Received on January 22, 1999; revised on April 14, 1999; accepted on April 14, 1999
Lipopolysaccharides (LPSs) from strains of Helicobacter pylori (442 and 471), which differed in stimulation of pepsinogen secretion, were isolated as water-soluble material of high-Mr, and as water-insoluble gels of low-Mr. Chemical and spectroscopic analyses of soluble LPS and oligosaccharides liberated from the gels led to proposed structures with Lewis (Le) antigen termini connected to N-acetyllactosaminoglycans of alternating 3-linked [beta]-D-Gal and 4-linked [beta]-D-GlcNAc residues with various laterally attached glycosyl substituents. The LPS of H.pylori 442 was similar to previously examined strains (NCTC 11637 and P466) in having partially glycosylated chains with [alpha]-L-Fuc units attached to O-3 of the majority of GlcNAc residues in Lexunits, and in chain termination with Lex or Ley determinants. In contrast, terminal Ley units occurred in LPS of H.pylori 471 and glycosaminoglycan chains carried a smaller proportion of [alpha]-L-Fuc units, but at O-6 of a majority of nonfucosylated GlcNAc residues, there was a novel type of branching with [alpha]-D-Gal substituents. Evidence for the branched regions was obtained from 1H-NMR spectra and from characterization of oligosaccharides formed by the action of endo-[beta]-galactosidase. Examination of oligosaccharides liberated from water-insoluble LPS gels of H.pylori 442 and 471 provided evidence for similar core OS structures to those from other H.pylori strains but interesting differences were observed.
Key words: Helicobacter pylori/lipopolysaccharides/Lewis antigens/structure determination/pepsinogen secretion
Lipopolysaccharides (LPSs) are an important group of bacterial cell surface carbohydrate components which often interact specifically with surface components of an infected host. In the case of Helicobacter pylori, the bacterium has been variously implicated as a causative agent of gastritis, gastric and duodenal ulcers, and gastric carcinoma (Cover and Blaser, 1995). H.pylori LPS possesses low endotoxic activity, induces a low immunological response, and has been implicated in a variety of biological interactions; these include an inhibitory effect on mucus glycosylation, interference with mucosal integrity, the stimulation of pepsinogen secretion, and a role in mediating adherence of the bacterium to laminin in the basement membrane (Moran, 1996a,b). These effects, however, were noted without regard to possible structural differences between H.pylori LPSs.
Evidence that such structural differences occur was obtained in studies in which H.pylori strains were differentiated by LPS electrophoretic patterns in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and in immunoblotting antigenic analyses with strain-specific antisera (Mills et al., 1992). In experiments of a different type (Borén et al., 1993), differences were reported in the binding of H.pylori strains to human gastric epithelial cells expressing the Lewisb (Leb) antigen. The binding of one such strain, H.pylori P 466 from a patient with dyspeptic symptoms, to gastric epithelium in situ was inhibited by the Leb antigen, whereas the MO19 strain, from an asymptomatic patient, did not bind to the epithelium (Borén et al., 1993; Falk et al., 1993). Although LPS was not implicated in the interaction or lack of interaction of these H. pylori strains with the gastric mucosa, these observations and those from the antigenic variant studies prompted a series of investigations of LPS from these strains.
The first study was of the LPS from the H.pylori type strain (NCTC 11637), provisionally assigned to serotype O:1 (Mills et al., 1992), and led to a proposed structure for the complete polysaccharide component of the smooth LPS comprising the O-antigen chain, a short intervening region and the inner core oligosaccharide (Aspinall et al., 1996). The most striking feature of the O-antigen region was the presence of extended chains with fucosylated and nonfucosylated N-acetyllactosamine, ->3-[beta]-D-Gal-(1->4)-[beta]-D-GlcNAc-(1- (LacNAc), units that mimic cell surface glycoconjugates on normal granulocytes (Fukuda et al., 1985). These chains were terminated by di- or trimeric Lewisx (Lex) determinants, which are also found in tumor-associated carbohydrate antigens in many adenocarcinomas (Hakomori, 1989) and are expressed in the normal gastric mucosa (Sakamoto et al., 1989).
Examination of LPS from H.pylori P466 and MO19 provided evidence for structural differences between these strains (Aspinall and Monteiro, 1996). LPS from strain P466, which bound to gastric mucosal surfaces expressing the Leb antigen, contained a partially fucosylated N-acetyllactosaminoglycan of rather shorter chain length than in the type strain and was terminated by the isomeric Lewis (Ley) determinant. The MO19 LPS was also terminated by the Ley determinant, contained the same core oligosaccharide (OS) region as in the P466 LPS, but the extended N-acetyllactosaminoglycan region was absent and replaced with oligomeric chains of D-glycero-[alpha]-D-manno-heptose (DD-Hep) residues, as a block in (1->3) linkages with single residues in (1->2) and (1->6) linkages in a hitherto undefined sequence. Furthermore, LPS from H.pylori serotype O:3 (Mills et al., 1992) has chains expressing both Lex and Ley epitopes which terminate extended partially fucosylated N-acetyllactosaminoglycan regions, and which in turn are linked to a similar heptan segment and thence to the inner core (Aspinall et al., 1997). Consequently the structural variations between LPS from different H.pylori strains in their expression of Lewis antigens have been utilized as an aid in serotyping (Simoons-Smit et al., 1996). Even so, the last words have not been spoken on H.pylori LPS structural variations. The lactosaminoglycan backbone chain in H.pylori UA861 has been shown to carry side-chains of single [alpha]-D-glucopyranose residues attached at O-6 of GlcNAc residues in replacement of most [alpha]-L-Fuc side-chains at O-3 (Monteiro et al., 1998a). The expression of type 1 Lewis blood group antigens has been detected using monoclonal antibodies and partial support for this further diversity has been obtained in chemical structural studies (Monteiro et al., 1998b).
A biological interaction in which LPS of some H.pylori strains has been implicated is the stimulation of pepsinogen secretion (Young et al., 1992). Elevated concentrations of serum pepsinogen I, the endocrine component of pepsinogen secreted by chief cells, are considered to be a major risk factor for both the development and recurrence of duodenal ulcers (Samloff et al., 1986). Infection with H.pylori occurs in about 90% of patients with duodenal ulcer disease, and eradication of infection is associated with a markedly decreased incidence of duodenal ulcer relapse (Kuipers et al., 1995). Furthermore, increased levels of serum pepsinogen I are found in more than 50% of duodenal ulcer patients (Richardson, 1990), but eradication of H.pylori is associated with a decrease of these levels (Wagner et al., 1991). H.pylori cell sonicates, but in particular LPS of duodenal ulcer H.pylori strains has been shown to induce pepsinogen secretion from guinea pig mucosa in Ussing chambers in vitro (Cave and Cave, 1991; Young et al., 1992; Moran and Aspinall, 1998). We report here a structural study from two strains, of which one (strain 442) does and the other (strain 471) does not stimulate pepsinogen.
Bacterial cell extracts were divided into soluble high-Mr LPS with extended outer chains and insoluble low-Mr LPS. Soluble LPS was treated with 1% acetic acid under mild conditions to cleave the sensitive glycosidic linkage from Kdo to lipid A with minimal loss of fucose residues, and gel permeation chromatography (GPC) yielded PS-1 of high-Mr. Similar treatment of insoluble LPS liberated oligosaccharide fractions, OS-1, OS-2, and OS-3 which were separated by GPC. Subsequently, later fractions from GPC of PS-1 were found to be a more abundant source of OS-2, of similar composition andstructure to that from the insoluble LPS. Heterogeneity was apparent in polysacccharide and oligosaccharide fractions, and compositional and linkage analysis gave only average values for constituent residues. However, since each type of constituent sugar, deoxyhexose (dHex), hexose (Hex), N-acetylhexosamine (HexNAc) and heptose (Hep), was of different mass, fast atom bombardment (FAB) spectra of pseudomolecular ions and fragment ions for permethylated derivatives, in conjunction with linkage-type information, provided precise estimates of composition, and often of the structure of individual components of mixtures. For LPS of each strain, compositional and linkage-type analyses are reported and give overall information on the outer regions of structure inclusive of repeating structures. In addition, chemical and/or enzymic degradations are described, and together with experiments on liberated oligosaccharide fractions of low Mr, reveal fine structure and/or connections between different regions.
Characterization of water-soluble LPS of high Mr from H.pylori 442
PS-1 (442) from mild acid treatment of water-soluble LPS had the following approximate sugar composition, L-Fuc, D-Gal, and D-GlcNAc in the molar ratio of 4:10:9, with smaller proportions (~2 each) of D-Glc, LD-Hep, and DD-Hep. Enantiomeric configurations of the constituent sugars were established by the chiral glycoside method (Leontein et al., 1978). The anomeric configurations of the principal sugar residues shown in the 1H-NMR spectrum were defined in 1H-1H COSY and 1-D TOCSY experiments as those of [alpha]-Fuc ([delta]H-1 5.05 ppm, J1,2 3.8 Hz), [beta]-Gal ([delta]H-1 4.45 ppm, J1,2 7.6 Hz), and [beta]-GlcNAc ([delta]H-1 4.60 ppm, J1,2 7.2 Hz), unresolved anomeric resonances in the [delta]H-1 range 5.0-5.2 were assigned to LD- and DD-Hep residues with the [alpha]-manno configuration. The 31P NMR spectrum for PS-1 (442) showed a single resonance at pD 6 assignable to a phosphoric monoester.
Linkage analysis performed on permethylated PS-1 (442) showed the presence of terminal Fuc, 3-linked Gal, 4-linked and 3,4-linked GlcNAc residues in the approximate molar ratio of 3:6:4:3, with approximately 1 molar proportion each of 4-linked Gal, and variously substituted Glc, DD-Hep, and LD-Hep units arising from the inner core region of the LPS (Table I). Fractional amounts of other sugar residues were also observed as constituents of structurally variable regions. The major constituents were consistent with the presence of an N-acetyllactosaminoglycan with 3-linked Gal and 4-linked GlcNAc residues with approximately half of the latter carrying at O-3 terminal Fuc residues. Confirmation that the backbone consisted of regular Gal(1->4)GlcNAc repeating units was obtained from a Smith degradation of PS-1 affording a linear degraded glycan PS-2 (442), for which supporting data was derived from linkage analysis (Table I) in conjunction with FAB-MS of the permethylated derivative (Figure 1). A series of fragment ions was observed arising from alternating sequences of 3-linked Gal and 4-linked GlcNAc residues in chains with Gal and GlcNAc terminal residues and consisting of up to five disaccharide repeating units. The removal of Fuc residues as side-chains during the Smith degradation showed that these were attached laterally to O-3 of GlcNAc residues. The delineation of the chain termini was less clearcut and required that the presence of 2-linked Gal residues in less than equimolar proportions in the parent glycan should be taken into account. FAB-MS data for permethylated PS-1 (442) showed fragment ions from chain termini at m/z 464 (Hex, HexNAc), 638 (dHex, Hex, HexNAc), 812 (dHex2, Hex, HexNAc). The detection of secondary ions at m/z 432 and 606 suggested their formation from ions at m/z 638 and 812, respectively, with loss of Fuc residues by [beta]-elimination from O-3 of GlcNAc. These observations were consistent with the presence of Lex and Ley determinants, and some nonfucosylated chain termini.
Fig. 1. Analysis of positive-ion FAB-MS data for the permethylated derivatives of O-antigen chain PS-1 from LPS of H.pylori 442, the products PS-2 from Smith degradation, and oligoglycosyladitols from the action of endo-[beta]-galactosidase on PS-1 followed by treatment with NaBD4. The subscript n refers to the number of LacNAc units in PS-2 arising from original fucosylated(x) and nonfucosylated(y) units.
Table I. Methylation linkage analysis of LPS (and PS-I), PS-2, PS-3, OS-2, OS-3, and OS-4 from H.pylori 442
Structural units from methylation analysis | Approx. molar ratiosa | |||||
LPS(PS-1) | PS-2 | PS-3 | OS-2 | OS-3 | OS-4 | |
O-Antigen region | ||||||
Fuc | 4 | 0 | tr | 1 | 0 | 0 |
Gal | 1 | 0.5 | 1 | tr | tr | 0 |
->2)-Gal | tr | 0 | 0 | 0 | 0 | 0 |
->3)-Gal | 4 | 4 | 4 | 0 | 0 | 0 |
GlcNAc | 0 | 1 | 0 | 0 | 0 | 1 |
->3)-GlcNAc | tr | tr | tr | 1 | 0 | 0 |
->4)-GlcNAc | 1 | 4 | 4 | tr | 0 | 0 |
-3/4)-GlcNAc | 3 | 0 | 0.5 | 0 | 0 | 0 |
Intervening and outer core regions | ||||||
Glc | 1 | 0 | 1 | 1 | 1 | 1 |
->3)-Glc | 0.5 | 0 | 1 | 0.5 | 0 | 0 |
->4)-Gal | 1 | 0 | 1 | 1 | 1 | 1 |
->7)DD-Hep | 1 | 0 | 1 | 1 | 0.5 | 1 |
->2/7)DD-Hep | 0.5 | 0 | 0.5 | 0.5 | 0 | 1 |
->2)LD-Hep | 0.5 | 0 | 0.5 | 1 | 1 | 1 |
Attempts to define inner regions of the O-antigen employed endo-[beta]-galactosidase to cleave selectively unbranched segments of N-acetyllactosaminoglycans (Kannagi et al., 1982). Enzymic digestion of PS-1 followed by treatment with NaBD4 yielded galactitol-terminated oligosaccharides for fractionation by GPC. The fractions were methylated for linkage analysis and for direct examination of the intact permethylated derivatives by FAB-MS. Despite incomplete separations of oligoglycosylalditols, but with knowledge of the specificity of the enzyme which yields at each site of action a 3-O-substituted galactitol-terminated oligosaccharide from the chain extrema, and from the internal regions, oligosaccharides with nonreducing GlcNAc end groups, the compositions of four glycosylalditols could be defined from the pseudomolecular ions [M + H]+ of their permethylated derivatives (Figure 1). Two oligosaccharides whose permethylated derivatives had pseudomolecular ions [M + H]+ at m/z 513 and 963 had terminal GlcNAc residues and thus arose from unbranched internal regions. The other two oligosaccharides contained Fuc residues. The permethylated glycosylalditol having [M + H]+ at m/z 891 showed a fragment ion at m/z 638 characteristic of the Lex terminus. The composition [GlcNAc2, Fuc, Gal, Galol] of the permethylated derivative with [M + H]+ at m/z 1136 required this glycosylalditol to have a terminal nonfucosylated GlcNAc residue and to result from internal cleavage of the chain. The formation of a fragment ion at m/z 883 of composition GlcNAc2, Fuc, Gal would therefore carry the Fuc residue at O-3 of the GlcNAc glycosyloxonium ion from which the ready loss of Fuc (206 amu) by [beta]-elimination was seen in the formation of an ion at m/z 677.
For the second enzymic degradation, treatment of PS-1 (442) with 5% aqueous acetic acid at 100° for 2 h yielded PS-3 (442), for which compositional analysis showed nearly complete defucosylation. The action of endo-[beta]-galactosidase on PS-3 (442) proceeded with erosion of the N-acetyllactosaminoglycan chain yielding OS-4 (442) which consisted of the innermost GlcNAc residue of the outer chain, the intervening DD-Hep residues and the core OS region (Table I). FAB-MS of permethylated OS-4 (442) confirmed the presence of a terminal GlcNAc residue from the glycosyloxonium ion at m/z 260, showed a fragment ion at m/z 508 indicative of the outer disaccharide GlcNAc-DDHep, and secondary ions at m/z 724 and 1132 which may be assigned to those resulting from cleavage of the DDHep-LDHep linkage in the inner core region followed by elimination of methanol (Figure 2). These results were consistent with a connection of the O-antigen chain through to a core OS region similar to that in the LPS from the type strain (NCTC 11637) (Aspinall et al., 1996).
Fig. 2. Structures for core oligosaccharide regions from H.pylori 442: (A) OS-2 (442) and in the hatched area OS-3 (442) from insoluble low Mr LPS; (B) OS-4 (442) formed from PS-1 of H.pylori 442 by defucosylation and the action of endo-[beta]-galactosidase; from a separate extraction (C) OS-2* (442) and (D) OS-3* (442). Selected 1H NMR data are for the oligosaccharides and FAB-MS data are for their permethylated derivatives.
Characterization of water-soluble LPS of high Mr from H.pylori strain 471
The examination of PS-1 (471) from the mild acid treatment of water-soluble LPS pointed to a structural feature not present in the 442 strain or in the type strain (NCTC 11637). The approximate sugar composition, L-Fuc, D-Gal, and D-GlcNAc in the molar ratio of 3:10:6, with smaller proportions (~2 each) of D-Glc, LD-Hep, and DD-Hep, showed Gal markedly in excess of the GlcNAc residues required in an alternating sequence in an N-acetyllactosaminoglycan backbone. Enantiomeric configurations of the constituent sugars were established by the chiral glycoside method (Leontein et al., 1978), and confirmed that those of Gal were only those of the D-enantiomer. The linkage analysis (Table II) showed several of the same building units as in H.pylori 442, notably terminal Fuc, 3-linked Gal, 4-linked and 3,4-linked GlcNAc residues. The most striking differences, however, were the high proportion of Gal end groups and the presence of 4,6-linked GlcNAc residues indicative of lateral attachment of Gal residues at O-6 of GlcNAc. The results indicated that the N-acetyllactosaminoglycan in this O antigen chain carried on some of the GlcNAc residues two types of glycosyl side-chains which were attached at different sites. The correctness of this interpretation was supported when PS-1 (471) was submitted to a Smith degradation (Figure 3) which yielded a linear degraded glycan PS-2(471). As for the corresponding glycan PS-2(442), evidence for regular Gal(1->4)GlcNAc repeating units was obtained from linkage analysis (Table II) in conjunction with FAB-MS of the permethylated derivative (Figure 3). Fragment ions were observed for alternating sequences of 3-linked Gal and 4-linked GlcNAc residues in chains consisting of up to five repeating units. This degradation also resulted in removal of Fuc residues whose linkage to O-3 of GlcNAc residues was apparent from the linkage analysis of PS-1 (471). As for permethylated PS-1 (442), FAB-MS data for permethylated PS-1 (471) showed fragment ions from chain termini at m/z 638 (dHex, Hex, HexNAc) and 812 (dHex2, Hex, HexNAc). The detection of secondary ions at m/z 432 and 606 pointed to their formation from ions at m/z 638 and 812 respectively with loss of Fuc residues by [beta]-elimination from O-3 of GlcNAc. The relationships of daughter to parent ions were confirmed in MS-MS experiments. These observations were consistent with the presence of Lex and Ley determinants, but the detection also of fragment ions at m/z 464 (Hex, HexNAc) and 668 (Hex2, HexNAc) pointed to some nonfucosylated chain termini.
Fig. 3. Analysis of positive-ion FAB-MS data for the permethylated derivatives of the O-antigen chain PS-1 from LPS of H.pylori 471, the products PS-2 from Smith degradation, and PS-3 from defucosylation. The subscript n refers to the number of LacNAc units in PS-2 arising from original glycosylated(x+y) and nonfucosylated(z) units.
Table II. Methylation linkage analysis of LPS (and PS-1), PS-2, PS-3, OS-2, OS-3 and a mixture of OGAs from H.pylori 471
Structural units from methylation analysis | Approx. molar ratiosa | |||||
LPS( PS-1) | PS-2 | PS-3 | OS-2 | OS-3 | OGAsb | |
O-Antigen region | ||||||
Fuc | 3 | 0.5 | tr | tr | 0 | + |
Gal | 6 | 0.5 | 6 | 0.5 | 0 | + |
->3)-Fuc | tr | 0 | 0 | 1 | 0 | |
->2)-Gal | tr | 0 | 0 | 0 | 0 | |
->3)-Gal | 5 | 4 | 5 | 0 | 0 | + |
->3)-Galol-1-d | + | |||||
GlcNAc | 0 | 1 | 1 | tr | 0 | + |
->3)-GlcNAc | 0 | 0 | 0 | 0.5 | 0 | |
->4)-GlcNAc | 1 | 4 | 1 | 0 | 0 | + |
->6)-GlcNAc | + | |||||
->3/4)-GlcNAc | 1 | 0 | tr | 0 | 0 | + |
->4,6)-GlcNAc | 3 | 0 | 3 | 0 | 0 | + |
Intervening and outer core regions | ||||||
Glc | 1 | 0 | 1 | 1.5 | 1 | |
->3)-Glc | 1 | 0 | 1 | 0.5 | 0 | |
->4)-Gal | 1 | 0 | 1 | 1.5 | 1 | |
->7)DD-Hep | 1 | 0 | 1 | 1 | 1 | |
->2/7)DD-Hep | tr | 0 | 0 | 1 | 0 | |
->2)LD-Hep | tr | 0 | 1 | 0 | 0.5 |
The anomeric configurations of the principal sugar residues shown in the 1H-NMR spectrum of PS-1 (471) were defined in 1H-1H COSY and 1-D TOCSY experiments as those of [alpha]-Fuc ([delta]H-1 5.01, J1,2 3.8 Hz), [beta]-Gal ([delta]H-1 4.45, J1,2 6.6 Hz), and [beta]-GlcNAc ([delta]H-1 4.68, J1,2 7.6 Hz), and unresolved anomeric resonances in the [delta]H-1 range 5.0-5.2 were assigned to LD-and DD-Hep residues with the [alpha]-manno configuration. In addition, an anomeric resonance at [delta]4.98 (J1,2 2.6 Hz) was assigned to the laterally attached [alpha]-D-Gal residues despite an atypical TOCSY spectrum in which H-2 and H-3 showed no vicinal couplings.
In order to remove uncertainty concerning the identity of the postulated [alpha]-D-Galp residues as single unit side-chains attached to C-6 of GlcNAc residues the following NMR experiments were performed on PS-3 (471) from which the majority of [alpha]-L-Fuc residues had been removed by selective hydrolysis with acetic acid. Assignments of proton resonances from 1H-1H COSY and 1-D TOCSY experiments for the residues in the repeating branched trisaccharide unit of PS-3 (471) are shown in Table III. The 13C NMR spectrum showed two barely resolved anomeric carbons at [delta]C 104.2 (JC,H 165 Hz) and a further anomeric carbon at [delta]C 100.0 (JC,H 171 Hz). Correlations of the carbon with proton resonances through an inverse 1H-13C-HMQC experiment showed that the resonances were, respectively, those of [beta]-D-GlcNAc, [beta]-D-Gal, and [alpha]-D-Gal residues. Two further correlations were of C-2 of the GlcNAc residue at the characteristic [delta]C 56 and, in conjunction with a related JMODXH spectrum, the down field shifted glycosylated C-6 of the 4,6-branched GlcNAc residue was observed at [delta]C 67.5. Supporting evidence for the galactosylated N-acetyllactosaminoglycan structure of PS-3 (471) was obtained from a 2D 1H-1H NOESY experiment which showed the following nuclear Overhauser contacts in the lactosaminoglycan backbone and for the lateral attachment of the [alpha]-D-Gal residue to C-6 of the [beta]-D-GlcNAc residue (Figure 4): (1) from H-1 ([delta] 4.68) of [beta]-GlcNAc to H-3 ([delta] 3.68) of [beta]-Gal; (2) from H-1([delta] 4.45) [beta]-Gal to H-4 ([delta] 3.60) of [beta]-GlcNAc; and (3) from H-1 ([delta] 4.98) of [alpha]-Gal to H-6 and H-6[prime] ([delta] 3.90, 4.15) of [beta]-GlcNAc. An intra-residue n.O.e. from H-1 ([delta] 4.98) to H-2 ([delta] 3.79) of the [alpha]-Galp residue was in accord with the assigned anomeric configuration.
Fig. 4. Structure of the repeating unit of the O-antigen region of LPS from H.pylori 471 with interpretations of selected nuclear Overhauser effect interaction in the 1H-NMR spectrum of defucosylated PS-3.
Table III. 1H- and selected 13C-NMR chemical shift data (p.p.m.) from 2D-COSY, 1D- and 2D-TOCSY, and HMQC for assignments of anomericaand ringb configurations of residues in PS-3 (471)
Residue | H-1 (J1,2, Hz) | H-2 | H-3 | H-4 | H-5 | H-6 | H-6[prime] | C-1 (JC-1,H-1, Hz) | C-2 | C-6 |
[beta]-Gal | 4.45 (6.6) | 3.55 | 3.68 | 4.09 | 3.60 | 3.68 | 3.69 | 104.19 (165) | ||
[beta]-GlcNAc | 4.68 (7.6) | 3.72 | 3.85 | 3.60 | 3.55 | 3.90 | 4.09 | 104.43 (165) | 56.0 | 67.5 |
[alpha]-Galc | 4.98 (2.6) | 3.79 | 3.95 | 4.16 | 100.00 (171) | 68.0 |
Information on the mode of attachment and distribution of [alpha]-Gal residues along the poly-LacNAc chain was sought from the action of endo-[beta]-galactosidase on PS-3 (471), from which a high proportion of the Fuc residues had been cleaved after treatment of PS-1 (471) with 5% acetic acid at 100°C for 2 h. The products from the action of the enzyme were treated with NaBD4, and although no individual oligosaccharides could be isolated, partial separation by GPC on Bio-Gel P-2 yielded mixtures of oligoglycosylalditols whose methylated derivatives were suitable for examination by FAB-MS and for linkage analysis. Linkage analysis revealed the exposure of 3-linked 2H-labeled galactitol termini at the "reducing" ends and, at the nonreducing ends, of terminal and 6-linked GlcNAc residues respectively from some of the formerly 4-linked and 4,6-branched residues. Without prior knowledge of the specificity of the enzyme towards the branched substrate, it was apparent that limited depolymerization had occurred with hydrolysis of some [beta]-galactosyl linkages to branched GlcNAc residues in the N-acetylglucosaminoglycan but with retention of the [alpha]-D-galactopyranosyl residues.
With knowledge of the constituent units, pseudomolecular ions [M+H]+ for the intact permethylated glycosylalditols defined the compositions and, in some cases, the sequences of sugar units may be advanced with confidence (Figure 5). Disaccharide 1, GlcNAc(1->3)Galol (m/z 513) arose from an unbranched region of the N-acetyllactosaminoglycan backbone. Two possible structures may be advanced for trisaccharide 2, Gal->GlcNAc->Galol (m/z 709), which could originate either from a Lewis antigen terminus which had undergone defucosylation, or from internal chain cleavage with retention of the [alpha]-galactosyl side-chain. Tetrasacccharide 3 (m/z 963) has a structure, (GlcNAc->Gal->GlcNAc->Galol), dictated by the alternating sequence of the backbone chain, whereas with present mass spectral data two possible structures for pentasaccharide 4 (m/z 1166) cannot be distinguished (Figure 5, OGA 4). Unambiguous structures may be advanced for hexasaccharides 5 (m/z 1370) and 6 (m/z 1340) (Figure 5, OGA 5 and OGA 6). The composition for 5 requires attachment of [alpha]-Gal residues as side-chains on both GlcNAc units. The formation of 5 with the apparent inability of the endo-[beta]-galactosidase to hydrolyze the [beta]-Gal linkage to this branched GlcNAc residue suggests that the specificity of the enzyme in densely branched regions of the galactosylated lactosaminoglycan cannot be defined without reference to changes in the backbone chain conformation with the progressive introduction of [alpha]-Gal side-chains at neighboring GlcNAc residues. A comparable, but not directly related, situation has been observed in relation to the action of endo-1,4-[beta]-D-mannanases on galactomannans (McCleary et al., 1985). The proposed structure for hexasaccharide 5 with the [alpha]-Gal residue at the distal terminus is the only possibility since endo-[beta]-galactosidase is unable to cleave bonds to fucosylated GlcNAc residues (Kannagi et al., 1982; Aspinall et al., 1996). The characterization of this hexasaccharide, which resulted from the less than complete defucosylation of PS-1, is of importance in giving evidence for the attachment of [alpha]-Gal and [alpha]-Fuc residues as side-chains in the same lactosaminoglycan O-antigen chains.
Fig. 5. Representative action of endo-[beta]-galactosidase on partially defucosylated PS-3 (471) with analysis of the FAB-MS of permethylated oligoglycosylallditols (OGAs 4, 5, and 6) from H.pylori 471.
The FAB-MS data also included the many glycosyloxonium ions whose formation would be expected from the above oligoglycosylalditols. Glycosyloxonium ions were observed at m/z 724, 928, and 1132 whose formation had been ascribed to the presence of the proposed OS-4 (442) (Figure 2B) formed from the defucosylated PS-3 (442) of the same H.pylori strain and previously from LPS of the H.pylori type strain (Aspinall et al., 1996). This less than complete degradative evidence for the connecting region between the innermost GlcNAc residue of the O-antigen and DD-Hep residues in the outer core, when taken in conjunction with that for the core OS-2 (471), lends support for a similar connection in H.pylori 471.
Examination of core oligosaccharides liberated from water-insoluble LPS
Treatment of the water-insoluble LPS from H.pylori 442 and 471 with aqueous 1% acetic acid in an identical manner as that for soluble LPS followed by GPC on Bio-Gel P-2 afforded in each case three fractions in quantities that allowed only partial characterization. The compositions and linkage analyses of the first fractions OS-1 (442) and OS-1 (471) showed that these were not significantly different from those of the respective water-soluble LPS of highest Mr and were not further examined. The compositions of OS-3 (442) and OS-3 (471) (Figure 2A) showed that GlcNAc was absent and together with linkage analyses pointed to inner core regions similar to those from LPS of other H.pylori strains. 1H-NMR assignments from 1H-1H COSY and 1-D TOCSY experiments for OS-3 and OS-2 (Table IV, Figure 2) confirmed the structure of the outer residues in OS-3, and served to define key resonances for the corresponding Glc and Gal residues in OS-2. The OS-3 fractions were heterogeneous, small in amount, and included both complete and incomplete molecules devoid of Glc and Gal residues, as shown by the presence of terminal nonreducing LD-and DD-Hep residues in the linkage analysis in amounts too small for inclusion in Table I. Furthermore, the presence of terminal Hep glycosyloxonium ions at m/z 263 and homologous oligomeric ions at m/z 479 and 759 in the mass spectra (FAB and ESI) of some permethylated oligosaccharides, e.g., OS-3* (442) (Figure 2D) pointed to several incomplete core molecules and could explain the observation of multiple resonances assigned to anomeric protons of [alpha]-Hep residues in the 1H NMR spectra for OS-3 in Table IV.
Table IV. 1H NMR chemical shift data (PPM) for assignments of anomerica and ringb configurations in OS-2 and OS-3 fractions from strains 442 and 471c
Residue | H-1 (J1,2, Hz) | H-2 | H-3 | H-4 | H-5 |
OS-2 | |||||
[alpha]-Hep | 5.35 (bsd) | 4.05 | |||
[alpha]-Fuc | 5.02 (bs) | 4.00 | 4.20 | ||
[alpha]-Hep | 4.99 (bs) | 4.15 | 4.30 | ||
[alpha]-Glc | 4.98 (3.4) | 3.78 | 3.95 | 4.12 | |
[alpha]-Glc | 4.92 (3.4) | 3.50 | 3.70 | 3.40 | 3.75 |
[beta]-GlcNAc | 4.60 (7.2) | 3.58 | 3.84 | ||
[beta]-GlcNAc | 4.54 (7.6) | 3.80 | 3.90 | ||
[beta]-Gal | 4.45 (7.6) | 3.55 | 3.70 | 3.98 | |
OS-3 | |||||
[alpha]-Hep | 5.35 (bs) | 3.90 | |||
[alpha]-Hep | 5.32 (bs) | 3.90 | |||
[alpha]-Hep | 4.90 (bs) | 3.96 | |||
[alpha]-Hep | 4.90 (bs) | 3.85 | |||
[alpha]-Glc | 4.82 (3.7) | 3.42 | 3.70 | 3.39 | 3.68 |
[beta]-Gal | 4.40 (7.4) | 3.50 | 3.68 |
Compositional and linkage analysis for OS-2 (442) showed this fraction to be that of lowest mass containing a GlcNAc residue. The fraction may be regarded as resulting from (1) chain extension of the linear core OS in OS-3 with a 3-linked Glc residue, and (2) the lateral chain attachment of a GlcNAc(1->7)-DD-Hep unit from which the major synthetic pathway would be O-antigen chain development. Most of the OS-2 (442) fraction carries the terminal trisaccharide unit, Fuc-(1->3)-GlcNAc-(1->7)-DD-Hep, as shown in the FAB-MS of permethylated OS-2, by the detection of a prominent fragment ion at m/z 682, whose presence was consistent with linkage analysis data, and may be regarded as resulting from premature fucosylation of some GlcNAc residues and may result in the terminal trisaccharide unit. Supporting evidence that this OS-2 (442) was indistinguishable from the corresponding oligosaccharide isolated from the type strain (Aspinall et al., 1996) was obtained in a 2D-NOESY experiment showing the following inter-residue connectivities: (1) in the side-chain from H-1 ([delta] 5.02) of [alpha]-Fuc to H-3 ([delta] 3.84) of [beta]-GlcNAc, from H-1 ([delta] 4.54) of [beta]-GlcNAc to H-7,7[prime] ([delta] 4.59) of a Hep residue (Müller-Loennies et al., 1994); and (2) in the extended core OS backbone from H-1 ([delta] 4.98) of the outer [alpha]-Glc residue to H-3 ([delta] 3.70) of the inner [alpha]-Glc residue (Figure 2A). Very similar results were obtained in the examination of the corresponding OS-2 fraction isolated from H.pylori 471. This fraction was similar to OS-2 (442), but differed in that the single Fuc residue escaped methylation at O-3 and suggested the presence of a glycosyl substituent. Similar observations have been made for 3-substituted Fuc residues in other H.pylori LPS derivatives, but in this, the most prominent example, NMR data from a 2D NOESY experiment on the two OS-2 samples failed to reveal evidence for any glycosyl substituent.
For the completion of the present investigation a new sample of OS-2 (442) for mass spectral analysis was generated from a fresh LPS isolate from H.pylori 442. This OS-2 (442) fraction proved to be deficient in the outer Gal and Glc residues of the core backbone. FAB-MS of the permethylated derivative showed a pseudomolecular ion [M+H]+ at m/z 1734 of composition dHex, HexNAc, Hep4, Kdo, with related ions [(M+H)-46]+ at m/z 1688, [(M+H)-116]+ at m/z 1618 and, in small amount, for a phosphorylated derivative at m/z 1828. The fragment ions defined the terminal trisaccharide unit, Fuc-(1->3)-GlcNAc-(1->7)-DD-Hep (m/z 682). Other heptose-terminated glycosyloxonium ions confirmed linkage sequences in the heptose region of the inner core with the formation of daughter ions by loss of methanol by [beta]-elimination from O-3 of oligomers not glycosylated at that site (Figure 2C). Similar results were obtained from electrospray ionization-mass spectrometry (ESI-MS) where the major ions in a group of different adducts included [M+H]+ at m/z 1734. Collision induced dissociation (CID) gave a series of daughter ions in accord with the structure shown for OS-2*(Figure 2C). The major ion [M+Na]+ at m/z 1084 in a second group of composition Hep3, Kdo corresponded to a hexose-deficient core oligosaccharide which may be designated OS-3*, and likewise showed fragment ions indicative of the linkage types at the glycosyloxonium ion centers (Figure 2D). In a series of experiments to be reported elsewhere, pseudomolecular ions were generated for unsubstituted and permethylated derivativesof OS-2 samples from the type strain (NCTC 11637), and H.pylori strains 442 and 471.In all samples, microheterogeneous populations of molecules were generated in ESI-MS with broadly similar compositions of dHex, HexNAc, Hex3-6, Hep4, P, Kdo, with the Hex3 molecules as the dominant components. These experiments showed that there were no detectable differences between the core OS regions of the various strains. Assuming that the core regions in the OS-2 fractions were also present in the extended chains of the LPS, structural differences in the high Mr LPS would be likely to occur in the O-antigen chains.
Correlations of differences in the antigenic properties of LPS from strains of H.pylori with structural diversity have pointed to the O-antigen chains or, if present, to heptan chains (Aspinall et al., 1997) as the loci for structural differences (Aspinall, 1988; Moran and Aspinall, 1998). The majority of H.pylori strains express Lewis antigens, Lex as in the type strain (Aspinall et al., 1996) and/or Ley as in H.pylori P466 strain, and both antigenic termini are found in the two strains now studied. However, LPSs from H.pylori 442 and 471, which respectively stimulate and fail to stimulate pepsinogen secretion, differ markedly in the O antigen chains. LPS from H.pylori 442 is essentially similar to that of the type strain (NCTC 11637) (Aspinall et al., 1996) and to strain P466 (Aspinall and Monteiro, 1996) in respect of the fucosylated N-acetyllactosaminoglycan repeating structure. In contrast, the H.pylori 471 LPS has provided the first example of branching with [alpha]-D-Gal residues as side chains at O-6 of a high proportion of the [beta]-D-GlcNAc residues in the O antigen. Anomalous 1H NMR parameters suggest that the presence of the [alpha]-D-Gal side-chain residues may cause mutual conformational distortion in those residues and in the lactosaminoglycan backbone. Analysis of oligosaccharides formed on treatment of the defucosylated glycan PS-3 with endo-[beta]-galactosidase gave evidence for structural regions with galactosylation of successive GlcNAc residues in OGA 5 (Figure 5). Fucosylation in the parent glycan has been found in Lewis antigen termini (mainly Ley) and at some internal GlcNAc residues, but not yet for such residues also bearing [alpha]-Gal side-chains in H.pylori 471. The presence in OGA 6 (Figure 5) of Fuc and Gal side-chains on successive GlcNAc residues shows that these units are constituents of the same glycan chains. Figure 6 summarizes the structures of the outer chain regions of LPS from H.pylori 442 (A) and H.pylori 471 (B) with similar attachment to the core oligosaccharide region (C).
Fig. 6. Structural comparison of the outer chain regions of LPS from H.pylori 442 (A) and H.pylori 471 (B) with similar attachment to the core oligosaccharide (C).
H.pylori 442 was isolated from a patient with duodenal ulceration, whereas strain 471 was from an asymptomatic patient. In a similar manner to H.pylori LPS previously tested, particularly that from duodenal ulcer strains, LPS of H.pylori 442 induced a 50-fold increase in pepsinogen secretion over base levels after 20 min incubation with guinea pig mucosa in situ (Moran and Aspinall, 1998). In contrast, H.pylori 471 did not induce significant secretion over base levels compared with controls. The observation that [alpha]-D-Gal residues as substituent residues to the N-acetyllactosaminoglycan chain of strain 471 could cause distortion of conformation, and hence explain why H.pylori 471 does not induce pepsinogen, is consistent with the finding that polymyxin B treatment of pepsinogen-inducing LPS diminishes pepsinogen secretion (Moran and Aspinall, 1998) due to conformational changes. Furthermore, in the latter study, isolated O-antigen chain, core OS and free lipid A did not induce pepsinogen secretion, again indicating that conformational changes important to stimulation may have occurred during isolation of individual LPS moieties.
However, low-Mr rough-form LPS derived from a pepsinogen-inducing H.pylori strain that usually produced smooth-form LPS with an O-antigen chain, induced the same level of pepsinogen secretion as the parental LPS (Moran and Aspinall, 1998). This indicated the involvement of the core OS when presented on lipid A in pepsinogen stimulation. A similar phenomenon has been observed with LPS from H.pylori 442 (A.P.Moran, unpublished observations). Although not yet observed, minor variations in the core OS regions of H.pylori LPS should not be precluded from a role in pepsinogen stimulation.
Overall, the results of this study document another type of O-antigen chain in H.pylori LPS, namely that of strain 471. This structure, with those already documented, provide a reference point for the examination of antigenic variants in this bacterium (Mills et al., 1992; Moran et al., 1992; Simoons-Smit et al., 1996) and provide further chemically characterized strains for unraveling the role of LPS in H.pylori pathogenesis (Moran, 1996b).
Except as listed below, experimental procedures, including NMR protocols and those for FAB-MS, analytical and spectroscopic methods, especially for compositional and linkage analysis using GLC-MS, and conditions for chemical and enzymic degradations, were those described in previous papers (Aspinall and Monteiro, 1996; Aspinall et al., 1996, 1997; Moran and Walsh, 1993). Typically, water-soluble LPS samples were heated in 1% aqueous acetic acid for 1 h at 100°C, and the liberated polysaccharides and oligosaccharides were separated by GPC on Bio-Gel P-2.
2D 1H NMR spectroscopy
2D Experiments were performed on a Bruker ARX 400 spectrometer in the Fourier transform mode with the following parameters: 2D COSY and 2D TOCSY [256×1024 data matrix, zero filled to 256 points, 64 scans per t1 value, 1.5 s for the recycle delay, unshifted for COSY but shifted sine-squared filtering in t1 and t2 for TOCSY, phase sensitive using TPPI]; for gsNOESY [256×2048 data matrix, zero filled to 512 data points, 64 scans per t1 value 1.5 s for the recycle delay, but shifted sine-squared filtering in t1 and t2 ].
Fast atom bombardment mass spectrometry (FAB-MS)
FAB-MS spectra were recorded with a VGZAB-SE instrument equipped with an Ion Tech saddle field gun. Permethylated glycans in methanol (1-2 µl) were loaded onto the target with a matrix (1 µl) of thioglycerol or 3:1 thioglycerol-glycerol. The samples were bombarded with xenon atoms (1.2 mA anode current, 8 keV anode potential), spectra were recorded with a VG 11-250 data system under the multichannel analyzing mode, and four to five scans were acquired. Resolution was set at 1500-2000 (10% valley definition), and CsI was used as calibrant. In some cases, the resolution was set lower in the high-mass range; thus, the average mass to charge was observed for the molecular ion. The molecular ions and fragment ions are reported as the nominal mass of the 12C-containing component (corresponding to the mass of integral atomic weights). The observed mass is about 1.5 amu higher in the 2000 mass range.
The interpretations of positive ion mass spectra of permethylated derivatives were as previously described (Dell et al., 1990). Spectra of permethylated glycans with reducing KDO termini showed that the major component of the molecular ion cluster corresponded to that calculated for [M+H]+ or other adduct ions. Related ions at [M+H]+ -46 and [M+H]+ -116 were sometimes present, and their co-occurrence aided in the identification of molecular ions. Evidence from FAB-MS for preferential cleavage at GlcNAc residues in permethylated type 2 blood group oligosaccharides and related gangliosides, followed by the [beta]-elimination of fucose resides from the derived fragment ions, provided the basis for assignments of structure for extended fucosylated LacNAc units, as exemplified previously for various glycoconjugates. The examples had included glycosphingolopids (Fukuda et al., 1985) and glycopeptides from human granulocytes (Spooncer et al., 1984) human leukocytes (Stroud et al., 1996), glycolipids in human adenocarcinoma (Hakomori et al., 1984), and in this laboratory LPSs from H.pylori (Aspinall et al., 1996, 1997).
Isolation of LPS and liberated core oligosaccharides
H.pylori strains 442 and 471, the former stimulating and the latter failing to stimulate pepsinogen (Young et al., 1992), were kindly made available to us by Dr. A.Lastovica (University of Cape Town Medical School) and were grown under microaerobic conditions as described previously (Moran et al., 1992).
The technical assistance of J.J.McGovern during growth of bacterial biomass is gratefully acknowledged. We also thank Dr. Jianyao Wang and associates, Mass Spectrometry Laboratory, Faculty of Medicine, University of Toronto for recording FAB and ESI mass spectra. Financial support from The Natural Sciences and Engineering Research Council of Canada and the Irish Health Research Board has been gratefully received.
CID, collision-induced dissociation; COSY, correlated spectroscopy; ESI-MS, electrospray ionization-mass spectrometry; FAB-MS, fast-atom-bombardment-mass spectrometry; GLC-MS, gas-liquid chromatography-mass spectrometry; GPC, gel permeation chromatography; HMQC, heteronuclear multiple quantum correlated spectroscopy; JMODXH, J-modulated spin echo (X-nuclei); LPS, lipopolysaccharide; Mr, molecular weight; NOESY, nuclear Overhauser enhancement spectroscopy; NMR, nuclear magnetic resonance; OS, oligosaccharide; PS, polysaccharide; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TOCSY, total correlated spectroscopy.
1To whom correspondence should be addressed