 194-200 Copyright © 1977 American Society for Microbiology
Structural Analysis of the Surface Polysaccharide of Staphylococcus aureus M DENG-FONG LIAU'* AND JOHN H. HASH Department of Microbiology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232
Received for publication 31 March 1977
The chemical structure of the surface polysaccharide from Staphylococcus M was investigated by a combination of methanolytic, hydrolytic, and chromatographic techniques. The repeating unit that was most consistent with the data was a hexasaccharide composed of N-acetyl-D-aminogalacturonic acid, N-acetyl-D-fucosamine, and taurine in molar ratios of 4:2:1. A disaccharide was isolated and characterized, by combined gas-liquid chromatography-mass spectrometry, as N-acetyl-D-aminogalacturonyl- (1 3)-N-acetyl-D-fucosamine. Taurine is linked to a carboxyl group of N-acetyl-n-aminogalacturonic acid via an amide bond. aureus
-+
HCl], Ott Chemical Co.; N,O-bis(trimethylsilyl)trifluoroacetamide with 1% chlorotrimethylsilane, Pierce; dansyl chloride (DANS-Cl; 5-dimethylaminonaphthalene-sulfonyl chloride), Calbiochem. Salt-free SPA was prepared by passing an aqueous solution of the polysaccharide through a column (1 by 10 cm) of AG50W-X8 (200 to 400 mesh, hydrogen form). The effluent was lyophilized and then dried in vacuo over P2O, for 2 days. From 50 mg of the salt form, 45 mg of the acid form was obtained. Amino acid analyses. Amino acid analyses were performed for the quantitation of taurine, fucosamine, galactosamine, and aminogalacturonic acid, all of which were resolved by a standard two-column system. Analyses were performed on a Beckman 121C automatic amino acid analyzer equipped with an Infotronics CRS-12AB integrator (9). In this programmed system, the neutral and acidics column is injected 80 min after the start of the basic column. Quantitative determination of taurine. SPA (5.0 MATERIALS AND METHODS mg) was hydrolyzed in vacuo in 1 ml of 4 N HCl at Unless otherwise indicated, the materials and 110°C for 4 h in sealed tubes. The sample was cooled, methods for the isolation and identification of the evaporated to dryness, and dissolved in 500 ,ul of components of the surface polysaccharide antigen water. Portions were diluted to 2.0 ml with 0.2 N (SPA) from S. aureus M are the same as previously citrate buffer (pH 2.2) for amino acid analysis. The described (2). Other materials used in this study color factor for taurine was determined from chrowere obtained from the following sources: AG50W- matography of authentic taurine under identical X8 resins, Bio-Rad Laboratories; thin-layer chro- conditions. Determination of aminogalacturonic acid and fumatographic (TLC) plates (no. 6064 cellulose, no. 6060 silica gel with fluorescent indicator), Eastman cosamine. Three methods were investigated for estiKodak Co.; coiled glass columns (1/8 inch by 6 feet mating the amount of aminogalacturonic acid and [0.32 by 183 cm]) filled with 3% OV-1 on Gas-Chrom fucosamine. The first method used was the methanolysis proQ (100 to 200 mesh) and coiled stainless-steel columns (1/8 inch by 6 feet [0.32 by 183 cm]) filled with cedure of Sandford et al. (7). SPA (5.0 mg) was GP 2% SP-1200/1% H3PO4 on Chromsorb WAW, Ap- methanolyzed in 0.5 ml of 1 M HCl in anhydrous plied Science Laboratories; [14C]methanol (4 mCi/ [14C]methanol (final specific activity, 16 gCi/mmol) mmol), New England Nuclear Corp.; carbodiimide in sealed tubes at 80°C for 20 h. After evaporation to [1- ethyl - 3- (3-dimethylaminopropyl)carbodiimide dryness, the residue was N-acetylated with acetic anhydride. In some cases, the sample was de-esteri1 Present address: Institute of Cancer Research, College fied with 1 ml of 25 mM KOH under nitrogen for 4 h. of Physicians & Surgeons, Columbia University, New It was then neutralized with 50 ,l of 0.5 N HCl and evaporated to dryness. The sample was reconstiYork, NY 10032.
We have previously reported the isolation and identification of the constituent components of the surface polysaccharide of Staphylococcus aureus M (2). This polysaccharide is composed of D-aminogalacturonic acid, D-fucosamine, and taurine. It thus differs from the surface polysaccharides of the S. aureus Smith diffuse variant, which is composed of D-aminoglucuronic acid and L-alanine (1), and S. aureus mutant T, which is composed of D-aminomannuronic acid and D-fucosamine (11). This report is concerned with structural analysis of the strain M surface polysaccharide. (This work was presented in part at the 59th Annual Meeting of the Federation of American Societies for Experimental Biology, Atlantic City, N.J., 1975.)
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VOL. 131, 1977
S. AUREUS M POLYSACCHARIDE
195
tuted in 250 ,ul of water, and samples were chromat- cooled, and the bottom end was immersed in a bath ographed on Whatman no. 1 filter paper (2.5 by 45 of dry ice and acetone to condense all of the acetic cm) for 20 h in the solvent n-butanol-ethanol-water acid liberated by hydrolysis. The hydrolysates were (4:1:5; vol/vol/vol, organic phase). The dried chro- then diluted to 2.0 ml with water, and samples were matograms were scanned for radioactivity on a chromatographed directly. N-acetylglucosamine Packard radiochromatogram scanner, model 7201, was used as a control, and acetic acid served as a and the radioactivity was estimated by integrating standard. the area under each peak. Amino-terminal analysis. The DANS procedure In the second method, the methanolysis products (8) was used to investigate amino-terminal groups were analyzed in an amino acid analyzer. In this in SPA. SPA (1 mg) was dissolved in 0.3 ml of 0.1 M case, N-acetylation and de-esterification were omit- NaHCO3 and evaporated to dryness in vacuo to reted from the sample treatment. SPA (2.0 mg) was move traces of ammonia. The residue was dissolved methanolyzed in 0.5 ml of 1 M HCI in anhydrous in 0.3 ml of ammonia-free water, to which an equal methanol in sealed tubes for 100 h as described volume of DANS-Cl solution (2.5 mg/ml in 50% aceabove. The sample was then evaporated to dryness, tone) was added. After 2 h at 37°C the reaction and the residue was dissolved in 2 ml of 0.2 N citrate mixture was evaporated to dryness, and excess buffer (pH 2.2) for direct amino acid analysis. DANS-Cl was extracted with 0.4 ml of toluene. The The third method involved converting aminoga- residue was dried and hydrolyzed in 1 ml of 6 N HCI lacturonic acid to galactosamine by reducing the at 110HC for 16 h. After removal of the HCI in vacuo, carboxyl groups in the SPA by the carbodiimide the residue was dissolved in 50 ,ul of water for procedures of Taylor and Conrad (10). To ensure TLC. Samples were chromatographed on silica gel complete reduction, the procedure was repeated. Re- thin-layer plates (with fluorescent indicator). The duced SPA was hydrolyzed in 4 N HCI at 110°C for 4 thin-layer plates were developed in methyl acetateh in sealed tubes in vacuo. Chromatography was by propanol-concentrated ammonium hydroxide (9:7:4, both TLC and an amino acid analyzer. Authentic vol/vol/vol) for 3 h. The plates were examined under aminogalacturonic acid and fucosamine are not ultraviolet light. commercially available for use as standards in calStructure of the disaccharide, compound X. culating a color factor for the amino acid analyzer. Compound X was isolated from SPA as previously Small quantities of each amino sugar were isolated described (2). From 140 mg of SPA, 7 mg of comfrom SPA by TLC, and the concentration ofeach was pound X was obtained (5% yield). It was methanolestablished by the Rondle and Morgan (6) procedure yzed in 0.5 ml of 1 M HCI in [14C]methanol, and the for amino sugars, with authentic glucosamine and products were separated by paper chromatography galactosamine as standards. These values were then as described for SPA. For amino acid analysis, the used to establish color factors for these compounds methanolysate was further hydrolyzed in 0.5 ml of 1 on the amino acid analyzer. N HCI in vacuo at 1100C for 1 h. Quantitative determination of N-acetyl groups. For combined GLC-mass spectrometry (GLC-MS), Two methods were employed for the quantitative which was the same as previously described (2), determination ofthe acetyl content of SPA. The first compound X (5 mg) was N-acetylated with acetic procedure used methanolysis with methanol hydro- anhydride and reduced to the corresponding alcohol chloride followed by colorimetric determination of with either sodium borohydride or sodium borodeuthe methyl acetate that was formed with the hy- teride. The sample was then trimethylsilylated with droxyamic acid procedure (3). SPA (10.0 mg) was N,O-bis(trimethylsilyl)-trifluoroacetamide-1% chlomethanolyzed in 1 ml of 1 N HCI in methanol at rotrimethylsilane. GLC was on a 3% OV-1 coiled 110°C for 4 h in sealed tubes. Methyl acetate was column, isothermal at 2300C. Mass spectra were collected by repeated distillation (3), and the absorb- taken with a scan speed of 4 to 6 s (ine 20 to 1,000). ance of the ferric-hydroxamate chelate was mea- All data were acquired and reduced with a PDP-12 sured in a Gilford 240 spectrophotometer at 520 nm. computer. N-acetylglucosamine was used as a control and ethyl acetate served as a standard. RESULTS The second method used gas-liquid chromatograEstablishing the saccharide unit through phy (GLC) by the procedures of Ottenstein and Bartits taurine content. Inasmuch as taurine is the ley (4). A Varian Aerograph 1800 gas chromatograph was used as previously described (2), and the only component of the SPA that is quantitacolumn packing was GP 2% SP-1200/1% H3PO4 on tively liberated and not partially destroyed by 80- to 100-mesh Chromsorb WAW. The carrier gas acid hydrolysis, it offers the best way of estabwas helium; the temperature of the molecular sepa- lishing the basic repeating unit of the polysacrator was 125°C, and that of the detector was 200°C. charide. Multiple amino acid analyses of careFormic, acetic, propionic, and butyric acids were fully prepared, salt-free SPA gave reproducible used as reference standards. These four compounds values of 3.71 pmol of taurine per 5.0 mg (dry were well separated and emerged in the sequence: weight). Therefore, there is 1 pmol of taurine acids. Each and formic, acetic, propionic, butyric was quantitated by integrating the area under its per 1,348 jAg of SPA, which is equal to the molecular weight of the repeating unit of the peak. SPA (5.0 mg) was hydrolyzed in 0.5 ml of 1 N HCI polysaccharide. Rough calculations show that at 110°C for 1 h in a sealed tube. The tube was the repeating unit must be hexasaccharide.
196
LIAU AND HASH
The calculated molecular weight of a hexasaccharide containing three residues each of Nacetylaminogalacturonic acid and N-acetylfucosamine plus one residue of taurine is 1,319. The precision of the taurine determination is sufficient to eliminate pentasaccharide and heptasaccharide repeating units, which have calculated molecular weights of 1,132 and 1,536, respectively. Amino-terminal analysis. An end-group analysis was performed for free amino groups in SPA by the DANS procedure. By TLC, DANS-OH and DANS-taurine have similar Rf values: 0.68 and 0.70, respectively. DANS-OH has a blue fluorescence, and DANS-taurine has a yellowish-green fluorescence; thus, the two can be distinguished. Dansylated SPA showed only the blue fluorescent spot atRf 0.68. Therefore, there are no free amino groups in the SPA that belong to either taurine or the amino sugars. The amino group of taurine either is linked to a carboxyl group of the aminogalacturonic acid or it is blocked by an acyl group. Acetyl content of SPA. Preliminary experiments with GLC showed that the acyl groups of SPA were acetyl groups. The single peak obtained from acid hydrolysates of SPA coincided with the retention time of authentic acetic acid, and a mixture of the two gave a single peak on co-chromatography. With the colorimetric procedure, the recovery of acetic acid from the N-acetylglucosamine control was 90%. Analyses with the colorimetric procedure gave an acetyl content of 4.0 ,umol/mg of SPA (17.20%). Based on a repeating unit molecular weight of 1,319, there are 5.3 acetyl residues per hexasaccharide unit. If this value is corrected by the value for recovery of acetic acid from N-acetylglucosamine (90%), the calculated value of 5.9 residues per hexasaccharide unit (19.11%) is obtained. Thus, there is one acetyl group per monosaccharide unit, and this finding is consistent with the fact that no free amino groups were found by the DANS reaction. For GLC, SPA hydrolysates were serially diluted, and portions of each dilution were applied directly to the gas chromatograph. Acetic acid was the sole substance found. By this procedure, the recovery of acetic acid from the Nacetylglucosamine control was 95%, and the recovery of acetic acid from SPA hydrolysates was 4.23 /imol/mg (dry weight) of SPA (18.19%). Correcting this value for the 95% recovery of acetic acid from the N-acetylglucosamine control, a value of 4.45 pmol/mg (19.14%) was obtained. This value calculated to 6.0 acetyl residues per hexasaccharide unit. Thus, it
J. BACTERIOL.
can be concluded that each monosaccharide unit is N-acetylated. Estimation of aminogalacturonic acid and fucosamine. The methanolysis procedure of Sandford et al. (7) leads to a mixture of a and (8 isomers of the corresponding glycosides. Sugars with free carboxyl groups are converted to methyl esters. Paper chromatography of SPA, methanolyzed with 1 M HCl in [14C]methanol, revealed several radioactive peaks (Table 1). There were significant amounts of incomplete methanolysis products near the origin and atRf 0.2 (peaks 1 and 2). Two peaks (3 and 5) were assigned to N-acetylaminogalacturonic acid glycoside methyl ester since these same two peaks were observed when a sample of authentic N-acetylaminogalacturonic acid was metha-
nolyzed under identical conditions. Saponification with KOH converted both compounds to the potassium salts, which migrated together near the origin. Peak 4 was assigned to Nacetylfucosamine glycoside since the same peak was obtained when authentic N-acetylfucosamine was methanolyzed under identical conditions. This compound was not affected by saponification. Because of incomplete methanolysis, the exact percent composition of each amino sugar cannot be established from these data. Aminogalacturonic acid was released from SPA in approximately 50% yield as the glycoside methyl ester. However, the ratios of aminogalacturonic acid and fucosamine can be deduced. If it is recalled that the compounds in peaks 3 and 5 each contain 2 mol of [14C]methyl groups, the ratio of the counts per minute of aminogalacturonic acid to fucosamine is 1.9 [i.e., 1/2 (8,000 + 38,000)/12,000]. After de-esterification, this value falls to 1.6, but the incomplete products at the origin complicate the calculation because they may also contain ester groups that are removed by de-esterification. Attempts were then made to determine the products of methanolysis with an amino acid analyzer. There were two peaks on the basic column at 35 and 44 min, which corresponded to fucosamine glycoside and aminogalacturonic TABLE 1. Methanolysis products of strain M SPA a Peak 1 2 3 4 5
Rf
cpm before de-
cpm after de-
0-0.1 0.2 0.52 0.59 0.66
esterification 15,000 23,000 8,000 12,000 38,000
22,000
esterification 34,000 12,000
a Peaks 1 and 2 are products of incomplete methanolysis. Peaks 3 and 5 are assigned to the methyl esters of Nacetylaminogalacturonic acid glycoside. Peak 4 is assigned to N-acetylfucosamine glycoside.
VOL. 131, 1977
acid glycoside methyl ester, respectively. On the neutral and acidics column, fucosamine glycoside emerged at 496 min, and the aminogalacturonic glycoside methyl ester emerged in two separate peaks at 606 and 629 min. There was no peak corresponding to aminogalacturonic acid (300 min). The methanolysate was hydrolyzed further with 1 N HCl at 1100C for 1 h in vacuo and rechromatographed. The peak at 44 min on the basic column and the two peaks on the neutral and acidics column at 606 and 629 min were absent; a peak at 300 min, corresponding to aminogalacturonic acid, was present. The quantities of aminogalacturonic acid glycoside methyl ester and fucosamine glycoside were calculated from the two peaks on the basic column. On methanolysis for 100 h, the molar ratio of aminogalacturonic glycoside methyl ester to fucosamine glycoside was 1.74:1. This ratio decreased to 1.34:1 when the methanolysis time was increased to 150 h. The acid hydrolysate of reduced SPA showed five compounds on TLC that corresponded to aminogalacturonic acid, compound X, taurine, galactosamine, and fucosamine. On the neutral and acidics column of the amino acid analyzer, taurine (141 min), aminogalacturonic acid (300 min), fucosamine (496 min), and compound X (500 min) were observed, as reported previously (2). A peak at 420 min, corresponding to authentic galactosamine, was also observed. Recoveries from the reduced SPA are shown in Table 2. No other peaks were observed, and the release of aminogalacturonic acid, fucosamine, galactosamine, and taurine is essentially quantitative. The losses incurred are those due to acid destruction of the liberated components. Under these conditions of hydrolysis, approximately 40% of the authentic aminogalacturonic acid was destroyed. The results indicate that reduced SPA consists of three galactosamine, two fucosamine, and one aminogalacturonic acid per taurine residue. From these results and the prior demonstration that the amino group of taurine is not free, it can be deduced that taurine is covalently attached to the carboxyl group of an N-acetylaminogalacturonic acid residue via an amide linkage. All of the results are consistent with the hypothesis that the molar ratios of N-acetylaminogalacturonic acid andN-acetylfucosamine in the native polymer are 2:1. Structure of disaccharide X. Milligram amounts of compound X were obtained from SPA as previously described (2). This compound was very resistant to acid hydrolysis; therefore, its composition, linkage, and sequence were determined by methanolysis fol-
S. AUREUS M POLYSACCHARIDE
197
TABLE 2. Amino acid analysis of reduced SPA a Compound
Retention time
(min) 141
nmol
Ratio
Nearest
integer
230 1.00 1 Taurine ....... Aminogalactu1 0.67 155 ronic acid .... 300 3 620 2.69 420 Galactosamine 2 400 1.74 Fucosamine ... 496 30 CompoundX .. 503 a Reduced SPA (325 gg) was hydrolyzed in 1 ml of 4 N HCl at 1100C for 4 h in sealed, evacuated tubes. Compound X was calculated with the fucosamine color factor.
lowed by amino acid analysis as well as by combined GLC-MS. Methanolysis ofcompound X with 1 M HCl in [14C]methanol followed by N-acetylation and paper chromatography showed the same peaks found in SPA (Table 1, peaks 3, 4, and 5) that corresponded to N-acetylfucosamine glycoside and N-acetylaminogalacturonic acid glycoside methyl ester. They were present in equimolar amounts. Acid hydrolysis of another methanolysate followed by TLC and amino acid analysis showed two compounds corresponding to fucosamine and aminogalacturonic acid, again in equimolar amounts. Combined GLC-MS proved that compound X was a disaccharide composed of aminogalacturonic acid and fucosamine. The mass spectrum of the derivative reduced with sodium borodeuteride and its fragmentation pattern are shown in Fig. 1. As observed previously with trimethylsilyl (TMS) derivatives of SPA components (2), the molecular ion was not present; rather, the largest ion observed was the M+-15 ion at 842 mass units. The molecular weight is thus 857 mass units for this derivative, which is in agreement with the calculated value for the monodeutero derivative. When the mass spectrometry was repeated with the derivative that had been reduced with sodium borohydride, an M+-15 ion of 841 mass units was obtained. The m/e of 434 (Fig. 1) corresponds to N-acetylaminogalacturonyl-(TMS)3+, whereas the mie of 408 corresponds to N-acetylfucosaminyl (TMS)3+-15. When the derivative was reduced with borohydride, the mie of 434 for N-acetylaminogalacturonyl-(TMS)3+ was unchanged, whereas the m/e for N-acetylfucosaminyl(TMS)3+-15 was shifted to 407. Therefore, the reducing end of the molecule is fucosamine. Peaks below m/e 450 result mainly from independent fragmentation of the amino sugar and amino sugar alditol units and provide information on how the monosaccharide units in the
198
LIAU AND HASH
J. BACTERIOL.
H OH
N-ACETYLATION REDUCTION WITH NaBD4 TRIMETHYLSILYLATION
434
COOTMS TMS
\
408
:104
D CHOTMS . .---H CNHCOCH3
175
-----------:219
H COTMS
H3CCONH
MW 857
NAc-FUCOSAMINITOL-(TMS)3
NAc-AGaI A-(TMS)3
100I
117
TMSO:H CH3
7r
FI5 Sv 434
U,
z
z w J
501-
344
4 -j LA /86
147 117
.n hI e
100
665
607
740
842
204
200
573
Ci
I 300
400
500 m/e
600
700
T0 800
--
900
FIG. 1. Mass spectra and fragmentation pattern of the reduced and trimethylsilylated compound X, aminogalacturonyl-(l 3)-fucosamine. -+
original disaccharide are linked (5). It was ob- Fig. 1, an ion of m/e 277 was not observed, but served that ions of m/e 104 and 175 (Fig. 1) were an ion of mle 219 was present. The structure of replaced with ions of m/e 103 and 174 when the the disaccharide is therefore aminogalactudisaccharide was reduced with NaBH4. The ronyl-(l 3)-fucosamine. ions of m/e 117 and 219 were unchanged. This fragmentation pattern is consistent with a 1 DISCUSSION 3 linkage. The only other alternative, a 1 4 linkage, should give ions of m/e 276 (reduced The results obtained from methanolytic, hywith NaBH4) or 277 (reduced with NaBD4), and drolytic, and combined GLC-MS studies permit the ion of m/e 219 should be absent. As shown in the conclusion that the repeating unit of strain --
--
VOL. 131, 1977
S. AUREUS M POLYSACCHARIDE
M capsular polysaccharide is a hexasaccharide composed of four residues ofN-acetyl-D-aminogalacturonic acid, two residues of N-acetyl-Dfucosamine, and one residue of taurine. The calculated molecular weight of this repeating unit is 1,349, in perfect agreement with the value found from the taurine content of the polysaccharide. Combined GLC-MS permitted the structure of compound X to be assigned unambiguously as N-acetyl-D-aminogalacturonyl-(1 -* 3)-Nacetyl-D-fucosamine. The assignment of the configuration of the linkage (a or 13) will have to await the isolation of larger quantities of this disaccharide. The stability of this compound to acid hydrolysis is due to the presence of two
199
amino groups immediately adjacent to the glycosidic bond of the disaccharide. As the acetyl groups are removed, the amino groups become protonated and prevent the approach of hydronium ions to the glycosidic bond. The same situation was found by Wu and Park (11) for the disaccharide aminomannuronyl-(1-b 3)-fucosamine. The reduced strain M polysaccharide proved to be easier to hydrolyze with aqueous acid, which permitted the molar ratios of the components to be established (Table 2). Data in Fig. 2 summarize the results obtained to date and pernit the hexasaccharide unit to be postulated as shown. At this time none of the configurational assignments are known. Because of the relatively easier release of aminogalactu-
N
80 C, 100 h
NaBH4
RATIO
RATIO
COOCH3 0
,C20H
OOCH3 NH2 Aminogolocturonic
Acid
Methyl Ester glycoside CH3
2
3 NH2
Galactosomine
COOH
0
OCH3 NH2
NH2 Fucosomine Glycoside
Aminogalacturonic Acid CH3
2 NH2
Fucosamine H2NCH2CH2 SO3M Taurine
I
FIG. 2. Proposed hexasaccharide repeating unit and degradation patterns from strain M polysaccharide. Compound X is shown in dotted brackets; the 1 -- 3 linkage was established by mass spectrometry. The positions of other linkages are not known, but it is speculated that they are 1 -I 4. The particular N-
acetylaminogalacturonic acid to which taurine is attached is not known.
200 LIAU AND HASH ronic acid by acid hydrolysis, it is speculated that the remaining glycosidic bonds in the polysaccharide are 1 -- 4. Taurine is an unusual component of this polysaccharide. It did not form a DANS derivative with DANS-Ci, and the position of this component was deduced from the fact that one of every four aminogalacturonic acid residues in the polysaccharide could not be esterified and reduced to galactosamine. On the average there is one taurine residue per hexasaccharide unit linked to a carboxyl group of an aminogalacturonic acid residue. The methanolysis procedure of Sandford et al. (7) was used to help elucidate the repeating unit of strain M polysaccharide. In contrast, however, to the results obtained with the yeast Y-6272 polysaccharide (8), where completemethanolysis occurred, the polysaccharide from strain M was more resistant to this procedure. Intermediate products were present after 100 h. Molar ratios of the constitutent amino sugars were based on the released components, but the total composition could not be established with this procedure. ACKNOWLEDGMENTS This investigation was supported by Public Health Service grant AI-06712 from the National Institute of Allergy and Infectious Diseases. We thank J. T. Watson of the Department of Pharmacology for mass spectrometric analyses and Lilah Clack for performing amino acid analyses. LITERATURE CITED 1. Hanessian, S., and T. H. Haskell. 1964. Structural studies on staphylococcal polysaccharide antigen. J.
J. BACTERIOL. Biol. Chem. 239:2758-2764. 2. Liau, D. F., M. A. Melly, and J. H. Hash. 1974. Surface polysaccharide from Staphylococcus aureus M that contains taurine, D-aminogalacturonic acid, and n. fucosamine. J. Bacteriol. 119:913-922. 3. Ludowieg, L., and A. Dorfman. 1950. A micromethod for the colorimetric determination of N-acetyl groups in acid mucopolysaccharides. Biochim. Biophys. Acta 38:212-218. 4. Ottenstein, D. M., and D. A. Bartley. 1971. Improved gas chromatography separation of free acids C2-C, in dilute solution. Anal. Chem. 43:952-955. 5. Radford, T., and D. C. DeJongh. 1972. Carbohydrates, p. 313-350. In G. R. Waller (ed.), Biochemical application of mass spectrometry. John Wiley & Sons, Inc., New York. 6. Rondle, C. J. M., and W. T. J. Morgan. 1955. Determination of glucosamine and galactosamine. Biochem. J. 61:586-589. 7. Sandford, P. A., P. R. Watson, and A. R. Jeanes. 1973. An extracellular microbial polysaccharide composed of 2-acetamido-2-deoxy-D-glucose and 2-acetamido-2deoxy-D-glucuronic acid: radiochemical and gas chromatographic analysis of the products of methanolysis. Carbohydr. Res. 29:153-164. 8. Seiler, N. 1970. Use of the dansyl reaction in biochemical analysis, p. 259-337. In D. Glick (ed.), Methods of biochemical analysis, vol. 18. Wiley-Interscience Publishers, New York. 9. Shih, J. W. K., and J. H. Hash. 1971. The N,O-diacetylmuramidase of Chalaropsis species. III. Amino acid composition and partial structural formula. J. Biol. Chem. 246:994-1006. 10. Taylor, R. L., and H. E. Conrad. 1972. Stoichiometric depolymerization of polyuronides and glycosaminoglycuronans to monosaccharides following reduction of their carbodiimide-activated carboxyl groups. Biochemistry 11:1383-1388. 11. Wu, T. C. M., and J. T. Park. 1971. Chemical characterization of a new surface antigenic polysaccharide from a mutant ofStaphylococcus aureus. J. Bacteriol.
108:874-884.
JOURNAL OF BACTERIOLOGY, JUlY 1977, P. 194-200 Copyright © 1977 American Society for Microbiology
Structural Analysis of the Surface Polysaccharide of Staphylococcus aureus M DENG-FONG LIAU'* AND JOHN H. HASH Department of Microbiology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232
Received for publication 31 March 1977
The chemical structure of the surface polysaccharide from Staphylococcus M was investigated by a combination of methanolytic, hydrolytic, and chromatographic techniques. The repeating unit that was most consistent with the data was a hexasaccharide composed of N-acetyl-D-aminogalacturonic acid, N-acetyl-D-fucosamine, and taurine in molar ratios of 4:2:1. A disaccharide was isolated and characterized, by combined gas-liquid chromatography-mass spectrometry, as N-acetyl-D-aminogalacturonyl- (1 3)-N-acetyl-D-fucosamine. Taurine is linked to a carboxyl group of N-acetyl-n-aminogalacturonic acid via an amide bond. aureus
-+
HCl], Ott Chemical Co.; N,O-bis(trimethylsilyl)trifluoroacetamide with 1% chlorotrimethylsilane, Pierce; dansyl chloride (DANS-Cl; 5-dimethylaminonaphthalene-sulfonyl chloride), Calbiochem. Salt-free SPA was prepared by passing an aqueous solution of the polysaccharide through a column (1 by 10 cm) of AG50W-X8 (200 to 400 mesh, hydrogen form). The effluent was lyophilized and then dried in vacuo over P2O, for 2 days. From 50 mg of the salt form, 45 mg of the acid form was obtained. Amino acid analyses. Amino acid analyses were performed for the quantitation of taurine, fucosamine, galactosamine, and aminogalacturonic acid, all of which were resolved by a standard two-column system. Analyses were performed on a Beckman 121C automatic amino acid analyzer equipped with an Infotronics CRS-12AB integrator (9). In this programmed system, the neutral and acidics column is injected 80 min after the start of the basic column. Quantitative determination of taurine. SPA (5.0 MATERIALS AND METHODS mg) was hydrolyzed in vacuo in 1 ml of 4 N HCl at Unless otherwise indicated, the materials and 110°C for 4 h in sealed tubes. The sample was cooled, methods for the isolation and identification of the evaporated to dryness, and dissolved in 500 ,ul of components of the surface polysaccharide antigen water. Portions were diluted to 2.0 ml with 0.2 N (SPA) from S. aureus M are the same as previously citrate buffer (pH 2.2) for amino acid analysis. The described (2). Other materials used in this study color factor for taurine was determined from chrowere obtained from the following sources: AG50W- matography of authentic taurine under identical X8 resins, Bio-Rad Laboratories; thin-layer chro- conditions. Determination of aminogalacturonic acid and fumatographic (TLC) plates (no. 6064 cellulose, no. 6060 silica gel with fluorescent indicator), Eastman cosamine. Three methods were investigated for estiKodak Co.; coiled glass columns (1/8 inch by 6 feet mating the amount of aminogalacturonic acid and [0.32 by 183 cm]) filled with 3% OV-1 on Gas-Chrom fucosamine. The first method used was the methanolysis proQ (100 to 200 mesh) and coiled stainless-steel columns (1/8 inch by 6 feet [0.32 by 183 cm]) filled with cedure of Sandford et al. (7). SPA (5.0 mg) was GP 2% SP-1200/1% H3PO4 on Chromsorb WAW, Ap- methanolyzed in 0.5 ml of 1 M HCl in anhydrous plied Science Laboratories; [14C]methanol (4 mCi/ [14C]methanol (final specific activity, 16 gCi/mmol) mmol), New England Nuclear Corp.; carbodiimide in sealed tubes at 80°C for 20 h. After evaporation to [1- ethyl - 3- (3-dimethylaminopropyl)carbodiimide dryness, the residue was N-acetylated with acetic anhydride. In some cases, the sample was de-esteri1 Present address: Institute of Cancer Research, College fied with 1 ml of 25 mM KOH under nitrogen for 4 h. of Physicians & Surgeons, Columbia University, New It was then neutralized with 50 ,l of 0.5 N HCl and evaporated to dryness. The sample was reconstiYork, NY 10032.
We have previously reported the isolation and identification of the constituent components of the surface polysaccharide of Staphylococcus aureus M (2). This polysaccharide is composed of D-aminogalacturonic acid, D-fucosamine, and taurine. It thus differs from the surface polysaccharides of the S. aureus Smith diffuse variant, which is composed of D-aminoglucuronic acid and L-alanine (1), and S. aureus mutant T, which is composed of D-aminomannuronic acid and D-fucosamine (11). This report is concerned with structural analysis of the strain M surface polysaccharide. (This work was presented in part at the 59th Annual Meeting of the Federation of American Societies for Experimental Biology, Atlantic City, N.J., 1975.)
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VOL. 131, 1977
S. AUREUS M POLYSACCHARIDE
195
tuted in 250 ,ul of water, and samples were chromat- cooled, and the bottom end was immersed in a bath ographed on Whatman no. 1 filter paper (2.5 by 45 of dry ice and acetone to condense all of the acetic cm) for 20 h in the solvent n-butanol-ethanol-water acid liberated by hydrolysis. The hydrolysates were (4:1:5; vol/vol/vol, organic phase). The dried chro- then diluted to 2.0 ml with water, and samples were matograms were scanned for radioactivity on a chromatographed directly. N-acetylglucosamine Packard radiochromatogram scanner, model 7201, was used as a control, and acetic acid served as a and the radioactivity was estimated by integrating standard. the area under each peak. Amino-terminal analysis. The DANS procedure In the second method, the methanolysis products (8) was used to investigate amino-terminal groups were analyzed in an amino acid analyzer. In this in SPA. SPA (1 mg) was dissolved in 0.3 ml of 0.1 M case, N-acetylation and de-esterification were omit- NaHCO3 and evaporated to dryness in vacuo to reted from the sample treatment. SPA (2.0 mg) was move traces of ammonia. The residue was dissolved methanolyzed in 0.5 ml of 1 M HCI in anhydrous in 0.3 ml of ammonia-free water, to which an equal methanol in sealed tubes for 100 h as described volume of DANS-Cl solution (2.5 mg/ml in 50% aceabove. The sample was then evaporated to dryness, tone) was added. After 2 h at 37°C the reaction and the residue was dissolved in 2 ml of 0.2 N citrate mixture was evaporated to dryness, and excess buffer (pH 2.2) for direct amino acid analysis. DANS-Cl was extracted with 0.4 ml of toluene. The The third method involved converting aminoga- residue was dried and hydrolyzed in 1 ml of 6 N HCI lacturonic acid to galactosamine by reducing the at 110HC for 16 h. After removal of the HCI in vacuo, carboxyl groups in the SPA by the carbodiimide the residue was dissolved in 50 ,ul of water for procedures of Taylor and Conrad (10). To ensure TLC. Samples were chromatographed on silica gel complete reduction, the procedure was repeated. Re- thin-layer plates (with fluorescent indicator). The duced SPA was hydrolyzed in 4 N HCI at 110°C for 4 thin-layer plates were developed in methyl acetateh in sealed tubes in vacuo. Chromatography was by propanol-concentrated ammonium hydroxide (9:7:4, both TLC and an amino acid analyzer. Authentic vol/vol/vol) for 3 h. The plates were examined under aminogalacturonic acid and fucosamine are not ultraviolet light. commercially available for use as standards in calStructure of the disaccharide, compound X. culating a color factor for the amino acid analyzer. Compound X was isolated from SPA as previously Small quantities of each amino sugar were isolated described (2). From 140 mg of SPA, 7 mg of comfrom SPA by TLC, and the concentration ofeach was pound X was obtained (5% yield). It was methanolestablished by the Rondle and Morgan (6) procedure yzed in 0.5 ml of 1 M HCI in [14C]methanol, and the for amino sugars, with authentic glucosamine and products were separated by paper chromatography galactosamine as standards. These values were then as described for SPA. For amino acid analysis, the used to establish color factors for these compounds methanolysate was further hydrolyzed in 0.5 ml of 1 on the amino acid analyzer. N HCI in vacuo at 1100C for 1 h. Quantitative determination of N-acetyl groups. For combined GLC-mass spectrometry (GLC-MS), Two methods were employed for the quantitative which was the same as previously described (2), determination ofthe acetyl content of SPA. The first compound X (5 mg) was N-acetylated with acetic procedure used methanolysis with methanol hydro- anhydride and reduced to the corresponding alcohol chloride followed by colorimetric determination of with either sodium borohydride or sodium borodeuthe methyl acetate that was formed with the hy- teride. The sample was then trimethylsilylated with droxyamic acid procedure (3). SPA (10.0 mg) was N,O-bis(trimethylsilyl)-trifluoroacetamide-1% chlomethanolyzed in 1 ml of 1 N HCI in methanol at rotrimethylsilane. GLC was on a 3% OV-1 coiled 110°C for 4 h in sealed tubes. Methyl acetate was column, isothermal at 2300C. Mass spectra were collected by repeated distillation (3), and the absorb- taken with a scan speed of 4 to 6 s (ine 20 to 1,000). ance of the ferric-hydroxamate chelate was mea- All data were acquired and reduced with a PDP-12 sured in a Gilford 240 spectrophotometer at 520 nm. computer. N-acetylglucosamine was used as a control and ethyl acetate served as a standard. RESULTS The second method used gas-liquid chromatograEstablishing the saccharide unit through phy (GLC) by the procedures of Ottenstein and Bartits taurine content. Inasmuch as taurine is the ley (4). A Varian Aerograph 1800 gas chromatograph was used as previously described (2), and the only component of the SPA that is quantitacolumn packing was GP 2% SP-1200/1% H3PO4 on tively liberated and not partially destroyed by 80- to 100-mesh Chromsorb WAW. The carrier gas acid hydrolysis, it offers the best way of estabwas helium; the temperature of the molecular sepa- lishing the basic repeating unit of the polysacrator was 125°C, and that of the detector was 200°C. charide. Multiple amino acid analyses of careFormic, acetic, propionic, and butyric acids were fully prepared, salt-free SPA gave reproducible used as reference standards. These four compounds values of 3.71 pmol of taurine per 5.0 mg (dry were well separated and emerged in the sequence: weight). Therefore, there is 1 pmol of taurine acids. Each and formic, acetic, propionic, butyric was quantitated by integrating the area under its per 1,348 jAg of SPA, which is equal to the molecular weight of the repeating unit of the peak. SPA (5.0 mg) was hydrolyzed in 0.5 ml of 1 N HCI polysaccharide. Rough calculations show that at 110°C for 1 h in a sealed tube. The tube was the repeating unit must be hexasaccharide.
196
LIAU AND HASH
The calculated molecular weight of a hexasaccharide containing three residues each of Nacetylaminogalacturonic acid and N-acetylfucosamine plus one residue of taurine is 1,319. The precision of the taurine determination is sufficient to eliminate pentasaccharide and heptasaccharide repeating units, which have calculated molecular weights of 1,132 and 1,536, respectively. Amino-terminal analysis. An end-group analysis was performed for free amino groups in SPA by the DANS procedure. By TLC, DANS-OH and DANS-taurine have similar Rf values: 0.68 and 0.70, respectively. DANS-OH has a blue fluorescence, and DANS-taurine has a yellowish-green fluorescence; thus, the two can be distinguished. Dansylated SPA showed only the blue fluorescent spot atRf 0.68. Therefore, there are no free amino groups in the SPA that belong to either taurine or the amino sugars. The amino group of taurine either is linked to a carboxyl group of the aminogalacturonic acid or it is blocked by an acyl group. Acetyl content of SPA. Preliminary experiments with GLC showed that the acyl groups of SPA were acetyl groups. The single peak obtained from acid hydrolysates of SPA coincided with the retention time of authentic acetic acid, and a mixture of the two gave a single peak on co-chromatography. With the colorimetric procedure, the recovery of acetic acid from the N-acetylglucosamine control was 90%. Analyses with the colorimetric procedure gave an acetyl content of 4.0 ,umol/mg of SPA (17.20%). Based on a repeating unit molecular weight of 1,319, there are 5.3 acetyl residues per hexasaccharide unit. If this value is corrected by the value for recovery of acetic acid from N-acetylglucosamine (90%), the calculated value of 5.9 residues per hexasaccharide unit (19.11%) is obtained. Thus, there is one acetyl group per monosaccharide unit, and this finding is consistent with the fact that no free amino groups were found by the DANS reaction. For GLC, SPA hydrolysates were serially diluted, and portions of each dilution were applied directly to the gas chromatograph. Acetic acid was the sole substance found. By this procedure, the recovery of acetic acid from the Nacetylglucosamine control was 95%, and the recovery of acetic acid from SPA hydrolysates was 4.23 /imol/mg (dry weight) of SPA (18.19%). Correcting this value for the 95% recovery of acetic acid from the N-acetylglucosamine control, a value of 4.45 pmol/mg (19.14%) was obtained. This value calculated to 6.0 acetyl residues per hexasaccharide unit. Thus, it
J. BACTERIOL.
can be concluded that each monosaccharide unit is N-acetylated. Estimation of aminogalacturonic acid and fucosamine. The methanolysis procedure of Sandford et al. (7) leads to a mixture of a and (8 isomers of the corresponding glycosides. Sugars with free carboxyl groups are converted to methyl esters. Paper chromatography of SPA, methanolyzed with 1 M HCl in [14C]methanol, revealed several radioactive peaks (Table 1). There were significant amounts of incomplete methanolysis products near the origin and atRf 0.2 (peaks 1 and 2). Two peaks (3 and 5) were assigned to N-acetylaminogalacturonic acid glycoside methyl ester since these same two peaks were observed when a sample of authentic N-acetylaminogalacturonic acid was metha-
nolyzed under identical conditions. Saponification with KOH converted both compounds to the potassium salts, which migrated together near the origin. Peak 4 was assigned to Nacetylfucosamine glycoside since the same peak was obtained when authentic N-acetylfucosamine was methanolyzed under identical conditions. This compound was not affected by saponification. Because of incomplete methanolysis, the exact percent composition of each amino sugar cannot be established from these data. Aminogalacturonic acid was released from SPA in approximately 50% yield as the glycoside methyl ester. However, the ratios of aminogalacturonic acid and fucosamine can be deduced. If it is recalled that the compounds in peaks 3 and 5 each contain 2 mol of [14C]methyl groups, the ratio of the counts per minute of aminogalacturonic acid to fucosamine is 1.9 [i.e., 1/2 (8,000 + 38,000)/12,000]. After de-esterification, this value falls to 1.6, but the incomplete products at the origin complicate the calculation because they may also contain ester groups that are removed by de-esterification. Attempts were then made to determine the products of methanolysis with an amino acid analyzer. There were two peaks on the basic column at 35 and 44 min, which corresponded to fucosamine glycoside and aminogalacturonic TABLE 1. Methanolysis products of strain M SPA a Peak 1 2 3 4 5
Rf
cpm before de-
cpm after de-
0-0.1 0.2 0.52 0.59 0.66
esterification 15,000 23,000 8,000 12,000 38,000
22,000
esterification 34,000 12,000
a Peaks 1 and 2 are products of incomplete methanolysis. Peaks 3 and 5 are assigned to the methyl esters of Nacetylaminogalacturonic acid glycoside. Peak 4 is assigned to N-acetylfucosamine glycoside.
VOL. 131, 1977
acid glycoside methyl ester, respectively. On the neutral and acidics column, fucosamine glycoside emerged at 496 min, and the aminogalacturonic glycoside methyl ester emerged in two separate peaks at 606 and 629 min. There was no peak corresponding to aminogalacturonic acid (300 min). The methanolysate was hydrolyzed further with 1 N HCl at 1100C for 1 h in vacuo and rechromatographed. The peak at 44 min on the basic column and the two peaks on the neutral and acidics column at 606 and 629 min were absent; a peak at 300 min, corresponding to aminogalacturonic acid, was present. The quantities of aminogalacturonic acid glycoside methyl ester and fucosamine glycoside were calculated from the two peaks on the basic column. On methanolysis for 100 h, the molar ratio of aminogalacturonic glycoside methyl ester to fucosamine glycoside was 1.74:1. This ratio decreased to 1.34:1 when the methanolysis time was increased to 150 h. The acid hydrolysate of reduced SPA showed five compounds on TLC that corresponded to aminogalacturonic acid, compound X, taurine, galactosamine, and fucosamine. On the neutral and acidics column of the amino acid analyzer, taurine (141 min), aminogalacturonic acid (300 min), fucosamine (496 min), and compound X (500 min) were observed, as reported previously (2). A peak at 420 min, corresponding to authentic galactosamine, was also observed. Recoveries from the reduced SPA are shown in Table 2. No other peaks were observed, and the release of aminogalacturonic acid, fucosamine, galactosamine, and taurine is essentially quantitative. The losses incurred are those due to acid destruction of the liberated components. Under these conditions of hydrolysis, approximately 40% of the authentic aminogalacturonic acid was destroyed. The results indicate that reduced SPA consists of three galactosamine, two fucosamine, and one aminogalacturonic acid per taurine residue. From these results and the prior demonstration that the amino group of taurine is not free, it can be deduced that taurine is covalently attached to the carboxyl group of an N-acetylaminogalacturonic acid residue via an amide linkage. All of the results are consistent with the hypothesis that the molar ratios of N-acetylaminogalacturonic acid andN-acetylfucosamine in the native polymer are 2:1. Structure of disaccharide X. Milligram amounts of compound X were obtained from SPA as previously described (2). This compound was very resistant to acid hydrolysis; therefore, its composition, linkage, and sequence were determined by methanolysis fol-
S. AUREUS M POLYSACCHARIDE
197
TABLE 2. Amino acid analysis of reduced SPA a Compound
Retention time
(min) 141
nmol
Ratio
Nearest
integer
230 1.00 1 Taurine ....... Aminogalactu1 0.67 155 ronic acid .... 300 3 620 2.69 420 Galactosamine 2 400 1.74 Fucosamine ... 496 30 CompoundX .. 503 a Reduced SPA (325 gg) was hydrolyzed in 1 ml of 4 N HCl at 1100C for 4 h in sealed, evacuated tubes. Compound X was calculated with the fucosamine color factor.
lowed by amino acid analysis as well as by combined GLC-MS. Methanolysis ofcompound X with 1 M HCl in [14C]methanol followed by N-acetylation and paper chromatography showed the same peaks found in SPA (Table 1, peaks 3, 4, and 5) that corresponded to N-acetylfucosamine glycoside and N-acetylaminogalacturonic acid glycoside methyl ester. They were present in equimolar amounts. Acid hydrolysis of another methanolysate followed by TLC and amino acid analysis showed two compounds corresponding to fucosamine and aminogalacturonic acid, again in equimolar amounts. Combined GLC-MS proved that compound X was a disaccharide composed of aminogalacturonic acid and fucosamine. The mass spectrum of the derivative reduced with sodium borodeuteride and its fragmentation pattern are shown in Fig. 1. As observed previously with trimethylsilyl (TMS) derivatives of SPA components (2), the molecular ion was not present; rather, the largest ion observed was the M+-15 ion at 842 mass units. The molecular weight is thus 857 mass units for this derivative, which is in agreement with the calculated value for the monodeutero derivative. When the mass spectrometry was repeated with the derivative that had been reduced with sodium borohydride, an M+-15 ion of 841 mass units was obtained. The m/e of 434 (Fig. 1) corresponds to N-acetylaminogalacturonyl-(TMS)3+, whereas the mie of 408 corresponds to N-acetylfucosaminyl (TMS)3+-15. When the derivative was reduced with borohydride, the mie of 434 for N-acetylaminogalacturonyl-(TMS)3+ was unchanged, whereas the m/e for N-acetylfucosaminyl(TMS)3+-15 was shifted to 407. Therefore, the reducing end of the molecule is fucosamine. Peaks below m/e 450 result mainly from independent fragmentation of the amino sugar and amino sugar alditol units and provide information on how the monosaccharide units in the
198
LIAU AND HASH
J. BACTERIOL.
H OH
N-ACETYLATION REDUCTION WITH NaBD4 TRIMETHYLSILYLATION
434
COOTMS TMS
\
408
:104
D CHOTMS . .---H CNHCOCH3
175
-----------:219
H COTMS
H3CCONH
MW 857
NAc-FUCOSAMINITOL-(TMS)3
NAc-AGaI A-(TMS)3
100I
117
TMSO:H CH3
7r
FI5 Sv 434
U,
z
z w J
501-
344
4 -j LA /86
147 117
.n hI e
100
665
607
740
842
204
200
573
Ci
I 300
400
500 m/e
600
700
T0 800
--
900
FIG. 1. Mass spectra and fragmentation pattern of the reduced and trimethylsilylated compound X, aminogalacturonyl-(l 3)-fucosamine. -+
original disaccharide are linked (5). It was ob- Fig. 1, an ion of m/e 277 was not observed, but served that ions of m/e 104 and 175 (Fig. 1) were an ion of mle 219 was present. The structure of replaced with ions of m/e 103 and 174 when the the disaccharide is therefore aminogalactudisaccharide was reduced with NaBH4. The ronyl-(l 3)-fucosamine. ions of m/e 117 and 219 were unchanged. This fragmentation pattern is consistent with a 1 DISCUSSION 3 linkage. The only other alternative, a 1 4 linkage, should give ions of m/e 276 (reduced The results obtained from methanolytic, hywith NaBH4) or 277 (reduced with NaBD4), and drolytic, and combined GLC-MS studies permit the ion of m/e 219 should be absent. As shown in the conclusion that the repeating unit of strain --
--
VOL. 131, 1977
S. AUREUS M POLYSACCHARIDE
M capsular polysaccharide is a hexasaccharide composed of four residues ofN-acetyl-D-aminogalacturonic acid, two residues of N-acetyl-Dfucosamine, and one residue of taurine. The calculated molecular weight of this repeating unit is 1,349, in perfect agreement with the value found from the taurine content of the polysaccharide. Combined GLC-MS permitted the structure of compound X to be assigned unambiguously as N-acetyl-D-aminogalacturonyl-(1 -* 3)-Nacetyl-D-fucosamine. The assignment of the configuration of the linkage (a or 13) will have to await the isolation of larger quantities of this disaccharide. The stability of this compound to acid hydrolysis is due to the presence of two
199
amino groups immediately adjacent to the glycosidic bond of the disaccharide. As the acetyl groups are removed, the amino groups become protonated and prevent the approach of hydronium ions to the glycosidic bond. The same situation was found by Wu and Park (11) for the disaccharide aminomannuronyl-(1-b 3)-fucosamine. The reduced strain M polysaccharide proved to be easier to hydrolyze with aqueous acid, which permitted the molar ratios of the components to be established (Table 2). Data in Fig. 2 summarize the results obtained to date and pernit the hexasaccharide unit to be postulated as shown. At this time none of the configurational assignments are known. Because of the relatively easier release of aminogalactu-
N
80 C, 100 h
NaBH4
RATIO
RATIO
COOCH3 0
,C20H
OOCH3 NH2 Aminogolocturonic
Acid
Methyl Ester glycoside CH3
2
3 NH2
Galactosomine
COOH
0
OCH3 NH2
NH2 Fucosomine Glycoside
Aminogalacturonic Acid CH3
2 NH2
Fucosamine H2NCH2CH2 SO3M Taurine
I
FIG. 2. Proposed hexasaccharide repeating unit and degradation patterns from strain M polysaccharide. Compound X is shown in dotted brackets; the 1 -- 3 linkage was established by mass spectrometry. The positions of other linkages are not known, but it is speculated that they are 1 -I 4. The particular N-
acetylaminogalacturonic acid to which taurine is attached is not known.
200 LIAU AND HASH ronic acid by acid hydrolysis, it is speculated that the remaining glycosidic bonds in the polysaccharide are 1 -- 4. Taurine is an unusual component of this polysaccharide. It did not form a DANS derivative with DANS-Ci, and the position of this component was deduced from the fact that one of every four aminogalacturonic acid residues in the polysaccharide could not be esterified and reduced to galactosamine. On the average there is one taurine residue per hexasaccharide unit linked to a carboxyl group of an aminogalacturonic acid residue. The methanolysis procedure of Sandford et al. (7) was used to help elucidate the repeating unit of strain M polysaccharide. In contrast, however, to the results obtained with the yeast Y-6272 polysaccharide (8), where completemethanolysis occurred, the polysaccharide from strain M was more resistant to this procedure. Intermediate products were present after 100 h. Molar ratios of the constitutent amino sugars were based on the released components, but the total composition could not be established with this procedure. ACKNOWLEDGMENTS This investigation was supported by Public Health Service grant AI-06712 from the National Institute of Allergy and Infectious Diseases. We thank J. T. Watson of the Department of Pharmacology for mass spectrometric analyses and Lilah Clack for performing amino acid analyses. LITERATURE CITED 1. Hanessian, S., and T. H. Haskell. 1964. Structural studies on staphylococcal polysaccharide antigen. J.
J. BACTERIOL. Biol. Chem. 239:2758-2764. 2. Liau, D. F., M. A. Melly, and J. H. Hash. 1974. Surface polysaccharide from Staphylococcus aureus M that contains taurine, D-aminogalacturonic acid, and n. fucosamine. J. Bacteriol. 119:913-922. 3. Ludowieg, L., and A. Dorfman. 1950. A micromethod for the colorimetric determination of N-acetyl groups in acid mucopolysaccharides. Biochim. Biophys. Acta 38:212-218. 4. Ottenstein, D. M., and D. A. Bartley. 1971. Improved gas chromatography separation of free acids C2-C, in dilute solution. Anal. Chem. 43:952-955. 5. Radford, T., and D. C. DeJongh. 1972. Carbohydrates, p. 313-350. In G. R. Waller (ed.), Biochemical application of mass spectrometry. John Wiley & Sons, Inc., New York. 6. Rondle, C. J. M., and W. T. J. Morgan. 1955. Determination of glucosamine and galactosamine. Biochem. J. 61:586-589. 7. Sandford, P. A., P. R. Watson, and A. R. Jeanes. 1973. An extracellular microbial polysaccharide composed of 2-acetamido-2-deoxy-D-glucose and 2-acetamido-2deoxy-D-glucuronic acid: radiochemical and gas chromatographic analysis of the products of methanolysis. Carbohydr. Res. 29:153-164. 8. Seiler, N. 1970. Use of the dansyl reaction in biochemical analysis, p. 259-337. In D. Glick (ed.), Methods of biochemical analysis, vol. 18. Wiley-Interscience Publishers, New York. 9. Shih, J. W. K., and J. H. Hash. 1971. The N,O-diacetylmuramidase of Chalaropsis species. III. Amino acid composition and partial structural formula. J. Biol. Chem. 246:994-1006. 10. Taylor, R. L., and H. E. Conrad. 1972. Stoichiometric depolymerization of polyuronides and glycosaminoglycuronans to monosaccharides following reduction of their carbodiimide-activated carboxyl groups. Biochemistry 11:1383-1388. 11. Wu, T. C. M., and J. T. Park. 1971. Chemical characterization of a new surface antigenic polysaccharide from a mutant ofStaphylococcus aureus. J. Bacteriol.
108:874-884.
