Eur. J. Biochem. 207,1063-1075 (1992)

0FEBS 1992

The structure of pneumococcal lipoteichoic acid Improved preparation, chemical and mass spectrometric studies Thomas BEHR’, Werner FISCHER’, Jasna PETER-KATALINIC’ and Heinz EGGE’

’ Institut fur Biochemie der Medizinischen Fakultat der Universitat Erlangen-Nurnberg, Federal Republic of Germany

*

Physiologisch Chemisches Institut der Universitat Bonn, Federal Republic of Germany

(Received March 18, 1992) - EJB 92 0381

Pneumococcal lipoteichoic acid was extracted and purified by a novel, quick and effective procedure. Structural analysis included methylation, periodate oxidation, Smith degradation, oxidation with Cr03, and fast-atom-bombardment mass spectrometry. Hydrolysis with 48% (by mass) HF and subsequent phase partition yielded the lipid anchor (I), the dephosphorylated repeating unit of the chain (11) and a cleavage product of the latter (111). The proposed structures are:

(I) Glc(p1

3)AATGal(Pl

-+

(11) Glc(p1

--f

(111) Glc(p1

+

3)AATGal(crl

+

3)Glc(al

-+

3)AATGal(al

--f

3)acy12Gro,

4)GalNAc(al

+ 4)GalNAc(al

-+

3)GalNAc(pl

-+

--f

1)ribitol and

3)GalNAc,

where AATGal is 2-acetamido-4-amino-2,4,6-trideoxygalactose, and all sugars are in the pyranose form and belong to the D-series. Alkaline phosphodiester cleavage of lipoteichoic acid, followed by treatment with phosphomonoesterase, resulted in the formation of I1 and IV, with IV as the prevailing species: (IV) Glc(p1

-+

3)AATGal(al

4)GalNAc(al 3)GalNAc(Pl 6 6 Chop 1 Chop --f

--f

--f

1)ribitol.

The linkage between the repeating units was established as phosphodiester bond between ribitol 5-phosphate and position 6 of the glucosyl residue of adjacent units. The chain was shown to be linked to the lipid anchor by a phosphodiester between its ribitol 5-phosphate terminus and position 6 of the non-reducing glucosyl terminus of I. The lipoteichoic acid is polydisperse: the chain length may vary between 2 and 8 repeating units and variations were also observed for the fatty acid composition of the diacylglycerol moiety. Preliminary results suggest that repeating units I1 and IV are enriched in separate molecular species. All species were associated with Forssman antigenicity, albeit to a various extent when related to the non-phosphocholine phosphorus. Owing to its unique structure, the described macroamphiphile may be classified as atypical lipoteichoic acid.

In 1943 pneumococcal Forssman antigen was identified by Goebel et al. as the first ‘lipocarbohydrate’ among Grampositive bacteria [l]. On the basis of its amphiphilic nature effected by covalently attached fatty acids [ 1,2] and its association with the plasma membrane, it was later classified as a choline-containing lipoteichoic acid [3]. The presence of glucose, N-acetylgalactosamine, ribitol, phosphorus, and choline led to the speculation that lipoteichoic acid might be Correspondence to W.Fischer, Institut fur Biochemie der Med.

structurally related to pneumococcal C polysaccharide, the homologous wall teichoic acid [l -41. However, except for two notes that the fatty acids comprise 6% (by mass) [l, 21,quantitative data have not been reported. Moreover, 2-acetamido-4-amino-2,4,6-trideoxy-~-galactose, a characteristic component of the teichoic acid [5, 61,has so far not been demonstrated in the lipoteichoic acid and the nature of its lipid anchor remains obscure. In the meantime the complete structure of the teichoic acid has been established

Fak., Univ. Erlangen-Nurnberg, Fahrstrasse 17, W-8520 Erlangen, 15, 61. Federal Republic of Germany Although serological studies showed cross reactivity beAbbreviations. AATCal, 2-acetamido-4-amino-2,4,6-trideoxy-~tween teichoic acid and lipoteichoic acid [7, 81, lipoteichoic galactose; Cho, choline; Gro, glycerol; abbreviations of fatty acids acid is the unique carrier of Forssman antigenicity, eliciting are illustrated by the following examples: 18 :0, octadecanoic acid; antibody formation against sheep erythrocytes in rabbits [l]. 9 - 18 : 1, octadecenoic acid with the double bond at position 9 (oleic By serological methods, all 83 known types of Streptococcus acid). Note. This work was carried out in partial fulfillment of the pneumoniae were shown to possess both Forssman antigen requirements for an MD degree from the University Erlangen- and C polysaccharide [8] which may therefore be considered Nurnberg, 1992,for T. Behr. as common antigens of pneumococci. On immunoelectron

1064 microscopy, the C polysaccharide was found to be distributed on the inside and outside of the cell wall, whereas the Forssman antigenic material appeared associated with the cytoplasmic membrane [9]. The potential role of lipoteichoic acid in the physiology of pneumococcal cells has been addressed in a number of studies. A role in regulating the activity of autolysin or in transmembrane transport of this enzyme has been postulated [lo141, whereby the phosphocholine residues were found to be essential [14, 151. Lipoteichoic acid and teichoic acid seem further to play a pathophysiological role as chemotaxins by the induction of meningeal inflammation [16]. In contrast to certain poly(g1ycerophosphate) lipoteichoic acids, pneumococcal lipoteichoic acid does not stimulate the production of interleukin lp, interleukin 6 or tumor necrosis factor CI in human monocytes [17]. An activation of the alternative complement pathway by pneumococcal lipoteichoic acid has been reported [18], whereas poly(g1ycerophosphate) lipoteichoic acids seem to activate the classical one [18a]. For understanding the biological properties on a molecular basis, knowledge of the lipoteichoic acid structure is required. An apparent reason why structural studies have not yet been performed lies in the time-consuming preparation of pneumococcal lipoteichoic acid which has not been changed essentially since 1943 [3, 141. We therefore developed a simple and effective isolation procedure and established the complete structure of the purified material.

EXPERIMENTAL PROCEDURES Materials DEAE-Sephadex was purchased from Pharmacia LKB GmbH (Freiburg, FRG), octyl-Sepharose from Sigma Chemie GmbH (Deisenhofen, FRG). Enzymes and cosubstrates were obtained from Boehringer Mannheim GmbH (Mannheim, FRG). Streptococcus pneumoniae R6, a derivative of the Rockefeller University laboratory strain R36 A, and a sample of pneumococcal Forssman antigen prepared by the previously described procedure [14] were kindly supplied by Dr R. Hakenbeck (Max-Planck-Institut fur Molekulare Genetik, Berlin, FRG). Quinovosamine was prepared from a uronic-acid-containing polysaccharide of Aerococcus viridans (gift of Dr B. Lindberg, Arrhenius Laboratory, University of Stockholm, Sweden) by reduction of the uronic acid residues (191, acid hydrolysis (2 M HCl, 100°C, 2.5 h), and cation-exchange chromatography. The D-configuration was established by degradation to L-serine [20] which was identified by HPLC using pre-column derivatization with o-phthaldialdehyde [21] and N-acetyl-L-cysteine instead of 3-mercaptopropionic acid, which allows separation of the chiral derivatives of D- and Lserine (Hannapel, E., unpublished work). Bacterial growth S . pneurnoniae R6 was grown in 20-1 batches which were stirred at 37°C without aeration. The growth medium contained in 1 1: 10 g glucose, 5 g meat extract, 10 g casein peptone, 3 g yeast extract, 17.5 g K2HP04, 10 mg CaC12; choline was present in the meat and yeast extract. After adjusting the pH to 7.8, the medium was sterilized by filtration. The bacteria were harvested in the late growth phase ( A 5 7 8zz 2) with a refrigerated continous-flow centrifuge. The

yield of bacteria was 2 medium.

0.7 g (X 2 CT,,-~,n = 19) wet mass/l

Extraction and purification of lipoteichoic acid The bacteria were suspended in 0.05 M sodium acetate pH 4.0 (0.5 g wet mass/ml) and disintegrated by ultrasonication in ice for three 3-min periods (Branson sonifier cell disruptor Bl5, 20 kHz, 150 W). After adjusting the pH to 5.5 with 1 M NaHCO,, 2 vol. MeOH and 1 vol. CHC13 were added and the mixture was stirred at room temperature for 3 h. After centrifugation, the supernatant was withdrawn, the pellet resuspended in 4 vol. 0.05 M sodium acetate pH 5.51 MeOH/CHC13 (0.8 :2.0 : 1.O, by vol.) and stirred overnight. After centrifugation the supernatants were combined in a separatory funnel and water and CHC1, were added to give CHCl,/MeOH/H,O in final proportions of 1.O: 1.O: 0.9 (by vol.). After phase separation, the lower layer and a bulky white precipitate at the interphase were removed. The latter was washed several times with water and, after centrifugation, the supernatants were added to the upper layer in the separatory funnel. The combined upper phases were extracted twice with chloroform, freed of methanol by rotary evaporation (bath temperature 20°C) and dialyzed against three 5-1 changes of distilled water. After concentration to a small volume (30 - 50 ml), propan-1-01 and 2 M sodium acetate pH 4.7 were added to concentrations of 15% (by vol.) and 0.05 M, respectively (buffer A). The crude extract (x1 mmol phosphorus) was loaded on a column (1.5 x 27 cm) of octylSepharose CL 4-B, pre-equilibrated in buffer A. It was eluted at 16 ml/h first with buffer A (200 ml) and then with a linear propan-1-01 gradient (15-65%, by vol., 250 ml each) in 0.05 M sodium acetate pH 4.7. Fractions (4 ml) were analyzed for phosphorus. The propanol concentration in the effluent was determined refractometrically. Appropriate fractions were combined, dialyzed against three 5-1 changes of distilled water and concentrated by rotary evaporation (x10 pmol phosphorus/ml). Purified preparations were stored at - 20 "C. Analytical procedures Carbohydrate [22], choline [23], formaldehyde [24], formic acid [25], glucose [26], glycerol [27], glycolaldehyde [28], hexosamine [29], nucleic acids [30], periodate [31], and phosphorus [32]were measured as in the references quoted. Ribitol and 2,5-anhydroribitol were quantitated as acetates by GLC using mannitol acetate as internal standard. Galactosamine was identified as the alditol acetate by GLC. For compositional analysis, lipoteichoic acid was N-acetylated, dephosphorylated with 48% (by mass) aqueous H F (2"C, 36 h) and, after drying, hydrolyzed with 2 M HCI (IOO'C, 2.5 h). For quantitation of fatty acids, prior to hydrolysis with HCI, pentadecanoic acid was added as internal standard. Fatty acids were extracted from the hydrolysate with light petroleum/CHCl, (4: 1, by vol.), converted into methyl esters with 10% (mass/vol.) BC13 in MeOH (9OoC, 15 min) and analyzed by GLC before and after catalytic hydrogenation [33]. For rapid determination of choline, hydrolysis with 48% H F was performed at room temperature for 3 h. Amino acids were released by hydrolysis with 6 M HC1(155"C, 60 min). GLC was performed on a Hewlett Packard 5840 A gas chromatograph equipped with a flame ionization detector and a cold injection system (Gerstel GmbH, Muhlheim/Ruhr, FRG). An HP 5 fused silica-gel capillary column (30 in, inner diameter 0.25 mm, film thickness 0.25 km) was used. Fatty

1065 acid methyl esters were separated at 135-25O0C, with a temperature rise of 6 "C/min, acetylated alditol acetates and their partially methylated derivatives at 195 -250 "C, with a temperature rise of 2 "C/min. GLC/MS analyses were performed on a Hewlett Packard 5890 A gas chromatograph connected with a mass spectrometer MS-D 5970. The temperature of the transfer line and ion source was 250"C, the ionization potential 70 eV. TLC was done on silica gel plates (Merck 60) with the following solvents: (A) CHC13/MeOH/H20 (65 :25 :4, by vol.); (B) CHCI3/MeOH/AcOH/H20(60: 12: 18: 5, by vol.); (C) propanol/25% (by mass) N H 4 0 H / H 2 0 (6: 3: 1, by vol.); (D) propanol/ethyl acetate/H20 (7: 2: 2, by vol.); (E) phenol/ AcOH/EtOH/H,O (75:4.5:4.5: 33, mass/vol./vol./vol.); (F) pyridine/ethyl acetate/AcOH/H20 ( 5 :5 : 1 :3, by vol.); (G) CHCl,/MeOH (9: 1, by vol.). Solvents A and B were used for the separation of lipids, solvents C - F for water-soluble compounds, solvent G for peracetylated oligosaccharides. Lipids were visualized with iodine vapour, carbohydrate with l-naphthol/H2S04 [34], amino groups with a ninhydrin reagent, polyols and N-acetylhexosamines with alkaline permanganate [35]. Fast-atom-bombardment mass spectrometry FAB-MS was performed in positive and negative ion mode as reported previously [36].

Ultraviolet-laser-induceddesorption/ionization mass spectroscopy

MeOH (1 :1, by vol.) and purified by phase partition. De-Oacetylation was performed in CHC13/MeOH/0.4 M aqueous NaOH (1 :2: 1, by vol.) at 37°C (15 min); phase separation was achieved by addition of CHCl, and water. The aqueous layer was deionized by passage through a small column of cation-exchange resin H and freeze-dried. +

Preparation of the phosphorylated repeating units Lipoteichoic acid (50 pmol phosphorus) was hydrolyzed in 0.3 M NaOH at 100°C for 2 h. The hydrolysate was passed through a small column of cation-exchange resin, NH;. After drying in vucuo, the products were dissolved in water, the pH was adjusted to 4 with acetic acid, and fatty acids were extracted with light petroleum/CHC13 (4:1, by vol.). The watersoluble products were N-acetylated, desalted, dissolved in water and applied to a column ( 0 . 8 16 ~ cm) of DEAESephadex A25. The column was preequilibrated with two column volumes each of 0.5 M and 0.05 M ammonium carbonate pH 8.2 and washed with water (25 ml) immediately before use. Elution (16ml/h) was performed with a linear gradient of 0 - 0.4 M NH4HC03, pH 8.2 (350 ml each) and fractions (4 ml) were analyzed for phosphorus and carbohydrate. Appropriate fractions were combined and freezedried. The salt-free products were dissolved in water, the pH was adjusted to 9.5 with ammonia, and the mixture was incubated at 37 "C overnight with alkaline phosphomonoesterase (10 U/ml). The enzyme was removed in a microconcentrator (Amicon; Witten, FRG), equipped with a Centricon membrane (cutoff 10 kDa, carefully washed free of glycerol).

This was performed on a Lamma 1000 laser microprobe using an Nd-YAG laser [37]. 3,5-Dinitrobenzoic acid was used as a solid matrix [38]. Accumulation of ten individual spectra was used for better signallnoise ratio. Mass was determined by centroiding.

N-Acetylation

Preparation of the lipid anchor and the dephosphorylated repeating unit

Deamination with HNOz

Water-soluble compounds were N-acetylated as described [39]. The lipid anchor was N-acetylated by the procedure of KozuliC et al. [40].

Free amino groups were deaminated with HNOz as deLipoteichoic acid (50 - 100 pmol phosphorus) was hy- scribed by Jennings et al. [6]. The reaction mixture was freezedrolyzed in a teflon vessel with 48% (by mass) H F (0.5 ml) at dried and desalted by passage through a small column of 2°C for 36 h. After drying over KOH in vacuo at 2"C, the cation-exchange resin, H +, overlaid by anion-exchange resin, lipid anchor and dephosphorylated chain fragments were acetate form. separated by phase partition in CHCI3/MeOH/0.05 M NH,HC03 (1 : 1: 0.9, by vol.). The organic layer was taken to Oxidation with C r 0 3 dryness, the residue dissolved in CHC1, and applied to a small Peracetylated oligosaccharides were treated with Cr03, column (0.5 x 3 cm) of silica gel (Iatrobeads 6RS-8060, Iatron Laboratories Inc, Tokyo, Japan). After washing with CHCI3, essentially as described by Laine and Renkonen [41]. the lipid anchor was eluted with CHC13/MeOH (1 : 1, by vol.). Monosaccharides that had survived oxidation, were measured The water-soluble products were reduced with NaBH4 after hydrolysis with 2 M HC1 (100'C, 2.5 h). Release of (10 mg/ml) at room temperature for 2 h. After oxidation of oxidized products (5-oxohexulosonic ester) was achieved by excess NaBH4 by acetone (100 pl), the mixture was passed treatment with NaBH4 in ethanol [42]. through a small column of cation-exchange resin (H' form) and taken to dryness with repeated additions of methanol. Methylation analysis The reduced material was peracetylated with acetic anhydride/ Prior to methylation, oligosaccharides and choline-phospyridine/acetic acid (2: 1:0.3, by vol.) at 37°C for 48 h. For FAB-MS analysis, peracetylation was preceded by deutero- phate-containing oligosaccharides were peracetylated as deN-acetylation [39], as described under Results. To the scribed above. The methylation procedure of Ciucanu and peracetylation mixture water and CHC13 were added, the Kerek was followed [433, except that after a reaction time of peracetylated oligosaccharides recovered from the organic 30 min a second portion of CH31 was added and the reaction layer and separated by preparative TLC on silica gel plates allowed to continue for another 30 min. Then solid NaOH (Merck 60) with solvent G. Bands were visualized with iodine was removed by centrifugation and washed with dry vapour and scraped off into glass tubes with teflon-lined screw dimethylsulfoxide. The supernatants were combined and caps. Compounds were extracted from silica gel with CHC13/ 2 vol. each of water and CHC13 were added. Phosphate-free

1066 compounds were recovered from the CHCI3 layer. Phosphorylated compounds appeared in the aqueous layer and were freed from solvent by freeze-drying. Partially methylated monosaccharides were released by hydrolysis with HC1 as detailed under Results. Prior to hydrolysis with HCl, dephosphorylation was achieved by treatment with 48% (by mass) HF (2"C, 36 h). Oxidation with N a I 0 4 and Smith degradation Lipoteichoic acid (20 pmol phosphorus) was oxidized in 0.05 M Na1O4 (3.2 ml) in the dark at 37°C. The reaction was followed spectrophotometrically [31]. After the oxidation was complete (4 h), ethyleneglycol(l50 pmol) was added and the reaction mixture extracted three times with CHC13(1 ml each). To the aqueous layer ethanol (13 ml) was added and the mixture was allowed to stand in ice for 30 min. Precipitated NaIO3 T U B E NUMBER was removed by centrifugation and the supernatant freed of ethanol by rotary evaporation. The recovery of phosphorus Fig. 1. Purification of lipoteichoic acid by hydrophobic interaction was 97%. chromatography. The crude extract from a 20-1 culture was Part of the water-soluble product (500 nmol phosphorus) chromatographed on a column of octyl-Sepharose as described under was incubated in 0.05 M sodium borate pH 9.5 (0.2 ml), con- Experimental Procedures. Lipoteichoic acid (220 pmol phosphorus) taining alkaline phosphatase (2 U), at 37 "C overnight and recovered from peak I1 (the actual phosphate values are half of those then analyzed for inorganic phosphate and total phosphorus. depicted) contained 90% of the onput choline. Peak I contained The major portion of the oxidation product was treated with nucleic acids (230 pmol phosphorus), unidentified phosphorus-conNaB2H4 (10 mg) at 37°C for 1 h. Then acetone (100 p1) was taining material (290 pmol), and contaminants which, on acid hydrolysis, yielded large amounts of glucosamine (255 pmol), alanine added and the mixture passed through a small column of (330 pmol), glutamate (233 pmol), lysine (141 pmol), and glycine cation-exchange resin, NH '4 form. Ammonium borate was (76 pmol). The material of tubes 110-125 was collected for characremoved from the effluent by evaporation with several ad- terization of lipoteichoic acid. ditions of MeOH. The reduced oxidation product was hydrolyzed in 0.1 M HCl at 37 "C for 24 h. After removal of HCl with Ag2C03, the mixture was added to DEAE-Sephadex acid isolated by the original procedure [14]. On phenol/water A25 (0.5 ml), HCO, form in a centrifuge-filtration device extraction, pneumococcal lipoteichoic acid partitioned into (Abitube, Abimed, Langenfeld, FRG), which was equipped the phenol layer, in contrast to other lipoteichoic acids (for with a polycarbonate membrane (pore size of 0.4 pm). Water review, see [44]).We therefore tried the Bligh-Dyer lipid extrac(2 x 2 ml) eluted 61% of the phosphorus, 0.2 M ammonium tion [45] and found pneumococcal lipoteichoic acid to be carbonate pH 8.2 (2 x 1 ml) 35%. The second eluate was readily soluble in the initial one-phase mixture and, on phase freeze-dried, trimethylsilylated [25 pl N,O-bis(trimethylsily1)- separation, to partition into the aqueous layer. This was also trifluoroacetamide, 3 p1 trimethylchlorosilane and 1.5 p1 the case when the procedure was applied to mechanically pyridine] and analyzed by GLC/MS. disintegrated pneumococci. The lipoteichoic acid in the aqueThe lipid-soluble oxidation product (see above) was ous layer separated from most of the co-extracted lipids which treated in the same way with the following modifications: partitioned into the chloroform layer. Residual lipid was re/I-elimination was performed in the presence of 0.05% (by moved from the aqueous layer by repeated extraction with mass) Triton X-100. Reduction with NaB2H4 (10mg) was chloroform. The lipid extract contained negligible amounts of done in 2 ml CHC13/methanol/water (1 :2: 1, by vol.) at 37°C choline-containing material. for 1 h. Then 4 M NaOH (50 pl) was added and the incubation The lipoteichoic acid was purified by hydrophobic interaccontinued for 30 min to hydrolyze fatty acid ester. After phase tion chromatography of crude extracts on octyl-Sepharose separation, the water-soluble product was desalted, subjected [30, 461. A typical experiment is depicted in Fig. 1. As shown to mild acid hydrolysis and analyzed for the presence of by choline determination, 90% of the lipoteichoic acid was glycerophosphoethylene glycol as described above. recovered from the column. It was free of nucleic acids and amino-acid-containing material. The yield from 1 1 bacterial culture was 11 pmol phosphorus corresponding to 12 mg Immunological procedures lipoteichoic acid. This value compares with 1 - 1.4 mg Antisera were raised in rabbits by repeated intravenous obtained from 1 1 culture by the original procedure [14], and injections of heat-killed mechanically disintegrated cells of 0.075 mg when this material was purified by chromatography S. pneumoniae R6 using a similar immunization schedule as on a Sepharose-TEPC 15 myeloma protein affinity column described earlier [l].Forssman reactivity of lipoteichoic acid was tested in a hemolysis inhibition assay and expressed in [161. arbitrary units as described by Goebel et al. [l]. Compositional analysis RESULTS Isolation of pneumococcal lipoteichoic acid The extraction procedure described in this report was based on pilot experiments with pneumococcal lipoteichoic

The purified lipoteichoic acid contained D-glUCOSe/Dgalactosamine 1 ribitoli phosphorus / choline / glycerol fatty acids in molar ratios of approximately 0.5 :0.9 :0.4: 1 .O: 0.6: 0.1 : 0.2. Glucose was incompletely released by acid hydrolysis, the value given was obtained by an anthrone pro-

1067 cedure [22]. The assignment of glucose to the D-SerieS was Deamination of the deacylated glycolipid with H N 0 2 libaccomplished enzymatically [26], that of galactosamine by erated glucose and Glc(a1 -+ 3)Gro which were identified as glycosidation with R( -)-2-butanol and GLC of the trifluoroacetates on GLC by cochromatography with stantrimethylsilyl derivatives [47, 481. dards. Glucose and GlcGro, quantitated as glucose before The constituent fatty acids were (mol% range of five prep- and after treatment with a-glucosidase, amounted to 0.26 and arations from separate bacterial cultures): 12:0 (2.9 - 8.2), 0.37 of the total glucose. These observations suggest that 14:0 (7.8-14.2), 47-16:l (2.8-4.9), 49-16:l (12.7-18.6), H N 0 2 treatment formed a 4-azohexosyl derivative situated 16:O (47.5-49.3), 49-1811 (2.5-4.1), 411-18:l (6.0-10.7), between the two glucosyl moieties and that hydrogen shifts 1810(3.3-6.6). from C-5 and C-3 to C-4 split off the glycosidic bonds at C-I In a search for the presence of 2-acetamido-4-amino-2,4,6- and C-3, respectively [49]. Further evidence for the proposed trideoxygalactose (AATGal), we followed the protocol of 4-azo-derivative comes from D-quinovosamine which was reLindberg et al. [49, 501. Deamination of the lipoteichoic acid leased by acid hydrolysis from the deamination product, but with HN02, followed by acid hydrolysis (2 M HCI, 100"C, not from the native compound. From these results we propose 2.5 h) yielded a sugar not present in the acid hydrolysate the structure Glc-AATGal-Glc-Gro. of the native compound. It was identified as 2-amho-2,6Methylation analysis yielded 2,3,4,6-tetra-O-methylgludideoxy-glucose (quinovosamine) by cochromatography with cose and 2,4,6-tri-O-methylglucose in equimolar amounts. an authentic sample on TLC (solvent C, F) and as the alditol Oxidation with C r 0 3 and subsequent treatment with NaB2H4 acetate by GLC/MS (primary fragments at m/z = 73, 144, in ethanol [42] released Glc(a1 + 3)Gro, glucitol and iditol. 159, 216, 288, 302). Quinovosamine was isolated by cation- As shown by GLC/MS, both polyols carried one and two exchange chromatography and assigned to the D-SerieS by deuterium atoms at C5 and C1, respectively. From these reglycosidation with R( -)-2-butanol and comparison with sults we conclude that the non-reducing glucosyl terminus and authentic R( -)-2-butyl-~-quinovosaminide and rac-2-butyl- the AATGal residue were oxidized and therefore in the pquinovosaminide by GLC of the trimethylsilyl derivatives [47, configuration. Attempts to detect epimeric alditol derivatives 481. When, after HNOz treatment, acid hydrolysis was pre- of AATGal failed. Collectively the complete structure of the ceded by reduction with NaBH4, two minor components were deacylated glycolipid (I) may be given as Glcp(/ll 3)detected as peracetylated derivatives by GLC having lower AATGalp(P1 + 3)Glcp(al -+ 3)Gro. retention times than peracetylated quinovosaminitol and mass The positive-ion FAB mass spectrum of the native glyspectra characteristic of 2-acetamido-2,4,6-trideoxyhexitol colipid yielded a major molecular ion (M + Na+) at m/z = acetates (primary fragments at m/z = 144, 173, 244). The for- 1099 which was shifted to m / z = 1435 by peracetylation (not mation of D-quinovosamine, together with these compounds, shown). Weaker molecular M Na' ions were observed at is indicative of the presence of 2-acetamido-4-amino-2,4,6-m/z = 1463, 1409, and 1407. After N-deuteroacetylation and trideoxy-D-galactose (49, 501. The decomposition of 4-amino- subsequent peracetylation, the major molecular M + Na' ion 4-deoxy sugars during acid hydrolysis has been established appeared at m/z = 1438 along with minor M Na' ions at m / z = 1466, 1412, and 1410 (Fig. 2). Together with the fatty earlier [2, 6, 49, 501. After HNOz deamination of the lipoteichoic acid, sub- acid composition described above, these molecular ions indisequent treatment with NaBH4 and acid hydrolysis, 2,5- cate at the diacylglycerol moiety the fatty acid combinations anhydrotalitol could not be detected, indicating the absence 16:0/16:1, 16:0/18:0, 16:0/14:0, and 16:1/14:0. These combinations also occur as diacylglycerol fragments at m/z = 549 of galactosamine with a non-acetylated amino group. (major), 577, 523, and 521. Of lower intensity are glycolipid ions arising after cleavage of the terminal glucose at mjz = Hydrolysis of lipoteichoic acid with H F 1086 (1114) (M-Glc' + 2 H') and mjz = 1068 (1096) (M + Hf- Glc-OH). The fragment ions from the non-reducFor structural analysis the glycolipid moiety and ing terminus at m/z = 331 + 289 + 169 (Glc') and 562 + 214 dephosphorylated repeating units were released from (Glc-AATGal') along with m/z = 850 of low intensity (Glclipoteichoic acid by treatment with aqueous 48% (by mass) AATGal-Glc '), confirm the postulated trisaccharide sequence H F and separated by phase partition (see Experimental Pro- of the sugar moiety. The high intensity of the daughter ion at cedures). Hydrolysis with aqueous 48% (by mass) H F cleaves m / z = 214 (m/z = 213 of peracetylated material) indicates the selectively phosphodiester and phosphomonoester bonds [Sl, particular lability of the p1-3 linkage in the Glc-AATGal unit. 521, except a few particularly acid-labile glycosidic linkages [6, 531. The structure of the phosphate-free repeating unit --f

+

+

Structure of the glycolipid moiety

The organic layer of the HF 'hydrolysate' contained a single unusually polar glycolipid. It could not be detected among membrane lipids in the crude lipid extract of S. pneumoniue by two-dimensional TLC (solvent A, B) (not shown). The glycolipid liberated from lipoteichoic acid reacted slowly with ninhydrin, and N-acetylation increased its chromatographic mobility relative to Gal(a1 + 2)Glc(al 3)acy12Gro, the major membrane glycolipid, from 0.39 to 0.97 (TLC, solvent A). Acid hydrolysis of the native compound yielded glucose, glycerol, and fatty acids in molar ratios of approximately 0.8 :1 .0 : 2.0. N-Acetylation prior to hydrolysis increased the glucosejglycerol molar ratio to 1.8. --f

The composition of the water-soluble HF 'hydrolysis' products of lipoteichoic acid was dependent on reaction time. Hydrolysis for 36 h released the total phosphorus as inorganic phosphate and liberated choline completely. As shown by TLC (solvent C) equal amounts of two oligosaccharides (11, 111) had formed along with ribitol, and approximately 25% of total hexosamine reacted in the Elson-Morgan reaction [29] without further hydrolysis. After treatment of lipoteichoic acid with H F for 120 h, oligosaccharide I1 was completely converted into oligosaccharide 111, the total ribitol was liberated and the reactivity of hexosamine in the Elson-Morgan reaction approached 50%. These results suggest that a 3- or 6-substituted N-acetylgalactosamine residue [54] forms the reducing terminus of oligosaccharide 111and is in oligosaccha-

1068 1000,

1

X 50

A.

I331

I I

I /

/

5; 2

1

I

I

HtNa' 0.

169-3317k:

1438

808 t

214

t

36

850,

562,

fl "

R

5 600

OR

w

2

1424

4

MtNa'

W

= $00

'

NAc H

' ,

I

L549 577

, 1466

M+H+

=1416/1444

M+Na+

=1438/1466

M+ ~ a +

=1546/1574

+GRoSH

200

MASS/CHARGE

Fig. 2. Structure of the glycolipid anchor (R, R, = H) and the positive-ion FAB mass spectra of its N-deuteroacetylated and peracetylated derivative (R = CH,CO; R, = C2H3CO). R2, R3,hydrocarbon chains of fatty acids: C15H31rC15H29/C15H31, C17H33(for less abundant fatty acid combinations, see text). (A) FAB-MS in thioglycerol as matrix without additions; (B) molecular ion area after addition of sodium acetate. GroSH, thioglycerol.

ride I1 linked to the ribitol moiety through a particularly acidlabile j-glycosidic bond [6]. The oligosaccharide mixture in the 36-h hydrolysate was treated with NaBH,. In order to locate the AATGal unit in the sugar sequence by FAB-MS, the sample was N-acetylated by a 2: 1 mixture of (CH3C0)20and (C2H,CO)20 and, after subsequent peracetylation with (CH3C0)20, separated by preparative TLC, as described under Experimental Procedures. Purified oligosaccharide I1 contained D-ghCOSe, N-acetylD-galactosamine, and ribitol in molar ratios of 1.O: 1.9 : 1.O. The positive-ion FAB mass spectrum of the peracetylated derivative, shown in Fig. 3, yielded molecular ions (M H') at m / z = 1453 (1456). The sugar sequence from the non-reducing terminus is documented by the ions at m/z= 331 + 289 + 169 (Glc'), 559 (562) + 21 1 (214) (GlcAATGal+) and 846 (849) 288 (Glc-AATGal-GalNAc'), and from the reducing end by rn/z=303 (ribitol'), 608 (ribitol-GalNAc-OH) . H' and 1123 (ribitol-GalNAcGalNAc-AATGal-OH) . H'. The daughter ion at m/z = 288,

+

--f

of high abundancy and free of 2H label, must necessarily arise from the trisaccharide at m/z = 846 (849) by cleavage of the al-4 glycosidic bond and reprotonation. Purified oligosaccharide 111-01 contained D-glucose, Nacetyl-D-galactosamine, and N-acetylgalactosaminitol in molar ratios of 1.O: 0.8 :1.O, as was shown after acid hydrolysis by GLC of the sugar acetates. Positive-ion FAB mass spectra of the peracetylated derivative revealed a molecular ion (M + Na') at m/z = 1259 and fragments at m/z = 331 (Glc'), 559 (Glc- AATGal'), 846 (Glc-AATGal-GalNAc'), and 374 (GalNAc-ol+) (not shown). All values were increased by 1 Da when oligosaccharide 111 was reduced with NaB2H4. These results are in agreement with a tetrasaccharide structure, derived from oligosaccharide I1 through cleavage of the glycosidic bond at the ribitol moiety during hydrolysis with HF. On oxidation with NaIO,, oligosaccharide I1 consumed 5 mol NaI04/mol glucose with the concomitant formation of 3 mol formic acid and 1 mol formaldehyde. These data indicate oxidation of the glucosyl and ribitol moiety, the former

1069 I00

'oSH

19 !6

-

x5 / /

9

800

/ /

>.

+

/

m

I I

w I-

5 600

GroSH

w

2 a Y 1

= 400

/ I

200

I562

,,

, , , , I. ,

7

1

500

400

169-331,

600

(21414562) (288)-(849) 211-559, 288-846,

700

1133.

,303

M+H+:1453 (1456)

MASS /CHARGE

Fig. 3. Structure of oligosaccharide I1 (R = H) and positive-ion FAB mass spectrum of the peracetylated derivative (R = Ac). Peracetylation was preceded by N-acetylation with a 2: 1 mixture of (CH3C0)20and (C2H3C0)20.GroSH, thioglycerol.

being oxidized at carbon atoms 2,3, and 4, the latter at carbon atoms 2, 3, 4, and 5(1). The glycosidic substitution of the ribitol moiety is therefore situated at C-l(5). Peracetylated oligosaccharide I1 was subjected to methylation analysis. The results, summarized in Table 1, suggest one of the N-acetylgalactosaminyl residues to be glycosidically substituted at position 4, the other at position 3. For analysis of the anomeric configurations, peracetylated oligosaccharide I1 was subjected to oxidation with CrOJ [41, 421. Analysis after acid hydrolysis revealed that total glucose and half of the N-acetylgalactosamine had been oxidized and were therefore linked by p-anomeric bonds. When the oxidation product was treated with NaBH4 in ethanol (see above), the release of ribitol and hexitols could be detected by TLC (solvent C, D). This result indicates that the Nacetylgalactosamine residue, attached to the ribitol moiety, has the P-configuration and the next one a. In order to evaluate the anomeric configuration of AATGal and to locate the 3and 4-substituted N-acetylgalactosaminyl residues within the oligosaccharide sequence, oligosaccharide 111-01was subjected to Smith degradation which yielded oligosaccharide IV

(Fig. 4). N-Acetylgalactosamine and 2-acetamido-2-deoxythreitol were liberated by acid hydrolysis (4 M HCI, 100°C, 8 h) and identified by GLC/MS as their acetates (Fig. 4). Therefore in oligosaccharide 111-01the glycosidic substitutions are at position 4 of the N-acetylgalactosaminyl and at position 3 of the N-acetylgalactosaminitol residue. On treatment with Cr03, peracetylated oligosaccharide IV remained unchanged, as was shown after de-0-acetylation by TLC (solvent D; alkaline permanganate stain), which indicates a-anomeric configuration for the AATGal residue and the adjacent N-acetylgalactosaminyl residue. Summarizing these results, we propose for the dephosphorylated repeating unit (11) the structure Glcp(B2 + 3)AATGalp(al -+ 4)GalNAcp(al -+ 3)GalNAcp(p1 -+ 1)ribitol (Fig. 3). Isolation and characterization of bis(phosphocho1ine)containing and phosphocholine-free repeating units Native lipoteichoic acid was subjected to alkaline hydrolysis in order to cleave phosphodiester bonds at adjacent hydroxyl groups through cyclic phosphate intermediates [55,56].

1070 Table 1. Methylated sugar components released by hydrolysis of permethylated oligosaccharide I1 in 4 M HCI at l0OT for 8 h and 16 h. After treatment for 16 h most of component A was converted into tri-0-methyl and di-0-methyl derivatives.

5

C

n

Amount after

Component

8h

h

I:

1.2

w

k Z

16h

0

m LL

mol/mol A 2,3,4,6-Tetra-0-methyl-glucose B 3,6-Di-0-methyl-2-N-methylacetamido2-deoxygalactose C 4,6-Di-0-methyl-2-N-methylacetamido2-deox ygalactose D 3-0-Methyl-2-N-methylacetamido2-deoxygalactose E 4-0-Methyl-2-N-methylacetamido2-deoxy galactose

CH,

1 .oo

Q

0

0.30

1 .oo

0.61

1.06

0.04

0.41

0.08

0.45

HOCH,

\

(vii)CrO,

CH'HOAC

,

D

P

2

Z 0

I I

a 10

30

50

70

TUBE NUMBER

(Vi )Ac,O

ACOCH I

,

0.1

V

N o oxidation

Fig. 4. Location of the glycosidic substitution at the N-acetylgalactosaminol residue of oligosaccharide 111-01 and determination of the anomeric configuration of the N-acetylgalactosaminyl and AATGal residue via degradation to oligosaccharide IV.

After hydrolysis in 0.3 M NaOH at 100°C for 2 h, the molar ratio of phosphomonoester to total phosphorus was 0.4 and approximately 90% of the choline residues had survived, as was shown by enzymatic determination after hydrolysis with HF. Neither before nor after treatment with phosphomonoesterase was free choline detected, indicating that phosphocholine linkages remained intact and the phosphomonoester resulted from hydrolysis of the intrachain phosphodiester bonds. The hydrolysis products were desalted, N-acetylated, and chromatographed on a column of DEAE-Sephadex A 25. The elution profile of carbohydrate and phosphorus is depicted in Fig. 5 , the composition of hydrolysis products in Fig. 6. Peak A contained the deacylated lipid anchor. Peak B was composed of uncleaved cyclic phosphate intermediates which, after a second alkaline treatment, cochromatographed with

Fig. 5. Separation of the alkaline hydrolysis products of lipoteichoic acid by column chromatography on DEAE-Sephadex A25. For details, see Experimental Procedures.

the major components of peak C (cf. Fig. 6). The material of peak D (18% of phosphorus) showed on analysis choline, glucose, galactosamine, phosphomonoester, and total phosphorus in molar ratios of 0.4: 0.3 :0.9: 0.4: 1.O. This composition, along with the release of oligosaccharides I1 and I11 by HF, suggests repeating units in which one of the phosphocholine residues was presumably converted during alkali hydrolysis into the vinyl derivative as discussed earlier by Poxton et al. [39]. The components of the major peak C (65% of phosphorus) were not uniform (Fig. 6). Up to tube 32 the molar ratio of phosphomonoester/choline/totalphosphorus/glucose was close to 1: 2: 3 : 1. In later fractions the ratio of choline to phosphorus rapidly decreased and approached zero, whereas the ratio of phosphomonoester to phosphorus approached 1. Appropriate fractions of peak C were combined, freed of salt by freeze-drying, treated with phosphomonoesterase and purified by ultrafiltration and passage through anion-exchange resin. After phosphornonoester cleavage, the major component of peak C contained glucose/N-acetylgalactosamine/ribitol/ 2,5-anhydroribitol/choline/phosphorus in a molar ratio of approximately 1.0:2.0:0.8:0.2:2.0:2.0 (oligosaccharide V), suggesting a bis(phosphocho1ine) derivative of oligosaccharide 11. TLC (solvent E) revealed a major component (SOY0 of total glucose) and ahead of it a minor one which, after preparative TLC, was shown to contain 2,5-anhydroribitol in place of ribitol. The formation of 2,5-anhydroribitol 1phosphate observed on alkaline hydrolysis of poly(ribito1phosphate) was suggested to occur by elimination of the phosphate ester bond at C5 through nucleophilic attack by the hydroxyl at C2 [57].Accordingly, in the present case, the bond at C5 of the 4,5-cyclic intermediate may have been eliminated. After treatment with phosphomonoesterase, the material from the later fractions of peak C (Fig. 5 ) was free of organic phosphorus and chromatographically identical with oligosaccharide I1 (TLC, solvents C, E). It was contaminated with small amounts of oligosaccharide V and its anhydroribitol derivative. After peracetylation and purification by TLC (solvent G), compositional analysis and permethylation confirmed the identity with oligosaccharide 11.

1071 may be ascribed to the bis(phosphocho1ine)-di(N-acetylgalactosaminy1)ribitol moiety. The fragmentation pattern did not allow us to decide whether the phosphocholine residues are attached to separate N-acetylgalactosaminyl residues or to the same. Methylation analysis of oligosaccharide V involved peracetylation, methylation, phosphate ester cleavage by HF, hydrolysis of the glycosidic bonds with HCl, and analysis by GLC/MS of the partially methylated alditol acetates. Hydrolysis with 4 M HC1 (lOO°C, 8 h) released 2,3,4,6tetra-0-methylglucose, 3-0-methyl-2-N-methylacetamido-2deoxygalactosamine, 4-O-methyl-2-N-methylacetamido-2deoxygalactosamine, and 2-N-methylacetamido-2-deoxygalactosamine in the ratio 1.00:0.24:0.44:0.10. After hydrolysis for another 8 h, 76% of the tetra-0-methyl-glucose had lost one of its methyl groups. The other components showed molar ratios of 0.9, 1.0, and 0.9 which suggests non-methylated galactosamine to be an artifact, and locates the phosphocholine residues at position 6 of each of the N-acetylgalactosaminyl residues.

Fig. 6. Major alkaline hydrolysis products of lipoteichoic acid, separated by column chromatography on DEAE-Sephadex (Fig. 5). Thin-layer chromatogram (silica gel, solvent C), stained with l-naphthol/H2SO4. Identification (see text): a, deacylated lipid anchor; b, cyclic phosphate intermediate of e (the two faster-moving spots in this lane are presumably the cyclic phosphate intermediates of c and d); c-e, phosphomonoester derivatives of oligosaccharide I1 (c), its bis(phosphocholine) derivative (e), and presumably its monophosphocholine derivative (d). Double spots (c - e) contain isomers which carry the phosphomonoester at C-4 and C-5 of the ribitol moiety. In spots c-e minor amounts of 4-phospho-2,5-anhydroribitol-containing derivatives were also present (for explanation see text).

The linkage between the repeating units and between the chain and the lipid anchor

N-Acetylated lipoteichoic acid was oxidized in 0.05 M NaIO, at 37 "C. The reaction, followed spectrophotometrically, was finished after 4 h. Per 1 mol non-phosphocholine phosphorus, approximately 4 mol periodate was consumed with the concomitant formation of 2 mol formic acid. These data suggest a connection of the repeating units by phosphodiester linkages between C-5(1) of the ribitol moiety and C-6 of the glucosyl residue of adjacent units. It should be noted that, when N-acetylation was omitted prior to oxidation, there was a rapid consumption of 2mol NaI04, whereafter the reaction slowed down and required 120 h for completeness. After complete oxidation, ethyleneglycol was added, and The molar ratio of repeating units carrying two the oxidation products of the chain and of the lipid anchor phosphocholine residues and none was 3.0 & 0.2 (2f c n - l )in were separated by addition of CHCI3. The ratio of phosphorus three lipoteichoic acid preparations from separate bacterial in the aqueous and CHC13 layer was 1 9 : l . The derivative batches. Repeating units carrying only one phosphocholine of the lipid anchor, recovered from the CHC13 layer, was residue were not rigorously identified but, if present, constitute characterized as follows. Treatment of a sample in 0.05 M very minor components (Fig. 6). sodium borate pH 9.5 containing 0.05% Triton X-100 (37"C, 18 h) released 86% of the total phosphorus as phosphomonoester, suggesting B-elimination of the phosphate group Structure of oligosaccharide V from C-6 of the oxidizable glucosyl moiety at the non-reducing On oxidation with periodate, oligosaccharide V consumed terminus (Fig. 2). Another sample was reduced with NaBZH4, 5 mol NaI04/mol glucose, with the concomitant formation of subsequently deacylated and freed of salts as described under 3 mol formic acid and 1 mol formaldehyde. These results are Experimental Procedures. The deacylation product contained identical with those obtained with oligosaccharide 11. They glucose, glycolaldehyde, glycerol, and phosphorus in the mosuggest that the two phosphocholine residues are situated lar ratio of approximately 1 :0.9 :2 : 1 (glucose and glycerol at the N-acetylgalactosaminyl residues if one considers that were measured after hydrolysis with 2 M HC1, 100°C, 2.5 h position 6 of the glucosyl residue is occupied by the and subsequent treatment with phosphomonoesterase; glycolaldehyde after hydrolysis with 0.1 M HC1, 37"C, 24 h). phosphodiester connecting the repeating units (see below). The molecular ion of N-acetylated oligosaccharide V in Mild acid hydrolysis (0.1 M HCl, 37"C, 24 h), which cleaves negative ion FAB-MS (Fig. 7) is represented by (M Cl-) at acetal bonds, but leaves phosphodiester and glycosidic bonds m/z = 1313, corresponding to oligosaccharide I carrying two essentially intact [58], liberated a phosphorus-containing comphosphocholine moieties. No ( M - H f ) - ion at m/z = 1277 pound which was isolated as described under Experimental was detected. A minor peak at m/z = 1295 is apparently the Procedures. As shown in Fig. 8, it was identified by GLC/ molecular ion (M' + Cl-) of the 2,5-anhydro-ribitol-contain- MS as tetrakis(trimethyl~ilyl)-[l-~H]glycero-3-phospho-2'ing derivative (see above). The fragments at m/z = 1263 11'-2H]ethyleneglycol by characteristic fragment ions at (M-15), 1245 (M'-15), andm/z=1192(M-87+1), 1174 m/z = 491 (M-CH3), 416 (M-Me3SiOH), 415 (M(M' - 87 1) are characteristic for choline residues, reflecting Me3Si02H), and 402 (M - Me3SiOCH2H). The monothe loss of CH3 and (CH2)2N+(CH3)3, respectively. The frag- deuterated glycerophosphate moiety is further indicated by ments of mjz = 923 (887 + 36) and at nzjz = 873 (923 1 5 0 ) the fragment at m/z = 462, the monodeuterated ethyleneglycol

+

+

1072

923

M+CI-: 1 3 1 3-50 -2 1295-

200

1218

1263 1245

1174

Jl I

6/ 4

873 923

743

MASS/ CHARGE

Fig. 7. Negative-ion FAB mass spectrum of the N-acetylated bis(phosphocho1ine)-carrying repeating unit (oligosaccharide V) and its corresponding fragmentation scheme.

Abundance 299

]?7

!

\

21 1

\

M-218-16 227

M'506

313

M-1f6-16

\,"

M-104

344

402

\

M-146

272

3 60

I

49 1

1, 200

400

300

500

Mass/Charge

Fig. 8. Electron impact mass spectrum of the trimethylsilylated derivative of glycerophosphoethylene glycol released by Smith degradation from the lipid- and water-soluble periodate oxidation products of lipoteichoic acid. For fragmentation of related trimethylsilylated phosphodiestercontaining compounds see [59].

phosphate moiety by the fragments at mjz = 360, 344 and 272 [58, 591. Clearly, the phosphorus-containing Smith degradation product comprises carbon atoms 4 and 5 of a ribitol and carbon atoms 4-6 of a hexopyranosyl moiety, indicating that the hydrophilic chain is attached through its ribitol 5phosphate terminus by a phosphodiester bond to C-6 of the non-reducing glucosyl terminus of the lipid anchor (Fig. 2). When the water-soluble oxidation products were subjected to mild alkaline treatment (0.05 M sodium borate pH 9.5,

37"C, 18 h), ,"-elimination converted 40% of the phosphorus into phosphomonoester. Reduction with NaB2H4 yielded a derivative which contained choline, glycerol, glycolaldehyde and phosphorus in the molar ratio 0.6: 0.43 :0.38 : 1 and only trace amounts of glucose. Mild acid hydrolysis (0.1 M HCI, 37 "C, 24 h) released two phosphorus-containing products which were separated on DEAE-Sephadex. Water eluted 61 % and 0.2M ammonium carbonate p H 8 35% of the phosphorus. The latter component was shown by GLC/MS of its

1073 0.8

I

on the deacylated glycolipid penta-, hexa-, and heptameric chains (calculated nominal values: 7056, 8334, and 9612, respectively). The broad signal between 15 and 20 kDa represents the 2 M Na' area, a common feature in this type of mass spectra [61]. Forssman antigenicity was determined by a hemolysis inhibition assay using sheep red cells and anti-pneumococcal antiserum raised in rabbits [l, 41. The results, depicted in Fig. 9, show that antigenicity, related to non-phosphocholine phosphorus, increased over most of the main peak reaching a maximum towards the end of the elution profile.

+

DISCUSSION

T U B E NUMBER

Fig. 9. Characterization of molecular lipoteichoic acid species, fractionated by column chromatography on octyl-Sepharose. Chromatography was performed as described under Experimental Procedures and the lipoteichoic acid was eluted with a propanol gradient as shown in Fig. 1. Abbreviations: Cho/P, molar ratio of choline to total phosphorus; P'/Gro, molar ratio of non-phosphocholine phosphorus to glycerol (chain length); kU/P, Forssman reactivity, determined in a hemolysis inhibition assay, expressed in units (defined by Goebel et al. [I])/pmol non-phosphocholine phosphorus.

trimethylsilylated derivative to be identical with glycerophospho-ethyleneglycol obtained from the lipid anchor. These findings confirm the above postulated location of the phosphodiester bond between the repeating units of the chain. Molecular species, chain length and Forssman antigenicity The elution profile of lipoteichoic acid from octylSepharose was analyzed as depicted in Fig. 9. Over most of the major peak the molar ratio of choline/phosphorus was close to 0.66, and alkaline hydrolysis released bis(phosph0cho1ine)-containing repeating units. In the lower descending part of the peak the molar ratio of choline/phosphorus decreased, approaching zero, and accordingly, in the alkali hydrolysate choline-free repeating units predominated (TLC as in Fig. 6). The chain length, estimated as molar ratio of non-phosphocholine phosphorus to glycerol, was close to 5 over the ascending part of the peak and then dropped to less than 2. The increase behind the minimum can not be interpreted in terms of chain length because in the later flat part of the elution profile the lipoteichoic acid was apparently contaminated with unidentified phosphorus-containing material (data not shown). Separation according to decreasing size of the hydrophilic head groups by hydrophobic interaction chromatography is not unexpected, because it has recently been demonstrated with various poly(g1ycerophosphate) lipoteichoic acids [60] (and Fischer, W., unpublished work). A sample of another lipoteichoic acid preparation, possessing bis(phosphocho1ine)-containing repeating units, was de-0-acylated, N-acetylated, and analyzed by positive-ion ultraviolet laser-induced desorption/ionization MS. The molecular ion area revealed three peaks at rnjz = 7027.2, 8337.2 (major) and 9623.5 (Fig. 10) corresponding to species carrying

The structure of pneumococcal lipoteichoic acid is summarized in Fig. 10 along with the mass spectrum of its de0-acylated and N-acetylated derivative. A tetrasaccharide, glycosidically linked to C-1 of ribitol, forms the repeating unit which is positively charged by the amino group of the AATGal residue. The repeating units are linked to each other by phosphodiester bonds between C-5 of the ribitol and C-6 of the glucosyl residue of adjacent repeating units. A phosphodiester bond links the hydrophilic chain to the nonreducing terminus of a positively charged unusual trihexosyldiacylglycerol structure which is the first glycolipid that contains an AATGal residue. In the predominating species both N-acetylgalactosaminyl residues of the repeating units are substituted at C-6 by phosphocholine residues (Fig. 9). A minor fraction of apparently shorter chain length is composed of phosphocholine-free repeating units (Fig. 10). Whether chains with both substituted and unsubstituted repeating units are also present requires further studies. It should be noted that pneumococcal lipoteichoic acid, prepared by Briese and Hakenbeck according to the original procedure [14], displayed a choline/phosphorus molar ratio of 0.64 (data not shown), in accord with repeating units each carrying two phosphocholine residues. Microheterogeneity concerns, in addition to substitution, the length of the hydrophilic chain and the fatty acid composition. As shown in Fig. 9, the chain length may vary between five and two repeating units, but with other preparations from separate bacterial batches, values between eight and four and between eight and two were observed. Using sophisticated analytical means, namely ultraviolet laser-induced desorption/ ionization MS, it became evident that the oligomer distribution of the bis(phosphocho1ine)-containing species of another lipoteichoic acid sample had its major component with six repeating units (Fig. 10). For the diacylglycerol moiety of the lipid anchor the fatty acid combinations 16:0/18:0, 16:0/16:1, 16:0/14:0, and 16: 1/14:0 were deduced from molecular ions and fragments observed in FAB-MS (Fig. 2 ) . The unusual behaviour of pneumococcal lipoteichoic acid on phenol/water extraction is not self-explanatory from its structure. For example, the poly(digalactosy1, galactosylglycerophosphate) lipoteichoic acid of Lactococcus garvieae containing 4- 12 repeating units is, albeit of similar length, on phenol/water extraction, completely recovered from the aqueous layer [63] (and Fischer, W., unpublished work). Pneumococcal lipoteichoic acid differs from all so far known lipoteichoic acids in three respects [64]. First, it contains a lipid anchor which does not occur in the free state among membrane glycolipids which have been identified in Pneurnococcus as Glc(a1 +3)acyl,Gro and Gal(al-+2)Glc-

1074

L

!

'

OH

NAc H

'

OH

M+Na+ 83,37.2

n

MOLECULAR ION CALCULATED 5

7056

M + Na+ FOUND 7027 8 3 37

7

9623

2M+Na+

2000

5000

15000

10000

20000

25000

M ASSYCHARGE

Fig. 10. Overall structure of pneumococcal lipoteichoic acid and the positive-ion ultraviolet laser-induced desorption/ionization MS of its de-0acylated and N-acetylated derivative. R1,Rz, hydrocarbon chains of fatty acids (for combinations, see text). The depicted D-ribitol5-phosphate structure is based on the established configuration in CDP-ribitol[62]. the putative biosynthetic precursor.

(21 -t3)acy12Gro [65, 661. Second, the hydrophilic chain con-

tains ribitol phosphate in place of glycerol phosphate and third, it is structurally closely related to wall teichoic acid, a so-far unique situation among Gram-positive bacteria. The structure of the repeating unit of wall teichoic acid, as proposed by Jennings et al., differs from that of lipoteichoic acid, described in the present study, only by carrying a single phosphocholine moiety and a non-N-acetylated galactosaminyl residue in linkage to ribitol[6]. In spite of this structural similarity, pulse/chase experiments with radiolabeled choline suggested no metabolic relationship at least between the completed forms of both polymers [67]. A major problem still unresolved is the immunodeterminant responsible for Forssman antigenicity of pneumococcal lipoteichoic acid. Forssman antigens, defined by their ability to induce rabbits to form hemolytic antibodies to sheep red blood cells, have been reported in a wide variety of animal organs and bacteria (for references, see [68]). The Forssman hapten glycosphingolipids from horse spleen and kidney [69], canine intestine and kidney [70],and sheep erythrocytes [68], as well as the Forssman reactive streptococcal group C polysaccharide [71], have in common a nonreducing GalNAc(ct1-+3)GalNAc~l terminus which has been established to be the immunodeterminant of Forssman reactivity. Recently pneumococcal lipoteichoic acid has been demonstrated to precipitate with anti-(streptococcal group C) antiserum which is specific for group C polysaccharide, and of 164 pneumococcal strains, examined for cross reactions with anti-(streptococcal group C) antiserum, 152 were positive [8]. --f

The present study shows that a GalNAc(al-t3)GalNAc~l+ moiety is also a structural element of pneumococcal lipoteichoic acid. Integrated into the chain and substituted with phosphocholine residues, it is unlikely to possess Forssman reactivity. Candidates for Forssman reactivity are rather incomplete terminal units or biosynthetic intermediates which may accumulate in later fractions of the elution profile from octyl-Sepharose (Fig. 9). We thank Dr Regine Hakenbeck (Berlin) for an initial sample of pneumococcal Forssman antigen, B. Stahl, Dr M. Karas, and Prof. F. Hillenkamp (Miinster) for the ultraviolet laser-induced desorptioni ionization mass spectrum, G. Distler (Erlangen) for GLC/MS analyses, Friederike Wartenberg and Prof. E. Hannappel (Erlangen) for amino acid analysis, and Birgitta Brunner and Edeltraud Ebnet for expert and reliable technical assistance. This work was supported by the Deutsche Forschun~s~emein.vchuft (Fi 21 8/4-8).

REFERENCES 1. Goebel, W. F., Shedlovsky, T., Lavin, G. I. & Adams, M. H. (1943) J . Biol. Chem. 148, 1- 15. 2. Brundish, D. E. & Baddiley, J. (1968) Biochem. J . 110, 573-5582. 3. Briles, E. B. &Tomasz, A. (1973) J . Biol. Chern. 248,6394-6397. 4. Fujiwara, M. (1967) Jpn J . Exp. Med. 37, 581 -592. 5. Watson, M. J. & Baddiley, J. (1974) Biochem. J . 137, 399-404. 6. Jennings, H. J., Lugowski, C. & Young, N . M. (1980) Biochemis-

try 19,4712-4719. 7. Goebel, W. F. & Adams, M. H. (1943) J . Exp. Med. 77, 435449.

1075 8. Ssrensen, U. B. S. & Henrichsen, J. (1987) J. Clin. Microbid. 25, 1854- 1859. 9. Ssrensen, U. B. S., Blom, J., Birch-Andersen, A. & Henrichsen, J. (1988) Infect. Immun. 56, 1890-1896. 10. Holtje, J. V. & Tomasz, A. (1975) Proc. Nut1 Acad. Sci. USA 72, 1690- 1694. 11. Holtje, J. V. & Tomasz, A. (1975) J . Bacteriol. 124, 10231024. 12. Holtje, J. V. & Tomasz, A. (1975) J. Biol. Chem. 250,6072 - 6076. 13. Tomasz, A., Westphal, M., Briles, E. B. & Fletcher, B. (1975) J . Supramol. Struct. 3, 1 - 16. 14. Briese, T. & Hakenbeck, R. (1985) Eur. J . Biochem. 146, 417427. 15. Horne, D. & Tomasz, A. (1985) J . Bacteriol. 161, 18-24. 16. Tuomanen, E., Liu, H., Hengstler, B., Zak, 0. & Tomasz, A. (1985) J . Infect. Diseases 151, 859-868. 17. Bhakdi, S., Klonisch, T., Nuber, P. & Fischer, W. (1991) lnfect. Immun. 59,4614-4620. 18. Hummell, D. S., Swift, A. J., Tomasz, A. & Winkelstein, J. A. (1985) Infect. Immun. 47, 384-387. 18a. Loos, M., Clas, F. & Fischer, W. (1986) Infect. Immun. 53,595GOO

JII.

19. Wilkinson, S. G. (1968) Biochim. Biophys. Acta 164, 148-156. 20. Jann, B. & Jann, K. (1968) Eur. J . Biochem. 5,173-177. 21. Hannappel, E., Kalbacher, EI. & Voelter, W. (1988) Arch. Biochem. Biophys. 260, 546- 551. 22. Ough, L. D. (1964) in Methods in carbohydrate chemistry (Wistler, R., ed.) vol. 4, pp. 91-98, Academic Press, New York, London. 23. Assman, G. & Schriewer, H. (1985) in Methods of enzymatic analysis (Bergmeyer, H. U., Bergmeyer, J. & Grassl, M., eds) 3rd edn, vol. 8, pp. 106- 111, Verlag Chemie, Weinheim. 24. Bok, S. H. & Demain, A. L. (1977) Anal. Biochem. 81, 18-20. 25. Schaller, K. H. & Triebig, G. (1984) in Methods of enzymatic analysis (Bergmeyer, H. U., Bergmeyer, J. & Grassl, M., eds) 3rd edn, vol. 6, pp. 668-672, Verlag Chemie, Weinheim. 26. Kunst, A,, Draeger, B. & Ziegenhorn, J. (1984) in Methods of enzymatic analysis (Bergmeyer, H. U., Bergmeyer, J. & Grassl, M., eds) 3rd edn, vol. 6, pp. 163-172, Verlag Chemie, Weinheim. 27. Niigele, U., Wahlefeld, A. W. & Ziegenhorn, J. (1985) in Methods of enzymatic analysis (Bergmeyer, H . U., Bergmeyer, J. & Grassl, M., eds) 3rd edn, vol. 8, pp. 2-12, Verlag Chemie, Weinheim. 28. Goedde, H. W. & Langenbeck, U. (1984) in Methods ofenzymatic analysis (Bergmeyer, H. U., Bergmeyer, J. & Grassl, M., eds) vol. 6, pp. 614-618, Verlag Chemie, Weinheim. 29. Strominger, J. L., Park, J. T. & Thompson, R. E. (1959) J . Biol. Chem.-234,3263 - 3268. 30. Fischer, W. (1991) Anal. Biochem. 194, 353-358. 31. Dixon, J. S. & Lipkin, D. (1954) Anal. Chem. 26, 1092- 1093. 32. Schnitger, H., Papenberg, K., Ganse, E., Czok, R., Biicher, T. & Adam, H. (1959) Biochem. 2,332,167-185, 33 , Kates, M. (1986) in Laboratory techniques in biochemistry and molecular biology (Burton, R. H. & van Knippenberg, P. H., eds) 2nd edn, p. 271, American Elsevier, New York. 34. Jacin, H. & Mischkin, A. R. (1965) J . Chromatogr. 18, 170-173. 35. Hay, G. W., Lewis, B. A. & Smith, F. (1963) J . Chromatogr. 11, 479 - 486. 36. Egge, H. & Peter-KataliniC, J. (1987) Mass Specfrom. Rev. 6, 331 -391. 37. Hillenkamp, F., Unsold, E., Kaufmann, R. & Nitsche, R. (1975) Nature 256, 119-120. 38. Karas, M., Bachmann, D., Bahr, U. & Hillenkamp, F. (1987) Int. J . Mass Spectrom. Ion Proc. 78, 53 - 68.

39. Poxton, I. R., Tarell, E. & Baddiley, J. (1978) Biochem. J . 175, 1033-1042. 40. KozuliC, B., Ries, B. & Mildner, P. (1979) Anal. Biochem. 94,3614

_I.

41. Laine, A. R. & Renkonen, 0. (1975) J . Lipid Res. 16, 102- 106. 42. Hoffman, J., Lindberg, B. & Svenson, S. (1972) Acta Chem. Scand. 26,661 -666. 43. Ciucanu, I. & Kerek, F. (1984) Carbohydr. Res. 131, 209-217. 44. Fischer, W. (1990) in Handbook of lipid research (Hanahan, D. J., ed.) vol. 6 (Kates, M., ed.) pp. 123 - 234, Plenum Press, New York and London. 45. Bligh, E. G. & Dyer, W. J. (1959) Can. J . Biochem. Physiol. 37, 911-917. 46. Fischer, W., Koch, H. U. & Haas, R. (1983) Eur. J. Biochem. 133, 523 - 530. 47. Gerwig, G. J., Kamerling, J. P. & Vliegenthart, J. F. G. (1978) Carbohydr. Res. 62, 349 - 357. 48. Gerwig, G. J., Kamerling, J. P. & Vliegenthart, J. F. G. (1979) Carbohydr. Res. 77, 1-7. 49. Lindberg, B., Lindqvist, B., Lonngren, J. & Powell, D. A. (1980) Carbohydr. Res. 78, 111 - 117. 50. Kenne, L., Lindberg, B. & Petersson, K. (1980) Carbohydr. Res. 78, 119-126. 51. Lipkin, D., Phillips, B. E. & Abrell, J. W. (1969) J . Urg. Chenz. 34,1539-1547. 52. Toon, P., Brown, P. E. & Baddiley, J. (1972) Biochem. J . 127, 399 - 409. 53. Fischer, W. (1987) Eur. J . Biochem. 165, 639-646. 54. Kuhn, R., Gauhe, A. & Baer, H. H. (1954) Chem. Ber. 87,11381141. 55. Baer, E. & Kates, M. (1950) J . Biol. Chem. 185, 61 5-623. 56. Ukita, T., Bales, N. A. & Carter, H. E. (1955) J. Biol. Chem. 216, 867 - 874. 57. Archibald, A. R. & Baddiley, J. (1966) in Advances in carbohydrate chemistry (Wolfrom, M. L. & Tipson, R. S., eds) vol. 21, pp. 323 - 375, Academic Press, New York. 58. Fischer, W. & Landgraf, H. R. (1975) Biochim. Biophys. Acta 380, 227 - 244. 59. Duncan, J. H., Lennarz, W. J. & Fenselau, C. C. (1971) Biochemistry 10, 927 - 932. 60. Leopold, K. & Fischer, W. (1992) Anal. Biochem. 201,350-355. 61. Karas, M. & Hillenkamp, F. (1988) Anal. Chem. 60, 22992301. 62. Baddiley, J., Buchanan, J. G. & Grass, B. (1957) J . Chem. Sor., 1869. 63. Koch, H. U. & Fischer, W. (1978) Biochemisfry 17, 52755281. 64. Fischer, W. (1988) in Advances ofmicrobialphysiology (Rose, A . H. & Tempest, D. W., eds) vol. 29, pp. 233-302, Academic Press, London. 65. Kaufman, B., Kundig, F. D., Distler, J. & Roseman, S. (1965) Biochem. Biophys. Res. Commun. 18, 312-318. 66. Brundish, D. E., Shaw, N. & Baddiley, J. (1967) Biochem. J . 104, 205 -21 1. 67. Briles, E. B. & Tomasz, A. (1975) J . Bacteriol. 122, 335-337. 68. Fraser, B. A. & Mallette, M. F. (1974) Zmmunochemistry 11, 581 593. 69. Siddiqui, B. & Hakomori, S. I. (1971) J . Biol. Chem. 246, 57665769. 70. Sung, S. J., Esselman, W. J. & Sweeley, C. C. (1973) J . Biol. Chem. 248,6528-6533. 71. Coligan, J. E., Fraser, B. A. & Kindt, T. J. (1977) J . Zmmunol. 118,6--11.