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J Biol Chem, Vol. 275, Issue 13, 9716-9724, March 31, 2000

Structural Relationships and Sialylation among Meningococcal L1, L8, and L3,7 Lipooligosaccharide Serotypes^*

J. McLeod Griffiss^§, Brenda L. Brandt^¶, Nancy B. Saunders^¶, and Wendell Zollinger^¶

From the  Centre for Immunochemistry and Department of Laboratory Medicine, University of California, San Francisco, California 94121 and the ^¶ Department of Bacterial Diseases, Walter Reed Army Institute of Research, Washington, D. C. 20307

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Eighteen of 34 endemic meningococcal case strains were of the L8 lipooligosaccharide (LOS) type; four of these were both L3 and L7 (L3,7), and seven were L1. L1 structures arose by alternative terminal Gal substitutions of lactosyl diheptoside L8 structures, as determined by electrospray ionization and other mass spectrometric techniques, and enzymatic and chemical degradations (Structures L1 and L1a).

The more abundant molecule, designated L1, had a trihexose globosyl  chain; the less abundant one, designated L1a, had a -lactosyl  chain and a parallel -lactosaminyl  chain. A P^k globoside (Gal14Gal14 Glc-R) monoclonal antibody bound 9/10 L1 strains, but a P₁ globoside (Gal14Gal14GlcNAc-R) mAb bound none of them. -Galactosidase caused loss of both L1 structures and creation of L8 structures; -galactosidase caused loss of the L8 determinant. The L1/P^k glycose was partially sialylated. Some LOS also had unsubstituted basal -GlcNAc additions. These structural relationships explain co-expression of L8, L1, and L3,7 serotypes.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The outer membrane lipooligosaccharides (LOS)¹ of Neisseria meningitidis have been divided into 11 serotypes (1), but this system of antigenic discrimination does not adequately account for the observed molecular heterogeneity of these glycolipids (2-5) nor for the fact that the several different LOS molecules made by each meningococcus may be of different serotypes. Monoclonal antibodies (mAbs) largely have replaced rabbit antisera as the LOS typing reagent (6, 7), but a set of mAbs that recognizes most of the important LOS antigens is not available, and many of the available mAbs lack a complete structural definition of their cognate epitopes (8-10). Furthermore, mAb LOS typing has been applied mostly to epidemiologically related strains (6, 7), which may have introduced biases that have led to underestimates of the prevalences of some LOS types among circulating organisms. For these reasons we have used a variety of mass spectrometric and standard immunochemical techniques to investigate the structural basis of LOS serotype heterogeneity and mAb binding among meningococci that caused sporadic disease. We began with the L1, L8, and L3,7 types.

Neisseria LOS structurally resemble human glycosphingolipids (GSL) which often are sialylated (2, 11, 12). Group B and C N. meningitidis endogenously sialylate lacto-N-neotetraose LOS structures (13-15). We now report that the  chain of the L1 LOS that mimics the human P^k globosyl GSL also is partially sialylated and that L1 strains make a second, alternative LOS structure that we have designated L1a. Whether the L1a structure also is sialylated was not determined.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Strains-- We have extensively characterized N. meningitidis strains 6940 (B:L1) the prototype L1 strain (16), and 126E (C:L1,8) (5, 17, 18), and 190I (B:L1,3,7) (19). We selected Neisseria lactamica strain 1207-0 and N. meningitidis strains 8529 (B:L3,7) and 8532 (B:L8) from the collections of the WRAIR. The lactamica was isolated from a pharynx and the meningococci from cerebrospinal fluids of Chilean children during an outbreak of group B meningococcal disease in Iquique (20).

We used LOS made by Neisseria gonorrhoeae strain MS11_mkD to mark the positions of LOS in SDS-PAGE gels (21). We used LOS made by N. gonorrhoeae strains 1291, and its pyocin-selected mutants, and by MS11_mkD as LOS M_r standards (22-24).

LOS Serotyping-- We used the SPRIA inhibition technique developed by Zollinger and Mandrell (1), with the original polyclonal rabbit antisera, to determine the LOS serotype(s) of 34 consecutive and unique blood and cerebrospinal fluid N. meningitidis isolates from epidemiologically unrelated pediatric patients with endemic disease in Houston, TX, from February 1977 through March 1978 (18, 19, 25, 26). This system arbitrarily sets 35% of the inhibition of a type-specific antiserum by the homologous prototype strain as the threshold criterion for a serotype designation (1). We then compared the L types of strains, defined by this criterion, with their binding of "serotype-specific" monoclonal antibodies (mAbs), as determined with use of a whole cell dot blot enzyme-linked immunosorbent assay as described (27).

Monoclonal Antibodies (mAbs)-- mAb 17-2-L1 was selected after immunization of mice with strain 6940 (L1) (28); we are designating it the L1 mAb in this report. The L3,7 mAb, 9-2-L379, was selected after immunization of mice with a mixture of three meningococcal strains that expressed several L-types, including L3,7 (28). The L8 mAb, 2-1-L8, has been characterized extensively; it binds the lactosyl -oligosaccharide of 3.6-kDa neisserial LOS (2, 21, 23, 29). mAbs that bind the P^k (Gal14Gal14Glc ceramide) (22, 30), P₁ (Gal14Gal14- GlcNAc-R), and Gal14Gal-R human GSL were bought from MonoCarb (Accurate Chemicals, Westbury, NY).

Preparation of LOS-- Bacteria were grown on supplemented GC agar in a CO₂ candle extinction jar, harvested, dehydrated with acetone, and stored at 4 °C until ready for use. We extracted LOS from rehydrated acetone-powdered organisms by a modification of the hot phenol/water method (31, 32).

Identification of the L1 LOS by SDS-PAGE-- We electrophoresed SDS-disaggregated LOS made by selected Neisseria through 11.5 inches of 13.1% polyacrylamide gel (SDS-PAGE), stained them with silver (33), and immunoblotted them with mAbs 17-2-L1 (L1), 2-1-L8 (L8), and 9-2-L379 (L3,7) (21, 34).

Mass and Composition of the L1 and L8 LOSs-- We estimated the mass of the L1 and L8 LOS by electrophoresing SDS-disaggregated LOS of meningococcal strain 126E, which makes both, with those of gonococcal strains, MS11_mkD and the 1291 series (3). MS11_mkD makes L8 and L3,7 LOS (21); 1291_b and 1291_c make L1 and L8 LOS, respectively (22), and 1291_wt makes an L3,7 LOS. We used mAbs to locate serotype-specific LOS in the gels.

We estimated the molecular weight of the 1291 LOS by assuming that they had diphosphoryl hexaacyl lipoidal moieties (13, 35, 36) and that the GlcNAc that is invariably linked to the second basal heptose (Hep₂) (2) is always O-acetylated (14, 37). The derived molecular weight of the diphosphoryl hexaacyl neisserial LOS lipoidal moiety is 1713 (35, 36).

The observed molecular weights of the (Kdo) oligosaccharides (OS) released by acid hydrolysis from LOS made by strain 1291 and its mutants are as follows: 1291_wt, 1679; 1291_a, 1517; 1291_b, 1476; 1291_c, 1314; 1291_d, 1152; and 1291_e, 990 (22). (The 1291_d and 1291_e mutants each make LOS with (Kdo) OS of molecular weights of 1152 and 990 but in different abundances (22). Although only ~20% of the molecules made by 1291_d have (Kdo) OS of M_r 1152, as compared with ~50% of those made by 1291_e (22), we assigned the smaller molecule to 1291_e to avoid confusion); we added the mass of Kdo (220) to each. The mass of the OS of the "5.4-kDa" LOS made by MS11_mkD (21) was calculated by adding the mass of GalNAc (203) to that of the 1291_wt OS (22, 24). The mass of each OS, minus the mass of H₂O lost by linkage to the lipoidal moiety (18), then was added to the calculated molecular weight of the lipoidal moiety, 1713. The calculated molecular weights of the native LOS were as follows: MS11_mkD, 3797; 1291_wt, 3594; 1291_a, 3432; 1291_b, 3391; 1291_c, 3229; 1291_d, 3067; and 1291_e, 2905.

The coefficient of correlation (r) between the electrophoretic mobilities of the gonococcal LOS and their derived molecular weights was 0.989. We used the function that described this correlation (M_r = (158.5  rd)/0.029) to calculate an estimated molecular weight for each of the four 126E LOS bands in the SDS-PAGE gel and then calculated the nominal triply charged ESI-MS negative ion that LOS of each of these estimated molecular weights would yield after deacylation. We used the formula shown in Equation 1,

(Eq. 1)

where 760 is the mass of the ester-linked laurate residues and 42 the mass of the  GlcNAc O-acetate groups that are removed during deacylation (10, 36).

We then sought each of these ions in the triply charged region of ESI-MS spectra of 126E LOS.

Electrospray Ionization Mass Spectrometry (ESI-MS)-- LOS were deacylated with anhydrous hydrazine, as described (38, 39). Heterogeneity among lipoidal moieties is accounted for by different numbers of ester-linked -OH-C₁₂ and C₁₂ fatty acids (31). Deacylation, therefore, yields a homogeneous lipoidal moiety that consists of two GlcN residues, two amide-linked -OH-C₁₄ fatty acid residues, and two phosphate residues (35); its average molecular weight is 953.0089 (36). The -GlcNAc O-acetate also is lost during deacylation.

We analyzed samples of O-deacylated LOS by ESI-MS with a VG Bio-Q mass spectrometer with an electrospray ion source. Scans were taken in the negative ion mode over the m/z range of 300-2000 at a resolution of ~500. LOS samples were dissolved in distilled water at a final concentration of 1-3 µg/µl, and 3-µl aliquots were injected via a Rheodyne injector into a constant stream of H₂O/acetonitrile (75:25%, v/v) containing 1% acetic acid as a solvent (36). A flow rate of 2-4 µl/min was maintained throughout the analysis with an Isco syringe pump. Mass calibration used horse heart myoglobin as a reference, and the software was supplied by VG Bio-Q; the instrument was tuned in the negative ion mode with sulfated cholecystokinin-8 (Peninsula Laboratories). Spectra were averaged over several scans and digitally smoothed (36). For relatively abundant peaks the accuracy was ±0.3 m/z; for poorly defined, low abundance peaks it was ±0.4-0.8 m/z.

O-Deacylated LOS form deprotonated species that are preferentially in charge state z = 3 and to a lesser extent in charge state z = 2 (36). Triply charged molecular ions appear as doublets, 6 mass units apart, because ions that have lost a water molecule (m/z = 18/3 = 6) are formed by variable lactonization of a Kdo residue (Kdo₂) during ionization (36); lactonized ions are indicated in the text by an asterisk.

Neisserial LOS OS are composed of hexoses (Hex), N-acetylhexosamines (HexNAc), heptose (Hep), Kdo, sialic acid (NeuNAc), phosphoethanolamine (PEA), and an O-acetyl group. Two Hep residues, two Kdo residues, and the O-acetylated -GlcNAc are conserved (2, 13, 14, 22, 24, 36). We used a computer program to generate all compositions that would have a molecular weight that was consistent with that of each O-deacylated LOS molecular ion that was resolved sufficiently in the 126E LOS ESI-MS spectrum. We considered only those compositions that were comprised of known LOS constituents, contained two Hep residues, two Kdo residues, an O-deacylated GlcNAc residue, and an O-deacylated, diphosphoryl lipoidal moiety and that would yield a triply charged ion that was within 1 m/z of each observed ESI-MS ion (36).

For predicting the molecular weights of the various compositions we used the following interval average mass values: H, 1.0082; H₂O, 18.0153; Hex, 162.1424; Hep, 192.1687; HexNAc, 203.1949; Kdo, 220.1791; NeuNAc, 291.2579; PEA, 123.0482; acetate, 42.0373; and O-deacylated lipid, 953.0089. The appropriate interval average mass values were added to that of the O-deacylated lipid. For calculating the molecular weights of native LOS, we added the interval nominal mass values to 1713, the nominal molecular weight of the conserved diphosphoryl hexaacyl lipoidal moiety (31, 35).

Release of OS from LOS-- OSs were released from LOS by hydrolysis with 1% acetic acid (100 °C, 2 h) (9). We centrifuged the hydrolysate twice for 20 min at 4 °C and 7000 rpm; the supernatant with released OS was lyophilized, redissolved in 52 mM pyridinium acetate (5-10 mg/ml), and separated by chromatography through 180 cm of Bio-Gel P-4 (<400 mesh) equilibrated with 52 mM pyridinium acetate. The eluate was monitored by refractometry and collected in 1.67-ml fractions.

Dephosphorylation-- We dephosphorylated LOS and OS by hydrolysis with HF. LOS and OS were reacted with a 48% (aqueous) HF solution (10 µg of LOS per µl) in the dark at 4 °C for 16-20 h (OS and O-deacylated LOS) or 36-40 h (intact LOS) (9). After evaporation of HF, the residues were resuspended in H₂O and lyophilized.

Carbohydrate Composition Analysis-- For composition analysis of neutral sugars, we dissolved 20 µg (~10 nM) of dephosphorylated OS in 200 µl of H₂O, added 200 µl of 4 M trifluoroacetic acid, and heated the mixture for 4.25 h at 100 °C (40). For quantitation of HexNAcs, we substituted concentrated HCl for 4 M trifluoroacetic acid (40). We evaporated the hydrolysates to dryness in a Speed-Vac concentrator, and we carried out monosaccharide separation and quantitation with use of high pH anion-exchange chromatography with pulsed amperometric detection, as described (39-41). To elute the monosaccharide components, the gradient was modified slightly as follows: (i) 20 mM NaOH for 22 min, (ii) linear to 50 mM NaOH in 10 min, (iii) linear to 100 mM NaOH and 100 mM sodium acetate in 3 min, and (iv) linear to 160 mM sodium acetate in 15 min (with NaOH kept constant at 100 mM). A monosaccharide mixture that contained known quantities of fucose, GalNH₂, GlcNH₂, galactose (Gal), and glucose (Glc) (Dionex, Sunnyvale, CA) was used as a standard. The authentic monosaccharide liberated from the OS of Salmonella typhimurium Ra mutant LPS was used as a standard for the identification of L-glycero-D-manno-heptose (41).

Carbohydrate Sequence and Linkage Analysis-- We used a combination of liquid secondary ion (LSIMS) and tandem (MS/MS) mass spectrometry and methylation analysis to confirm compositions and determine the sequences and linkages of the component sugars (13, 22). Oligosaccharides were dissolved in H₂O, and aliquots were transferred to a stainless steel probe tip that had been charged with approximately 1 µl of a glycerol/thioglycerol (1:1) matrix. O-Deacylated HF-treated LOS were first dissolved in H₂O/methanol (1:1). Samples for LSIMS were analyzed with a Kratos MS-50 double focusing mass spectrometer operating at a mass resolution of 1500-2000 (m/m, 10% valley). Samples were sputtered and ionized with a Cs⁺ ion primary beam of 10 keV; secondary ions were accelerated at 6-8 kV. Scans were acquired at 300 s/decade (22). Ultramark 1621 was used manually to calibrate spectra to an accuracy of better than ±0.2 Da.

MS/MS spectra were gotten with a four-sector Kratos Concept II HH mass spectrometer with an optically coupled diode array detector on MS-2 (22). Spectra were acquired in the negative mode; a Cs⁺ ion primary beam energy of 18 keV was used to sputter and ionize the oligosaccharides. The deprotonated molecular ions (M  H) were selected in MS-1 and passed through to the helium collision cell floated at 2 keV, and pressure was adjusted to attenuate the precursor molecular ion by approximately 65%. Fragment ions were separated in MS-2 with use of a magnet jump with successive 4% mass windows and a constant B/E ratio. Fragment ions were detected on a one-inch optically coated diode array detector, and masses were assigned automatically by a Kratos Mach 3 data system that used CsI as an external reference for both MS-1 and MS-2.

The mass spectra were interpreted on the basis of the established structures of gonococcal LOS that bind mAbs 2-1-L8 (23) and 9-2-L379 (24). Nominal masses, based on the isotopically pure ¹²C component of the natural isotopic distribution, are given for ions.

Enzymatic Degradations of LOS-- We used enzymatic degradations to assign anomeric configurations (10, 23, 24). We bought the following glycosidases from Sigma: -galactosidase from Aspergillus niger; -galactosidase from A. niger; -N-acetylglucosaminidase from jack beans; and neuraminidase from Clostridia perfringens.

Sialic acid residues were removed from LOS by treatment with neuraminidase (13). We suspended 50 µg of purified LOS in 25 µl of phosphate-buffered saline (pH 6.0) and mixed the suspension with an equal volume of the same buffer that also contained 50 milliunits of active enzyme or the same buffer that contained 50 milliunits of enzyme that had been inactivated at 100 °C for 5 min. We incubated the reaction mixtures at 37 °C for 2 h and then stopped the reaction by adding 50 µl of SDS-PAGE sample buffer (34).

We suspended - and -galactosidases and -N-acetylglucosaminidase in 200 mM sodium acetate buffer of pH 4.5; -galactosidase and -N-acetylglucosaminidase were predialyzed in the buffer to remove ammonium sulfate in the enzyme preparations (8). We added appropriate amounts of each enzyme in NaAc buffer (10 µl; 2 units) to a 20-µg solution of LOS in 40 µl of 0.2% sodium taurodeoxycholate (Sigma). We kept the reaction mixture at 37 °C for 16 h and then inactivated the enzymes by boiling the mixture for 5 min. We analyzed the effects of the enzymes by SDS-PAGE and immunoblot (9).

Structual assignments were confirmed by referring to published structures of gonococcal (22-24, 42) and meningococcal LOS (10, 13-15, 23, 29, 37).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Identification of the L1 LOS-- The three reference L1 strains each shared two LOS components; the faster migrating (lower) bound mAb 17-2-L1 (Fig. 1). This LOS was the next higher 126E LOS (LOS₃) from the LOS that bears L8 (second component from the bottom, or LOS₂). The N. lactamica strain, 1207-0, bound mAb 17-2-L1 to an LOS that was slightly smaller than the meningococcal L1 LOSs. N. meningitidis strains 8529 and 8532, which are not L1, did not make LOS of these electrophoretic mobilities and did not bind mAb 17-2-L1. Strain 190I made two additional and higher molecular weight LOS, the smaller of which bound the L3,7 mAb (Fig. 1). We assigned the L1 epitope to LOS₃ of strain 126E.

View larger version (63K): [in this window] [in a new window]   Fig. 1.   Identification of the L1 LOS and a type-specific mAb. A, SDS-PAGE separation; B, immunoblot of LOS made by N. meningitidis L1 (190I, 126E and 6940), L3,7 (8529), and L8 (8532) strains and by an N. lactamica strain (1207-0). N. gonorrhoeae MS11mkD provided LOS molecular weight markers. The meningococcal L1 strains each shared two LOS components that were not made by 8529 or 8532 (A). The faster migrating components (rd = LOS3 of 126E (5)) bound mAb 17-2-L1 (L1; B) and migrated just above LOS2 of 126E (A) which bound mAb 2-1-L8 (B). The lactamica also made an LOS that bound mAb 17-2-L1 (B), but it was not visible in A. 190I, which also is L3,7, made an LOS that migrated with the "4.5-kDa" gonococcal LOS (21) and with LOS made by 8529 and the lactamica strain (A); these LOS bound mAb 9-2-L379 (L3,7; B).

LOS Serotypes of Endemic Strains (Table I)-- Each of the 34 endemic strains made some amount of more than one "type-specific" LOS; only one was of a single SPRIA inhibition L-type (L2). L-type was not a uniformly distributed attribute. L5 was not found, and 25 strains were L3 (1), L7 (2), or both (22). L-type also was not an independent attribute (degrees of freedom = 11, ² = 19.9-43.5, p = 0.05 to <0.0001 for the independence of L1, L2, L3, L4, L7, and L8 from all other L-types; degrees of freedom = 11, ² = 18.3, p = 0.07 for the independence of L6 from all other L-types).

Almost all strains made some amount of L8; only 3/34 strains inhibited the L8 antiserum <10% in the SPRIA inhibition assay. Eighteen (53%) strains met the formal criterion for L8. L8 expression was positively associate with L1 expression (² = 7.8, p = 0.005) and negatively associate with L2, L4, and L6 (² = 11.8, p = 0.0006; ² = 6.2, p = 0.01, and ² = 5.3, p = 0.02, respectively).

Seven (21%) strains were L1, as determined by SPRIA inhibition; mAb 17-2-L1 bound to the six L1 strains that had SPRIA inhibitions of >50%, as well as to four strains that inhibited the L1 antiserum <35% (10-33%). SDS-PAGE and immunoblot analysis of LOS made by strains 7952 (10% L1 inhibition) and 8024 (33% L1 inhibition) (Fig. 2) confirmed that these strains made L1 but in lesser amounts than the formal L1 strains. Three L1 strains that also were L3,7 by SPRIA inhibition bound mAb 9-2-L379 to the L3,7 LOS strongly; the L3 strain, 8022, bound it only weakly (Fig. 2). Nine of 11 L1 strains (SPRIA inhibition and/or mAb binding) also were L8 (Table I).

View larger version (55K): [in this window] [in a new window]   Fig. 2.   L1 expression by endemic case strains. LOS of strains 7981 (L1,3,7,8), 7982 (L1,8), 7992 (L1,8), and 8022 (L1,3,7,8), which inhibited the L1 SPRIA antiserum by 60-93%, were electrophoresed (A) with those of strains 7952 (L3,4,6,7,8) and 8024 (L2,3) (10% and 33% L1 SPRIA inhibition, respectively), and immunoblotted (B) with three LOS mAbs, 9-2-L379 (L3,7), 17-2-L1 (L1), and 2-1-L8 (L8). The position of each mAb-stained LOS band in B is shown to the right of the gel by labeled arrowheads. Gonococcal MS11mkD and meningococal 126E LOS provided size markers. All L1 strains save 7981 made abundant LOS that migrated with LOS3 of 126E (A) and bound mAb 17-2-L1 (B); 7981 (77% L1 SPRIA-Inhibition) made only a little of this LOS. Strains 7952 and 8024 made enough L1 LOS to bind the L1 mAb (B) but not enough to be seen clearly in A. Strains 7982 and 8022 also made an LOS that migrated with LOS4 of 126E (A). L3,7 strains made a higher molecular weight doublet (faintly visible for strain 8022, A), the lower component of which bound the L3,7 mAb (B); 8024, an L3 strain, made an abundant doublet but bound the mAb poorly. L8 strains with SPRIA inhibitions >50% (strains 7982, 7992, and 8022) made LOS that weakly bound the L8 mAb.

Identification of the L1 and L8 LOS in ESI-MS Spectra-- Since strain 126E is both L1 and L8, we used its LOS to compare the L1 and L8 structures. Fig. 3 shows the electrophoretic mobility of the four visible 126E LOS components relative to those of the gonococcal LOS molecular weight "standards," and Table II gives their estimated molecular weights. A comparison of the triply charged, deacylated ESI-MS ion that LOS of each of these molecular weights would yield and the corresponding observed ion in the ESI-MS spectrum (Fig. 4) also are given in Table II.

View larger version (46K): [in this window] [in a new window]   Fig. 3.   Estimation of 126E LOS molecular weights. A, 126E LOS was electrophoresed with gonococcal LOS that have established structures (21-24) (see "Experimental Procedures" for details). The coefficient of correlation between the electrophoretic mobilities of the gonococcal LOS and their derived masses was 0.989. The molecular weights of the L3,7 gonococcal LOS and of the four 126E LOS components, as calculated by the function that described the correlation, are given in the margin. B is an immunoblot of A with three LOS mAbs, 9-2-L379 (L3,7), 17-2-L1 (L1), and 2-1-L8 (L8). The position of each mAb-stained LOS band in B is shown to the right of the gel by labeled arrowheads. Gonococcal MS11mkD and mAb 9-2-L379 bound LOS from all of the strains except 126E. mAb 17-2-L1 bound 1291b and 126E LOS3; mAb 2-1-L8 bound LOS of MS11, 1291c, and 126E.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Negative ion ESI-MS of triply charged 126E LOS. One Kdo residue variably lactonized during ionization, causing the loss of an H2O molecule (m/z 6) and the molecular ions to appear as doublets. The many molecular species could be resolved into series, or families, that differed only in their basal structures. The most abundant series (ions A-C and F) had a single basal HexNAc and one PEA residue and are filled. A variant (PEA)1 series that had two, rather than one, basal HexNac residues (m/z +67.7) was in low abundance at m/z 923.5 and 1027.6. A second, reasonably abundant series of ions lacked a basal PEA (PEA)0 (m/z 41) but had either one (cross-hatched; E) or two (diagonal lines; D and H) basal HexNAc residues. Series with two basal PEA (PEA)2 residues (G) and with a single Kdo residue were difficult to resolve. There were at least five sialylated species; they are identified by *.

In general, there was good concordance between the calculated and observed ESI-MS ions. Ions that were within 6 m/z of the calculated values for the two fastest migrating SDS-PAGE LOSs, LOS₁ and LOS₂, were easily identified (A and B, respectively); they were the highest abundance ions in their respective regions of the spectrum. The most abundant ion in the entire spectrum, C, had the closest mass to that expected for the most abundant 126E LOS, LOS₃ (862 m/z versus 844 m/z). The expected ion for the highest mass, or slowest migrating, 126E LOS, LOS₄ (889 m/z), was the same as that of the D ion in the ESI-MS spectrum (889.1 m/z), but the D ion was present in much lower abundance than would be expected for LOS₄, and its mass was less than that of either the E or F ion, both of which were present in equal or greater abundance than the D ion. The F ion (959.1 m/z) was the highest mass ion that was present in good abundance; its +97 m/z = 3 suggested that it arose by the addition of sialic acid (M_r 291) to the 862.2 m/z C ion. By assigning the F ion to LOS₄, the relative abundances of the A, B, C, and F ions matched the relative densities of the four 126E LOSs that were visible in SDS-PAGE.

The compositions of the A, B, C, and F ions were readily deduced from the ESI-MS spectrum (Fig. 4), and the compositions of their oligosaccharides are given in Table II. The composition of the OS of the B, or LOS₂, ion, was consistent with the lactosyl diheptoside L8 structure. The C ion (LOS₃; L1) was a single Hex residue larger than the B ion (Hex)₃; the A ion (LOS₁) was a Hex residue smaller (Hex)₁. The F ion (LOS₄) was the sialylated form of the C ion.

Resolution of the ESI-MS Spectrum (Fig. 4)-- The ESI-MS spectrum contained several prominent ions in addition to those that could be assigned to the four LOS that were visible in SDS-PAGE and many other low abundance and sometimes incompletely resolved ions. The multiple molecular species could be resolved into series, each of which had the same pattern of one, two, or three hexose constituents as the ABC molecular series, but each of which had different basal constituents (Table III). The (Hex)₃ molecule of each series usually also was present as a sialylated molecule.

The most abundant ABCF series had a single HexNAc residue ((HexNAc)₁) and one PEA residue ((PEA)₁). The (Hex)₃ D (883.1* m/z) and H (979.9* m/z) ions lacked a basal PEA ((PEA)₀) but had a second HexNAc residue ((HexNAc)₂); the H ion arose by sialylation of the D molecule (+97 m/z). The 54 m/z (Hex)₂ and the 108 m/z (Hex)₁ ions of this second prominent series were present at 829.4* and 781.6 m/z, respectively, albeit in low abundances. The presence, at 727 m/z, of a very low abundance of (Hep)₂(HexNAc)₂(Kdo)₂ ion in this series confirmed that the second HexNAc was a basal addition. The relative abundances of the four largest molecules of the ABCF and DH basal series were quite similar, as judged by signal height. The sialylated F and H ions accounted for 30 and 26%, respectively, of their respective molecular series and their non-sialylated (Hex)₃ precursors, C and D, for 49 and 47%, respectively.

Although (HexNAc)₂ molecules were most abundant as the (PEA)₀ series, they also were found with one or two PEA residues (Table III and Fig. 4). Ions representing the (HexNAc)₂(PEA)₁ series were present at 923.5* m/z ((Hex)₃) and 1027.6 m/z (NeuNAc(Hex)₃); the G ion (963.6* m/z) was the (Hex)₃ molecule of the (HexNAc)₂(PEA)₂ basal series. Smaller ions in the latter series were not resolved from ions of more abundant series. The E ion could have been either the sialylated (Hex)₃ molecule of the (HexNAc)₁(PEA)₀ basal series or the (Hex)₂ ion of the (HexNAc)₂(PEA)₂ series (Table III); desialylation (see below) showed it to be the former. The (Hex)₃ ion of this series was at m/z 815.

LOS Sialylation-- We confirmed that L1 LOSs were partially sialylated by treating those of various strains with neuraminidase (Fig. 5). Neuraminidase degradation enhanced the electrophoretic mobility of the largest LOS made by each of the L1 N. meningitidis strains and increased the densities of their L1 LOSs. Strain 190I partially sialylated both its M_r 3338 L1 LOS and its M_r 3595 L3,7 LOS. ESI-MS analysis showed that neuraminidase degradation of 126E LOS caused the loss of the E, F, and H ions, as well as that of the 1027.6 ion (Fig. 6), thus confirming that these ions arose by sialylation of lower mass precursors. A, B, C, and D ions were preserved.

View larger version (68K): [in this window] [in a new window]   Fig. 5.   Effect of neuraminidase treatment. SDS-PAGE (A) and immunoblot (B) of native (A lanes) and neuraminidase-treated (B lanes) LOS. B was immunoblotted with three LOS mAbs, 9-2-L379 (L3,7), 17-2-L1 (L1), and 2-1-L8 (L8). The position of each mAb-stained LOS band in B is shown to the right of the gel by labeled arrowheads. The electrophoretic mobility of the largest LOS made by each of the N. meningitidis strains was increased by neuraminidase treatment. Neuraminidase did not affect LOS of N. gonorrhoeae (MS11) or N. lactamica (1207-0), species that cannot endogenously sialylate their LOS (13, 53).

View larger version (19K): [in this window] [in a new window]   Fig. 6.   ESI-MS spectra of native (left panel) and neurominidase-treated (right panel) 126E LOS. Ion designations are those used in Fig. 4. The presumptively sialylated E, F, and H ions are lost after neuraminidase treatment; the A-C and D ions remain. The fact that neuraminidase treatment caused the heights of the C and D ions to increase proportionately relative to those of the A and B ions confirmed that the former were partially sialylated. The position of the G ion is marked in the desialylated spectrum; it was not in sufficient abundance to be regarded as a signal.

Analysis of Released Oligosaccharides-- OS released from 126E LOS by mild acid hydrolysis contained galactose and glucose in the molar ratio of 3:2 and GlcNAc as the sole HexNAc. The 3:2 Gal/Glc ratio in the mixture of OSs is consistent with the addition of galactose to lactosyl (Gal14Glc) molecules. This was confirmed by methylation analysis of the released OSs that showed the presence of Gal and GlcNAc as terminal non-reducing residues and of 1,4-Glc and 1,4-Gal as internal hexose residues. These linkages would be expected if Gal residues were added 14 to the now internal Gal residue of the lactosyl moiety of (Hex)₂ L8 molecules to form (Hex)₃ L1 molecules. Most of the Hep residues were tri-linked, as expected (Hep₁, 1,3,4-linked; Hep₂, 1,2,3-linked), but 1,2-Hep from (PEA)₀ basal series molecules was present in low abundance.

The released OSs eluted from a P-4 polyacrylamide column as two peaks of equal volume (7.5 ml); each 1.67-ml fraction was analyzed by LSIMS. A (H) molecular ion of mass 1475 was present in all nine fractions. The composition of this ion, (Hex)₃(Hep)₂(PEA)₁(HexNAc)₁(Kdo)₁, was consistent with that of the ESI-MS C ion. The (PEA)₀(HexNAc)₂ basal series analogue of this (Hex)₃ species (ESI-MS ion D) also was present, at m/z = 1555, but only in the last two fractions. The abundance of this m/z 1555 ion was not sufficient for MS/MS analysis.

A (H) molecular ion from the loss of one Hex residue from the m/z 1475 ion (m/z 1313; ESI-MS B ion) appeared in the second volume. The m/z 1151 molecular ion for the ESI-MS A ion appeared in the later fractions of the second volume, slightly before the m/z 1555 ion.

Tandem Mass Spectrometry-- Tandem LSIMS spectra were gotten to confirm structural assumptions. MS/MS spectra of m/z 1475 molecular ions and comparison of the spectra of native and dephosphorylated ions revealed the presence of two isobaric (Hex)₃ ions, shown as a composite M_r 1638 molecule with the fragmentation sites shown in Fig. 7. The more abundant M_r 1476 molecule partitioned into the first P4 volume only; its three Hex residues were in sequence (Hex HexHexHep₁), as shown by the presence of ^1,5A/X₃ (m/z 458/1017), Y₃ (m/z 988), and Z₃ (m/z 972) fragmentation ions in MS/MS spectra (Fig. 7). Its O-acetylated HexNAc residue was linked to C₂ of the second Hep residue (B/Y₁ fragmentation ions; m/z 246/1229), which was phosphorylated on C₃ (^1,3A₂ (m/z 427) and ^1,5A₂ (m/z 531) fragmentation ions). We designated it L1.

View larger version (24K): [in this window] [in a new window]   Fig. 7.   Negative ion MS/MS CID fragmentation ion spectral analysis of (M  H) 126E m/z 1475 OS precursor ion. Two different m/z 1475 structures are depicted as a composite, Mr 1638, structure with alternative non-reducing hexose termini within brackets. Brackets also enclose the -GlcNAc O-acetate that is variably present after acid hydrolysis. The site of PEA substitution of Hep2 is shown by an asterisk. Potential cleavage sites (- - -) are designated according to a modification of the nomenclature (22) proposed by Domon and Costello (54) in which  and  denote the precedence of branching, not chain length. As Hep2 is attached to C3 of Hep1, it precedes the C4-attached -Glc residue and defines the  branch; this usage is distinct from the use of , , and  to denote the three glycose chains that can arise from the basal region of neisserial LOS, a usage that was based on the original Domon and Costello (54) proposal rather than precedence of branching (2). Assignment of A-C fragments is for the composite structure that would have two triglycose branches and an m/z of 1637. The A-C designations of fragments from the diglycose branches of the two m/z 1475 ions would be reduced by 1 (e.g. B3B2). Spectra were obtained for precursor ions found in each of nine 1.67 ml of OS-containing P-4 volumes before and after HF hydrolysis to remove PEA residues. Nominal masses for precursor and fragmentation CID fragments were used.

Both P4 volumes contained a second m/z 1475 ion, in lower abundance, that had two Hex residues linked to Hep₁ in sequence and a third Hex residue linked to C₄ or C₆ of its O-acetylated HexNAc residue, as shown by the presence of ^0,2A/X₂ (m/z 323/1152), ^1.3A/X₃ (m/z 589/886), and ^1,5A/X₃ (m/z 693/782) fragmentation ions that each differed by m/z 162 from their respective L1 A/X fragmentation ions (Fig. 7). We designated this molecule, with an elongated  chain, L1a.

Negative ion MS/MS CID spectra of m/z 1313 ions confirmed the expected HexHexHep₁(Hep₂(PEA)(HexNAc))Kdo L8 structure.

Glycosidase Degradations-- Hydrolysis of 126E LOS with -galactosidase caused the total collapse of LOS₃ into LOS₂ and the loss of the L1 epitope (Fig. 8). The now more abundant LOS₂ bound mAb 2-1-L8. Degradation of strain 6940 LOS with -galactosidase also caused the total loss of its L1 LOS and the appearance of a very prominent, and previously inapparent, LOS that bound mAb 2-1-8. These results confirmed that all components of LOS₃ arose by the addition of -Gal residues to LOS₂ molecules and that L1 is determined by an -Gal substitution of the L8 structure.

View larger version (60K): [in this window] [in a new window]   Fig. 8.   Effect of treatment with glycosidases. 126E LOSs that had been treated with either -galactosidase (126Eb), -galactosidase (126Ec), -N-acetylglucosaminidase (126Ed), or mock-treated (126Ea) were electrophoresed (A) and then immunoblotted (B) with three LOS mAbs, 9-2-L379 (L3,7), 17-2-L1 (L1), and 2-1-L8 (L8). The position of each mAb-stained LOS band in B is shown to the right of the gel by labeled arrowheads. Strain 6940 LOS was treated with either -galactosidase (6940a), -galactosidase (6940b), -N-acetylglucosaminidase (6940c), or -galactosidase followed by -galactosidase (6940d) before electrophoresis (C). Each exoglycosidase would remove its respective terminal glycose substrate and uncover its precursor.

Addition of Gal14 to an LOS lactosyl moiety would create an -OS with the same structure as that of the human P^k GSL oligosaccharide. A P^k mAb bound strains 126E, 190I, 6940 and the six Houston L1 strains that bound mAb 17-2-L1. In contrast, only strains 126E, 6940, and 8022 (93% L1 SPRIA inhibition), and the gonococcal 1291_b LOS, bound a Gal14Gal-R mAb as well as they bound the P^k mAb, and only strain 1291_b bound a P₁ mAb (Gal14Gal14GlcNAc-R). These results confirm the Gal14Gal14Glc structure of the L1 LOS  chain and that it is this structure that binds mAb 17-2-L1.

-Galactosidase hydrolysis of 126E LOS caused the partial loss of LOS₂ and a marked increase in LOS₁ (Fig. 8), thus confirming that LOS₂ terminates with a -Gal residue. The binding of mAb 2-1-L8, however, was not affected visibly, either because hydrolysis was incomplete or because the L1a molecule, which would run as LOS₂ after removal of the terminal -Gal residue from its  chain (Table IV), bound the mAb.

View this table: [in this window] [in a new window]   Table IV L8 and alternative L1 LOS structures L8 structures are from published studies (21-23) and the present data; the L8 lactosyl structure is in boldface. mAb 2-1-L8 requires a single PEA substitution and the absence of the -GlcNAc substitution. Whether lactosyl diheptoside molecules with -GlcNAc substitutions remain L8 is not known. Note that the alternative Hep2 substitutions are in brackets.

-Galactosidase degradation of strain 6940 LOS caused the partial loss of LOS₄ and LOS₃ and the appearance of prominent LOS₂ and LOS₁ molecules, such that the treated 6940 LOS had the same SDS-PAGE profile as the native 126E LOS. Thus, the principal difference between the LOS of these two L1 strains appeared to be the presence of terminal -Gal residues on higher molecular weight LOS of strain 6940. Treatment of this same LOS with -galactosidase after prior hydrolysis with -galactosidase caused the appearance of a much more prominent LOS₁, as would be expected if the -galactosidase removed a terminal -Gal residue from an L1 molecule and uncovered a precursor -Gal residue (Fig. 4).

Hydrolysis of 126E LOS with -N-acetylglucosaminidase caused a marked increase in the density of LOS₂ and in its binding of mAb 2-1-L8. Since GlcNAc was the only HexNAc detected in 126E LOS, the second HexNAc in the (HexNAc)₂ basal series must be a terminal GlcNAc as the -anomer. -N-Acetylglucosaminidase degradation of strain 6940 LOS caused the appearance of a new LOS₂ and a reduction in the densities of higher molecular weight LOS, some of which also must have been (HexNAc)₂ basal series molecules with terminal -GlcNAc residues.

Structures for the different basal series L8 and the two alternative L1 molecules could be derived from these data, and published structures and are given in Table IV.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

These studies reveal both quantitative and qualitative differences in LOS immunotype expression and find that meningococal LOS are more heterogeneous, and LOS serotyping less discriminatory, than previously thought.

To our knowledge, this is the first application of LOS serotyping to strains that caused discrete, sporadic disease during a prolonged period in a single location and that were of diverse protein serotypes (19, 26, 43). With use of mAbs, Jones et al. (6) found that 97% of hyperendemic ET-5 complex strains isolated from cases in the United Kingdom expressed the L3,7 immunotype and that only 13% of these invasive "L3,7,9" strains also expressed "L1,8,10." In contrast, 70% of contemporaneously carried but non-invasive ET-5 complex strains expressed the L1,8,10 immunotype, and only 24% expressed L3,7,9 alone. We found that L3 and L7 also were expressed commonly by the diverse strains (19, 26, 43) that cause endemic disease (25/34; 74%), but these endemic L3,7 strains always expressed other LOS types, as well, including L8 (14) and L1 (3).

The structural basis for the association between L8, L1, and L3,6,7 is readily apparent, as the latter two arise by alternative, and mutually exclusive, additions to the lactosyl diheptoside L8 structure as follows: L1 by the addition of an -Gal residue to create a globoside-like structure, and L3,7 by the addition of lactosamine to form the lacto-N-neotetraosyl oligosaccharide of paragloboside. Thus, L8 structures act as "toggles" or biosynthetic decision points. Some strains only add -Gal (126E, strain 6940), whereas others only add -GlcNAc (strain 8529 and others), and still others add both (190I and 8022). Whether a strain types as L8 depends on how many of its L8 LOS molecules remain unsubstituted. This biosynthetic relationship between lactosyl, globosyl, and paraglobosyl molecules also has been noted among gonococcal LOS (22-24).

The 126E L1 LOS structure was found previously with use of NMR spectroscopy (37), but our application to this strain's LOS of the mass spectrometric techniques that we developed to structure gonococcal LOS (22, 36, 44) enabled us to appreciate additional structures. It is reasonable to expect that still more structures will be found as more neisserial LOSs are studied. For example, terminal -Gal residues on higher molecular weight LOSs of strain 6940 were not expected and are not explained by known meningococcal LOS structures.

ESI-MS proved to be particularly useful (36). By electrophoresing 126E LOS with gonococcal LOS whose structures have been established (21-24), we could estimate the molecular weights of 126E LOS components with sufficient accuracy to align their SDS-PAGE profile with their ESI-MS spectrum and gain structural information about SDS-PAGE-visualized components. SDS-PAGE resolved 126E LOS into only four components, even with use of longer gels to maximize resolution (45); ESI-MS resolved these same LOS into more than 20 molecules of various abundances that had six different basal regions, four  chains, and an extended  chain.

The LPS made by Salmonella isogenic mutants that we used as molecular weight standards in the past (3) systematically overestimate the molecular weight of neisserial LOS, presumably because of the different branching patterns of the two sorts of glycolipids (42). Our previous estimates of the size of the L1 and L8 LOSs were too great by ~610 (4000 versus 3391) and ~370 (3600 versus 3229), respectively (3, 5). We now can replace the LPS-based estimates of the size of the most abundant LOSs that can be visualized in SDS-PAGE with more exact masses.

The different PEA basal series were not unexpected (46). The site of the second PEA substitution is not known, but meningococcal LOSs may have single PEA substitutions at C₆ and C₇ of Hep₂ (47-49), so one of these two sites may bear the second PEA residue. Kulshin et al. (50) reported that a diphosphoryl meningococcal LOS lipoidal moiety was substituted at both ends with PEA. Lipid PEA substitutions would have confounded interpretation of the ESI-MS spectra, but we have found no evidence of pyrophosphoryl groups on deacylated neisserial LOS.

Neither the presence of an isobaric L1a molecule nor that of a -GlcNAc basal region addition was expected. Di Fabio et al. (37) reported that the OS released by acid hydrolysis from their preparation of strain 126E LOS eluted from 90 cm of P-4 in a single broad peak that could be narrowed by deacylation with NH₄OH and that strain 126E made only a single LOS molecule with the (GlcNAc)₁(PEA)₁ L1 structure. One explanation why Di Fabio et al. (37) missed the heterogeneity of 126E LOS would be that NMR spectroscopy is not sensitive to the presence of structurally related oligosaccharides in a mixture (51). In addition, we were able to resolve 126E OS into two fractions with use of 180 cm of P-4.

The L1a structure was confirmed by the presence in tandem spectra of A/X fragmentation ions that differed by m/z 162. That these were  and not  fragments was clear from the difference in mass between in-line Hex (m/z 162) and OAcHexNAc (m/z 245) residues and the contribution of the O-acetate moiety (m/z 42) to the ^0,2A/X₂ fragment. The site of Gal substituion of the -GlcNAc could not be unambiguously assigned in the absence of ^2,4A₂ fragments, but as in previous studies (22), tandem spectra confirmed O-acetylation of C₃ of the -GlcNAc, which leaves only C₄ and C₆ as possible sites. Methylation analysis did not resolve this question, as di-linked GlcNAc was not seen, presumably because of the relatively low abundance of L1a molecules. C₆ substitutions of neisserial LOS Hex residues have not been found, whereas C₄ substitutions are common, so we assigned the Gal residue to C₄. This assignment is consistent with the unambiguous assignment of the L1 additional Hex to C₄ of the preceding Hex.

Extensions from the  GlcNAc, although predicted (2), have not been reported previously. Because the L1 complexes of both 126E and 6940 were entirely degraded by -galactosidase, we assigned the -anomeric configuration to the additional Gal residue of the L1a structure. This would create an -lactosaminyl  chain in parallel with a -lactosyl  chain (Table IV and Fig. 7).

The linkage of the basal region -GlcNAc could not be determined because of the low abundance of the m/z 1555 LSIMS ion. C₃ of Hep₂ normally is substituted, either with PEA (14, 22-24, 37, 48) or an -Glc residue (42, 47, 49), so this would be a likely site of -GlcNAc substitution, as well. This supposition is supported by the absence of a PEA residue on the most abundant (HexNAc)₂ basal series and the predominance of 1,3,4- and 1,2,3- tri-linked Hep residues. We presume that phosphoethanolaminylated (HexNAc)₂ molecules had Hep₂ C₆ or C₇ PEA substitutions (48, 49), but such species were in such low abundance that their tetra-linked Hep residues would have been missed.

Sialylated L1 LOS also were unexpected. Di Fabio et al. (37) would have missed sialylation of 126E LOS, because acid hydrolysis of LOS desialylates the released OS. Mass spectrometric analysis of intact LOS, however, could not distinguish between the two L1 isomers nor determine whether both, or only the more abundant L1 isomer, were partially sialylated. Regardless, sialogloboside-like LOSs now can be added to sialoparagloboside-like ones in the list of human glycoconjugates that neisserial LOS mimic.

Strain 190I, an epidemic strain that expresses a unique bactericidal serotype (19), made both L1 and L3,7 LOS and partially sialylated both molecular species. It will be interesting to learn whether the same enzyme sialylates both molecules and, if so, whether the kinetics of sialylation differ.

Strains 126E and 6940 LOS had very different SDS-PAGE profiles, primarily due to the absence of LOS₁ and LOS₂ (L8) from the LOS of strain 6940. Both strains used basal region -GlcNAc substitutions, but strain 6940 also used a -Gal substitution that was not used by 126E. Treatment of strain 6940 LOS with -galactosidase resulted in an SDS-PAGE profile that was nearly the same as that of 126E LOS. Solving strain 6940 LOS structures will require additional analyses.

In summary, strains of N. meningitidis use different kinases and glycosyltransferases to substitute lactosyl diheptoside LOS in a variety of ways, some mutually exclusive and some not, that create a set of structurally related glycolipids. The use of multiple sets of modifying enzymes by a strain results in expression of more than one LOS type. When multiple LOS types are expressed, one may predominate, reflecting preferential use of the responsible enzymes. Whereas the relative abundances of the different LOS that an individual strain can make is a fairly stable attribute of laboratory grown organisms (6, 52), they can vary greatly among different strains of the same L-type. Thus LOS type is neither a stable nor discrete phenotype.

How these organisms express LOS in vivo, and whether certain LOS molecules that are uncommonly made by in vitro grown organisms are necessary for pathogenesis, remains to be explored.

	ACKNOWLEDGEMENTS

We thank May Fong for administering the grants and preparing the manuscript. Mass spectra provided by the UCSF Mass Spectrometry Facility (A. L. Burlingame, Director) were supported by the Biomedical Research Technology Program of the National Center for Research Resources, National Institutes of Health Grant BRTP RR01614 and by National Science Foundation Grant DIR 8700766.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants AI 21171 and AI 21620, Thrasher Research Fund Grant 2802-0, and the Research Service of the Department of Veterans Affairs (all to J.McL. G.). This is Paper 95 from the Centre for Immunochemistry.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ To whom correspondence should be addressed: VAMC (111W1), 4150 Clement St., San Francisco, CA 94121. Tel.: 415-476-5371; Fax: 415-221-7542; E-mail: crapaud@vacom.ucsf.edu.

	ABBREVIATIONS

The abbreviations used are: LOS, lipooligosaccharides; mAb, monoclonal antibody; GSL, glycosphingolipids; PAGE, polyacrylamide gel electrophoresis; Kdo, 3-deoxy-D-manno-oct-2-ulosonic acid; ESI-MS, electrospray ionization-mass spectrometry; LSIMS, liquid secondary ion-mass spectrometry; OS, oligosaccharide; Hep, heptose; Hex, hexose; HexNAc, N-acetylhexosamine; PEA, phosphoethanolamine.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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