Complex Carbohydrate Research Center, The University ofGeorgia, 220 Riverbend Road, Athens, GA, USA, 2Laboratoryof Immunology, National Institute on Deafness and Other CommunicationDisorders, S. Research Court, Rockville, MD 20850, USA,and 3Center for Biologics Evaluationand Research, Food and Drug Administration, 8800 Rockville Pike, Bethesda,MD 20892, USA
Received on April 20, 1999. revisedon June 10, 1999; accepted on June 10, 1999.
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Abstract |
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Hex-(14)-[Glc]0-4-(1
4)-D,D-Hep-(1
6)-Glc-(1
4)-L,D-Hep-(1
5)-Kdo
3
1
L,D-Hep
2
1
(Hex)01-(12)-L,D-Hep
where Hex is either a Glc or Gal residue.
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Introduction |
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NTHi does not produce a lipopolysaccharide (LPS) with an O-antigenpolysaccharide. Like other H.influenzae, NTHi expressesheterogenous populations of LOSs which differ by the addition ordeletion of glycoses, phosphates (P), and phosphoethanolamine (PEA)substituents (2Capagnari et al.,1987; 16
Patrick et al.,1987, 1989; 18
Phillips et al., 1992). Expression of these heterogeneousoligosaccharide (OS) epitopes in NTHi is important in hostpathogeninteractions and therefore is an essential target for regulatingthe virulence potential of this organism (18
Phillips et al., 1992). Mouse monoclonal antibodies toNTHi 9274 LOS bound to 90 % of 100 NTHi clinical isolateswhich indicates that at least a number of the various NTHi 9274LOS structures must be relatively conserved among these isolates(8
Gu et al., 1996a).Furthermore, these monoclonal antibodies were bactericidal to 30% ofthese isolates (unpublished observations). In addition, detoxifiedLOS conjugates from strain 9274 induced bactericidal antibodiesagainst both homologous and heterologous strains in rabbits (9
Gu et al., 1996b), and conferredprotection against NTHi otitis media in chinchillas (10
Gu et al., 1997).
The LOSs of several H.influenzae serotype band NTHi strains have been previously studied (18Phillips et al., 1992, 1993; 13
Masoud et al., 1997). Glucose (Glc), galactose(Gal), L-glycero-D-manno-heptose (L,D-Hep), and 3-deoxy-D-manno-2-octulosonic acid (Kdo)are the common sugar constituents found in these LOSs. In addition,some LOSs contain N-acetylglucosamine(GlcNAc). Phosphate, PEA, and acetyl groups are also found as commonnonglycosidic constituents. The single Kdo residue present in H.influenzae LOSs has been found to be phosphorylatedin both the serotype b strain (11
Helander et al., 1988; 19
Phillips et al., 1993) and in an NTHi strain (18
Phillips et al., 1992).Previous structural studies (18
Phillips et al., 1992, 1993; 13
Masoud et al., 1997) showed that
-L,D-Hepp-(1
2)-
-L,D-Hepp-(1
3)-
-L,D-Hepp-(1
5)-Kdop (i.e., HepIII-HepII-HepI-Kdo) is a common LOSinner core element for H.influenzae strains. Structuralvariation of these LOSs is due to the various OSs attached to HepI,HepII, and/or HepIII (13
Masoud et al., 1997), to the phosphate and phosphoethanolamine(PEA) substituents on HepII and HepIII (13
Masoud et al., 1997), and, more recently, toa phosphocholine-glucosyl moiety attached to HepIII (22
Risberg et al., 1997). Unlike other H.influenzae, thisreport shows that the inner core region of NTHi 9274 LOS containsfour heptosyl and one hexosyl residues. Three of the heptosyl residuesare L,D-Hep (HepI, II, and III), and the fourth isa D-glycero-D-manno-heptosyl residue, D,D-Hep(HepIV). Additional OSs of various sizes are attached to HepIIIand/or HepIV. The presence or absence of phosphate andphosphoethanolamine (PEA) groups also contributes theheterogeneity of NTHi 9274 LOS.
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Results |
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A sample of the LOS preparation was de-O-acylatedby treatment with anhydrous hydrazine and analyzed by MALDI-TOF MS.The spectrum (not shown) showed that the LOS preparation was extremelyheterogeneous consisting of a mixture of at least 25 molecules rangingin size from m/z 2600 to 3600. The OSsreleased by mild acid hydrolysis were also analyzed by MALDI-TOFMS. Again, the results (spectrum not shown) showed that the heterogeneityobserved in the de-O-acylated LOS sample was alsoobserved in the OSs in that a minimum of 17 molecules were detectedwith a the mass range between m/z 1400and 2400. The differences in masses between the various moleculesindicated that the heterogeneity was due to variation in the numberof hexosyl residues, as well as in phosphate, and PEA substituents.
Analysis of the HF-treated OSs from NTHi 9274 LOS
In order to further characterize the structures of these OSs,it was necessary to reduce the heterogeneity of the OS preparation. Thus,the phosphate and PEA substituents were removed by treatment withaqueous HF and the resulting OSs (HFOSs) were purified by Bio-GelP4 column chromatography. Two fractions, HFOSa, and HFOSb, elutedjust after the void volume of the column, the major fraction beingHFOSb. Proton NMR analysis was performed on the OS preparation priorto (Figure 1), and after (not shown) aqueousHF treatment. The NMR spectrum is quite complex due to the mixtureof OSs present. However, HF treatment resulted in the disappearanceof the PEA proton resonances at 3.2.The spectrum also shows a complex pattern of proton resonances between
1.8 and 2.3 consistent with the H-3 protonsof Kdo and
-acetylgroups. The spectrum of HFOSb (data not shown) also contained resonancesindicative of the H-3 methylene protons of anhydro Kdo (e.g., 4,8-anhydro Kdo)between
2.9 and 3.3 (Carlson and Krishnaiah1992). These anhydro Kdo methylene protons are exchangeable with deuterium(from D2O) and were, therefore, greatly reduced in intensitycompared with those of a normal Kdo residue. Thus, the mild acidreleased OSs and HFOSs, contain both normal, and anhydro Kdo residues.
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Tandem MS/MS analysis of the permethylatedHFOS from NTHi 9274 LOS
The HFOS samples were permethylated and major molecular ionswere analyzed by tandem MS/MS in order to determine thepossible glycosyl sequences of some of the LOS molecules presentin this strain. The permethylated HFOSs were first characterizedby FAB-MS and ESI-MS analyses in the positive mode. The FAB-MS spectrumis shown in Figure 2. The observed m/z valuesfor molecular ions of the permethylated HFOSs are shown in Table and compared with calculated values. Forboth FAB-MS and ESI-MS, four series of ammoniated and/orsodiated molecular ions were found; two major series of higher intensities,and two minor series. One major ion series corresponded to OSs havingions of m/z values of 1695.0, 1899.0,2103.2, and 2308 due to molecules of compositions givenin Table
; each molecule having ananhydro Kdo at its reducing end (Figure 2,structure b of Scheme B). The second major seriesof ions was due to OSs having m/z valuesof 1723.0, 1927.1, 2131.4, and 2336.5 and having the compositionsgiven in Table
with a modified Kdomoiety at their reducing ends. While the structure of this modifiedKdo residue is not known with certainty, the sequential methylation procedureused may have resulted in either structure a (Figure 2 Scheme A) or structure d (Figure 2, Scheme B). A third minor series of ions indicatedmolecules which may have a modified Kdo of structure c (Figure 2, Scheme B). Each of these series of ions isdue to five different molecules, HFOS1 to HFOS5 corresponding topermethylated Hex1-5Hep4Kdo1 anddiffering only in the type of Kdo modification. A fourth minor seriesof ions of m/z 1465, 1669, and 1873 consistingof Hex1-3Hep4 and a modified Kdowhich can not be accounted for by the schemes shown in Figure 2. Two other expected ions, those for permethylatedHFOS6 and HFOS7 (see Table
), werenot observed since they had masses above the maximum detectable ionof m/z 2400.
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Discussion |
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In addition to the uniqueness of the D,D-Hep-Glcinner core extension in NTHi 9274 LOSs, there are other significant featuresof the various oligosaccharide substitutions to this extended innercore. In the case of other reported H.influenzae LOSs,whenever an R2 oligosaccharide is present it is attached toO3 of the 2-linked L,D-heptosyl residue (HepII).The absence of any 2,3-linked L,D-Hep togetherwith the MS/MS data show that none of the various NTHi9274 LOS molecules have an R2 oligosaccharide substitution.The absence of an R2 oligosaccharide has also been reportedfor H.influenzae RM.11828 (22Risberg et al., 1997), H.influenzae AH1-3 (23
Schweda et al., 1995a),and NTHi 2019 LOSs (18
Phillips et al., 1992).
Structural variation of H.influenzae LOSs isreported to be due to the various OSs attached to this "innercore" structure at HepI, HepII, or HepIII (i.e., R1,R2, and R3 of Figure 6).These OSs are reported to consist of a single glucosyl residue,a phosphocholine substituted glucosyl residue, lactosyl or diglucosyl disaccharides,the p epitope (
-Gal-1
4-ß-Gal-1
4-ß-Glc-), truncatedlacto-N-neotetraose, and sialylated versions ofthe lacto-N-neotetraose (18
Phillips et al., 1992, 1993, 1996; 13
Masoud et al., 1997; 22
Risberg et al., 1997). The fact that NTHi 9274 OSslack GlcNAc indicates that none of the variant LOS molecules fromthis strain contains lacto-N-neotetraose or its sialylatedversion. Also, the absence of a 4-linked galactosyl residue rulesout the presence of the p
epitope structurein these various LOS molecules.
While the more extended oligosaccharide structures such as lacto-N-neotetraose and the p epitopeare not present in NTHi 9274 LOS, the data suggest that the NTHi9274 LOS molecules contain many of the more truncatedstructural features reported to be present in other H.influenzae LOSs.The presence of terminally and 4-linked Glc indicates that someof the some of the NTHi 9274 LOS molecules have the frequently reported Glc-1
4-Glc- disaccharide structural featureas the R1 oligosaccharide. Likewise, the presenceof terminally linked Gal and 4-linked Glc may indicate that someof these NTHi 9274 LOS molecules could contain a lactosyl substitutionat the R1 position; however, it is also possible thatthe terminally linked Gal is totally accounted for by the hexosylresidue that is linked to HepIII, i.e., R3. It is clearfrom the MS/MS data that when NTHi 9274 LOS molecules containR3, it exists as a single hexosyl residue. This is alsotrue for other H.influenzae LOSs in which the HepIIIis reported to be glycosylated at O2 by Gal (13
Masoud et al., 1997), or by Glc (22
Risberg et al., 1997). Again, the presence ofterminally linked Gal and Glc supports the possibility that therecould be versions of the NTHi 9274 LOS in which R3 iseither Glc or Gal. The various possible R1 and R3 OSsfor NTHi 9274 LOS molecules are summarized in Table
. These structures are consistent with thelinkages reported in Table
, and withthe MS/MS spectra shown in Figures 35. The presence of significant levels of bothterminally linked D,D-Hep and terminally linked L,D-Hep suggest that there are a significant numberof molecules without R1 or R3 OSs which maynot be fully accounted for by the MS data. These molecules may behave been lost during purification of the HFOSs. Purification oflarger amounts of OSs is in progress in order to obtain more completesequence information on these variable OSs.
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Taken together, the hexosyl, phosphate, and PEA variation revealthat the NTHi 9274 LOS preparation is extremely heterogeneous.The presence of numerous LOS molecules containing various truncatedoligosaccharides that are common to many H.influenzae LOSsmay explain why antibodies to NTHi 9274 LOS react with the majorityof NTHi clinical isolates and show bactericidal activity to theseisolates (9Gu et al., 1996b).The exact glycosyl sequence of the R1 OSs of the NTHi9274 LOSs is under further investigation.
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Material and methods |
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Preparation of LOS OSs
The LOS preparation (20 mg) from NTHi strain 9274 was hydrolyzedin aqueous 1% acetic acid (10 ml) for 2 h at 100°C. Thehydrolyzate was centrifuged at 10,000 x g for 20 min, and the supernatant was collected.The pellet was washed once with 5 ml of water and collected by centrifugation.The wash water was added to the original supernatant, and the remaining lipidA was extracted with diethyl ether (three times, 5 ml volumes eachtime). The aqueous phase containing the OSs was lyophilized. Thelyophilized OSs were dissolved in 0.5 ml of water, filtered withMicrofilterfuge tubes containing 0.45 µmpore size Nylon-66 membrane filters, applied to a Bio-Gel P-4 column (70 x 1.6 cm), and eluted with deionizedwater containing 1% 1-butanol. Fractions were assayed forcarbohydrate by the phenol-sulfuric acid assay, and the OS peakswere pooled and lyophilized.
De-O-acylation of LOS
The LOS sample of NTHi 9274 was de-O-acylatedaccording to the procedure of Helander et al. (11Helander et al., 1988). Approximately8 mg of LOS were incubated with 1 ml of anhydrous hydrazine for20 min at 37°C. The solution was cooledto 20°C, and 5 ml of chilledacetone were added drop-wise to precipitate the de-O-acylatedLOS. The sample was then centrifuged at 12,000 x g for 20 min at 4°C.The supernatant was removed, and the pellet was washed again withcold acetone and centrifuged. The precipitated de-O-acylatedLOS was then resuspended in 1 ml of water and lyophilized.
Treatment of LOS and OS with aqueous HF
The LOS and OS samples (8 mg) were each placed in 1.5 ml polypropylenetubes. The samples were treated with cold aqueous 48% hydrogenfluoride (HF) (100 µl) and kept for24 h at 4°C (12Kenne et al., 1993). The HF was removed by flushingunder a stream of air followed by addition of diethyl ether (600 µl) and drying with a stream of air.This step was repeated three times. The dry pellet was dissolvedin water and lyophilized. The lyophilized dephosphorylated sampleswere dissolved in 0.5 ml of water, and filtered with Microfilterfugetubes containing 0.45 µm pore size Nylon66 membrane filters. The HF-treated OS sample (HFOS) was then appliedto a Bio-Gel P-4 column (70 x 1.6 cm),and eluted with water containing 1% 1-butanol. Fractionswere assayed for carbohydrate by the phenol-sulfuric acid assay,and the OS peaks were pooled and lyophilized.
Glycosyl composition analyses
Glycosyl composition analysis of LOS and OS samples (0.5 mg each)was performed by hydrolysis in 2 M trifluoroaceticacid (0.5 ml) in a closed vial at 120°Cfor 3 h. The glycoses in the hydrolyzate were reduced with NaBH4,acetylated, and analyzed by gas liquid chromatography (GLC) andby combined GLC-mass spectrometry (MS) (31Yorket al., 1985). For the determination ofKdo, the LOS sample (0.5 mg) was dried in vacuum, and methanolyzedin 1 ml of methanolic 2 M HCl at 80°Cfor 4 h. The released methyl glycoside residues were trimethylsilylated, andanalyzed by GLC-MS (31
York et al.,1985). The absolute configurations of the glycoses presentin the OS from NTHi were determined by GLC-MS analysis of the trimethylsilylated (S/R)-2-butylglycosides (6
Gerwig et al.,1979). GLC and GLC-MS analyses were performed on fusedsilica capillary columns (length, 30 m; inner diameter, 0.32 mm)with helium as the carrier. A DB-5 column (J & W Scientific)was used for aminoglycosyl alditol acetate derivatives, and an SP2330column (Supelco, Bellefonte, PA) was used for the neutral glycosylalditol acetate derivatives. The DB-5 column was also used for theanalysis of trimethylsilyl methyl or 2-butyl glycosides. GLC equipment consistedof HP5890 gas chromatograph equipped with a flame ionization detector(Hewlett-Packard). GLC-MS (EI) was performed using a Hewlett-Packard5970 MSD.
Glycosyl linkage analyses
Glycosyl linkage analyses were carried out using a modified NaOH-method(Ciucanu and Kerek 1984). Samples (1 mg each) were dissolved indimethylsulfoxide (100 µl), powdered NaOH(100 mg) was added, and the reaction mixture was stirred rapidlyfor 30 min. Methylation was performed by the sequential additionsof methyl iodide (40, 40, and 80 µl)at 10 min intervals. After an additional 20 min stirring,the methylated glycans were recovered in the organic phase afteraddition of chloroform (0.5 ml) and 1 M sodiumthiosulfate (1 ml). The extraction with chloroform was repeatedtwo more times, the organic phases were combined and dried witha stream of nitrogen. The permethylated product residue was suspendedin deionized water and was further purified by reverse-phase chromatographyon a Sep-Pak C18 cartridge as previously described (29Waeghe et al., 1983). Methylated glycans were hydrolysedwith 2 M trifluoroacetic acid (120°C,3 h), reduced with NaBH4 or NaB(2H)4,acetylated, and analyzed by GLC and GLC-MS (31
Yorket al., 1985). For the determination ofKdo linkages, the permethylated LOS was methanolized in 1 ml of methanolic2 N HCl at 80°C for4 h, and the released methyl glycoside residues were acetylatedand analyzed by GLC-MS (5
Edebrink et al., 1994).
Lipid A purification
Lipid A was released from the LPS (10 mg) by hydrolysis in aqueous1% acetic acid (5 ml) at 100°Cfor 2 h. The precipitated lipid A was collected by centrifugation(10,000 x g). The precipitatewas resuspended with water (2 ml) and partitioned into chloroform(2 ml). The chloroform layer containing purified lipid was concentratedto dryness.
Fatty acid analysis
Total fatty acids were released by methanolysis of lipid A with methanolic M HCl at 80°Cfor 16 h, and were trimethylsilylated. The resulting fattyacid methyl esters were analyzed by GLC-MS (31Yorket al., 1985). Ester and amide-linked fatty acidswere distinguished by preferential release of the ester-linked fattyacids using anhydrous sodium methoxide (30
Wollenweberand Rietschel, 1990).
Mass spectrometry
Fast atom bombardment mass spectrometry (FAB-MS) was performedin the positive mode with a JEOL (Tokyo, Japan) SX/SX 102Atandem mass spectrometer, which was operated at 10 kV acceleratingpotential. Ions were produced by FAB with xenon, using a JEOL FABgun operated at 6 kV in a conventional FAB ion source. Spectra acquiredfor the first MS are averaged profile data of three scans as recordedby a JEOL XMS data system. These spectra were acquired from 200to 2000 m/z at a rate that would scanthe mass range from 0 to 2500 in 1 min. A filtering rate of 100Hz was used in acquiring these spectra. The samples were dissolvedin water, and 1 µl aliquots were mixedwith an equal volume of the FAB matrix, thioglycerol, on the probetip. The MS-MS spectrum was obtained by using a linked scan (B/Econstant) at a rate that would scan the mass range from 5 to 2400in 1 min with a filtering of 300 Hz.
Matrix-assisted laser desorption ionization time-of-flight massspectrometry (MALDITOF-MS) was performed on an LDI 1700XP spectrometer(Linear Scientific, Reno, NV) The instrument was operated at anaccelerating voltage of 30 kV and an extractor voltage of 9 kV.The pressure was 2.1 x 106 torr. Thesample was ionized with a nitrogen laser (
= 337nm) with a pulse width of 3 ns and a 4 to 7.5 µJpulse. The samples were dissolved in water, and the matrix (2,4-dihydroxybenzoic acid)-samplemixture (12 µl) was appliedto the probe and quickly dried under vacuum. The sample solutionwas serially dried with matrix to obtain optimal sensitivity. Themass spectra were recorded from m/z 400to 10,000. The spectra are the average of 100250 acquisitions.A mixture of maltooligosaccharides was used as the calibrationstandard.
The MALDI magnetic sector experiment was performed on the firsttwo sectors of a JEOL (Tokyo, Japan) SX/SX102A tandem four-sectormass spectrometer with a point detector located at the end of MS1.In the MALDI configuration, the FAB gun is replaced by a JEOL MALDIkit (JUS-MALDI, JEOL USA Inc., Peabody, MA), which includes a nitrogen laserof 337 nm wavelength, 3 ns pulse width, and 250 µJmaximum pulse energy. This laser was operated at a pulse rate of 20Hz. MS1 was operated at 10 kV, full accelerating voltage, for allexperiments except the survey scan of the maltooligosaccharidemixture was used as the calibration standard, where the acceleratingvoltage was lowered to 6 kV to enable the magnetic field to scanup to 4000 m/z units. The spectra wereacquired at a rate that would scan the mass range 02400 in1 min with 300 Hz filtering. Spectra are averaged profile data asrecorded by a JEOL complement data system.
Electrospray ionization mass spectrometry (ESI-MS) was performedwith a SCIEX API-III mass analyzer operated in the positive ionmode with an orifice potential of 50 V. Spectra are the accumulationof 1015 scans collected over the mass range of 4002000.The sample was dissolved in methanol at a final concentration of2 µg/µl,and pumped into the mass spectrometer at a rate of 3 µl/min.
Nuclear magnetic resonance spectroscopy (NMR)
Samples were exchanged several times with D2O, dissolvedin D2O, and analyzed at 298 K, using a Bruker AM500 spectrometer. Chemicalshifts were measured relative to sodium 3-trimethylsilylpropionate-2,2,3,3-d4 ( 0.00).
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Acknowledgments |
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Footnotes |
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References |
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