Richards Institute for Biological Sciences, National Research Council, Ottawa, ON, Canada The amino acid glycine is identified as a component of the inner core oligosaccharide in meningoc
Trang 1Identification and localization of glycine in the inner core
Andrew D Cox, Jianjun Li and James C Richards
Institute for Biological Sciences, National Research Council, Ottawa, ON, Canada
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
studies confirmed and extended this observation locating the glycine residue at the 7-position of the HepII molecule in L3 and L4 immunotype strains
Keywords: Neisseria meningitidis; lipopolysaccharide; glycine; NMR; mass spectrometry
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 meningitidisto 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
lpt3that 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
LPS In earlier studies, for ease of interpretation of otherwise complexdata, 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 meningitidisimmunotype 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 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 °C, 5 h) [11] yielding 200 mg in each case O-deacylated LPS was prepared as described previously [13]
in 50% yield from the LPS Core oligosaccharides were prepared according to the following procedure LPS was hydrolysed at 100°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 50% yield
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, SussexDrive, 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)
Trang 2described previously [14] The 2D 13C-1H HMBC ex
peri-ment was acquired on a Varian Inova 500 spectrometer and
was of the order of 15 h The13C–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 t2was 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 a.m.u higher, of approximately equal intensity was 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 complexmixture (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.
Trang 3fragment 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
a similar series of ions (Fig 2B) to those observed for L3 galEcore 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
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.
Strain
Observed Ions (m/z) Molecular Mass (Da)
Relative intensity Proposed composition (M-H) – (M-2H) 2– Observed Calculated
Trang 4also 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
glycine residue is attached to the core oligosaccharide via
an ester linkage
In order to confirm and further characterize the presence
of the glycine residue, NMR studies were performed Initial experiments on commercial glycine suggested the1H 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 complexmixture than the core oligosaccha-ride from L4 galE A13C-1H HSQC experiment (Fig 3A) revealed a cross-peak with a1H resonance of 3.56 p.p.m and a13C resonance of 42.4 p.p.m., in excellent agreement with the glycine standard A 13C-1H HMBC ex periment (Fig 3B) produced a cross-peak at 1H resonance at 3.56 p.p.m and a13C 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.
Trang 5with relay between the carbonyl carbon and the CH2
protons of the glycine moiety, thus confirming the
identi-fication of glycine
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 substisubsti-tutes 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 CH2protons appear as
a sharp singlet at 3.56 p.p.m in the1H 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 the1H 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
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 galEoligosaccharide 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 the31P-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- 13 C- 1 H-HMBC NMR
spectra of the core oligosaccharide from
N meningitidis strain L3 galE illustrating the
-CH 2 - group of the glycine residue and the
-CH 2 -CO 2 - connectivity within the glycine
residue, respectively.
Table 2 1 H NMR assignment of HepII residue from N meningitidis L3 galE core oligosaccharide The spectrum was recorded at 25 °C relative
to HOD at 4.77 p.p.m.
a Data for the O-deacylated core oligosaccharide.
Fig 4 Region of the 2D-31P-1H-HMQC-TOCSY
NMR spectrum of the core oligosaccharide from
N meningitidis strain L4 galE.
Trang 6HepII 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
galEoligosaccharide 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
D I S C U S S I O N
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
Shigellaspecies [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
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
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
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) Structural basis of the Neisseria meningitidis immunotypes 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.
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.
Trang 712 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.
13 Lysenko, E., Richards, J.C., Cox, A.D., Stewart, A., Martin, A.,
Kapoor, M & Weiser, J.N (2000) The position of
phosphor-ylcholine on the lipopolysaccharide of Haemophilus influenzae
affects binding and sensitivity to C-reactive protein-mediated
killing Mol Microbiol 35, 234–245.
14 Cox, A.D., Li, J., Brisson, J.-R., Moxon, E.R & Richards, J.C.
(2002) Structural analysis of the lipopolysaccharide from Neisseria
meningitidis strain BZ157 galE: localisation of two
phosphoetha-nolamine residues in the inner core oligosaccharide Carbohydr.
Res in press.
15 Rahman, M.M., Kahler, C.M., Stephens, D.S & Carlson, R.W (2001) The structure of the lipooligosaccharide (LOS) from the a-1,2-N-acetyl glucosamine transferase (rfaK NMB ) mutant strain CMK1 of Neisseria meningitidis: implications for LOS inner core assembly and LOS-based vaccines Glycobiol 11, 703–709.
16 Gamian, A., Mieszala, M., Katzenellenbogen, E., Czarny, A., Zal, T & Romanowska, E (1996) The occurrence of glycine in bacterial lipopolysaccharides FEMS Immunol Med Microbiol.
13, 261–268.
17 Li, J., Bauer, S.H.J., Mansson, M., Mox on, E.R., Richards, J.C & Schweda, E.K.H (2001) Glycine is a common constituent of the inner-core in Haemophilus influenzae lipopolysaccharide Glyco-biology 11, 1009–1015.
18 Vinogradov, E & Radziejewska-Lebrecht, J (2000) The structure
of the carbohydrate backbone of the core-lipid A region of the lipopolysaccharide from Proteus mirabilis serotype O28 Carbo-hydr Res 329, 351–357.