Linkage analysis showed major proportions of 3-substituted GalNAc, 4-substituted galac-tose and terminal galacgalac-tose Table 2, and the anomeric con®guration of the latter was determin
Trang 1Characterization of glycosphingolipids from Schistosoma mansoni
eggs carrying Fuc(a1±3)GalNAc-, GalNAc(b1±4)[Fuc(a1±3)]GlcNAc-and Gal(b1±4)[Fuc(a1±3)]GlcNAc- (Lewis X) terminal structures
Manfred Wuhrer1,*, Sven R Kantelhardt1,*, Roger D Dennis,1Michael J Doenhoff2, GuÈnter Lochnit1
and Rudolf Geyer1
1 Institute of Biochemistry, University of Giessen, Germany; 2 School of Biological Sciences, University of Wales, Bangor, Wales, UK
The carbohydrate moieties of glycosphingolipids from eggs
of the human parasite, Schistosoma mansoni, were
enzy-matically released, labelled with 2-aminopyridine (PA),
fractionated and analysed by linkage analysis, partial
hydrolysis, enzymatic cleavage, matrix-assisted laser
desorption/ionization time-of-¯ight mass spectrometry and
nano-electrospray ionization mass spectrometry Apart
from large, highly fucosylated structures with ®ve to seven
HexNAc residues, we found short, oligofucosylated species
containing three to four HexNAc residues Their structures
have been determined as Fuc(a1±3)GalNAc(b1±4)[ Fuc
(a1±3)]GlcNAc(b1±3)GalNAc(b1±4)Glc-PA, GalNAc(b1±
4)[Fuc(a1±3)]GlcNAc(b1±3)GlcNAc(b1±3)GalNAc(b1±4)
Glc-PA, Fuc(a1±3)GalNAc(b1±4)[Fuc(a1±3)]GlcNAc(b1±
4) GlcNAc(b1±3)GalNAc(b1±4)Glc-PA, and Fuc(a1±3)
GalNAc(b1±4)[ Fuc(a1±2) Fuc(a1±2)Fuc(a1±3)]Glc
NAc(b1±3)GlcNAc(b1±3)GalNAc(b1±4)Glc-PA The last
structure exhibits a trifucosyl sidechain previously identi®ed
on the cercarial glycocalyx These structures stress the importance of 3-fucosylated GalNAc as a terminal epitope
in schistosome glycoconjugates To what degree these gly-cans contribute to the pronounced antigenicity of S mansoni egg glycolipids remains to be determined In addition, we have identi®ed the compounds GlcNAc(b1±3)GalNAc(b1± 4)Glc-PA, Gal(b1±4)[Fuc(a1±3)]GlcNAc(b1±3) GalNAc (b1±4)Glc-PA, the latter of which is a Lewis X-pentasac-charide identical to that present on cercarial glycolipids, as well as Gal(b1±3)GalNAc(1±4)Gal(1±4)Glc-PA, which corresponds to asialogangliotetraosylceramide and is most probably derived from the mammalian host
Keywords: ceramide glycanase; internal fucose; oligo-saccharide structural analysis; Schistosoma mansoni egg glycolipids
Schistosomiasis is caused by parasitic blood ¯ukes of the
genus Schistosoma and affects 200 million people
world-wide During infection, schistosomal glycoconjugates play
important roles in host±parasite pathological interactions
[1,2] Schistosomes produce a variety of complex
carbohy-drate structures, many of which are highly fucosylated [2]
These glycans are conjugated to proteins and/or lipids in
different life-cycle stages and vary in their recognition by the
immune system For Schistosoma mansoni, the Lewis X
epitope has been found on egg and cercarial glycoproteins
[3±5] and cercarial glycolipids [6] Although this epitope is shared with the mammalian host, S mansoni infection serum contains cytolytic antibodies directed against this epitope [7] Besides Lewis-X-carrying glycolipids, S man-soni expresses highly antigenic glycolipids mainly in the egg and the cercarial stage, and to a lesser extent in the adult stage [8,9] The stage-associated expression of carbohydrate structures on S mansoni glycolipids is paralleled by changes
in glycolipid ceramide structures during the life-cycle [10] The highly antigenic glycolipids are speci®cally recog-nized by schistosome infection serum and not by other helminth infection sera, which makes them possible sero-diagnostic antigens [11] They share a fucose-containing epitope with keyhole limpet haemocyanin (KLH) [12], which is consistent with the use of KLH for the diagnosis of schistosomiasis [13] The induction of signi®cant titres of antibodies against these glycolipids is associated with the onset of patency [11], and the IgE-response against worm glycolipids may play a role in mediating resistance to
S mansoni reinfection after praziquantel treatment [14] All schistosome glycosphingolipids share the unique core structure, GalNAcb4Glc1±1ceramide, which was ®rst described by Makaaru et al [15] Cercarial glycolipids were found to be dominated by short-chained carbohydrate moieties expressing Lewis X (Gal(b1±4)[Fuc(a1±3)]Glc-NAcb1-) and pseudo-Lewis Y (Fuc(a1±3)Gal(b1±4)[Fuc (a1±3)]GlcNAc(b1-)epitopes[6],whereasthestructuralchar-acterization of unfractionated complex glycosphingolipids from S mansoni eggs has revealed the terminal structure
Correspondence to R Geyer, Biochemisches Institut am Klinikum,
UniversitaÈt Giessen, Friedrichstrasse 24, D-35392 Giessen, Germany.
Fax: + 49 641 99 47409, Tel.: + 49 641 99 47400,
E-mail: Rudolf.Geyer@biochemie.med.uni-giessen.de
Abbreviations: Cer, ceramide; dHex, deoxyhexose; ESI,
electrospray-ionization; Fuc, fucose; Gal, galactose; GalNAc,
N-acetylgalactos-amine; Glc, glucose; GlcNAc, N-acetylglucosN-acetylgalactos-amine; Hex, hexose;
HexNAc, N-acetylhexosamine; KLH, keyhole-limpet hemocyanin;
LSIMS, liquid secondary-ion mass spectrometry; mAb, monoclonal
antibody; Man, mannose; PA, 2-aminopyridine; PGC, porous
graphitic carbon.
Enzymes: a- L -fucosidase (EC 3.2.1.51); b- D -galactosidase (EC
3.2.1.23); ceramide glycanase (oligoglycosylceramideglycohydrolase;
EC 3.2.1.23).
*Note: these authors contributed equally to this work.
(Received 31 August 2001, revised 8 November 2001, accepted 14
November 2001)
Trang 2Fuc(1±2)Fuc(1±3)GalNAc (b1- attached to a series of
up to three )4)[Fuc(1±2)Fuc(1±3)]GlcNAc(b1-repeats and
one )4)[ Fuc(1±2) Fuc(1±3)]GlcNAc(b1-unit, which
is linked to )3)GalNAc(b1±4)Glc(1±1)ceramide, i.e the
Schisto-core [16]
In the present study, we have chosen an approach that
differs from the one employed in the aforementioned and
other investigations of glycolipid structures from S mansoni
eggs [16,17] in which complex mixtures of
glycosphingo-lipids have been analysed mainly by liquid secondary-ion
mass spectrometry (LSIMS) using various chemical
deri-vatizations Similar to previous studies on glycolipids from
Caenorhabditis elegans [18] and S mansoni cercariae [6], we
have enzymatically removed the ceramide moieties and
fractionated the released oligosaccharide chains before
structural characterization by mass spectrometry, chemical
and enzymatic degradation and linkage analysis Using this
strategy, structural information on the carbohydrate
moi-eties of individual glycolipids from S mansoni eggs is
obtained
M A T E R I A L S A N D M E T H O D S
Glycolipid puri®cation and fractionation
S mansoni egg glycolipids were puri®ed by organic solvent
extraction, saponi®cation, desalting and anion-exchange
chromatography as described previously [6] Neutral
gly-colipids were fractionated on a silica-gel cartridge (Waters,
Eschborn, Germany) as outlined elsewhere [19] by step-wise
elution with chloroform/methanol (80 : 20, v/v),
chloro-form/methanol/water (65 : 25 : 4) and
chloroform/metha-nol/water (10 : 70 : 20), and analysed by HPTLC orcinol/
H2SO4-staining and HPTLC-immunostaining as described
previously [6] The ®rst fraction contained ceramide
monohexoside and dihexoside and was not further
analysed The second fraction was positive in
HPTLC-immunostaining and was used for preparation of
PA-oligosaccharides For HPTLC-immunostaining, the mAbs
M2D3H, G11P and C1C7 were provided by Q Bickle,
Department of Infectious and Tropical Diseases, London
School of Hygiene and Tropical Medicine, England, while
the mAb 290-2E6 [20] was provided by A M Deelder,
Leiden University Medical Center, the Netherlands
Preparation and separation of PA-oligosaccharides
The glycan moieties were released from glycolipids using
recombinant ceramide glycanase (endoglycoceramidase II
from Rhodococcus sp.; Takara Shuzu Co., Otsu, Shiga,
Japan) and separated from uncleaved glycolipids and free
ceramides on a reverse-phase cartridge [6] Released
oligo-saccharides were labelled with 2-aminopyridine (PA) and
excess reagent was partitioned with chloroform [21]
PA-oligosaccharides were fractionated on an amino-phase
HPLC column (4.6 ´ 250 mm, Nucleosil-Carbohydrate;
Macherey and Nagel, DuÈren, Germany) at a ¯ow rate of
1 mLámin)1at room temperature and detected by
¯uores-cence (310/380 nm) [21] The column was equilibrated with
200 mMaqueous triethylamine/acetic acid, pH 7.3 :
aceto-nitrile (25 : 75, v/v) A gradient of 25±60% aqueous
triethylamine/acetic acid buffer was applied within a
60-min period and the column was run isocratically for a
further 10 min Peak fractions were collected and lyophil-ized Heterogeneous fractions were resolved further on a porous graphitic carbon (PGC)-column (4.6 ´ 100 mm, Hypercarb; Hypersil, Runcorn, UK) at a ¯ow rate of
1 mLámin)1at room temperature with ¯uorescence detec-tion (310/380 nm) The column was equilibrated with
20 mMtriethylamine/acetic acid, pH 5.0 A gradient from
0 to 30% acetonitrile was applied in 50 min Individual peak fractions were collected and lyophilized
MALDI-TOF MS and ESI MS MALDI-TOF MS was performed on a Vision 2000 (Ther-moFinnigan, Egelsbach, Germany) equipped with a UV nitrogen laser (337 nm) as described previously [6] The instrument was operated in the positive-ion re¯ectron mode throughout using 6-aza-2-thiothymine (Sigma) as matrix ESI MS was performed with an Esquire 3000 ion-trap mass spectrometer (Bruker Daltoniks, Bremen, Germany) equipped with an off-line nano-ESI source A 2±5 lL aliquot
of PA-oligosaccharides in methanol/water (1 : 1, v/v) or glycolipids in chloroform/methanol/water (10 : 20 : 3) was loaded into a laboratory-made, gold-coated glass capillary and electrosprayed at a voltage of 700±1000 V using N2as dry-gas (120 °C, 4 Lámin)1) The skimmer voltage was set to
30 V,exceptforFig 7AandB,where55Vwereapplied.For each spectrum 20±100 repetitive scans were averaged The accumulation time was between 5 and 50 ms All MS/MS experiments were performed with helium as collision gas Enzymatic and chemical degradation
PA-oligosaccharides were treated with either a-fucosidase from bovine kidney (4 mUálL)1; Roche Diagnostics, Mannheim, Germany) or with b-galactosidase from bovine testes (4 mUálL)1; Roche Diagnostics) on the MALDI-TOF MS target [22] Enzymes were dialysed for 4 h against
25 mMammonium acetate solution adjusted to pH 5.0 for a-fucosidase and pH 4.0 for b-galactosidase After measurement of the educts by MALDI-TOF MS using 6-aza-2-thiothymine matrix, dialysed enzymes (2 lL) were added undiluted to sample aliquots on the target and spots were analysed again by MALDI-TOF MS after incubation overnight at 37 °C For chemical defucosylation, dried samples were treated with 48% HF at 4 °C overnight (modi®ed from [23]) HF was removed by a stream of nitrogen
Monosaccharide composition and linkage analysis After hydrolysis in 4M aqueous tri¯uoroacetic acid at
100 °C for 4 h and labelling with anthranilic acid, mono-saccharides were determined by HPLC and ¯uorescence detection [9,24] For linkage analysis, PA-oligosaccharides were permethylated with methyl iodide after deprotonation with lithium methylsul®nyl carbanion [25] and hydrolysed (4M aqueous tri¯uoroacetic acid, 100 °C, 4 h) Partially methylated alditol acetates obtained after sodium boro-hydride reduction and peracetylation were analysed by capillary GC followed by ¯ame ionization detection or chemical ionization mass spectrometry (single ion monitor-ing), using a moving needle injector, fused silica bonded phase capillary columns of different polarity (60-m DB-1
Trang 3and 30-m DB-210; ICT, Bad Homburg, Germany) and
helium as carrier gas as detailed elsewhere [26]
R E S U L T S
Preparation of glycolipids
Glycolipids were isolated from S mansoni eggs analogously
to the study performed on S mansoni cercarial glycolipids
[6] and analysed by HPTLC (Fig 1) Orcinol/H2SO4
-staining (lane 1) revealed some major, slow-migrating
compounds and a weaker staining for smaller, minor
components Both murine S mansoni infection serum (lane 2) and four monoclonal antibodies (lanes 3±6) visualized antigenic glycolipids Murine infection serum and the mAbs M2D3H, G11P and C1C7 exhibited a similar pattern, whereas the mAb 290±2E6, which is known to recognize mono- and difucosylated structures like GalNAc(b1±4) [ Fuc(a1±2)Fuc(a1±3)]GlcNAc(1- [20], displayed a com-pletely different pattern The murine infection serum and mAb M2D3H were especially ef®cient in detecting the fast-migrating glycolipids (lanes 2 and 3), which orcinol/H2SO4 -staining revealed to be present in only low amounts We expected these minor, fast-migrating, antigenic glycolipids not to be covered by the structural characterization of whole mixtures of complex glycolipids [16,17] and therefore decided to fractionate these compounds as their corre-sponding PA-oligosaccharides and to structurally charac-terize the individual species
Preparation and separation of PA-oligosaccharides Glycans were released from the ceramide moieties by ceramide glycanase treatment of complex egg glycolipids For the separation of uncleaved glycolipids and ceramides from the released oligosaccharides, the sample was frac-tionated on a reverse-phase cartridge Released oligosac-charides were collected as the combined ¯ow-through and wash fractions, while the uncleaved glycolipids and cera-mide moieties were obtained by elution with organic solvents Released glycans and uncleaved glycolipids were quantitated by monosaccharide composition analysis (Table 1), showing an overall ef®cacy of over 95% glycan release Released oligosaccharides were analysed by MAL-DI-TOF MS (Fig 2C) and the obtained pattern was very similar to the patterns observed for the intact glycolipids in ESI- and MALDI-TOF MS (Fig 2A,B) This indicated that the released oligosaccharides were representative for the glycans of the major complex egg glycolipids The released oligosaccharides were pooled and labelled with the ¯uores-cent tag, 2-aminopyridine (PA) PA-oligosaccharides were fractionated by amino-phase HPLC (Fig 3A) Collected fractions (1 to 25; hereafter, fractions denoted by number only) were screened by MALDI-TOF MS and assessed for monosaccharide content by composition analysis (Tables 2 and 3) Fractions 1 to 7 were not found to contain carbohydrate Starting with 8, MALDI-TOF MS revealed several compounds for most of the fractions (Fig 4) In
-CTH
-CTetH
Fig 1 HPTLC of S mansoni egg glycolipids S mansoni egg
glycoli-pids were resolved with chloroform/methanol/0.25% aqueous KCl
(50 : 40 : 10, v/v/v) and visualized by orcinol/H 2 SO 4 staining (lane 1)
or immunostaining using a pool of eight murine S mansoni infection
sera (lane 2) and the mAbs M2D3H (1 : 20 000; lane 3), G11P (1 : 200;
lane 4), C1C7 (1 : 200; lane 5) and 290±2E6 (1 : 50; lane 6) CTH and
CtetH mark the migration positions of globotriaosyl- and
globotet-raosylceramide standards, respectively.
Table 1 Ecacy of ceramide glycanase cleavage of the complex egg glycolipids shown by monosaccharide composition analysis Complex egg glycolipids were cleaved with ceramide glycanase and fractionated on a reverse-phase cartridge The aqueous fractions of two experiments were combined (water fraction) as well as the organic solvent-eluted fractions (organic solvent fraction) and compared by composition analysis to the starting complex egg glycolipids The amounts of monosaccharides are given in micrograms and their relative ratios are normalized to GalNAc 2.0 in parentheses.
Monosaccharide Egg glycolipidfraction
Released monosaccharide (lg) Water fraction Organic solvent fraction Released monosaccharide (%)
Trang 4order to reduce peak heterogeneity and obtain as far as
possible pure compounds, the amino-phase fractions 8 to 14
were subfractionated by PGC-HPLC (Fig 3B,C)
Subfrac-tions (designated, for example, 12-5 for subfraction 5 of
fraction 12) were again screened by MALDI-TOF MS (cf
insets in Fig 3B,C and Table 3)
Structural elucidation of individual PA-glycans
Individual PA-glycans were analysed by composition
anal-ysis, linkage analanal-ysis, ESI MS/MS, as well as chemical and
enzymatic degradation followed by a second linkage analysis ESI MS fragments were assigned according to the nomenclature introduced by Domon & Costello [27] Anomeric con®gurations were in some cases determined enzymatically (Fig 5D,G), but generally assigned based on the results of egg glycolipid CrO3oxidation, which indicated b-anomeric linkages for GlcNAc and GalNAc and a-anomeric linkage for fucose as described recently [9] The major compound in 8 as judged from MALDI-TOF
MS (Fig 4) was Hex3HexNAc1PA After rechromatogra-phy by PGC-HPLC, it was detected in 8-11 by
MALDI-Fig 2 Mass spectrometry of unfractionated complex glycolipids and derived oligosaccha-rides from S mansoni eggs (A) ESI MS and (B) MALDI-TOF MS of complex S mansoni egg glycosphingolipids (C) MALDI-TOF MS
of oligosaccharides released from S mansoni egg glycosphingolipids.
Trang 5TOF MS ESI MS2of this compound revealed the sequence
Hex-HexNAc-Hex-Hex-PA and thus indicated a
non-Schisto-core (Fig 6A) Linkage analysis showed major
proportions of 3-substituted GalNAc, 4-substituted
galac-tose and terminal galacgalac-tose (Table 2), and the anomeric
con®guration of the latter was determined by MALDI-TOF
MS/on-target enzymatic cleavage with b-galactosidase from
bovine testes (data not shown) Taken together, the
structure was found to be Gal(b1±3)GalNAc(1±4)Gal(1±4)
Glc-PA (Table 4), which probably corresponds to the
host-derived glycolipid asialogangliotetraosylceramide As a
minor component in 8-11, the proton adduct of Hex1
Hex-NAc2PA was detected by ESI MS at m/z 664.9, and its
sequence was determined by ESI MS2 to be
HexNAc-HexNAc-Hex-PA (data not shown) Linkage analysis of
fraction 8-11 indicated minor amounts of terminal GlcNAc
Based on the assumption of a Schisto-core structure, this
leads to the structure GlcNAc(b1±3)GalNAc(b1±4)Glc-PA
(Table 4) for this minor compound Fraction 9 was not
analysed further, but its major compound
Hex-NAc2Hex2PA ([M + Na]+ at 849.5; Fig 4) might be
identical with the PA-tetrasaccharide
Galb3GlcNAcb3Gal-NAcb4Glc-PA derived from S mansoni cercarial
glycoli-pids [6] For 10-10, linkage analysis before and after fucose
removal by HF-treatment allowed the localization of the
fucose at the 3-position of GalNAc and the assignment of
the structure (Tables 2 and 4) In the case of 11-7, terminal fucose and galactose (Table 2) had a- and b-anomeric con®guration, respectively, as determined by on-target enzymatic cleavage with bovine kidney a-fucosidase and b-galactosidase from bovine testes (data not shown) Linkage analyses before and after preparative removal of fucose by HF-treatment (Table 2) and ESI MS/MS (Fig 6B,C) indicated 11-7 to have the Lewis X-containing structure Gal(b1±4)[Fuc(a1±3)]GlcNAc(b1±3)GalNAc (b1±4)Glc-PA
12-5 was found to contain fucose both in the 3-position of GalNAc and in the 3-position of GlcNAc (cf linkage analysis, Table 2) ESI MS2 (Fig 7B) showed the two fucosylated HexNAc residues to be adjacent to each other (B3ion at m/z 721.5) One of the fucoses of 12-5 could be removed enzymatically, while the second was only removed
by HF-treatment (Fig 5B,E,H) These accumulated data for 12-5 resulted in the structure Fuc(a1±3)GalNAc(b1±4) [Fuc(a1±3)]GlcNAc(b1±3)GalNAc(b1±4)Glc-PA (Table 4) The species 13-4 exhibited both terminal GalNAc and terminal fucose (Table 2) Loss of HexNAc in ESI MS2(Y4a
at 1036.1; Fig 8B) corroborated the presence of a terminal HexNAc Loss of a further HexNAc residue appeared only together with the loss of fucose (Y3at m/z 687.0 but no signal
at m/z 833), which indicated fucose to be linked to the subterminal HexNAc residue Bovine kidney a-fucosidase
Fig 3 HPLC separation of egg
glycolipid-derived PA-oligosaccharides (A) Separation
on an amino-phase column Elution positions
of the PA-labelled dextran hydrolysate
stan-dards of dierent chain-length are indicated by
arrows Fractions devoid of
carbohydrate-positive material are marked by asterisks (*).
Rechomatography of fractions 11 (B) and 12
(C) an a PGC-column and analysis of the
major peaks by MALDI-TOF MS (insets) n,
minor compound identical in mass with
10-10.
Trang 6did not act on 13-4, but the fucose could be removed by
HF-treatment (Fig 5) Taken together, the data showed 13-4 to
represent the structure shown in Table 4
Subfractionation of 14 led to the resolution of
difucosy-lated 14-2 and 14-3, trifucosydifucosy-lated 14-4 and tetrafucosydifucosy-lated
14-5 species ESI MS2analysis of 14-5 (Fig 9B) showed the
four fucose residues to be linked to the two outermost
GalNAc and/orGlcNAc residues (B4 ion at m/z 1013.2)
Three of the fucose residues are attached to one of these
HexNAc residues (Y4bB4at m/z 664.9) to form a Fuc(a1±2)
Fuc(a1±2)Fuc(a1±3)GlcNAc unit (see linkage analysis,
Table 2), and this ion could lose one or two fucose residues
on further fragmentation (Fig 9C) The HexNAc, which
carried this oligofucosyl chain is not outermost, as indicated
by the Y6aY4bion at m/z 1182.8 (Fig 9B) An ion at m/z
1328, which would have indicated the loss of one fucose and
one HexNAc (Y4b) was not registered in the 14-5 MS2
shown in Fig 9B, but was detected in similar MS2
experiments as a minor ion (not shown), thus corroborating the location of the trifucosyl chain on the second and not on the outermost HexNAc Taken together with the linkage analysis data (Table 2), the structure of 14-5 could
be elucidated as Fuc(a1±3)GalNAc(b1±4)[Fuc(a1±2) Fuc (a1±2)Fuc(a1±3)]GlcNAc(b1±3)GlcNAc(b1±3) GalNAc (b1±4) Glc-PA (Table 4) The simultaneous presence of isobaric structural isomers in this fraction could not be excluded Similarly, ESI MS2of the 14-4 [M + Na + H]2+
ion at m/z 766 (data not shown) yielded a [Hex-NAc2dHex3+ Na]+ion at 867.0 Da, indicating the three fucoses to be linked to adjacent HexNAc residues For 14-4, the loss of one fucose and one HexNAc resulted in an intense fragment ion at m/z 1182.0, which showed that the
Table 2 Analysis of PA-oligosaccharides by MALDI-TOF MS, ESI MS, composition analysis (CA) and linkage analysis (LA) Masses were determined by MALDI-TOF MS and ESI MS (*) and were rounded to the ®rst decimal place The type of pseudomolecular ion is given in brackets and the calculated, monoisotopic masses in parentheses 10-10HF, fraction 10-10 after HF-treatment, etc t-Fuc, terminal fucose; 4-Gal, 4-substituted galactose, etc.
Fraction Measured mass (Theoretical mass)[Pseudomolecular ion] Deduced composition Key structural data
8-11 808.0 (808.3) [M + Na] + ; 404.5*
(404.7) [M + H + Na] 2+ Hex 3 HexNAc 1 PA
Hex 1 HexNAc 2 PA LA: t-Gal, 4-Gal, 3-GalNAc, t-GlcNAc 664.9 (665.3) [M + H] +
10-10 1036.1 (1036.4) [M + Na] + Hex 1 HexNAc 3 dHex 1 PA CA: GlcN:GalN (1.1 : 2.0)
LA: t-Fuc, 4-GlcNAc, 3-GalNAc 10-10HF 890.9 (890.4) [M + Na] + Hex 1 HexNAc 3 PA LA: t-GalNAc, 4-GlcNAc, 3-GalNAc 11-7 995.6 (995.4) [M + Na] + Hex 2 HexNAc 2 dHex 1 PA CA: GlcN:GalN:Gal:Fuc
(1.1 : 1.2 : 0.9 : 1.0) LA: t-Fuc, t-Gal; 3-GalNAc; 3,4-GlcNAc 11-7HF 827.2* (827.4) [M + H] + Hex 2 HexNAc 2 PA LA: t-Gal; 4-GlcNAc; 3-GalNAc 12-5 1182.5 (1182.5) [M + Na] + ;
591.6* (591.7) [M + H + Na] 2+ Hex 1 HexNAc 3 dHex 2 PA LA: t-Fuc; 3-GalNAc; 3,4-GlcNAc 12-5HF 445.6* (445.7) [M + H + Na] 2+ Hex 1 HexNAc 3 PA LA: t-GalNAc; 4-GlcNAc; 3-GalNAc 13-4 1240.6 (1239.6) [M + Na] + ;
620.5* (620.3) [M + H + Na] 2+ Hex 1 HexNAc 4 dHex 1 PA LA: t-Fuc; t-GalNAc; 3-GlcNAc;
3-GalNAc; 3,4-GlcNAc 13-4HF 547.3* (547.2) [M + H + Na] 2+ Hex 1 HexNAc 4 PA LA: t-GalNAc; 4-GlcNAc, 3-GlcNAc;
3-GalNAc 14-2 1384.4 (1385.5) [M + Na] + Hex 1 HexNAc 4 dHex 2 PA LA: t-Fuc; 4-GlcNAc; 3-GalNAc;
3,4-GlcNAc 14-3 1384.4 (1385.5) [M + Na] + Hex 1 HexNAc 4 dHex 2 PA CA: GlcN:GalN:Fuc (2.1 : 2.0 : 1.9)
LA: t-Fuc; 3-GlcNAc; 3-GalNAc; 3,4-GlcNAc
14-3HF 547.0* (547.2) [M + H + Na] 2+ Hex 1 HexNAc 4 PA LA: t-GalNAc; 4-GlcNAc; 3-GlcNAc;
3-GalNAc 14-4 1530.8 (1531.6) [M + Na] + ;
766.5* (766.3) [M + H + Na] 2+ Hex 1 HexNAc 4 dHex 3 PA CA: GlcN:GalN:Fuc (2.1 : 2.0 : 2.3)
LA: t-Fuc; 2-Fuc; 3-GlcNAc;
3-GalNAc; 3,4-GlcNAc 14-5 839.1* (839.3) [M + H + Na] 2+ Hex 1 HexNAc 4 dHex 4 PA CA: GlcN:GalN:Fuc (2.2 : 2.0 : 3.2)
LA: t-Fuc; 2-Fuc; 3-GlcNAc;
3-GalNAc; 3,4-GlcNAc 14-5HF 1094.5 (1093.5) [M + Na] + Hex 1 HexNAc 4 PA LA: t-GalNAc; 4-GlcNAc; 3-GlcNAc;
3-GalNAc
15 1385.8 (1385.5) [M + Na] + Hex 1 HexNAc 4 dHex 2 PA CA: GlcN:GalN:Fuc (2.1 : 1.8 : 2.0)
LA: t-Fuc; 4-GlcNAc; 3-GlcNAc; 3-GalNAc; 3,4-GlcNAc
15HF 1094.6 (1093.5) [M + Na] + Hex 1 HexNAc 4 PA LA: t-GalNAc; 4-GlcNAc; 3-GlcNAc;
16-4 1587.4 (1588.6) [M + Na] + Hex 1 HexNAc 5 dHex 2 PA CA: GlcN:GalN : Fuc (2.9:2 : 2.8) 16-5 1876.7 (1880.7) [M + Na] + Hex 1 HexNAc 5 dHex 4 PA CA: GlcN:GalN : Fuc (3.2:2 : 4.3)
Trang 7outermost HexNAc carried only one fucose residue.
Together with the linkage analysis data, this allowed the
deduction of the structure shown in Table 4 For 14-2 and
14-3, MALDI-TOF MS indicated similar compositions as
for 15 Linkage analysis, however, revealed a difference in
the substitution positions at the monosubstituted GlcNAc
While 14-2 contained only 4-substituted and 14-3 only
3-substituted GlcNAc, 15 exhibited approximately equal
amounts of 3- and 4-substituted GlcNAc (Table 2) Linkage
analysis of 15 before and after HF showed fucose to be
linked to the 3-position of GalNAc and to the 3-position of
3,4-disubstituted GlcNAc ESI MS2of 15 (Fig 7D) showed
a B3ion at m/z 721.0, which was the same branched terminal
group as 12-5 (Table 4) As for 12-5, also in the case of 15,
a-fucosidase from bovine kidney could remove only one
fucose from this difucosylated terminal group (Fig 5G)
Taken together the structure of 15 was shown to be
Fuc-(a1±3)GalNAc(b1±4)[Fuc(a1±3)]GlcNAc(b1±3/4)Glc NAc
(b1±3)GalNAc(b1±4)Glc-PA, containing either a
3-substi-tuted or 4-substi3-substi-tuted GlcNAc unit (Table 4)
Characterization of large PA-glycans
Due to the pronounced heterogeneity and increasing
complexity of fractions 16 to 25, we could only partially
characterize these compounds by MALDI-TOF MS
(Fig 4), carbohydrate composition and linkage analysis
(Table 3) MALDI-TOF MS revealed compositions with an
increasing number of HexNAc residues The highest peak in
this region, 21 (Fig 3), contained as its major compound
Hex1HexNAc6dHex7PA, which is consistent with the results
of MALDI-TOF MS and ESI MS analyses of total egg
glycosphingolipids and released glycans (Fig 2) While 19
to 22 contained almost exclusively PA-oligosaccharides with
six HexNAc residues, species with ®ve HexNAc were most
intense in 16 to 18 Likewise, 23 to 25 were dominated by
PA-oligosaccharides with seven HexNAc residues, which
have not been described in previous studies [16,17] Linkage
analyses of the intact PA-oligosaccharides revealed
3-substituted GalNAc as the only GalNAc species
through-out and fucose as the only terminal sugar (Table 3) Linkage analysis of 24 after fucose removal by HF-treatment resulted in the loss of all fucose species and the appearance
of 3-substituted and terminal GalNAc in similar amounts, while all GlcNAc residues were converted to 4-substituted GlcNAc (data not shown) This shows GalNAc to be the outermost HexNAc in 24 The composition analyses of 18
to 25 imply that all complex PA-oligosaccharides contain an average of 2 GalNAc residues Of the ®ve HexNAc residues
in 18, approximately two are GalNAc and three are GlcNAc Of the six HexNAc residues in 19 to 22, approximately two are GalNAc and four are GlcNAc, and for the species in 24, the average HexNAc composition
is two GalNAc and ®ve GlcNAc residues This supports the hypothesis, that there is one GalNAc residue both at the reducing and at the nonreducing end of the HexNAc chain, the ®rst being involved in the Schisto-core structure, while the core of the HexNAc chain would appear to consist of GlcNAc residues throughout This is consistent with the structures proposed by Khoo et al [16] Concerning fucosylation, there exists a vast heterogeneity, as exempli®ed
by the detection of Hex1HexNAc6dHex2)8in 21 Several fractions showed a 2-substituted fucose/terminal fucose ratio of approximately 1 : 1 (Table 3), which could be explained by the occurrence of difucosylated chains throughout However, based on the observation of a trifucosylated HexNAc in 14-5 and odd fucose numbers per molecule, e.g in 21, it can be assumed that trifucosylated HexNAc residues may also occur in some of these complex PA-oligosaccharides Taken together, the partial character-ization of these larger PA-oligosaccharides after amino-phase fractionation gave a detailed overview of the HexNAc chain-lengths and fucosylation heterogeneity, which was not obtained by FAB-MS analyses of the entire mixture of glycolipids [16,17]
D I S C U S S I O N
In this study, individual S mansoni egg glycolipid structures have been elucidated Seven of the determined
Table 3 Composition and linkage analyses of the large PA-oligosaccharides derived from complex glycolipids of S mansoni eggs As for composition analysis, molar ratios based on GalNAc 2.0 were determined after hydrolysis and reverse-phase chromatography of the anthranilic acid-derivatized components The components were quanti®ed by application of a standard mixture for determination of the individual detection response factors PA-Glc conjugates were not registered For linkage analysis, the partially methylated monosacharide derivatives obtained after hydrolysis, reduction and peracetylation were analysed by GC/MS and GC using chemical ionization in conjunction with single-ion monitoring and
¯ame-ionization detection, respectively Results are expressed as peak ratios of the alditol acetates after ¯ame-ionization detection on the basis of 3-GalNAc 2.0 for HexNAc species and t-Fuc 1 for fucose species For fraction 25, peak ratios were determined by GC/MS due to the limited amount of material 4-GlcNAc, 4-substituted GlcNAc, etc.
Fraction
Trang 8Fig 4 MALDI-TOF MS analysis of the HPLC-fractionated PA-oligosaccharides Fractions 8 to 25 (8 to 25) of the amino-phase separated PA-oligosaccharides (Fig 3) were analysed by MALDI-TOF MS as their sodium (8 to 19) or lithium adducts (20 to 25) Fucose increments are indicated by arrows #, proton adduct; s, potassium adduct; *, contaminant.
Trang 9structures consist of fucosylated HexNAc chains based on
the Schisto-core structure They are in agreement with the
structure proposed by Khoo et al [16], which is Fuc(1±2)
Fuc(1±3)GalNAc(1±4)([Fuc(1±2)Fuc(1±)3]GlcNAc (1±4))1)3
[ Fuc(1±2) Fuc(1±3)]GlcNAc(1±3)GalNAc (1±4)
Glc-(1±1)Cer The reported role of 3-fucosylated GalNAc as the
major structural motif at the nonreducing end of the
HexNAc chain [16] is supported by our analyses of
individual PA-oligosaccharides (10-10, 12-5, 15, 14-4 and
14-5) and by the linkage analyses of larger
PA-oligosac-charides, in particular 24, before and after HF-treatment
Recently, the terminal motif GalNAc(b1±4)GlcNAc- has
been identi®ed as a good acceptor for fucosylation by
S mansoni egg extracts, leading to a heterogeneity of
fucosylated products, which parallels our ®nding of various
oligofucosylated terminal structures [28]
Our data, however, do not support the proposed Fuc(a1±
4)[Fuc(a1±3)]GlcNAc-terminal motif, or the interdigitation
of the HexNAc chain by fucose residues [17], because
terminal GlcNAc was never generated by defucosylation in
any of the PA-oligosacchride fractions studied, and MS
analyses of HF-treated individual PA-oligosaccharides only
revealed the loss of fucose and not of HexNAc residues
Our data require an extension of the existing picture
While the structure outlined by Khoo et al [16] comprises
HexNAc4-HexNAc6 chains, the structures 10-10 and 12-5
show that fucosylated glycolipids with HexNAc3-chains
also exist Together with the Hex1HexNAc2PA compound
found in 8-11, these carbohydrate chains obviously repre-sent the Ômissing linkÕ between the Schisto-core and the extended structures described [16] Likewise, 23 to 25 are dominated by HexNAc7species, which also have not been detected in the FAB-MS analyses [16,17] Secondly, we have found trifucosyl sidechains on 14-5, which have so far only been described for O-glycans derived from the cercarial glycocalyx [29] and not for egg glycolipids [16] The high relative amount of 2-Fuc in the larger PA-oligosaccharides further indicates that trifucosyl sidechains are also likely to occur in the glycolipids with HexNAc5-HexNAc7 chains Thirdly, 13-4 shows that incomplete fucosylation may also occur, leading in low amounts to terminal GalNAc residues involved in GalNAc(b1±4)[Fuc(a1±3)]GlcNAc(b1- units, as have been described for N-glycans of adult worm glyco-proteins [30] Fourthly, the characterization of the HexNAc backbone revealed in some cases a deviation from the GalNAc(1±4)(GlcNAc(1±4))1)3GlcNAc(1±3)GalNAc(1±4) Glc(1±1)ceramide basic structure described by Khoo et al [16], as we found this motif only in one of the elucidated structures (14-2, which is identical to one compound in 15), while four structures (13-4, 14-3, which is identical to a second compound in 15, 14-4 and 14-5) showed the motif±GalNAc(1±4)GlcNAc(1±3)GlcNAc(1±3)GalNAc(1± 4) Glc(1±.The presence of internal 3-substituted GlcNAc residues is in agreement with previous studies on the carbohydrate structure of a cercarial Lewis-X-containing ceramide hexahexoside [6]
Fig 5 Fucose removal from individual PA-oligosaccharides by a-fucosidase or HF-treatment MALDI-TOF MS of 13-4 before (A) and after (B) HF-treatment, intact 12-5 (C), 12-5 after a-fucosidase treatment (D), 12-5 after HF-treatment (E), intact 15 (F), 15 after a-fucosidase treatment (G), and 15 after HF-treatment (H).
Trang 10Apart from the dominant glycolipids with fucosylated
HexNAc chains, we characterized in this study the glycan of
a Lewis X-containing glycolipid (11-7), which we previously
have shown to be the dominating glycolipid of S mansoni
cercariae [6] Though this result shows that Lewis X
glycolipids are not absolutely restricted to the cercarial
and schistosomular life-cycle stage, they are drastically
downreguated in the egg and thus have not been detected by
HPTLC-overlay [9]
The ®nding of a PA-oligosaccharide corresponding to asialogangliotetraosylceramide (8-11) structurally identi®es
a glycolipid which the parasite most probably has taken up from the host MALDI-TOF MS of the corresponding intact glycolipid after silica-gel puri®cation showed that its ceramides contain more than 40 carbon atoms (data not shown), which indicated the presence of long-chain fatty acids and thus differed from the typical ceramides of complex egg glycolipids, which are dominated by
C20-Fig 6 ESI MS/MS of 8-11 and 11-7 (A) ESI
MS 2 of the 8-11 [M + Na + H] 2+ precursor ion at m/z 404.5 (B) ESI MS 2 of the 11-7 [M + H] + precursor ion at m/z 973.5 and (C) subsequent ESI MS 3 fragmentation of the ion
at m/z 511.9, which corresponds to the Lewis
X trisaccharide unit ,, sodium adduct; #, proton adduct.
Table 4 Proposed structures of S mansoni egg glycolipid-derived PA-oligosaccharides.
Fuc(a1±3)GalNAc(b1±4)[Fuc(a1±3)]GlcNAc(b1±3)GlcNAc(b1±3)GalNAc(b1±4)Glc-PA 15 + 14-3 Fuc(a1±3)GalNAc(b1±4)[Fuc(a1±3)]GlcNAc(b1±4)GlcNAc(b1±3)GalNAc(b1±4)Glc-PA 15 + 14-2 Fuc(a1±3)GalNAc(b1±4)[Fuc(a1±2)Fuc(a1±3)]GlcNAc(b1±3)GlcNAc(b1±3)GalNAc(b1±4)Glc-PA 14-4 Fuc(a1±3)GalNAc(b1±4)[Fuc(a1±2)Fuc(a1±2)Fuc(a1±3)]GlcNAc(b1±3)GlcNAc(b1±3)GalNAc(b1±4)Glc-PA 14-5