Báo cáo khoa học: Identification and localization of glycine in the inner core lipopolysaccharide of Neisseria meningitidis

doi:10.1046/j.1432-1033.2002.03131.x

Eur. J. Biochem. 269, 4169–4175 (2002) (cid:2) FEBS 2002

Identification and localization of glycine in the inner core lipopolysaccharide of Neisseriameningitidis

Andrew D. Cox, Jianjun Li and James C. Richards

Institute for Biological Sciences, National Research Council, Ottawa, ON, Canada

studies confirmed and extended this observation locating the glycine residue at the 7-position of the HepII molecule in L3 and L4 immunotype strains.

lipopolysaccharide;

Keywords: Neisseria meningitidis; glycine; NMR; mass spectrometry.

The amino acid glycine is identified as a component of the inner core oligosaccharide in meningococcal lipopolysac- charide (LPS). Ester-linked glycine residues were consis- tently found by mass spectrometry experiments to be located on the distal heptose residue (HepII) in LPS from several strains of Neisseria meningitidis. Nuclear magnetic resonance

for ease of

interpretation of LPS. In earlier studies, otherwise complex data, the majority of structural analyses on meningococcal core oligosaccharides had been per- formed following O-deacylation and/or dephosphorylation. Naturally, base-labile residues that may have been present in the native LPS molecule would have been removed by such procedures. Previous studies had identified O-acetyl groups in the core oligosaccharide of some immunotype strains [3,5,7,8]. In this study we identify and structurally characterize the presence of ester-linked glycine residues in the core oligosaccharide of meningococcal LPS.

M A T E R I A L S A N D M E T H O D S

Growth of organism and isolation of LPS

N. meningitidis immunotype strains L3 galE (NRCC #4720) and L4 galE (NRCC #4719) and clinical strains BZ157 galE B5+ (NRCC #6094) and NGH15 B5+ and B5– (NRCC #6092 and 6093) were all grown in a 28-L fermenter as described previously [11] yielding (cid:1) 100 g wet wt. of cells from each growth. Strains BZ157 and NGH15 are from the culture collection of E. R. Moxon. LPS was extracted by the hot phenol/water method as described previously and purified from the aqueous phase by ultracentrifugation (45K, 4 (cid:1)C, 5 h) [11] yielding (cid:1) 200 mg in each case. O-deacylated LPS was prepared as described previously [13] in (cid:1) 50% yield from the LPS. Core oligosaccharides were prepared according to the following procedure. LPS was hydrolysed at 100 (cid:1)C for 2 h in 2% acetic acid. Insoluble material was removed by centrifugation (6000 g, 20 min.) and the supernatant solution was lyophilized yielding core oligosaccharide in (cid:1) 50% yield.

The LPS of Neisseria meningitidis contains a core oligosaccharide unit with an inner core di-heptose-N- acetyl-glucosamine backbone, wherein the two L-glycero- D-manno-heptose (Hep) residues can provide a point of attachment for the outer core oligosaccharide residues [1]. Meningococcal LPS has been classified into 12 distinct LPS immunotypes (L1-L12), originally defined by monoclonal antibody (mAb) reactivities [2], but further defined by structural analyses. The structures of LPS from immuno- types L1/6 [3,4], L2 [5], L3 [6], L4/7 [7], L5 [8] and L9 [9] have been elucidated. The structural basis of the immuno- typing scheme is governed by the location of a phospho- ethanolamine (PEtn) moiety on the distal heptose residue (HepII) at either the 3- or 6-position or absent. The length and nature of oligosaccharide extension from the proximal heptose residue (HepI) and the presence or absence of a glucose sugar at HepII also dictates the immunotype. The enzyme UDP glucose-4-epimerase (GalE) is essential for N. meningitidis to synthesize UDP-Gal for incorporation of galactose into its LPS and is encoded by the gene galE [10]. The absence of galactose residues in the conserved inner core structure of meningococcal LPS has led to the utilization of mutants defective in the enzyme, resulting in the truncation of the LPS’s oligosaccharide chain at the glucose residue at HepI, and galE mutants have been used by our group in order to derive mAbs to inner core LPS epitopes [11]. mAb B5 was identified in this way, which had an absolute requirement for PEtn at the 3-position of HepII. Subsequently this mAb was used to identify the gene lpt3 that is responsible for the transfer of the PEtn residue to the 3-position of HepII [12]. During the course of these studies we examined the core oligosaccharide of a clinical isolate NGH15, which revealed additional O-linked residues that had not previously been identified in meningococcal

Mass spectrometry

All ES-MS and CE-MS analyses were carried out as described previously [12].

NMR spectroscopy

Nuclear magnetic resonance experiments were performed on Varian INOVA 500, 400 and 200 NMR spectrometers as

Correspondence to A. D. Cox, Institute for Biological Sciences, National Research Council, 100, Sussex Drive, Ottawa, ON K1A 0R6, Canada. Fax: + 1 613 952 9092, Tel.: + 1 613 991 6172, E-mail: Andrew.Cox@nrc.ca Abbreviation: LPS, lipopolysaccharide. (Received 15 April 2002, revised 25 June 2002, accepted 24 July 2002)

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described previously [14]. The 2D 13C-1H HMBC experi- ment was acquired on a Varian Inova 500 spectrometer and was of the order of 15 h. The 13C–1H coupling constant was 140 Hz, with a sweep width in the F2 (1H) dimension of 10.0 p.p.m. and in the F1 (13C) dimension of 230 p.p.m. Water presaturation during the relaxation delay was 1.0 s, acquisition time in t2 was 0.205 s, and 80 increments with 256 scans per increment were obtained.

R E S U L T S

Mild acid hydrolysis of immunotype L3 galE and L4 galE LPS afforded core oligosaccharides that were initially examined by ES-MS (Fig. 1). Several ions were observed and the compositions for each glycoform are listed in Table 1. Typical ions differing by 18 a.m.u. were observed for each glycoform and corresponded to reducing end anhydro and intact Kdo species due to rearrangements of the Kdo molecule during hydrolysis. L3 galE core oligo- saccharide consisted of two sets of ions differing by 57 a.m.u. (Fig. 1A). The ion at m/z 1110 corresponds to a composition of Hex, 2Hep, HexNAc, PEtn, Kdo as would be expected for the galE mutant, as further extension beyond the first glucose (GlcI) at the proximal heptose residue (HepI) is precluded due to the unavailability of

galactose in this genetic background. A second ion, 57 intensity was a.m.u. higher, of approximately equal observed at m/z 1167. A mass of 57 a.m.u. corresponds to the amino acid glycine (Gly) that has been reported previously in the LPS of several Gram-negative bacteria [15] but not in N. meningitidis. The ES-MS of L4 galE core oligosaccharide reflected a more complex mixture (Fig. 1B). In addition to glycoforms corresponding to the presence or absence of glycine, species with and without an O-acetyl group and the PEtn residue were also observed (Table 1). As indicated in Table 1 for L4 galE core oligosaccharide, the ions at m/z 969 and 987 correspond to the expected core oligosaccharides without the PEtn moiety. Ions at m/z 1011 and 1029 correspond to the same core oligosaccharides but with an additional O-acetyl group. Ions at m/z 1092 and 1110 correspond to the expected inner core structure without any modifications corresponding to a composition of Hex, 2Hep, HexNAc, PEtn, Kdo. The final two sets of ions correspond to the addition of an O-acetyl group alone (m/z 1134,1152) and both an O-acetyl group and a glycine moiety (m/z 1191, 1209) to the expected inner core structure. To locate the glycine residue in the inner core oligosaccha- ride of L3 galE, MS-MS studies were performed in positive ion mode, as the positive functionalities available on the glycine and ethanolamine moieties enable more stable

Fig. 1. Transformed electrospray mass spec- trum of N. meningitidis strains (A) L3 galE core oligosaccharide (B) L4 galE core oligo- saccharide obtained in negative ion mode.

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Table 1. Negative ion ES-MS data and proposed compositions of core oligosaccharide from N. meningitidis strains. L3 galE, L4 galE, NGH15 B5– and B5+ and BZ157 galE B5+. Average mass units were used for calculation of molecular mass based on proposed composition as follows: Glc, 162.15; Hep, 192.17; GlcNAc, 203.19; Kdo, 220.18; PEtn, 123.05. Gly, 57.05; OAc, 42.00.

Observed Ions (m/z) Molecular Mass (Da)

(M-2H)2– Observed Calculated (M-H)– Proposed composition Strain Relative intensity

L3 galE

1091.4 1109.3 1148.5 1166.4 – 554.2 – 582.5 1092.3 1110.5 1149.5 1167.4 1092.9 1110.9 1149.9 1167.9 0.3 1.0 0.5 0.9 Glc, GlcNAc, 2Hep, PEtn, aKdo Glc, GlcNAc, 2Hep, PEtn, Kdo Gly, Glc, GlcNAc, 2Hep, PEtn, aKdo Gly, Glc, GlcNAc, 2Hep, PEtn, Kdo L3 galE O-deac 1091.5 1109.4 – 554.2 1092.6 1110.5 1092.9 1110.9 0.2 1.0 Glc, GlcNAc, 2Hep, PEtn, aKdo Glc, GlcNAc, 2Hep, PEtn, Kdo L4 galE

968.3 986.4 1010.2 1028.1 1091.5 1109.3 1133.4 1151.4 1190.3 1208.3 – – – – – – – – – – 969.3 987.3 1011.4 1029.1 1092.4 1110.4 1134.3 1152.3 1191.4 1209.3 969.8 987.8 1011.8 1029.8 1092.9 1110.9 1134.9 1152.9 1191.9 1209.9 0.2 0.2 0.4 0.6 0.3 0.3 0.8 0.9 1.0 0.5 Glc, GlcNAc, 2Hep, aKdo Glc, GlcNAc, 2Hep, Kdo OAc, Glc, GlcNAc, 2Hep, aKdo OAc, Glc, GlcNAc, 2Hep, Kdo Glc, GlcNAc, 2Hep, PEtn, aKdo Glc, GlcNAc, 2Hep, PEtn, Kdo OAc, Glc, GlcNAc, 2Hep, PEtn, aKdo OAc, Glc, GlcNAc, 2Hep, PEtn, Kdo Gly, OAc, Glc, GlcNAc, 2Hep, PEtn, aKdo Gly, OAc, Glc, GlcNAc, 2Hep, PEtn, Kdo NGH15 B5-

– – – – – – 747.6 756.4 768.7 777.6 796.8 806.0 1496.9 1514.9 1539.3 1557.5 1595.9 1614.4 1497.3 1515.3 1539.3 1557.3 1596.3 1614.3 0.5 0.4 0.4 1.0 0.3 0.4 3Hex, 2HexNAc, 2Hep, aKdo 3Hex, 2HexNAc, 2Hep, Kdo OAc, 3Hex, 2HexNAc, 2Hep, aKdo OAc, 3Hex, 2HexNAc, 2Hep, Kdo Gly, OAc, 3Hex, 2HexNAc, 2Hep, aKdo Gly, OAc, 3Hex, 2HexNAc, 2Hep, Kdo NGH15 B5+

– – – – 1678.8 – – – 808.9 818.0 830.0 837.5 839.0 846.5 858.5 867.5 1620.0 1638.0 1662.6 1677.0 1680.1 1694.9 1718.9 1737.0 1620.3 1638.3 1662.3 1677.3 1680.3 1695.3 1719.3 1737.3 0.1 0.3 0.3 0.1 1.0 0.1 0.3 0.4 3Hex, 2HexNAc, 2Hep, PEtn, aKdo 3Hex, 2HexNAc, 2Hep, PEtn, Kdo OAc, 3Hex, 2HexNAc, 2Hep, PEtn, aKdo Gly, 3Hex, 2HexNAc, 2Hep, PEtn, aKdo OAc, 3Hex, 2HexNAc, 2Hep, PEtn, Kdo Gly, 3Hex, 2HexNAc, 2Hep, PEtn, Kdo Gly, OAc, 3Hex, 2HexNAc, 2Hep, PEtn, aKdo Gly, OAc, 3Hex, 2HexNAc, 2Hep, PEtn, Kdo BZ157 galE B5+

a similar series of ions (Fig. 2B) to those observed for L3 galE core oligosaccharide. In addition to a similar location for the glycine residue being identified, the O-acetyl group of the L4 galE oligosaccharide was also localized in this experiment by virtue of an ion at m/z 246 that corresponds to the N-acetyl-glucosamine residue bearing an O-acetyl group. This ion can be compared to the ion at m/z 204 in the fragmentation of L3 galE oligosaccharide (Fig. 2A) that does not contain an O-acetyl group. ES-MS analyses were

fragment ions to be formed (Fig. 2). Fragmentation of the positively charged ion at m/z 1169 of L3 galE oligosaccha- ride (Fig. 2A) produced a series of ions due to consecutive losses of glycose residues as indicated. A product ion at m/z 373 was diagnostic for the location of the glycine residue at the distal heptose residue (HepII), as this ion corresponds to Gly-HepII-PEtn. In the same way the glycine moiety was localized in the L4 galE core oligosaccharide, wherein fragmentation of the positively charged ion at m/z 1193 gave

1011.3 1028.4 – – 1133.5 1151.5 1190.4 1208.7 1256.6 1274.7 1313.6 1331.5 – – 545.2 554.1 566.3 575.4 594.7 – 628.0 636.9 – – 1011.7 1029.4 1092.6 1110.3 1134.5 1152.7 1191.4 1209.8 1257.7 1275.8 1314.6 1332.6 1011.8 1029.8 1092.9 1110.9 1134.9 1152.9 1191.9 1209.9 1257.9 1275.9 1314.9 1332.9 0.1 0.2 0.2 0.2 0.7 1.0 0.4 0.3 0.6 0.6 0.6 0.3 OAc, Glc, GlcNAc, 2Hep, aKdo OAc, Glc, GlcNAc, 2Hep, Kdo Glc, GlcNAc, 2Hep, PEtn, aKdo Glc, GlcNAc, 2Hep, PEtn, Kdo OAc, Glc, GlcNAc, 2Hep, PEtn, aKdo OAc, Glc, GlcNAc, 2Hep, PEtn, Kdo Gly, OAc, Glc, GlcNAc, 2Hep, PEtn, aKdo Gly, OAc, Glc, GlcNAc, 2Hep, PEtn, Kdo OAc, Glc, GlcNAc, 2Hep, 2PEtn, aKdo OAc, Glc, GlcNAc, 2Hep, 2PEtn, Kdo Gly, OAc, Glc, GlcNAc, 2Hep, 2PEtn, aKdo Gly, OAc, Glc, GlcNAc, 2Hep, 2PEtn, Kdo

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glycine residue is attached to the core oligosaccharide via an ester linkage.

also performed on two other meningococcal strains of clinical origin and the compositions of the glycoforms observed are listed in Table 1. In each case where glycine was identified, MS-MS studies located this residue to the HepII molecule (data not shown).

The glycine residue was assumed to be ester-linked because it had not been observed in O-deacylated material examined from these samples [6,7]. Base-labile ester-linked residues are readily removed under the alkaline conditions for O-deacylation. To confirm this, O-deacylated LPS from L3 galE was hydrolysed with 2% acetic acid in order to afford the O-deacylated core oligosaccharide. ES-MS analysis for this sample revealed a simplified spectrum without ions corresponding to glycine con- taining glycoforms (Table 1), thus confirming that the

In order to confirm and further characterize the presence of the glycine residue, NMR studies were performed. Initial experiments on commercial glycine suggested the 1H and 13C resonances of the -CH2- group were at 3.56 and 41.5 p.p.m., respectively. The core oligosaccharide from L3 galE was chosen for NMR studies as the MS data had indicated a less complex mixture than the core oligosaccha- ride from L4 galE. A 13C-1H HSQC experiment (Fig. 3A) revealed a cross-peak with a 1H resonance of 3.56 p.p.m. and a 13C resonance of 42.4 p.p.m., in excellent agreement with the glycine standard. A 13C-1H HMBC experiment (Fig. 3B) produced a cross-peak at 1H resonance at 3.56 p.p.m. and a 13C resonance at 172.5 p.p.m. consistent

Fig. 2. Positive ion capillary electrophoresis- electrospray mass spectrum of O-deacylated LPS from N. meningitidis strains (A) L3 galE core oligosaccharide MS/MS of m/z 1169; (B) L4 galE core oligosaccharide MS/MS of m/z 1193. Fragmentation pathways of the core oligosaccharides are illustrated by indication of the molecules lost from the core oligosac- charide to give the resulting fragment ions. Diagnostic ions that localized the glycine residue and the O-acetylation status of the GlcNAc residue are indicated in the inset figures.

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with relay between the carbonyl carbon and the CH2 protons of the glycine moiety, thus confirming the identi- fication of glycine.

of the H-4 1H resonance between the O-deacylated and native core oligosaccharide. This is not the case as 1H resonances for each spin-system from H-1 to H-5 are virtually identical and therefore provides further evidence that the glycine residue is located at an exocyclic position, presumably the 7-position of the HepII residue.

To confirm the location of the glycine moiety at the HepII residue, 31P-1H HMQC and 31P-1H HMQC-TOCSY experiments (Fig. 4) were performed on O-deacylated L4 galE oligosaccharide. Oligosaccharide derived from the L4 immunotype LPS was chosen for this analysis because of the inherent difficulties in accessing the exocyclic protons in a heptosyl spin-system from the anomeric proton resonance. The HepII residue of immunotype L4 LPS is substituted at the 6-position by a PEtn residue and therefore this configuration was taken advantage of in 31P-1H NMR experiments [7]. The 31P-1H HMQC experiment revealed cross-peaks from the 31P-resonance of the PEtn molecule at the 6-position of HepII to 1H-resonances at 4.58 p.p.m. which is characteristic for substitution of the 6-position of

Fig. 3. Regions of the (A) 2D-13C-1H-HSQC NMR and (B) 2D-13C-1H-HMBC NMR spectra of the core oligosaccharide from N. meningitidis strain L3 galE illustrating the -CH2- group of the glycine residue and the -CH2-CO2- connectivity within the glycine residue, respectively.

Table 2. 1H NMR assignment of HepII residue from N. meningitidis L3 galE core oligosaccharide. The spectrum was recorded at 25 (cid:1)C relative to HOD at 4.77 p.p.m.

Sugar Residue H1 H2 H3 H4 H5

a Data for the O-deacylated core oligosaccharide.

NMR provided evidence for the exact location of the glycine moiety on the HepII residue of the inner core oligosaccharide. There are only a few positions available for attachment on this substituted residue in the meningococcal LPS inner core. Linkage to HepI occurs from the anomeric position of HepII, the N-acetyl-glucosamine residue substi- tutes HepII at the 2-position and PEtn substitutes HepII at the 3-position in immunotype L3 and at the 6-position in immunotype L4. Ring formation occurs at position 5 in this pyranose sugar. There are therefore only two locations available for the glycine moiety namely the 4-position or the 7-position of the HepII residue common to both immuno- types. If the glycine residue was linked to the 4-position of HepII one might expect the CH2 protons of the glycine molecule to be split by the plane of symmetry from the carbohydrate ring. However, as the CH2 protons appear as a sharp singlet at 3.56 p.p.m. in the 1H spectrum, this would suggest that they are behaving as equivalent protons and have not been affected by the pyranose ring. This behaviour is more consistent with an exocyclic location, where the additional freedom would enable these protons to appear equivalent. Additional evidence that the 4-position is not the location of the glycine moiety was obtained when the chemical shifts of the 1H resonances of the HepII molecule were compared for the core oligosaccharide and O-deacy- lated core oligosaccharide from L3 galE (Table 2). Clearly if O-deacylation removed the glycine group from the 4-position one would expect a change in the chemical shift

HepII HepIIa 5.52 5.51 4.37 4.37 4.40 4.40 4.13 4.13 3.70 3.70

Fig. 4. Region of the 2D-31P-1H-HMQC-TOCSY NMR spectrum of the core oligosaccharide from N. meningitidis strain L4 galE.

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4.00 p.p.m., which when compared to the sharp singlet at 3.56 p.p.m. observed for the -CH2- protons of the glycine moiety in meningococcal LPS points to an exocyclic location for the glycine residue in N. meningitidis LPS.

The genetic control of the elaboration of glycine is not understood, nor is the propensity of the bacterium to display this residue in a clinical environment or in a variety of growth conditions. Some researchers have speculated that the amino acid may protect the core oligosaccharide against host glycosidases during infection or could be involved in modifying the net charge of the LPS molecule [15]. However, the rationale behind the incorporation of glycine into the core oligosaccharide remains unclear, but one could expect that in a particular niche this structure may confer an advantage upon the bacterium, thus aiding its competitiveness in maintaining an infection.

A C K N O W L E D G E M E N T S

HepII with PEtn and resonances at 3.32 and 4.18 p.p.m. diagnostic for the ethanolamine protons distal and proximal to the phosphorus atom, respectively [7]. The 31P-1H HMQC-TOCSY experiment revealed additional cross peaks at 3.85 and 3.75 p.p.m. consistent with nonsubstituted H-7 1H-resonances [7]. When these experiments were performed on L4 galE oligosaccharide the 31P-1H cross- peaks observed were identical, apart from an addi- tional 1H-resonance at 4.38 p.p.m. consistent with the presence of a substituent at the 7-position of HepII. Signals indicative of the presence and absence of substituents at the 7-position of HepII in the L4 galE oligosaccharide, are consistent with the nonstoichiometric substitution of glycine as indicated by ES-MS experiments. This data therefore confirmed that the glycine residue was attached at the 7-position of the HepII molecule in L4 galE oligosaccharide. The similarity of the NMR data for the glycine residue in L3 galE oligosaccharide and BZ157 B5+ galE oligosaccharide, and MS data for other strains that elaborate glycine would suggest that in meningococcal LPS the glycine moiety is consistently located at the 7-position of the HepII residue.

We thank Don Krajcarski for ES-MS, Suzon Larocque for NMR assistance and Doug Griffith for cell growth.

R E F E R E N C E S

D I S C U S S I O N

1. Kahler, C.M. & Stephens, D.S. (1998) Genetic basis for bio- synthesis, structure, and function of meningococcal lipooligo- saccharide (endotoxin). Crit Rev. Microbiol. 24, 281–334.

2. Scholten, R.J., Kuipers, B., Valkenburg, H.A., Dankert, J., Zollinger, W.D. & Poolman, J.T. (1994) Lipo-oligosaccharide immunotyping of Neisseria meningitidis by a whole-cell ELISA with monoclonal antibodies. J. Med. Microbiol. 41, 236–243. 3. Di Fabio, J.L., Michon, F., Brisson, J. & Jennings, H.J. (1990) Structure of L1 and L6 core oligosaccharide epitopes of Neisseria meningitidis. Can J. Chem. 68, 1029–1034.

4. Wakarchuk, W.W., Gilbert, M., Martin, A., Wu, Y., Brisson, J.R., Thibault, P. & Richards, J.C. (1998) Structure of an alpha- 2,6-sialylated lipooligosaccharide from Neisseria meningitidis immunotype L1. Eur J. Biochem. 254, 626–633.

5. Gamian, A., Beurret, M., Michon, F., Brisson, J.R. & Jennings, H.J. (1992) Structure of the L2 lipopolysaccharide core oligo- saccharides of Neisseria meningitidis. J. Biol. Chem. 267, 922–925. 6. Pavliak, V., Brisson, J.R., Michon, F., Uhrin, D. & Jennings, H.J. (1993) Structure of the sialylated L3 lipopolysaccharide of Neis- seria meningitides. J. Biol. Chem 268, 14146–14152.

7. Kogan, G., Uhrin, D., Brisson, J.R. & Jennings, H.J. (1997) the Neisseria meningitidis immunotypes Structural basis of including the L4 and L7 immunotypes. Carbohydr. Res. 298, 191– 199.

8. Michon, F., Beurret, M., Gamian, A., Brisson, J.R. & Jennings, H.J. (1990) Structure of the L5 lipopolysaccharide core oligo- saccharides of Neisseria meningitides. J. Biol. Chem. 265, 7243– 7247.

9. Jennings, H.J., Johnson, K.G. & Kenne, L. (1983) The structure of an R-type oligosaccharide core obtained from some lipopoly- saccharides of Neisseria meningitidis. Carbohydr. Res. 121, 233– 241.

This paper has described another structural variation to the inner core oligosaccharide of N. meningitidis LPS. The identification and localization of the amino acid glycine substituting the HepII molecule has revealed the potential for further complexity at this inner core residue. From a survey of the meningococcal immunotype strains, variation of substituents and substitution patterns at the HepII residue is an important feature. HepII can carry a Glc residue at the 3-position, the presence of which is phase- variable due to the homopolymeric tract in the glucosyl- transferase-encoding gene, lgtG [12]. PEtn residues can be present at either the 3- or 6-postion, absent or at both the 3- and 6-positions simultaneously in some glycoforms [6,7,14,15]. In this report we have identified yet another structural variation at this HepII residue, namely the elaboration of the amino acid glycine, and it is interesting to note that in strain BZ157 galE a glycine residue is elaborated at this HepII residue even in glycoforms that contain two PEtn moieties (data not shown). It is important to note that incorporation of glycine is not simply a consequence of the galE mutation. The LPS from the parent strain of immunotype L3 and its lgtB and lgtA mutants also elaborated a glycine residue (data not shown). Glycine has been identified in the LPS of several Gram-negative bacteria including Escherichia, Salmonella, Hafnia, Citrobacter and Shigella species [16], but in each case the relevance of this finding is unclear. Recently glycine was identified as a common component in the LPS of Haemophilus influenzae [17]. However in H. influenzae the glycine residue is not found consistently in one location in the inner core oligosaccharide as appears to be the case for N. meningitidis. Depending on the strain of H. influenzae glycine could be found at each of the heptose residues of the inner core tri- heptosyl group or on the Kdo residue. Glycine has also been localized in the core oligosaccharide of Proteus mirabilis serotype O28 [18]. The glycine moiety was found to be amide-linked to the amino group of a glucosamine residue. Interestingly in this arrangement the -CH2- protons of the glycine moiety are split and are found at 3.80 and

10. Jennings, M.P., van der Ley, P., Wilks, K.E., Maskell, D.J., Poolman, J.T. & Moxon, E.R. (1993) Cloning and molecular analysis of the galE gene of Neisseria meningitidis and its role in lipopolysaccharide biosynthesis. Mol. Microbiol. 10, 361–369. 11. Plested, J.S., Makepeace, K., Jennings, M.P., Gidney, M.A., Lacelle, S., Brisson, J., Cox, A.D., Martin, A., Bird, A.G., Tang, C.M., Mackinnon, F.G., Richards, J.C. & Moxon, E.R. (1999) Conservation and accessibility of an inner core lipopoly- saccharide epitope of Neisseria meningitidis. Infect Immun 67, 5417–5426.

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12. Mackinnon, F.G., Cox, A.D., Plested, J.S., Tang, C.T., Make- peace, K., Coull, P.A., Wright, J.C., Chalmers, R., Hood, D.W., Richards, J.C. & Moxon, E.R. (2002) Identification of a gene (lpt-3) required for the addition of phosphoethanolamine to the lipopolysaccharide inner core of Neisseria meningitidis and its role in mediating susceptibility to bactericidal killing and opsonopha- gocytosis. Mol. Microbiol. 43, 931–943.

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