Structural characterization of a novel branching pattern in the lipopolysaccharide from nontypeable Haemophilus influenzae Martin Ma ˚ nsson 1 , Derek W. Hood 2 , E. Richard Moxon 2 and Elke K. H. Schweda 1 1 Clinical Research Centre, Karolinska Institutet and University College of South Stockholm, Novum, Huddinge, Sweden; 2 Molecular Infectious Diseases Group and Department of Paediatrics, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK Structural analysis of the lipopolysaccharide (LPS) from nontypeable Haemophilus influenzae strain 981 has been achieved using NMR spectroscopy and ESI-MS on O-deacylated LPS and core oligosaccharide (OS) material as well as by ESI-MS n on permethylated dephosphorylated OS. A heterogeneous glycoform population was identified, resulting from the variable length of the OS branches attached to the glucose residue in the common structural element of H. influenzae LPS, L -a- D -Hepp-(1fi2)- [PEtnfi6]- L -a- D -Hepp-(1fi3)-[b- D -Glcp-(1fi4)]- L -a- D -Hepp- (1fi5)-[PPEtnfi4]-a-Kdop-(2fi6)-Lipid A. Notably, the O-6 position of the b- D -Glcp residue was either substituted by PCho or the disaccharide branch b- D -Galp-(1fi4)- D -a- D -Hepp, while the O-4 position was substituted by the globotetraose unit, b- D -GalpNAc-(1fi3)-a- D -Galp-(1fi4)- b- D -Galp-(1fi4)-b- D -Glcp, or sequentially truncated ver- sions thereof. This is the first time a branching sugar residue has been reported in the outer-core region of H. influenzae LPS. Additionally, a PEtn group was identified at O-3 of the distal heptose residue in the inner-core. Keywords: Haemophilus; lipopolysaccharide; phosphocho- line; structural analysis; ESI-MS n . Haemophilus influenzae is a Gram-negative pathogen that routinely colonizes the human upper respiratory tract and which can be found both in encapsulated (types a–f) and unencapsulated (nontypeable) forms. While the incidence of disease caused by H. influenzae type b (invasive diseases, including meningitis and pneumonia) has been greatly reduced in recent years as a result of the development of conjugate vaccines, there exists no vaccine against nontype- able H. influenzae (NTHi). NTHi strains are a common cause of otitis media and respiratory tract infections [1] and its lipopolysaccharide (LPS) molecule has been shown to be important for colonization and bacterial persistence during infection. H. influenzae LPS is composed of a membrane- anchoring lipid A moiety linked by a single phosphorylated 3-deoxy- D -manno-oct-2-ulosonic acid (Kdo) residue to a variable core oligosaccharide (OS) portion. The carbo- hydrate regions provide targets for recognition by host immune responses and some of these OS epitopes mimic human antigens, suggesting that the bacteria may use these structures to evade the host immune system [2,3]. Moreover, the OS portion of H. influenzae LPS has a propensity for reversible switching of terminal epitopes (phase variation), which is a major factor in the generation of the vast intrastrain LPS heterogeneity usually found [4]. This heterogeneity is thought to be an advantage to the bacteria, allowing them to better confront different host compart- ments and microenvironments and to survive the host immune response [5]. The availability of the complete genome sequence of H. influenzae strain Rd [6] has facili- tated a comprehensive study of LPS biosynthetic loci in the homologous strain RM118 [7] and in the type b strains Eagan (RM153) and RM7004 [8]. Gene functions have been identified that are responsible for most of the steps in the biosynthesis of the OS portion of their LPS molecules. Molecular structural studies of LPS from H. influenzae strains [9–23] have resulted in a structural model consisting of a conserved phosphoethanolamine (PEtn)-substituted triheptosyl inner-core moiety (labelled HepI–HepIII) in which each of the heptose residues can provide a point for attachment of OS chains or noncarbohydrate substituents (Fig. 1). Notably, HepIII and the b- D -Glcp residue that is linked to HepI (labelled GlcI) have an especially wide range of alternatives in the substitution pattern. HepIII has been found to be substituted by a b- D -Glcp residue (either at O-2 [13] or O-3 [16]) or a b- D -Galp residue (either at O-2 [10] or O-3 [19]). Analysis of LPS from lpsA mutants established in a number of strain backgrounds supports a role for LpsA in each of the alternative glycose substitutions of HepIII. HepIII has also been found to be substituted by the Correspondence to E. Schweda, University College of South Stockholm, Clinical Research Centre, NOVUM, S-141 86 Huddinge, Sweden. Fax: + 46 8585 838 20, Tel.: + 46 8585 838 23, E-mail: elke.schweda@kfc.ki.se Abbreviations: CE, capillary electrophoresis; PCho, phosphocholine; PEtn, phosphoethanolamine; PPEtn, pyrophosphoethanolamine; Hep, heptose; D , D -Hep, D -glycero- D -manno-heptose; L , D -Hep, L -glycero- D -manno-heptose; Hex, hexose; HexNAc, N-acetylhexos- amine; HMBC, heteronuclear multiple-bond correlation; lipid A-OH, O-deacylated lipid A; Kdo, 3-deoxy- D -manno-oct-2-ulosonic acid; LPS, lipopolysaccharide; LPS-OH, O-deacylated LPS; MS n , multiple step tandem mass spectrometry; Neu5Ac, N-acetylneuraminic acid; NTHi, nontypeable Haemophilus influenzae; OS, oligosaccharide; AnKdo-ol, reduced anhydro Kdo. (Received 27 February 2003, revised 13 May 2003, accepted 16 May 2003) Eur. J. Biochem. 270, 2979–2991 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03675.x noncarbohydrate substituents Ac (either at O-2 [16] or O-3 [15]), Gly [16,24], P (atO-4[11])andPEtn[9].ForGlcI,the O-4 position has been found to be substituted by a b- D -Galp residue [9] or a b- D -Glcp residue [10], while in other strains, O-6 was substituted by phosphocholine (PCho) [13], D -glycero- D -manno-heptose ( D , D -Hep) [14] or L -glycero- D - manno-heptose ( L , D -Hep) [21]. In several strains, a disubsti- tution-pattern of GlcI has been observed, including b- D - Glcp (at O-4)/PCho (at O-6) [17], b- D -Galp (at O-4)/PCho (at O-6) [20], Ac (at O-4)/PCho (at O-6) [18] and Ac (at O-4)/ L , D -Hep (at O-6) [21]. In each strain analysed, PCho addition has been shown to be directed by the products of the lic1 locus. Our recent studies have focussed on the structural diversity of LPS expression, and the genetic basis for that diversity, in a representative set consisting of 25 NTHi clinical isolates obtained from otitis media patients [25]. In the present investigation we report on the structural analysis of LPS from one of these isolates (NTHi strain 981), which provides evidence for a novel branching pattern at GlcI. Experimental procedures Bacterial culture and preparation of LPS NTHi strain 981 was obtained from the Finnish Otitis Media Study Group and is an isolate obtained from the middle ear [25]. Bacteria were grown in brain-heart infusion broth supplemented with haemin (10 lgÆmL )1 ), NAD (2 lgÆmL )1 )andN-acetylneuraminic acid (Neu5Ac) (25 lgÆmL )1 ). LPS was extracted by the phenol/chloro- form/light petroleum method, as described previously [16]. Chromatography Gel filtration chromatography and GLC were carried out as described previously [16]. Preparation of OS material O-Deacylation of LPS. O-Deacylation of LPS was achieved with anhydrous hydrazine, as described previously [16,26]. Mild acid hydrolysis of LPS. Reduced core OS material was obtained after mild acid hydrolysis (1% aqueous acetic acid, pH 3.1, 100 °C, 2 h) and simultaneous reduction (borane-N-methylmorpholine complex, 20 mg) of LPS (120 mg). The insoluble lipid A (62 mg) was separated by centrifugation and the water-soluble part was repeatedly chromatographed on a P-4 column, giving a major (OS-1, 8.5 mg) and a minor (OS-2, 3.7 mg) OS-containing fraction. Dephosphorylation of OS. Dephosphorylation of OS material was performed with 48% aqueous HF, as described previously [18]. Mass spectrometry GLC-MS and ESI-MS were performed as described previously [16,21]. ESI-MS n on permethylated dephos- phorylated OS was performed using a Finnigan-MAT LCQ ion trap mass spectrometer (Finnigan-MAT; San Jose, CA, USA) in the positive ion mode. The samples were dissolved in methanol/water (7 : 3) containing 1 m M NaOAc to a concentration of about 1 mgÆmL )1 ,andwere injected into a running solvent of identical composition at 10 lLÆmin )1 . NMR spectroscopy NMR spectra were obtained at 25 °C(OS)or20°C [O-deacylated LPS (LPS-OH)] either on a Varian UNITY 600 MHz spectrometer or on a JEOL JNM-ECP500 spectrometer, using the previously described experiments [16,18,21]. Analytical methods Sugars were identified as their alditol acetates, as previously described [27]. Methylation analysis was performed as described previously [16]. The relative proportions of the various alditol acetates and partially methylated alditol acetates obtained in sugar- and methylation analyses correspond to the detector response of the GLC-MS. Permethylation of dephosphorylated OS was performed in the same way as in the methylation analyses [16], but without the prior acetylation step. The absolute configura- tions of the hexoses were determined by the method devised by Gerwig et al. [28]. The total content of fatty acids was analysed as described previously [29]. Results Characterization of LPS NTHi strain 981 was cultured in liquid media and the LPS was extracted using the phenol/chloroform/light pet- roleum method. Compositional sugar analysis of the LPS Fig. 1. 5 Structural model of the lipopolysaccharide from Haemophilus influenzae. R 1 ¼ H, phosphocholine (PCho), D -glycero- D -manno- heptose ( D , D -Hep) or L -glycero- D -manno-heptose ( L , D -Hep); R 2 ,R 4 , R 5 ¼ H, glucose (Glc), galactose (Gal) or acetate (Ac) 6 ;R 3 ¼ HorGlc, Y ¼ Gly, P or phosphoethanolamine (PEtn). Note that substitution with Gly has also been reported at HepI, HepII and Kdo [24]. 2980 M. Ma ˚ nsson et al. (Eur. J. Biochem. 270) Ó FEBS 2003 sample indicated D -glucose (Glc), D -galactose (Gal), 2-amino-2-deoxy- D -glucose (GlcN), 2-amino-2-deoxy- D -galactose (GalN), D -glycero- D -manno-heptose ( D , D -Hep) and L -glycero- D -manno-heptose ( L , D -Hep) in the ratio 25:26:18:1:5:25,asidentifiedbyGLC-MSoftheir corresponding alditol acetate and 2-butyl glycoside deriva- tives [28]. In previous investigations the LPS was found to contain ester-linked glycine [24] and a low level of Neu5Ac [30]. On treatment of the LPS with anhydrous hydrazine under mild conditions, water-soluble O-deacylated LPS (LPS-OH) was obtained. ESI-MS data (Table 1) indicated a heterogeneous mixture of glycoforms consisting of two subpopulations: a major subpopulation in which the glycoform compositions comprised three heptoses and, to agreatextent,PCho (Hep3-glycoforms); and a minor subpopulation with compositions comprising four heptoses but lacking PCho (Hep4-glycoforms). Quadruply charged ions were observed at m/z 640.5/671.3 (minor), 680.8/ 711.5 (major), 721.4/751.7 and 772.3/802.9, corresponding to Hep3-glycoforms with the compositions PChoÆHex 2 Æ Hep 3 ÆPEtn 2)3 ÆP 1 ÆKdoÆLipid A-OH, PChoÆHex 3 ÆHep 3 Æ PEtn 2)3 ÆP 1 ÆKdoÆLipid A-OH, PChoÆHex 4 ÆHep 3 ÆPEtn 2)3 ÆP 1 Æ KdoÆLipid A-OH and PChoÆHexNAc 1 ÆHex 4 ÆHep 3 ÆPEtn 2)3 Æ P 1 ÆKdoÆLipid A-OH, respectively. Minor quadruply charged ions were also found at m/z 639.8/670.3, consistent with Hep3-glycoforms that were lacking PCho with the compo- sitions Hex 3 ÆHep 3 ÆPEtn 2)3 ÆP 1 ÆKdoÆLipid A-OH. Quadruply charged ions at m/z 646.9/677.8, 687.7/718.4 (minor) and 728.1/758.8 (major) corresponded to Hep4-glycoforms with the compositions Hex 2 ÆHep 4 ÆPEtn 2)3 ÆP 1 ÆKdoÆLipid A-OH, Hex 3 ÆHep 4 ÆPEtn 2)3 ÆP 1 ÆKdoÆLipid A-OH and Hex 4 Æ Hep 4 ÆPEtn 2)3 ÆP 1 ÆKdoÆLipid A-OH, respectively. Characterization of OS Core OS material was obtained after partial acid hydrolysis of LPS with dilute aqueous acetic acid. The OS material was repeatedly chromatographed on a P-4 column with inter- mediate selection for the Hep3- and Hep4-glycoforms (by ESI-MS). This procedure resulted in a major fraction (OS-1, 8.5 mg) in which the Hep3-glycoforms accounted for about 95%, and a minor fraction (OS-2, 3.7 mg) in which the Hep4-glycoforms accounted for about 80% (Table 2). In the ESI-MS spectrum of OS-1 (Fig. 2A), doubly charged ions at m/z 684.9 (minor), 766.0 (minor), 847.2 (major) and 928.3, corresponded to the respective compositions PChoÆ Hex 1)4 ÆHep 3 ÆPEtn 2 ÆAnKdo-ol, while ions at m/z 949.1 (very minor) and 1029.9 were consistent with the composi- tions PChoÆHexNAc 1 ÆHex 3)4 ÆHep 3 ÆPEtn 2 ÆAnKdo-ol, res- pectively. In the ESI-MS spectrum of OS-2 (Fig. 2B), doubly charged ions at m/z 698.4 (minor), 779.6 (major), 860.6, 941.8 (major) and 1022.5 (very minor) were consistent with the respective compositions Hex 1)5 ÆHep 4 ÆPEtn 2 ÆAnK- do-ol. In both OS fractions, minor peaks were observed corresponding to the above-mentioned compositions but additionally containing glycine or a phosphate group. Minor peaks could also be observed corresponding to glycoforms containing only one PEtn group or containing one PEtn group and a phosphate group. In order to obtain sequence and branching information, the OS fractions were dephosphorylated and permethylated and subjected to ESI-MS n [21,31]. Sodiated adduct ions were observed in the MS spectra (positive mode) corres- ponding to the compositions Hex 1)4 ÆHep 3 ÆAnKdo-ol, Hex- NAc 1 ÆHex 3)4 ÆHep 3 ÆAnKdo-ol, Hex 1)5 ÆHep 4 ÆAnKdo-ol and HexNAc 1 ÆHex 5 ÆHep 4 ÆAnKdo-ol (Tables 3 and 4). The Table 1. Negative ion ESI-MS data and proposed compositions for O-deacylated lipopolysaccharide (LPS-OH) of nontypeable Haemophilus influ- enzae (NTHi) strain 981. Average mass units were used for calculation of molecular mass values based on proposed compositions, as follows: Hex (hexose), 162.14; HexNAc (N-acetylhexosamine), 203.19; Hep (heptose), 192.17; Kdo (3-deoxy- D -manno-oct-2-ulosonic acid), 220.18; P (phos- phate), 79.98; PEtn (phosphoethanolamine), 123.05; PCho (phosphocholine), 165.13 and lipid A-OH (O-deacylated lipid A), 953.02. Relative abundance was estimated from the area of molecular ion peak relative to the total area (expressed as percentage). Peaks representing less than 3% of the base peak were not included in the table. Observed ions (m/z) Molecular mass (Da) Relative abundance (%) Proposed composition (M-4H) 4– (M-3H) 3– Observed Calculated 639.8 853.4 2563.2 2562.2 1 Hex 3 ÆHep 3 ÆPEtn 2 ÆP 1 ÆKdoÆLipid A-OH 670.3 893.7 2684.6 2685.3 2 Hex 3 ÆHep 3 ÆPEtn 3 ÆP 1 ÆKdoÆLipid A-OH 640.5 854.1 2565.6 2565.2 1 PChoÆHex 2 ÆHep 3 ÆPEtn 2 ÆP 1 ÆKdoÆLipid A-OH 671.3 894.7 2688.2 2688.2 2 PChoÆHex 2 ÆHep 3 ÆPEtn 3 ÆP 1 ÆKdoÆLipid A-OH 680.8 907.8 2726.8 2727.3 17 PChoÆHex 3 ÆHep 3 ÆPEtn 2 ÆP 1 ÆKdoÆLipid A-OH 711.5 948.9 2849.8 2850.4 26 PChoÆHex 3 ÆHep 3 ÆPEtn 3 ÆP 1 ÆKdoÆLipid A-OH 721.4 962.0 2889.3 2889.5 3 PChoÆHex 4 ÆHep 3 ÆPEtn 2 ÆP 1 ÆKdoÆLipid A-OH 751.7 1003.2 3011.7 3012.5 4 PChoÆHex 4 ÆHep 3 ÆPEtn 3 ÆP 1 ÆKdoÆLipid A-OH 772.3 1029.7 3092.6 3092.7 3 PChoÆHexNAc 1 ÆHex 4 ÆHep 3 ÆPEtn 2 ÆP 1 ÆKdoÆLipid A-OH 802.9 1070.8 3215.5 3215.7 5 PChoÆHexNAc 1 ÆHex 4 ÆHep 3 ÆPEtn 3 ÆP 1 ÆKdoÆLipid A-OH 646.9 862.8 2591.5 2592.2 4 Hex 2 ÆHep 4 ÆPEtn 2 ÆP 1 ÆKdoÆLipid A-OH 677.8 903.9 2715.0 2715.3 4 Hex 2 ÆHep 4 ÆPEtn 3 ÆP 1 ÆKdoÆLipid A-OH 687.7 917.0 2754.4 2754.4 1 Hex 3 ÆHep 4 ÆPEtn 2 ÆP 1 ÆKdoÆLipid A-OH 718.4 958.6 2878.2 2877.4 1 Hex 3 ÆHep 4 ÆPEtn 3 ÆP 1 ÆKdoÆLipid A-OH 728.1 971.0 2916.2 2916.5 9 Hex 4 ÆHep 4 ÆPEtn 2 ÆP 1 ÆKdoÆLipid A-OH 758.8 1012.2 3039.4 3039.6 17 Hex 4 ÆHep 4 ÆPEtn 3 ÆP 1 ÆKdoÆLipid A-OH Ó FEBS 2003 Structural analysis of LPS from NTHi strain 981 (Eur. J. Biochem. 270) 2981 monosaccharide sequence and branching for the different glycoforms were obtained following collision-induced dis- sociation (CID) of the glycosidic bonds [21,31]. Through the ion mass distinction between reducing, nonreducing and internal fragments resulting from the bond ruptures [21,31], the topology could be determined for nearly all composi- tions found in the MS profiling spectra (Tables 3 and 4). In those cases, where the same glycoform was found in both OS-1 and OS-2 (Table 2), experiments showed that identical structures were present in both fractions. For the major Hep3-glycoform with the composition Hex 3 ÆHep 3 ÆAnKdo-ol ([M + Na] + 1671.8 Da), ion selec- tion and collisional activation of the precursor ion at m/z 1671.8 provided the MS 2 spectrum shown in Fig. 3. The fragmentation pattern was consistent with two nonreducing terminal branches attached to a disubstituted heptose (HepI)whichislinkedtotheterminalAnKdo-ol residue. The ions at m/z 1409.7 and 1161.6 corresponded to losses of terminal-Hep (t-Hep) and t-Hep–Hep, respectively, which indicated the presence of a HepIII–HepII-disaccharide branch. Furthermore, ions at m/z 1453.7, 1249.6 and 1045.5, corresponding to the respective loss of t-Hex, t-Hex–Hex and t-Hex–Hex–Hex, indicated the presence of a Hex–Hex–Hex-trisaccharide branch. Loss of the terminal AnKdo-ol residue was also observed from the ion at m/z 1393.6. The assigned structure was confirmed by MS 3 experiments on selected product ions (data not shown). The structures of the other Hep3-glycoforms were obtained in an analogous manner (data not shown) and for all glycoforms the hexoses were found to be members of a linear chain attached to HepI (Table 3). For the major Hep4-glycoform with the composition Hex 4 ÆHep 4 ÆAnKdo-ol ([M + Na] + 2124.0 Da), ions in the MS 2 spectrum (precursor ion [M + 2Na] 2+ 1073.5 Da) (Fig. 4A) at m/z 1861.8 (loss of t-Hep) and 1613.8 (loss of t-Hep–Hep) indicated the presence of a HepIII–HepII- branch attached to HepI, as was also found in the Hep3- glycoforms (Tables 3 and 4). An OS extension consisting of four hexose residues and one heptose residue was indicated to be linked to HepI from the occurrence of an ion at m/z 1045.6 (counterpart at m/z 1101.6) correspond- ing to loss of the entire Hex4Hep moiety. The fragmen- tation pattern clearly indicated this OS extension not to be arranged in a linear chain as fragment ions were found at m/z 1905.8 (loss of t-Hex), 1701.7 (loss of t-Hex–Hex) and 1657.7 (either loss of t-Hex–Hep or t-Hep–Hex), consis- tent with the presence of a Hex–Hex-disaccharide branch and a second disaccharide branch that either could be Hex–Hep- or Hep–Hex- (the former alternative was shown, see below). Ions resulting from double glycosidic bond cleavage could also be observed at m/z 1395.7 (e.g. loss of t-Hep–Hep/t-Hex) and at m/z 1191.5 (loss of t-Hep–Hep/t-Hex–Hex). An MS 3 experiment on m/z 1613.8 (Fig. 4B) gave fragment ions at m/z 1101.6 (coun- terpart at m/z 535.3), as a result of glycosidic cleavage between the Hex4Hep moiety and HepI. Ions at m/z 1395.7, 1191.6 and 1147.4, corresponding to the respective loss of t-Hex, t-Hex–Hex and t-Hex–Hep, indicated that the Hex4Hep moiety consisted of two nonreducing ter- minal disaccharide branches (Hex–Hex- and Hex–Hep-) attached to a disubstituted hexose. To confirm the assigned structure, an MS 4 experiment on m/z 1191.6 was per- formed (Fig. 4C), which showed the expected ions at m/z 973.5 (loss of t-Hex) and 725.3 (loss of t-Hex–Hep). The topology of the other Hep4-glycoforms were determined in a similar manner (data not shown). For the higher molecular mass Hep4-glycoforms (Hex3–Hex5 and Hex- NAc1Hex5), the hexose residue attached to HepI was found to be disubstituted with a Hex–Hep-branch and a second branch containing hexoses (Table 4). Table 2. Negative ion ESI-MS data and proposed compositions for oligosaccharide (OS)-1 and (OS)-2 derived from the lipopolysaccharide (LPS) of nontypeable Haemophilus influenzae (NTHi) strain 981. Average mass units were used for calculation of molecular mass values based on proposed compositions as follows: Hex (hexose), 162.14; HexNAc (N-acetylhexosamine), 203.19; Hep (heptose), 192.17; AnKdo-ol (reduced anhydro Kdo), 222.20; PEtn (phosphoethanolamine), 123.05; and PCho (phosphocholine), 165.13. Relative abundance was estimated from the area of molecular ion peak relative to the total area (expressed as a percentage). Minor peaks were observed corresponding to the proposed compositions but additionally containing glycine or a phosphate group. Minor peaks could also be observed corresponding to glycoforms containing only one PEtn group or containing one PEtn group and a phosphate group. ND, not detected. Observed ions (m/z) (M-2H) 2) Molecular mass (Da) Relative abundance (%) Proposed compositionObserved Calculated OS-1 OS-2 764.8 1531.6 1531.2 ND 9 Hex 3 ÆHep 3 ÆPEtn 2 ÆAnKdo-ol 684.9 1371.8 1372.1 3 3 PChoÆHex 1 ÆHep 3 ÆPEtn 2 ÆAnKdo-ol 766.0 1534.0 1534.2 5 5 PChoÆHex 2 ÆHep 3 ÆPEtn 2 ÆAnKdo-ol 847.2 1696.4 1696.4 70 3 PChoÆHex 3 ÆHep 3 ÆPEtn 2 ÆAnKdo-ol 928.3 1858.6 1858.5 11 ND PChoÆHex 4 ÆHep 3 ÆPEtn 2 ÆAnKdo-ol 949.1 1900.2 1899.6 Trace a ND PChoÆHexNAc 1 ÆHex 3 ÆHep 3 ÆPEtn 2 ÆAnKdo-ol 1029.9 2061.8 2061.7 8 ND PChoÆHexNAc 1 ÆHex 4 ÆHep 3 ÆPEtn 2 ÆAnKdo-ol 698.4 1398.8 1399.1 ND 3 Hex 1 ÆHep 4 ÆPEtn 2 ÆAnKdo-ol 779.6 1561.2 1561.3 ND 36 Hex 2 ÆHep 4 ÆPEtn 2 ÆAnKdo-ol 860.6 1723.2 1723.4 ND 7 Hex 3 ÆHep 4 ÆPEtn 2 ÆAnKdo-ol 941.8 1885.6 1885.5 3 34 Hex 4 ÆHep 4 ÆPEtn 2 ÆAnKdo-ol 1022.5 2047.0 2047.7 Trace Trace Hex 5 ÆHep 4 ÆPEtn 2 ÆAnKdo-ol 1124.4 2250.8 2250.9 Trace ND HexNAc 1 ÆHex 5 ÆHep 4 ÆPEtn 2 ÆAnKdo-ol a Trace amounts, defined as peaks representing less than 3% of the base peak. 2982 M. Ma ˚ nsson et al. (Eur. J. Biochem. 270) Ó FEBS 2003 Methylation analysis of dephosphorylated OS-1 indi- cated terminal-Gal (t-Gal), 4-substituted-Gal (4-Gal), 4-Glc, 3-Gal, t- L , D -Hep, 4,6-disubstituted-Glc (4,6-Glc), 4- D , D -Hep, 2- L , D -Hep and 3,4- L , D -Hep in the relative proportions 24 : 5 : 31 : 2 : 9 : 3 : 3 : 10 : 13 together with trace amounts of t-Glc and t-GalN. The methylation analysis of intact OS-1 showed significantly decreasing amounts of 2- L , D -Hep and t- L , D -Hep, which could be derived from the presence of PEtn substituents at HepII and HepIII, respectively (see below). Methylation analysis of dephosphorylated OS-2 showed t-Gal, 4-Glc, 6-Glc, t- L , D - Hep, 4,6-Glc, 4- D , D -Hep, 2- L , D -Hep and 3,4- L , D -Hep in the ratio 32 : 15 : 2 : 10 : 5 : 15 : 10 : 11 together with trace amounts of t-Glc, 4-Gal, 3-Gal and t- D , D -Hep. The increas- ing amounts (compared to dephosphorylated OS-1) of t-Gal, 4- D , D -Hep, 6-Glc and 4,6-Glc indicated that the structural element Hex–Hep–Hex–(–) in the Hep4-glyco- forms was probably Gal-(1fi4)- D , D -Hep-(1fi4,6)-Glc, and this was confirmed and detailed by NMR spectroscopy (see below). Characterization of OS fractions and LPS-OH by NMR The 1 H NMR resonances of OS fractions and LPS-OH were assigned by 1 H– 1 H chemical shift correlation experi- ments (DQF-COSY and TOCSY). Subspectra correspond- ing to the individual glycosyl residues were identified on the basis of spin-connectivity pathways delineated in the 1 H chemical shift correlation maps, the chemical shift values, and the vicinal coupling constants. From the glycoform compositions of the different OS fractions indicated by ESI-MS (Table 2), the spin-systems could more easily be identified as originating from either the Hep3- or the Hep4- glycoform population. The 13 C NMR resonances of OS material and LPS-OH were assigned by heteronuclear 1 H– 13 C chemical shift correlation in the 1 H detected mode (HSQC). The chemical shift data obtained for the Hep3- and Hep4-glycoforms are presented in Tables 5 and 6, respectively, and are consistent with each D -sugar residue being present in the pyranosyl ring form. Further evidence for this conclusion was obtained from NOE data, which also served to confirm the anomeric configurations of the linkages and, together with a heteronuclear multiple-bond correlation (HMBC) experiment on OS-2, determined the monosaccharide sequence and branching pattern. Characterization of the Kdo-lipid A-OH region. ESI-MS data (Table 1), fatty acid compositional analysis (yielding 3-hydroxytetradecanoic acid) and NMR experiments on LPS-OH (data not shown, giving similar results as for NTHi strains 486 [16] and 1003 [18]), indicated the presence of the common Kdo-(2fi6)-lipid A-OH element in NTHi strain 981. As observed previously [16,18], two spin-systems Table 3. Structures of the Hep3-glycoforms of nontypeable Haemophilus influenzae (NTHi) strain 981, as indicated by ESI-MS n on permethylated dephosphorylated oligosaccharide (OS). ND, not determined. 8 Hex1 Hex2 Hex3 Hex4 HexNAc1Hex3 HexNAc1Hex4 Hex-Hep-AnKdo-ol Hex 2 -Hep-AnKdo-ol Hex 3 -Hep-AnKdo-ol Hex 4 -Hep-AnKdo-ol ND HexNAc-Hex 4 -Hep-AnKdo-ol || | | | Hep Hep Hep Hep Hep || | | | Hep Hep Hep Hep Hep Fig. 2. Negative ion ESI-MS spectra of oligosaccharide (OS)-1 (A) and OS-2 (B) derived from the lipopolysaccharide (LPS) of nontypeable Haemophilus influenzae (NTHi) strain 981 showing doubly charged ions. (A) The peak at m/z 684.9 corresponds to a glycoform with the com- position, phosphocholine (PCho)ÆHex 1 ÆHep 3 Æphosphoethanolamine (PEtn) 2 ÆAnKdo-ol. (B) The peak at m/z 698.4 corresponds to a gly- coform with the composition Hex 1 ÆHep 4 ÆPEtn 2 ÆAnKdo-ol. Minor peaks were observed corresponding to the compositions proposed in Table 2 but additionally containing glycine (indicated by · )ora phosphate group (indicated by °). Minor peaks could also be observed corresponding to glycoforms containing only one PEtn group (indi- cated by e) or containing one PEtn group and a phosphate group (indicated by +). Ó FEBS 2003 Structural analysis of LPS from NTHi strain 981 (Eur. J. Biochem. 270) 2983 could be traced for the single a-linked Kdo residue, probably owing to the partial occurrence of PEtn attached to the phosphate group at O-4 of Kdo [11,13,16]. Structure of the core region of the Hep3-glycoforms. In the 1 H NMR spectrum of OS-1 (Fig. 5A), anomeric resonances of HepI–HepIII were observed at d 5.89–5.82 (1H, not resolved), 5.24–5.22 (1H, not resolved) and 5.15– 5.01 (1H, not resolved). The Hep ring systems were identified on the basis of the observed small J 1,2 values, and their a-configurations were confirmed by the occurrence of single intraresidue NOE between the respective H-1 and H-2 resonances, as observed previously [16,18]. An intense signal from methyl protons of an N-acetyl group was obser- ved at d 2.04, which correlated to a 13 Csignalatd 22.7 in the HSQC spectrum. A crosspeak from the ester-linked glycine substituent was observed at d 4.00/40.7 (in the HSQC spectrum) as a result of correlation between the methylene proton and its carbon. Several signals for methylene protons of AnKdo-ol were observed in the DQF-COSY and TOCSY spectra of OS-1 in the region d 2.26–1.67, as observed and explained previously [10,16,21]. The mono- saccharide sequence within the inner-core region, as indicated by ESI-MS n (described above), was confirmed and detailed from transglycosidic NOE connectivities between the proton pairs HepIII H-1/HepII H-2, HepII H-1/HepI H-3 (LPS-OH and OS-1) and HepI H-1/Kdo H-5 and H-7 (LPS-OH), evidencing the sequence as L -a- D - Hepp-(1fi2)- L -a- D -Hepp-(1fi3)- L -a- D -Hepp-(1fi5)-a-Kdop. Relatively large J 1,2 values ( 7.8 Hz) of the anomeric resonances observed at d 4.64, 4.62, 4.55, 4.52 and 4.46, indicated that each of the corresponding residues had the b-anomeric configuration. The residues with anomeric signals at d 4.94 (J 4.0 Hz) and 4.91 (J 4.0 Hz) were identified as having the a-anomeric configuration. The assignments of the anomeric configurations were also supported by the occurrence of intraresidue NOE between the respective H-1, H-3 and H-5 resonances (b-configur- ation) or between H-1 and H-2 (a-configuration). On the basis of the chemical shift data and the large J 2,3 , J 3,4 and Table 4. Structures of the Hep4-glycoforms of nontypeable Haemophilus influenzae (NTHi) strain 981, as indicated by ESI-MS n on permethylated dephosphorylated oligosaccharide (OS). 9 Hex1 Hex2 Hex3 Hex4 Hex5 HexNAc1Hex5 Hex Hex a Hex Hex Hex || | | | Hep Hep Hep Hep Hep Hep || | | | | Hex-Hep-AnKdo-ol Hex-Hep-AnKdo-ol Hex-Hex-Hep-AnKdo-ol Hex 2 -Hex-Hep-AnKdo-ol Hex 3 -Hex-Hep-AnKdo-ol HexNAc-Hex 3 -Hex-Hep-AnKdo-ol || | | | | Hep Hep Hep Hep Hep Hep || | | | | Hep Hep Hep Hep Hep Hep Hep b | Hex 2 -Hex-Hep-AnKdo-ol | Hep | Hep a Major isomer of the Hex3 glycoform (estimated from the intensity of the fragments in MS 2 experiments). b Minor isomer of the Hex3 glycoform (estimated from the intensity of the fragments in MS 2 experiments). Fig. 3. ESI-MS 2 analysis of permethylated dephosphorylated oligosac- charide (OS) derived from the lipopolysaccharide (LPS) of nontypeable Haemophilus influenzae (NTHi) strain 981. The product ion spectrum is shown of [M + Na] + m/z 1671.8, corresponding to a glycoform with the composition Hex 3 ÆHep 3 ÆAnKdo-ol. The proposed structure is shown in the inset. 2984 M. Ma ˚ nsson et al. (Eur. J. Biochem. 270) Ó FEBS 2003 J 4,5 values ( 9 Hz), the residues with anomeric shifts of d 4.55 and 4.64 could be attributed to the 4-Glc (GlcI and GlcII) identified by methylation analysis. On the basis of low J 3,4 and J 4,5 values (<4 Hz) and chemical shift data, the residues with anomeric resonances at d 4.46, 4.94, 4.52, 4.91 and 4.62 were attributed to the t-Gal (GalI and GalII), 4-Gal (GalI*), 3-Gal (GalII*) and t-GalNAc (GalNAc) identified by linkage analysis. Signals for the methyl protons of PChowereobservedat d 3.23 and characteristic spin-systems for ethylene protons from this residue and from the two PEtn residues were found at d 4.41/3.69 (PCho), 4.15/3.29 (PEtnI) and 4.17/ 3.29 (PEtnII). 1 H– 31 P NMR correlation studies (Fig. 6) demonstrated PChotobelocatedatGlcIandthetwoPEtn residues to be situated at HepII and HepIII, respectively. Intense 31 P resonances from phosphodiesters were observed at d 0.33, )0.50 and )0.62. Correlations between the former signal and the signals from H-6 of HepII (d 4.55) and the methylene proton pair of PEtnI (d 4.15) in the 1 H– 31 P HMQC experiment evidenced substitution by PEtn at O-6 of HepII. The second PEtnresiduewasdemonstratedtobe linked to O-3 of HepIII as the 31 P resonance at d )0.50 correlated to the signals from H-3 of HepIII (d 4.33) and the methylene proton pair of PEtnII (d 4.17). Correlations between the signal at d )0.62 and the signals from the H-6 protons of GlcI (d 4.30) and the methylene protons of PCho (d 4.41) established the PCho substituent to be located at O-6 of this residue. The occurrence of interresidue NOE connectivities between the proton pairs GalI H-1/GlcII H-4, GlcII H-1/ GlcI H-4 and GlcI H-1/HepI H-4 and H-6 established the presence of the tetrasaccharide unit b- D -Galp-(1fi4)- b- D -Glcp-(1fi4)-[PChofi6]-b- D -Glcp-(1fi4)- L -a- D -Hepp- (1fi). This element was shown to be further chain elongated by an a- D -Galp residue because transglycosidic NOE between GalII H-1/GalI* H-4 and GalI* H-1/GlcII H-4 were observed. Further chain extension by a b- D -GalpNAc residue was evidenced from the occurrence of interresidue NOE between GalNAc H-1/GalII* H-3 and GalII* H-1/ GalI* H-4. From the combined data, the structure in Fig. 7 is proposed for the fully extended globotetraose-containing Hep3-glycoform (HexNAc1Hex4) of NTHi strain 981. It was thus established that the Hex4 and Hex3 glycoforms are sequential truncations of this structure. It is also likely that the Hex2 and Hex1 glycoforms are further sequential truncations, which is consistent with the occurrence of small amounts of t-Glc in the methylation analysis. Indeed, two weak crosspeaks are observed in the COSY spectrum at d 4.57/3.50 and 4.56/3.38, which possibly arise from the two b- D -Glcp residues appearing as terminal residues; however, this could not be confirmed because of a significant overlap of signals. Structure of the core region of the Hep4-glycoforms. In the 1 H NMR spectrum of OS-2 (Fig. 5B), anomeric resonances of HepI–HepIII were observed at d 6.01–5.87 (HepII, not resolved), 5.17–5.03 (HepI, not resolved) and 5.14/5.10 (HepIII, not resolved). The chemical shift values for the other resonances of HepI–HepIII (data not shown) were similar to the corresponding values in the Hep3- glycoforms. Transglycosidic NOE connectivities (data simi- lar to that of the Hep3-glycoforms) confirmed the sequence of the characteristic triheptosyl unit. Anomeric signals of the fourth heptose were identified at d 5.06/4.94 (HepIV, not resolved) and single intraresidue NOE between H-1 and H-2 confirmed its a-configuration. This residue could be attributed to the 4- D , D -Hep identified in the methylation analysis on the basis of chemical shift data (Table 6). 1 H– 31 P NMR correlation studies (data similar to that of the Hep3-glycoforms) demonstrated the PEtn substituents to be located at O-6 of HepII and at O-3 of HepIII, as previously observed (see above). Relatively large J 1,2 values ( 7.8 Hz) of the anomeric resonances observed at d 4.57, 4.54, 4.53 (very weak), 4.51, 4.50, 4.49 and 4.48, indicated each of the corresponding residues to have the b-anomeric configuration. The residue with an anomeric signal at d 4.95 (very weak, J 4.0 Hz) was identified as having the a-anomeric configuration. The anomeric configurations were also confirmed from Fig. 4. ESI-MS n analysis of permethylated dephosphorylated oligosac- charide (OS) derived from the lipopolysaccharide (LPS) of nontypeable Haemophilus influenzae (NTHi) strain 981. (A) MS 2 spectrum of [M + 2Na] 2+ m/z 1073.5, corresponding to a glycoform with the composition Hex 4 ÆHep 4 ÆAnKdo-ol. The proposed structure is shown in the inset in B. (B) MS 3 spectrum of the fragment ion at m/z 1613.8. (C) MS 4 spectrum of the fragment ion at m/z 1191.6 7 . Ó FEBS 2003 Structural analysis of LPS from NTHi strain 981 (Eur. J. Biochem. 270) 2985 intraresidue NOE, as described earlier (see above). The residues with anomeric shifts of d 4.50, 4.54 and 4.57 could be attributed to the 6-Glc (GlcI), 4,6-Glc (GlcI*) and 4-Glc (GlcII) identified by methylation analysis, on the basis of the chemical shift data and the large J 2,3 , J 3,4 and J 4,5 values ( 9 Hz). On the basis of low J 3,4 and J 4,5 values (<4 Hz) and chemical shift data, the residues with anomeric resonances at d 4.48, 4.95, 4.51/4.49 and 4.53 were attributed to the t-Gal (GalI, GalII and GalIII) and 4-Gal (GalI*) identified by linkage analysis. Interresidue NOE were observed between the proton pairs GalIII H-1 (d 4.51)/HepIV H-4 (d 3.92), HepIV H-1 (d 4.94)/GlcI H-6A, H-6B and GlcI H-1/HepI H-4 and H-6, which established the tetrasaccharide unit b- D -Galp-(1fi4)- D -a- D -Hepp-(1fi6)-b- D -Glcp-(1fi4)- L -a- D -Hepp-(1fi of the Hex2 glycoform. The structure of the Hex4 glycoform was concluded from interresidue NOE between the proton pairs GalIII H-1 (d 4.49)/HepIV H-4 (d 3.90), HepIV H-1 (d 5.06)/GlcI* H-6A, H-6B, GlcI* H-1/HepI H-4 and H-6 and between the proton pairs GalI H-1/GlcII H-4, GlcII H-1/ GlcI* H-4, which evidenced the hexasaccharide unit b- D - Galp-(1fi4)-b- D -Glcp-(1fi4)-[b- D -Galp-(1fi4)- D -a- D -Hep p-(1fi6)]-b- D -Glcp-(1fi4)- L -a- D -Hepp-(1fi. This monosac- charide connectivity was also confirmed by transglycosidic correlations in an HMBC experiment, where correlations were seen between GalIII H-1/HepIV C-4, HepIV Table 5. 1 Hand 13 C NMR chemical shifts for Hep3-glycoforms of oligosaccharide (OS)-1 derived from the lipopolysaccharide (LPS) of nontypeable Haemophilus influenzae (NTHi) strain 981. Data were recorded in D 2 Oat25 °C. Signals corresponding to N-acetyl- D -galactosamine (GalNAc) and phosphocholine (PCho) methyl protons and carbons occurred at 2.04/22.7 and 3.23/54.5 p.p.m., respectively. Pairs of deoxyprotons of AnKdo-ol (reduced anhydro 3-deoxy- D -manno-oct-2-ulosonic acid) were identified in the DQF-COSY spectrum at 2.26–1.67 p.p.m. Residue Glycose unit H-1/C-1 H-2/C-2 H-3/C-3 H-4/C-4 H-5/C-5 H-6 A /C-6 H-6 B H-7 A /C-7 H-7 B HepI fi3,4)- L -a- D -Hepp-(1fi 5.01–5.15 a 3.96–4.04 a 3.96–4.01 a 4.28 b – c 4.09–4.11 a,b –– 97.1–98.6 a 70.8–70.9 a 72.3–72.4 a 74.1 – 68.2 – HepII fi2)- L -a- D -Hepp-(1fi 5.82–5.89 a 4.23–4.25 a 3.89–3.90 a 3.93 3.77 4.55 3.70 3.89 6 › PEtn 98.4–98.7 a 79.6 69.8 66.7 71.8 75.0 63.0 HepIII L -a- D -Hepp-(1fi 5.22–5.24 a 4.24–4.26 a 4.33 3.87 – – – – 3 › PEtn 101.6 70.2 76.6 65.8 – – – GlcI fi4)-b- D -Glcp-(1fi 4.55 3.51 3.60 3.70 3.68 4.30 4.30 6 › PCho 103.4 73.9 75.0 78.5 74.1 64.1 GlcII fi4)-b- D -Glcp-(1fi 4.64 3.30 3.64 3.72 3.61 3.84 3.93 102.7 73.6 74.9 78.5 75.0 60.2 GalI b- D -Galp-(1fi 4.46 3.54 3.66 3.92 3.72 d –– 103.4 71.4 73.0 69.0 75.8 – GalI* e fi4)-b- D -Galp-(1fi 4.52 3.58 3.74 4.03 3.78 d –– 103.7 71.3 72.6 77.8 75.9 – GalII a- D -Galp-(1fi 4.94 3.83 3.90 4.02 4.35 3.70 – 100.8 69.0 69.8 69.4 71.3 – GalII* e fi3)-a- D -Galp-(1fi 4.91 3.89 3.95 4.25 4.38 3.68 – 100.9 68.1 79.2 69.5 70.8 – GalNAc b- D -GalpNAc-(1fi 4.62 3.94 3.75 3.94 3.67 d –– 103.8 53.0 – 68.2 75.4 – PEtnI 4.15 3.29 62.5 40.5 PEtnII 4.17 3.29 62.5 40.5 PCho 4.41 3.69 60.1 66.5 Gly 4.00 – 40.7 a Several signals were observed for HepI, HepII and HepIII owing to heterogeneity in the AnKdo moiety. b H-4/H-6 of HepI were identified at d 4.28/4.09–4.11 by NOE from GlcI. c –, not obtained owing to the complexity of the spectrum. d Tentative assignment from NOE data. e Residue marked with * denotes a further substituted analogue of the corresponding residue without *. 2986 M. Ma ˚ nsson et al. (Eur. J. Biochem. 270) Ó FEBS 2003 H-1/GlcI* C-6, GalI H-1/GlcII C-4 and GlcII H-1/GlcI* C- 4. This element was shown to be further chain extended by an a- D -Galp residue, providing the Hex5 glycoform, as transglycosidic NOE between GalII H-1/GalI* H-4 and GalI* H-1/GlcII H-4 were observed. From the combined data, the structure in Fig. 8 is proposed for the Hex5 glycoform in the Hep4-glycoform population of NTHi strain 981. The Hex3 and Hex1 glycoforms are probably sequential truncations of the established structures, which is consistent with the low amounts of t-Glc and t- D , D -Hep found in the methylation analysis. A weak crosspeak in the COSY spectrum at d 4.55/3.36 may arise from the terminal Table 6. 1 Hand 13 C NMR chemical shifts for Hep4-glycoforms of oligosaccharide (OS)-2 derived from the lipopolysaccharide (LPS) of nontypeable Haemophilus influenzae (NTHi) strain 981. Data were recorded in D 2 Oat25°C. Chemical shift values for resonances of HepI-HepIII, PEtn and Gly were similar to the corresponding values in the Hep3-glycoforms (see Table 5). Residue Glycose unit H-1/C-1 H-2/C-2 H-3/C-3 H-4/C-4 H-5/C-5 H-6 A /C-6 H-6 B H-7 A /C-7 H-7 B GlcI fi6)-b- D -Glcp-(1fi 4.50 3.51 3.42 3.57 3.56 3.81 4.09 103.6 74.0 77.4 70.2 74.4 65.3 HepIV a fi4)- D -a- D -Hepp-(1fi 4.94 4.07 3.88 3.92 4.17 – b –– 99.3 69.9 70.4 78.8 71.2 – – 5.06 4.05 3.80 3.90 4.28 – – – 100.6 69.2 70.4 80.2 71.4 – – GalIII a b- D -Galp-(1fi 4.51 3.57 3.68 3.93 3.76 c –– 103.6 71.5 73.1 69.1 76.1 – 4.49 3.78 3.67 3.94 3.77 c –– 104.0 71.4 73.1 69.1 76.1 – GlcI* d fi4,6)-b- D -Glcp-(1fi 4.54 3.58 – 3.65 3.65 3.90 4.16 103.6 74.0 – 77.7 73.8 66.1 GlcII fi4)-b- D -Glcp-(1fi 4.57 3.40 3.52 3.79 3.85 3.87 3.91 102.5 73.5 75.2 78.1 74.1 60.1 GalI b- D -Galp-(1fi 4.48 3.74 3.66 3.93 3.74 c –– 103.7 71.2 73.1 69.1 75.9 – GalI* d fi4)-b- D -Galp-(1fi 4.53 3.81 3.78 4.05 – – – 103.8 – 72.8 77.9 – – GalII a- D -Galp-(1fi 4.95 3.84 3.91 4.03 – – – – 69.0 – 69.3 – – a Two spin-systems were found for the residue probably as a result of differences in the chemical environments of the various glycoforms. The first spin-system is attributed to the Hex2 glycoform, while the second spin-system is attributed to the Hex4 and Hex5 glycoforms. b –, not obtained owing to the complexity of the spectrum. c Tentative assignment from NOE data. d Residue marked with * denotes a further substituted analogue of the corresponding residue without *. Fig. 5. The 600-MHz 1 H NMR spectra of oligosaccharide (OS)-1 (A) and OS-2 (B) derived from the lipopolysaccharide (LPS) of nontypeable Haemophilus influenzae (NTHi) strain 981 showing the anomeric regions. (A) Anomeric resonances that are characteristic for the Hep3- glycoforms are labelled. Also indicated is an ethylene proton signal from phosphocholine (PCho) at d 4.41. (B) Anomeric resonances that are characteristic for the Hep4-glycoforms are labelled. Fig. 6. Part of the 500-MHz heteronuclear 1 H– 31 PHMQCspectrumof oligosaccharide (OS)-1 derived from the lipopolysaccharide (LPS) of nontypeable Haemophilus influenzae (NTHi) strain 981. Assignments are labelled. Ó FEBS 2003 Structural analysis of LPS from NTHi strain 981 (Eur. J. Biochem. 270) 2987 b- D -Glcp residue in the major isomer of the Hex3 glycoform (Table 4), but, as a result of overlapping signals, this could not be confirmed. Given the structure of the HexNAc1Hex5 glycoform by ESI-MS n (Table 4), a terminal b- D -GalpNAc residue is assumed in analogy of the Hep3-glycoforms. Discussion This investigation has shown that LPS from NTHi strain 981 contain either three heptoses (Hep3-glycoforms) or four heptoses (Hep4-glycoforms). In both of these glycoform subpopulations, either the fully assembled globotetraose unit [b- D -GalpNAc-(1fi3)-a- D -Galp-(1fi4)-b- D -Galp- (1fi4)-b- D -Glcp] or sequentially truncated versions thereof were found to be linked to O-4 of the b- D -Glcp residue (labelled GlcI) that is attached to HepI of the conserved triheptosyl inner-core moiety. This is the first example of an H. influenzae strain expressing globotetraose in this mole- cular environment. However, recently the H. influenzae type b strain, RM7004, was reported to express a trun- cated epitope, the globoside trisaccharide (globotriose) [a- D -Galp-(1fi4)-b- D -Galp-(1fi4)-b- D -Glcp]inthesame molecular environment [22]. Additionally, in that strain, the globotriose epitope was expressed as a terminal unit of a tetrasaccharide extension from HepII, the same tetrasac- charide that previously had been found in the type b strain, RM153, at that location [11]. A third molecular environ- ment for the globotriose epitope has been reported for H. influenzae strain RM118 (Rd) [13] and several NTHi isolates [21], where globotriose/globotetraose are present as terminal OS extensions at HepIII. The globotriose epitope is found on many human cells (p k blood group antigen), and in H. influenzae is thought to be important for virulence by acting as a mimic of these host structures, thus allowing the organism to evade some element of the host immune response [5,32]. For strain RM118, the glycosyltransferases involved in the assembly of its globotetraose side-chain were recently identified [7]. The lpsA gene was shown to be involved in the addition of a b- D -Glcp residue in a 1,2-linkage to initiate chain extension from HepIII. Furthermore, the lic2A, lgtC and lgtD genes were shown to encode glycosyltransferases involved in sequential addi- tion of b-1,4-linked Galp (Lic2A), a-1,4-linked Galp (LgtC) and b-1,3-linked GalpNAc (LgtD), resulting in the fully assembled globotetraose structure. Both lic2A and lgtC are phase-variable genes [32,33] as is lex2 [34], which is the gene responsible for the addition of a b- D -Glcp residue in a 1,4-linkage to GlcI in strain RM7004 (R. Aubrey, A. D. Cox, K. Makepeace, J. C. Richards, E. R. Moxon & D. W. Hood, unpublished results). Genetic analysis has indicated the presence of lex2, lic2A, lgtC and lgtD gene homologues in NTHi strain 981 (data not shown), and although it would Fig. 7. Structure proposed for the fully extended Hep3-glycoform (HexNAc1Hex4) of nontypeable Haemophilus influenzae (NTHi) strain 981. Fig. 8. Structure proposed for a Hep4-glycoform (Hex5) of nontypeable Haemophilus influenzae (NTHi) strain 981. 2988 M. Ma ˚ nsson et al. (Eur. J. Biochem. 270) Ó FEBS 2003 [...]... Structural analysis of lipopolysaccharide oligosaccharide epitopes expressed by non-typeable Haemophilus in uenzae strain 176 Carbohydr Res 337, 409–420 Cox, A. D., Hood, D.W., Martin, A. , Makepeace, K.M., Deadman, M.E., Li, J., Brisson, J.-R., Moxon, E.R & Richards, J.C (2002) Identification and structural characterization of a sialylated lacto-N-neotetraose structure in the lipopolysaccharide of Haemophilus. .. (1992) Structural characterization of the cell surface lipooligosaccharides from a nontypable strain of Haemophilus in uenzae Biochemistry 31, 4515–4526 10 Schweda, E.K.H., Hegedus, O.E., Borrelli, S., Lindberg, A. A., Weiser, J.N., Maskell, D.J & Moxon, E.R (1993) Structural studies of the saccharide part of the cell envelope lipopolysaccharide from Haemophilus in uenzae strain AH1-3 (lic3+) Carbohydr... [16,18,40,41] The lic 3A gene, which has been shown to encode a sialyltransferase that adds Neu5Ac in an a- 2,3linkage to lactose in other H in uenzae strains, is present in strain 981 (data not shown) Sialyllactose is known to contribute to the resistance of H in uenzae strains to the killing effect of normal human serum [41] Ó FEBS 2003 ˚ 2990 M Mansson et al (Eur J Biochem 270) Acknowledgements The authors... Zahringer, U (1988) Chemical structure of ¨ the lipopolysaccharide of Haemophilus in uenzae strain I-69 Rd–/ b+ Eur J Biochem 177, 483–492 ˚ 30 Bauer, S.H.J., Mansson, M., Hood, D.W., Richards, J.C., Moxon, E.R & Schweda, E.K.H (2001) A rapid and sensitive procedure for determination of 5-N-acetyl neuraminic acid in lipopolysaccharides of Haemophilus in uenzae: a survey of 24 nontypeable H in uenzae... located at HepIII; however, for the majority of strains, the molecular environment has not yet been established [12,14,40] The present investigation locates the additional PEtn group to O-3 of HepIII In the type b strain, RM153, a phosphate group was identified at O-4 of HepIII [11] MS analysis has indicated the presence of a phosphate group in several other strains [12–14], but, owing to the low abundance... residues in the outer-core regions of its LPS Structural studies have shown the LPS to have much in common with H in uenzae LPS, including an identical inner-core region (apart from an absence of PEtn at HepII) and lipid A [36] The D,D-Hep residue has been shown to be 1,6-linked to a b-D-Glcp residue attached to HepI (as in H in uenzae LPS, see above) and interrupts what otherwise would have been sialyl-lacto-N-neotetraose... & Jeanes, A (1965) Quantitative determination of monosaccharides as their alditol acetates by gas liquid chromatography Anal Chem 37, 1602–1604 28 Gerwig, G.J., Kamerling, J.P & Vliegenthart, J.F (1979) Determination of the absolute configuration of monosaccharides in complex carbohydrates by capillary G.L.C Carbohydr Res 77, 1–7 29 Helander, I.M., Lindner, B., Brade, H., Altmann, K., Lindberg, A. A.,... & Hansen, E.J (1986) Antigenic and phenotypic variations of Haemophilus in uenzae type b lipopolysaccharide and their relationship to virulence Infect Immun 51, 69–79 5 Weiser, J.N & Pan, N (1998) Adaption of Haemophilus in uenzae to acquired and innate humoral immunity based on phase variation of lipopolysaccharide Mol Microbiol 30, 767–775 6 Fleischmann, R.D., Adams, M.D., White, O., Clayton, R .A. ,... Richards, J.C (2001) Structural analysis of the lipopolysaccharide from the nontypable Haemophilus in uenzae strain SB 33 Eur J Biochem 268, 5278–5286 ˚ Mansson, M., Hood, D.W., Li, J., Richards, J.C., Moxon, E.R & Schweda, E.K.H (2002) Structural analysis of the lipopolysaccharide from nontypeable Haemophilus in uenzae strain 1003 Eur J Biochem 269, 808–818 Schweda, E.K.H., Li, J., Moxon, E.R & Richards,... Structural analysis of LPS from NTHi strain 981 (Eur J Biochem 270) 2989 seem reasonable that these genes are responsible for the expression of the globotetraose epitope, and truncated versions thereof, in this strain, this remains to be determined Genetic analysis has also shown that the lpsA gene may be nonfunctional, which is consistent with the lack of OS extension at HepIII (M Deadman, personal . Structural characterization of a novel branching pattern in the lipopolysaccharide from nontypeable Haemophilus in uenzae Martin Ma ˚ nsson 1 ,. of conjugate vaccines, there exists no vaccine against nontype- able H. in uenzae (NTHi). NTHi strains are a common cause of otitis media and respiratory tract