Báo cáo khoa học: Repeats of LacdiNAc and fucosylated LacdiNAc on N-glycans of the human parasite Schistosoma mansoni doc

Repeats of LacdiNAc and fucosylated LacdiNAc on N-glycans of the human parasite Schistosoma mansoni ´ Manfred Wuhrer, Carolien A M Koeleman, Andre M Deelder and Cornelis H Hokke Biomolecular Mass Spectrometry Unit, Department of Parasitology, Center of Infectious Diseases, Leiden University Medical Center, The Netherlands Keywords mass spectrometry; parasite; terminal N-acetylgalactosamine; trematode Correspondence M Wuhrer, Department of Parasitology, Leiden University Medical Center, P.O Box 9600, 2300 RC Leiden, the Netherlands Fax: +31 71 526 6907 Tel: +31 71 526 5077 E-mail: m.wuhrer@lumc.nl (Received 15 September 2005, revised 11 November 2005, accepted 21 November 2005) doi:10.1111/j.1742-4658.2005.05068.x N-Glycans from glycoproteins of the worm stage of the human parasite Schistosoma mansoni were enzymatically released, fluorescently labelled and analysed using various mass spectrometric and chromatographic methods A family of 28 mainly core-a1–6-fucosylated, diantennary N-glycans of composition Hex3)4HexNAc6)12Fuc1)6 was found to carry dimers of N,N¢diacetyllactosediamine [LacdiNAc or LDN; GalNAc(b1–4)GlcNAc(b1-] with or without fucose a1–3-linked to the N-acetylglucosamine residues in the antennae {GalNAc(b1–4)[±Fuc(a1–3)]GlcNAc(b1–3)GalNAc(b1– 4)[±Fuc(a1–3)]GlcNAc(b1-} To date, oligomeric LDN and oligomeric fucosylated LDN (LDNF) have been found only on N-glycans from mammalian cells engineered to express Caenorhabditis elegans b4-GalNAc transferase and human a3-fucosyltransferase IX [Z S Kawar et al (2005) J Biol Chem 280, 12810–12819] It now appears that LDN(F) repeats can also occur in a natural system such as the schistosome parasite Like monomeric LDN and LDNF, the dimeric LDN(F) moieties found here are expected to be targets of humoral and cellular immune responses during schistosome infection The most common complex-type N-glycans of mammals contain N-acetyllactosamine-type antennae [LacNAc; Gal(b1–4)GlcNAc(b1-], which can be substituted in various positions by fucoses, sialic acids, sulfate, glucuronic acid, etc LacNAc-type antennae are also seen in other eukaryotes [1–5] The arising terminal structures can be antigenic [2,6] or may act as ligands of lectins [7,8] They may modify cell–cell interaction, for example in development [9–12] and cancer [13–15] An alternative antennary structure is N,N¢-diacetyllactosediamine [LacdiNAc or LDN; GalNAc(b1– 4)GlcNAc(b1-], which may likewise be modified by various substituents such as fucose, sulfate or sialic acid LDN-based motifs have been found on N- or O-glycans of various mammalian glycoproteins including seminal plasma glycodelin [16], thyrotropin [17] and tissue factor pathway inhibitor [18], but are also expressed by various pathogens, including the human parasite Schistosoma mansoni [2] Glycans with LDN and LDNF {GalNAc(b1–4)[Fuc(a1–3)]GlcNAc(b1-} expressed by S mansoni are targets of the humoral immune responses of the host [19], and LDN-containing glycoconjugates of schistosomes may be ligands for galectin-3-mediated immune recognition [7] In humans, biosynthesis of LDN can occur by two different b1–4-N-acetylgalactosaminyltransferases: b4GalNAc-T3, which is expressed in stomach, colon and testes at high levels [20]; and b4GalNAc-T4, which is transcribed in ovaries and brain tissues [21] From the nematode Caenorhabditis elegans, a b1–4-galactosaminyltransferase involved in LDN has likewise been identified [22] The expression of this enzyme in Chinese hamster ovary (CHO) Lec8 cells has led to the production of N-glycans with LDN repeats, which Abbreviations 2AB, 2-aminobenzamide; CHO, Chinese hamster ovary cells; F, deoxyhexose; H, hexose; IT, ion-trap; LacNAc, Gal(b1–4)GlcNAcb1-; LC, liquid chromatography; LDN or LacdiNAc, N,N¢-diacetyllactosediamine; LDNF, GalNAc(b1–4)[±Fuc(a1–3)]GlcNAc(b1-; N, N-acetylhexosamine; PNGase F, peptide N-glycosidase F FEBS Journal 273 (2006) 347–361 ª 2005 The Authors Journal compilation ª 2005 FEBS 347 S mansoni N-glycans with dimeric LacdiNAc M Wuhrer et al A B Fig MALDI-TOF-MS of N-glycans released from S mansoni adult worms (A) Low mass range; (B) high mass range; ·8, intensities eight times enlarged; F, deoxyhexose; H, hexose; N, N-acetylhexosamine N-glycan species containing eight or more HexNAc residues are labelled in bold-type were converted to LDNF repeats by coexpression of human a1–3-fucosyltransferase IX [23] The mammalian b1–3-N-acetylglucosaminyltransferase(s) which contribute(s) to the synthesis of this alternating chain have yet to be identified [23] Whereas LDN repeats have thus been registered after expressing a b4-GalNAc transferase in a heterologous system, oligo- or poly LDN units have hitherto not been described for natural sources Here, we describe the expression of dimeric, in part fucosylated LDN {GalNAc(b1–4)[±Fuc(a1–3)]GlcNAc(b1–3)GalNAc(b1–4)[±Fuc(a1–3)]GlcNAc(b1-} on N-glycans of S mansoni adults Results Evidence for N-glycans with chains of four HexNAc residues N-Glycans were released from a total (glyco)protein preparation of S mansoni adult worms using peptide 348 N-glycosidase F (PNGase F) treatment and analysed by MALDI-TOF-MS (Fig 1) Almost all groups of glycans thus characterized had compositions in accordance with the published structures of N-glycans from S mansoni adult worms [2–5] (Fig 1) Glycans of composition H2)10N2 were interpreted as being oligomannosidic structures with an additional terminal glucose residue for the H10N2 species H2)4N2F1 represents a group of core-a1–6-fucosylated, paucimannosidic N-glycans H3N4F0)2 and H3N6F1)3 are in agreement with complex-type structures containing one or two LDN or fucosylated LDN (GalNAc(b1–4)[Fuc(a1– 3)]GlcNAc(b1-; LDNF) antennae Glycans with compositions of H4N6F1, H4N5F0)3, H5N6F1)4, H4N4F1)2, H5N5F0)2 and H3N5F1 were interpreted as containing two or more LDN and ⁄ or partially truncated LacNAc antennae H4N3F1)2, H5N4F0)3, H6N5F0)4, H7N6F1 most likely correspond to hybrid-type structures with 1, 2, or LacNAc and ⁄ or Lewis X antennae, although there are no detailed data in the literature to substantiate this FEBS Journal 273 (2006) 347–361 ª 2005 The Authors Journal compilation ª 2005 FEBS M Wuhrer et al S mansoni N-glycans with dimeric LacdiNAc Intensity A BPC EIC 1017 EIC (MS/MS) 632 400 800 1200 1400 1605.5 H3N4F1A B 1524.5 H3N5 1460.5 1321.4 H3N4 1402.5 H3N3F1A 1199.4 H3N2F1A 1000 1256.4 1008.5 906.9 600 1017.5 H3N6F1A# 916.1 944.4 814.4 842.9 713.4 N2F1A Time (min) 741.9 510.2 N1F1A 364.2 N1A Intensity 429.2 N2 632.3 N3 MS2 (1017) H3N6F1A 20 16 1118.4 H3N3 12 1600 m /z Fig RP-nano-LC-MS ⁄ MS of 2-aminobenzamide-labelled N-glycans from adult S mansoni (A) Chromatogram indicating the presence of H3N6F1A N-glycan species (double sodiated; EIC 1017) leading to a fragment ion at m ⁄ z 632 which corresponds to sodiated N3 (B) Fragment ion spectrum of the H3N6F1A N-glycan species A, 2-aminobenzamide; BPC, base peak chromatogram; EIC, extracted ion chromatogram; F, deoxyhexose; H, hexose; N, N-acetylhexosamine; double-headed arrow with continuous line, N-acetylhexosamine; double headed arrow with dashed line, deoxyhexose; #, double-sodiated species One particular group of glycans, however, which showed compositions of H3N8)12F1)5 (Fig 1), attracted our attention by its high N-acetylhexosamine content which led to the hypothesis that they contain elongated N-acetylhexosamine stretches on their antennae To test this hypothesis, RP-nano-LC-ESI-IT-MS ⁄ MS was performed on the N-glycans of S mansoni adult worms after labelling with 2-aminobenzamide (2AB; Fig 2) MS ⁄ MS data, obtained in the automatic mode, were screened for fragment ions that indicate oligo-N-acetylhexosamine stretches A fragment ion corresponding to three N-acetylhexosamine residues (m ⁄ z 632) was found for the precursor at m ⁄ z 1017 ([M + 2Na]2+) corresponding to the N-glycan H3N6F1A (A ¼ 2-aminobenzamide) The fragment ion spectrum (Fig 2B) indicated that at least part of the N-glycans of this composition contained N-acetylhexosamine chains of three or more residues Because the MALDI-TOF-MS profile indicated that many of the N-glycans with high N-acetylhexosamine content were of low abundance, we chose a twodimensional HPLC approach to allow MS ⁄ MS analysis of most of the species In the first dimension, the 2AB-labelled N-glycans were separated by normalphase HPLC on an amide column (Fig 3) Individual peak fractions were analysed by MALDI-TOF-MS and separated in the second dimension by RP-nanoLC-IT-MS with automatic acquisition of MS ⁄ MS data In order to obtain extensive MS ⁄ MS data of both sodium and proton adducts, each peak fraction was analysed by nano-LC-MS both with and without addition of sodium hydroxide to the running solvents, resulting in the registration ⁄ fragmentation of FEBS Journal 273 (2006) 347–361 ª 2005 The Authors Journal compilation ª 2005 FEBS 349 S mansoni N-glycans with dimeric LacdiNAc M Wuhrer et al Fig Normal-phase HPLC separation of 2-aminobenzamide-labelled N-glycans from adult S mansoni Peaks are labelled with fraction numbers and glycan compositions of the major 2-aminobenzamide-labelled species detected Retention times of 2-aminobenzamide-labelled partial hydrolysate of dextran are indicated in italics in the upper part of the figure All fractions were subjected to RP-nano-LC-MS ⁄ MS Fractions containing species with a fragment ion at m ⁄ z 632 corresponding to sodiated N3 and ⁄ or an ion at m ⁄ z 813 corresponding to protonated N4 are labelled with (+) An overview of the N-glycan species giving rise to these characteristic ions is given in Table F, deoxyhexose; H, hexose; N, N-acetylhexosamine 350 A Intensity predominantly sodium adducts or proton adducts, respectively For glycans of composition H3N6F1A, which contain, at least in part, N-acetylhexosamine stretches (Fig 2), this two-dimensional HPLC approach resolved three isomers eluting in normal-phase fractions 24 and 26 (Fig 4) The MS ⁄ MS data obtained for these isobaric N-glycans (Fig 5) indicated that isomers and were diantennary N-glycans with two LDN antennae differing only in the fucose attachment site, i.e core-(a1–6)-fucosylation for isomer and antenna-fucosylation for isomer (Figs and 5) Isomer 3, however, exhibited a stretch of four N-acetylhexosamine residues, which was tentatively interpreted as a diLDN unit [GalNAc(b1–4)GlcNAc(b1–3)GalNAc(b1–4)GlcNAc(b1-] Overall screening of the two-dimensional LC-MS ⁄ MS data set revealed a large group of N-glycans exhibiting fragment ions indicative of HexNAc chains, as summarized in Table For many of the N-glycan species, MS ⁄ MS data were obtained for sodium adducts Fr 24 A A Fr 26 15 20 Time (min) Fig RP-nano-LC-MS ⁄ MS of normal-phase HPLC fractions 24 and 26 Extracted ion chromatograms of m ⁄ z 1017 displayed for fractions 24 and 26 indicated three isomers separated by the twodimensional HPLC system The assigned structures are based on the MS ⁄ MS spectra shown on Fig Yellow square, N-acetylgalactosamine; blue square, N-acetylglucosamine; green circle, mannose; red triangle, fucose; A, 2-aminobenzamide FEBS Journal 273 (2006) 347–361 ª 2005 The Authors Journal compilation ª 2005 FEBS S mansoni N-glycans with dimeric LacdiNAc 1697.9 1587.4 1524.4 1605.5 1402.4 1321.4 1199.3 1210.3 1256.3 1118.3 944.3# 1053.2 1008.9# 800 1459.4 1256.5 1524.5 1605.5 1118.4 1053.3 1008.4# A A A A A 1460.5 1400 1649.3 1524.5 1321.3 1344.2 1200 1605.5 1402.5 1000 1256.4 1200.4 1118.3 1053.5 1008.9# 916.2# 944.3# 835.3# 671.4 510.2 567.1 600 814.6# 842.7# 713.1# 632.2 429.1 364.3 400 A A A 916.3# 814.8# 567.2 364.1 A isomer A 944.3# 843.0# 741.2# 429.1 Intensity 639.7# A A C 842.8# 590.2 510.2 A isomer A A 741.6# 429.1 639.6# 364.1 346.1 B A 1459.5 713.2# A A 814.3# isomer A 916.3# M Wuhrer et al 1600 m /z Fig Fragment ion spectra (nano-LC-ESI-IT-MS ⁄ MS) of the three isomers displayed in Fig The deduced structures are boxed Yellow square, N-acetylgalactosamine; blue square, N-acetylglucosamine; green circle, mannose; red triangle, fucose; A, 2-aminobenzamide; doubleheaded arrow with continuous line, N-acetylhexosamine; double-headed arrow with dashed line, deoxyhexose; #, double-sodiated fragment as well as proton adducts, with both fragment ion spectra revealing valuable structural information including the HexNAc chain lengths (exemplified in Fig 6) In the case of the protonated species, the length of the HexNAc chain was clearly indicated by the intense protonated HexNAc4 (m ⁄ z 813), whereas the protonated HexNAc3 fragment was less abundant (m ⁄ z 610; Fig 6A), indicating a particular lability of the HexNAc-Hex linkage Although the fragment ion spectrum of the sodiated species likewise exhibited HexNAc3 and HexNAc4 ions (m ⁄ z 632 and 835), the intensity ratios were the reversed The innermost linkage of the antenna HexNAc chain appeared to be particularly labile, resulting in an intense HexNAc3 peak and a low-abundance signal for the HexNAc4 sodium adduct (Fig 6B) For some species, at least part of the HexNAc chains seemed to be fucosylated as indicated by the corresponding reporter ions (e.g m ⁄ z 778 for sodiated N3F1) In all cases the length of the HexNAc chain was deduced to be four HexNAc residues, and none of the fragment ion spectra indicated species with HexNAc chains of three or five HexNAc residues Detailed characterization of N-glycans Few of the identified N-glycans with chains of four HexNAc were obtained in sufficient quantities and purity to allow more detailed structural characterization: the glycan species H3N8F4A detected in fraction 39 (Table 1) was purified by preparative RP-HPLC (not shown) and subjected to permethylation Fragment ion analysis of permethylated H3N8F4A using FEBS Journal 273 (2006) 347–361 ª 2005 The Authors Journal compilation ª 2005 FEBS 351 S mansoni N-glycans with dimeric LacdiNAc M Wuhrer et al Table Analytical data for S mansoni adult worm N-glycans exhibiting partially fucosylated diLDN antennae Compositions are given in terms of hexose (H), N-acetylhexosamine (N), and deoxyhexose (fucose; F) Fragment ions were determined by nano-LC-ESI-IT-MS ⁄ MS selecting sodiated and ⁄ or protonated precursors n d., not done Composition HPLC fractions Ions registered by nano-LC-ESI-IT-MS (m ⁄ z) b + Characteristic fragmentions (m ⁄ z) 2+ H3N6F1 H3N6F2 26 37 1893.1 [M + Na] ; 1017.4 [M +2Na] 2038.9b [M + Na]+; 1068.6 [M +2H]2+ H3N6F3 39 2185.2b [M + Na]+; 1141.6 [M +2H]2+; 1163.4 [M +2Na]2+ H3N7F1 27 2096.1b [M + Na]+; 1196.9 [M +2H]2+; 1218.9 [M +2Na]2+ H4N7F4 40 2696.0b [M + Na]+; 932.1 [M +3H]3+ H3N8F1 30 H3N8F2 37 H3N8F3 37 2299.0b [M + Na]+; 1198.5 [M +2H]2+; 1220.5 [M +2Na]2+ 2445.1b [M + Na]+; 1271.5 [M +2H]2+; 1293.5 [M +2Na]2+ 2591.6b [M + Na]+; 896.9 [M +3H]3+; 918.8 [M +3Na]3+ H3N8F4 38–40 2737.5b [M + Na]+; 945.6 [M +3H]3+; 967.4 [M +3Na]3+; 1439.6 [M +2Na]2+ H3N9F1 37 H3N9F2 37 H3N9F3 39 2502.2b [M + Na]+; 1300.0 [M +2H]2+; 1321.9 [M +2Na]2+ 2648.4b [M + Na]+; 916.0 [M +3H]3+; 937.7 [M +3Na]3+ 2795.0b [M + Na]+; 964.6 [M +3H]3+; 986.4 [M +3Na]3+ H3N10F1 H3N10F2 38 38 2705.7b [M + Na]+; 1423.5 [M +2Na]2+; 2851.5b; 1005.4 [M +3Na]3+ H3N10F3 40 2996.5b [M + Na]+; 1032.3 [M +3H]3+ H3N10F4 40–42 3143.9b [M + Na]+; 1081.2 [M +3H]3+; 1102.8 [M +3Na]3+ 352 Fig 5C 407 (N2)a, 553 (N2F1)a, 813 (N4)a, 778 (N3F1) 510.2 (N1F1A), 778 (N3F1), 553 (N2F1)a, 610 (N3)a, 699 (N2F2)a, 756 (N3F1)a, 813 (N4)a, 959 (N4F1)a, 1106 (N4F2)a 488.1 (N1F1A)a, 813 (N4)a, 632 (N3), 835 (N4), 1218.4 (H2N3F1A)a 407 (N2)a, 512 (H1N1F1)a, 813 (N4)a, 959 (N4F1)a Fig n.d 510 (N1F1A), 575 (N2F1), 778 (N3F1), 989 [M +2Na]2+ (H3N5F2A), 1549 (H3N3F2A) 496 (N1F2)a, 699 (N2F2)a, 813 (N4)a, 1106 (N4F2)a, 510 (N1F1A), 632 (N3), 778 (N3F1), 1062 [M +2Na]2+ (H3N5F3A), 1164 [M +2Na]2+ (H3N6F3A), 1338 [M +2Na]2+ (H3N7F4A), 1810 (H3N4F2A), 1900 (H3N4F3A); see also Fig n.d 813 (N4)a, 959 (N4F1)a, 510 (N1F1A), 778 (N3F1) 510 (N1F1A), 699 (N2F2)a, 813 (N4)a, 575 (N2F1), 632 (N3), 778 (N3F1), 981 (N4F1), 1090 [M +2Na]2+ (H3N6F2A), 1810 (H3N5F1A) n.d 510.2 (N1F1A), 632 (N3), 778 (N3F1), 981 (N4F1), 1192 [M +2Na]2+ (H3N7F1A), 1753 (H3N4F2A), 1809 (H3N5F1A), 488 (N1F1A)a, 813 (N4)a, 959 (N4F1)a 510 (N1F1A), 813 (N4)a, 699 (N2F2)a, 1252 (N4F3)a, 575 (N2F1), 778 (N3F1), 1753 (H3N4F2A), 1899 (H3N4F3A) Proposed structural features core-(a1–6)-fucosylation, LDN-LDN- antenna no core-fucosylation, LDNF-LDNF- antenna core-(a1–6)- fucosylation, LDNF-LDNF- antenna core-(a1–6)- fucosylation, LDN- LDN- antenna, antenna of a single HexNAc residue (truncated) core-(a1–6)- fucosylation, LDNF-LDNF- antenna, Lewis X- antenna core-(a1–6)- fucosylation, LDN- LDN- antenna, LDN- antenna LDN-LDN- antenna, LDN- antenna, both possibly fucosylated core-(a1–6)- fucosylation, LDNF- LDNF- antenna, LDN- antenna core-(a1–6)- fucosylation, LDNF- LDNF- antenna, LDNF- antenna LDN-LDN- antenna, LDN- antenna, antenna of a single HexNAc residue (truncated) core-(a1–6)- fucosylation, LDNF-LDN- antenna, antenna of a single HexNAc residue (truncated) core-(a1–6)-fucosylation, LDNF-LDNF- antenna, antenna of a single HexNAc residue (truncated) LDN-LDN antennae, possibly fucosylated core-(a1–6)-fucosylation, LDN-LDN- antennae, one of them singly fucosylated core-(a1–6)-fucosylation, LDN-LDN- antennae, core-(a1–6)-fucosylation, LDNF-LDNF- antenna, LDN-LDNF- antenna FEBS Journal 273 (2006) 347–361 ª 2005 The Authors Journal compilation ª 2005 FEBS M Wuhrer et al S mansoni N-glycans with dimeric LacdiNAc Table (Continued) Composition HPLC fractions Ions registered by nano-LC-ESI-IT-MS (m ⁄ z) Characteristic fragment ions (m ⁄ z) H3N10F5 42, 43 3289.5b [M + Na]+; 1129.8 [M +3H]3+; 1151.5 [M +3Na]3+ H3N11F1 37 2909.0b [M + Na]+; 1002.3 [M +3H]3+; 1024.4 [M +3Na]3+ H3N11F2 39, 40 1051.4 [M +3H]3+; 1072.8 [M +3Na]3+ H3N11F3 40, 41 1099.9 [M +3H]3+; 1121.8 [M +3Na]3+ H3N11F4 42, 43 1148.6 [M +3H]3+; 1170.5 [M +3Na]3+ H3N11F5 43 1219.2 [M +3Na]3+ H3N12F1 H3N12F2 39 40 3111.8b [M + Na]+; 1092.1 [M +3Na]3+; 3257.8b [M + Na]+; 1118.8 [M +3H]3+; 1140.8 [M +3Na]3+ H3N12F3 41, 42 3403.7b [M + Na]+; 1167.6 [M +3H]3+; 1189.5 [M +3Na]3+ H3N12F4 43 1238.1 [M +3Na]3+ H3N12F5 43 1265.1 [M +3H]3+; 1286.9 [M +3Na]3+ H3N12F6 43 1335.6 [M +3Na]3+ 813 (N4)a, 902 (N3F2)a, 1106 (N4F2)a, 510 (N1F1A), 575 (N2F1), 778 (N3F1), 1897 (H3N4F3A) 813 (N4)a; 1380 (H3N3F1A)a; 510 (N1F1A), 429 (N2), 632 (N3), 1119 [M +2Na]2+ (H3N8F1A), 1221 [M +2Na]2+ (H3N9F1A), 1322 [M +2Na]2+ (H3N10F1A), 1809 (H3N5F1A), 1931 (H3N7) 488 (N1F1A)a, 553 (N2)a, 813 (N4)a, 1170 [M +2H]2+ (H3N7F2A)a, 1301 [M +2H]2+ (H3N9F1A)a, 1374 [M +2H]2+ (H3N9F2A)a, 1731 (H3N4F2A)a, 1294 [M +2Na]2+ (H3N8F2A), 1090 [M +2Na]2+ (H3N6F2A), 1323 [M +2Na]2+ (H3N9F1A), 1810 (H3N5F1A), 1955 (H3N5F2A), 1752 (H3N4F2A) 813 (N4)a, 959 (N4F1)a, 510 (N1F1A), 575 (N2F1), 778 (N3F1), 1293 [M +2Na]2+ (H3N8F2A), 1954 (H3N5F2A) 813 (N4)a, 699 (N2F2)a, 1252 (N4F3)a, 510 (N1F1A), 575 (N2F1), 778 (N3F1), 1955 (H3N5F2A) 510 (N1F1A), 429 (N2), 713 (N2F1A), 778 (N3F1), 2101 (H3N5F3A) n.d 510 (N1F1A), 813 (N4)a, 959 (N4F1)a, 1120 [M +2H]2+ (H3N8F1A)a, 1177 (H3N2F1A)a, 1273 [M +2H]2+ (H3N8F2A)a, 1584 (H3N4F1A)1 510 (N1F1A), 575 (N2F1), 632 (N3),778 (N3F1), 2159 (H3N6F2A) 510 (N1F1A), 575 (N2F1), 713 (N2F1A), 778 (N3F1), 2158 (H3N6F2A), 2304 (H3N6F3A) 813 (N4)a, 699 (N2F2)a, 575 (N2F1), 778 (N3F1), 2304 (H3N6F3A) 510 (N1F1A), 575 (N2F1), 778 (N3F1), 2451 (H3N6F4A) a Protonated fragment b Proposed structural features core-(a1–6)-fucosylation, LDNF-LDNFantennae core-(a1–6)-fucosylation, LDN-LDN- antennae, antenna of a single HexNAc residue (truncated) core-(a1–6)-fucosylation, partially fucosylated LDN-LDN- antennae, antenna of a single HexNAc residue (truncated) core-(a1–6)-fucosylation, partially fucosylated LDN-LDN- antennae, antenna of a single HexNAc residue (truncated) core-(a1–6)-fucosylation, partly fucosylated LDN-LDN- antennae, antenna of a single HexNAc residue (truncated) core-(a1–6)-fucosylation, LDNF-LDNFantennae, antenna of a single HexNAc residue (truncated) – core-(a1–6)-fucosylation, LDN-LDN- antenna, LDNF-LDN- antenna, LDN- antenna core-(a1–6)-fucosylation, LDNF-LDN antenna, LDN-LDN- antenna, LDNF- antenna core-(a1–6)-fucosylation, partially fucosylated LDN-LDN- antennae, LDN(F)- antenna no core-fucosylation, LDNF-LDNF- antennae, LDNF- antenna core-(a1–6)-fucosylation, LDNF-LDNFantennae, LDNF- antenna Sodium adducts of the native glycans were registered by MALDI-TOF-MS (average masses) FEBS Journal 273 (2006) 347–361 ª 2005 The Authors Journal compilation ª 2005 FEBS 353 S mansoni N-glycans with dimeric LacdiNAc M Wuhrer et al A 1000 800 1400 1600 2193.6 1786.8 1381.1 1300.5 1179.6 1225.8 1200 1800 1119.4# 2000 2200 1000 A 1400 1600 1800 1995.0 1930.7 1402.5 1459.3 1524.5 1147.7 1211.8 1256.4 1200 1605.5 1662.5 1727.5 1808.6 A # 1017.4# 1046.3# 593.6 600 713.2 753.2 805.6 842.8# 915.7# 835.2 632.2 400 A 510.3 364.2 A 1097.3# 996.1# 914.2# 610.3 732.4 800 MS/MS (m /z 1220) [M+2Na]2+ 429.2 Intensity 600 1990.7 813.4 407.1 A 400 B A A 1584.6 M S/ MS (m /z 1198) A [M+2H]2+ 2000 m /z Fig Fragment ion spectra of the double-protonated (A) and double-sodiated (B) N-glycan species H3N8F1A Spectra where obtained by RP-nano-LC-ESI-IT-MS ⁄ MS of the 2-aminobenzamide-labelled glycans of fraction 30 The deduced structure is boxed Yellow square, N-acetylgalactosamine; blue square, N-acetylglucosamine; green circle, mannose; red triangle, fucose; A, 2-aminobenzamide; double-headed arrow with dashed line, deoxyhexose; #, double-sodiated fragment nano-LC-MS ⁄ MS indicated the attachment of one fucose at the subterminal HexNAc with a second fucose linked to the fourth HexNAc of the chain (Fig 7) These data are in agreement with a fucosylated version of the diLDN structure, namely a diLDNF motif {GalNAc(b1–4)[Fuc(a1–3)]GlcNAc(b1–3)GalNAc(b1–4)[Fuc(a1–3)]GlcNAc(b1-} as the antenna structure In order to corroborate these findings, the permethylated N-glycan species H3N8F4A was subjected to linkage analysis, i.e hydrolysis, reduction and peracetylation, followed by GC-MS analysis of the obtained partially methylated alditol acetates using electron-impact ionization (Fig 8) In addition to 2-substituted mannose, 3,6-disubstituted mannose and 4-substituted GlcNAc, which are in accordance with the trimannosyl core structure, linkage analysis revealed 354 terminal Fuc as well as the following N-acetylhexosamine variants: terminal GalNAc, 3-substituted GalNAc and 3,4-disubstituted GlcNAc (Fig 8) Notably, terminal GlcNAc was not detected In conclusion, linkage analysis data were in line with the postulated LDNF and diLDNF antennae (Figs and 9) Furthermore, in order to obtain detailed information about the attachment of diLDN to upper and ⁄ or lower branch antennae for the above-mentioned isomer (Figs and 5C), fraction 26 was subjected to a-mannosidase treatment, and partial removal of hexose from the H3N6F1A isomer was indicated by MALDI-TOF-MS (not shown) In order to determine the substitution position of the b-linked core mannose in isomer after removal of the terminal mannose, the sample was subjected to permethylation and linkage FEBS Journal 273 (2006) 347–361 ª 2005 The Authors Journal compilation ª 2005 FEBS S mansoni N-glycans with dimeric LacdiNAc 600 800 1000 A A 1676.8 1900.1 1964.9 1632.9 -N 1400 1113.8 1200 1090.5 -N4F2 1300.7 1365.7 1400.6 1423.3 1455.5 -NFA 1503.2 400 1172.2 1197.1 250.2 1600 1800 2000 22 00 MS3 (1182→1423) A A 500 1000 1950.9 1423.7 1500 A 2144.2 1899.1 m /z 1479.9 A 1321.1 -H 1161.3 1197.7 1286.4 1293.1 1116.5 946.6 1054.5 1082.6 857.8 701.4 767.0 687.4 636.3 A 2210.0 Intensity 200 A and 1010.1 1027.6 687.4 946.5 701.4 636.4 442.1 A 956.4 -N2F MS2 (1182) A B 1096.3 -N M Wuhrer et al 2000 Fig Fragment ion analysis of the triple-sodiated, permethylated N-glycan H3N8F4A (A) Fragment ion spectrum obtained by RP-nano-LCESI-IT-MS ⁄ MS of the permethylated 2-aminobenzamide-labelled glycan from subfraction 39–4 (B) MS3 of the double-charged precursor at m ⁄ z 1423 The deduced structure is boxed Yellow square, N-acetylgalactosamine; blue square, N-acetylglucosamine; green circle, mannose; red triangle, fucose; A, 2-aminobenzamide analysis (not shown) In addition to 3,6-disubstituted mannose and 2-substituted mannose, which belong to the conventional trimannosyl core, 3-subsituted mannose and 6-substituted mannose were detected in a ratio of  : These monosaccharide derivatives arise from isomer after removal of the terminal a-linked mannose This would indicate that isomer is a mixture of monoantennary structures carrying the diLDN motif on the lower branch (a1–3-linked mannose;  30%) and on the upper branch (a1–6-linked mannose;  70%) Furthermore, terminal GalNAc, 4-substituted GlcNAc, 3-substituted GalNAc and 3,4-disubstituted GlcNAc were registered, which is in accordance with the postulated antennae structures of isomer and isomer occurring as a mixture in fraction 26 (Figs and 5B,C) Proposed structures Whereas the two N-glycans of composition H3N6F1 (isomer 3) and H3N8F4 (see above) were studied in detail revealing diLDN antennae and diLDNF antennae, respectively, the structural data obtained for the other N-glycans listed in Table comprised mainly nano-LC-MS ⁄ MS analyses Because the whole group of N-glycans exhibited consistent patterns of character- FEBS Journal 273 (2006) 347–361 ª 2005 The Authors Journal compilation ª 2005 FEBS 355 S mansoni N-glycans with dimeric LacdiNAc M Wuhrer et al A terminal GalNAc 3,4-disubstituted GlcNAc terminal GlcNAc 4-substituted GlcNAc 3-substituted GalNAc * * * * 40 Time (min) 45 Fig Linkage analysis of the N-glycan H3N8F4A The extracted-ion chromatogram of m ⁄ z 158 indicates the HexNAc species, which were identified based on retention times and electron-impact fragmentation patterns The elution position of terminal GlcNAc as determined with an authentic standard is indicated by an arrow The 2AB-labelled, innermost GlcNAc is not detected in linkage analysis Yellow square, N-acetylgalactosamine; blue square, N-acetylglucosamine; green circle, mannose; red triangle, fucose; A, 2-aminobenzamide; *, contaminant istic fragment ions revealing chains of four HexNAc residues as antenna structures carrying up to two fucose residues per antenna (Table 1), we postulate that these glycans also have antennae based on the diLDN motif Structures are proposed as shown in Fig based on both the data summarized in Table 1, and the detailed analysis of compounds H3N6F1 (isomer 3) and H3N8F4 Discussion We describe a very heterogeneous group of N-glycans expressed by adult worms of the human parasite S mansoni, which feature repeats of LDN units These units can also carry a fucose in the 3-position of N-acetylglucosamine residues, resulting in repetitive LDNF units Repetitive LDN and LDNF structures have not previously been described in natural sources Indications that such structures might occur in C elegans were given by the heterologous expression of a C elegans b1–4-N-acetylgalactosaminyltransferase in CHO Lec cells leading to the production of oligoLDN chains and, upon coexpression of an a1–3-fucosyltransferase, oligo-LDNF chains [23] Whereas Kawar et al [23] found up to seven LDN units and up to five LDNF units per N-glycan chain, we found up to five LDN(F) units on schistosome N-glycans, with a maximum of four HexNAc residues in a row [diLDN(F) antenna structures] The differences in chain 356 length might be due to the acceptor specificities of the involved b1–3-N-acetylglucosaminyltransferases, which have yet to be characterized in the case of schistosomes as well as the CHO cells used for the expression of the C elegans GalNAc T [23] The occurrence of LDN(F) repeats parallels the poly(LacNAc) and poly(Lewis X) chains found on N-glycans from total S mansoni worm glycoproteins [2–5,24], O-glycans of the circulating cathodic antigen of S mansoni worms [2–5,25] as well as on glycoproteins and glycolipids of granulocytes [26,27] The structural similarities of partially fucosylated LDN repeats and LacNAc repeats might be paralleled by the enzymatic repertoire recruited for these biosyntheses: both the b3-GlcNAc transferase and the a3-Fuc transferase are likely to be shared by the two biosynthetic pathways Many carbohydrate epitopes from schistosomes, such as FLDN(F) {Fuc(a1–3)GalNAc(b1–4)[± Fuc(a1–3)]GlcNAc(b1-)}, LDN(F), and Lewis X, have been shown to be antigenic and several also appear to be targets for the innate immune system through recognition by human lectins [2–5,7,8,19,28–31] In particular, LDN(F) units, repeats of which are described here, have been shown to be target of the host immune response in schistosomiasis [19,29,38,39] Despite the pronounced structural similarity between epitopes such as Lewis X and LDNF, the N-acetyl substitution at the 2-position of the Gal residue being the only difference, antibodies to Lewis X or LDNF FEBS Journal 273 (2006) 347–361 ª 2005 The Authors Journal compilation ª 2005 FEBS M Wuhrer et al S mansoni N-glycans with dimeric LacdiNAc GalNAc(β1-4)GlcNAc(β1-3)GalNAc(β1-4)GlcNAc(β1-2)Man(α1-6/3) ±Fuc(α1-3) ±Fuc(α1-3) Fuc(α1-6) Man(β1-4)GlcNAc(β1-4)GlcNAcβ1-Asn H3N6F1-3 Man(α1-3/6) GalNAc(β1-4)GlcNAc(β1-3)GalNAc(β1-4)GlcNAc(β1-2)Man(α1-6/3) Fuc(α1-6) Man(β1-4)GlcNAc(β1-4)GlcNAcβ1-Asn H3N7F1 GlcNAc(β1-2)Man(α1-3/6) GalNAc(β1-4)GlcNAc(β1-3)GalNAc(β1-4)GlcNAc(β1-2)Man(α1-6/3) Fuc(α1-3) Fuc(α1-3) Fuc(α1-6) Man(β1-4)GlcNAc(β1-4)GlcNAcβ1-Asn H4N7F4 Gal(β1-4)GlcNAc(β1-2)Man(α1-3/6) Fuc(α1-3) GalNAc(β1-4)GlcNAc(β1-3)GalNAc(β1-4)GlcNAc(β1-2)Man(α1-6/3) ±Fuc(α1-3) ±Fuc(α1-3) Fuc(α1-6) Man(β1-4)GlcNAc(β1-4)GlcNAcβ1-Asn H3N8F1-4 GalNAc(β1-4)GlcNAc(β1-2)Man(α1-3/6) ±Fuc(α1-3) GalNAc(β1-4)GlcNAc(β1-3)GalNAc(β1-4)GlcNAc(β1-2)Man(α1-6) ±Fuc(α1-3) ±Fuc(α1-3) Fuc(α1-6) Man(β1-4)GlcNAc(β1-4)GlcNAcβ1-Asn H3N10F1-5 GalNAc(β1-4)GlcNAc(β1-3)GalNAc(β1-4)GlcNAc(β1-2)Man(α1-3) ±Fuc(α1-3) ±Fuc(α1-3) Fig Summary of deduced structures exhibit exclusive specificity Using surface plasmon resonance, it was shown that an Lewis X monoclonal antibody selectively bound to a Lewis X neoglycoprotein, but not to LDNF, and vice versa (Fig in Van Remoortere et al [36]) Moreover, in a chimpanzee S mansoni-infection model, antibodies against Lewis X showed a time course which was markedly different from those observed for anti-LDN and anti-LDNF sera [19] Finally, a recent study in schistosome-infected mice has indicated an intense humoral immune response to dimeric and trimeric Lewis X compared with monomeric Lewis X [37] Differential recognition FEBS Journal 273 (2006) 347–361 ª 2005 The Authors Journal compilation ª 2005 FEBS 357 S mansoni N-glycans with dimeric LacdiNAc M Wuhrer et al of monomeric, dimeric and trimeric Lewis X was also shown with various monoclonal antibodies [37] In conclusion, the rather minor structural variations found on schistosome glycoconjugates can have a profound effect on their immunological recognition Thus, differences in antigenicity between monomeric LDN(F) and oligomers thereof can be foreseen, and the LDN(F) repeats described here are expected to be immunogenic and involved in host–parasite interaction Based on the findings of Kawar et al [23], LDN and LDNF repeats might not be restricted to the trematode S mansoni, but are likely to be found also on glycoproteins of C elegans and possibly other nematodes Furthermore, future experiments will answer the question whether mammals likewise produce these oligo-LDN(F) antenna structures This question should be addressed by studying both the fine specificities of potentially involved enzymes as well as by structural studies of the N-glycosylation of LDN-expressing tissues [20,21] MALDI-TOF-MS Glycan samples were analysed by MALDI-TOF-MS using an Ultraflex mass spectrometer (Bruker Daltonics, Bremen, Germany) in the positive reflectron mode with 6-aza-2-thiothymine (5 mgỈmL)1; Sigma, St Louis, MO) as matrix Labelling and fractionation Glycans released with PNGase F were tagged with 2AB by reductive amination as outlined previously [40] The reaction mixture was applied to a carbon cartridge (Alltech, Deerfield, IL), and the 2AB-labelled glycans were eluted with mL of 25% acetonitrile The acetonitrile content was reduced under a stream of nitrogen, and the samples were lyophilized Fractionation by normal-phase HPLC Experimental procedures (Glyco-)protein extraction S mansoni worms were obtained by perfusion of infected hamsters and stored at )80 °C until use Adult worms were homogenized in mL of water (1 vol.Ỉ100 mg)1 wet weight of worms) In order to delipidize samples, methanol and chloroform were sequentially added (5 vol each) The supernatant was removed after centrifugation, and the extraction was repeated In order to extract (glyco-)proteins, the pellet was suspended in phosphate-buffered saline (35 mm sodium phosphate, pH 7.6, 0.85% NaCl) SDS and 2-mercaptoethanol were added to final concentrations of 1% (w ⁄ v) and 0.5%, respectively The samples were incubated for 10 at 100 °C, allowed to cool to room temperature, and Chaps (Fluka/Sigma-Aldrich, Zwijndrecht, The Netherlands) was added to a final concentration of 1% (w ⁄ v) Samples were centrifuged, and supernatants were subjected to PNGase F treatment These experiments were carried out in accordance with EC Council Directive (89/609/EEC) and after approval of the animal experiment commitee (DEC) of the Leiden University Medical Center Glycan release S mansoni (glyco-)proteins were incubated with PNGase F (2 m100 mg)1 wet weight; Roche Diagnostics, Mannheim, Germany) overnight at 37 °C For the purification of the released glycans, samples were first applied to a reverse-phase cartridge (500 mg of Bakerbond octadecyl; Baker, Phillipsburg, NJ) Flow-through and wash (5 mL of water) were then applied to a carbon cartridge (150 mg Carbograph; 358 Alltech, Deerfield, IL) After washing with water (5 mL), glycans were eluted with 25% aqueous acetonitrile (5 mL) Released glycans were detected by MALDI-TOF-MS 2AB-labelled glycans were fractionated by normal-phase HPLC on a TSK-Amide 80 column (4 · 250 mm; Tosohaas, Montgomeryville, PA) at 0.4 mLỈmin)1 Solvent A was mm formic acid adjusted to pH 4.4 with ammonia, which is a modification of a previously published separation system [41] Solvent B was 20% of solvent A in acetonitrile The following gradient conditions were used: at time t ¼ min, 100% solvent B; t ¼ 152 min, 52.5% solvent B; t ¼ 155 min, 0% solvent B; t ¼ 162 min, 0% solvent B; and t ¼ 163 min, 100% solvent B The total run time was 180 Samples were injected in 80% acetonitrile Because of the large amounts of material injected, fluorescence was detected at 280 nm ⁄ 500 nm instead of the routinely used 360 nm ⁄ 425 nm in order to avoid saturation of the detector Fractions were collected manually and analysed by MALDI-TOF-MS Fractionation by RP-HPLC 2AB-labelled glycans were fractionated by RP-HPLC on a Hypersil ODS lm (2 · 250 mm; Thermo Electron Corp., Waltham, MA) at 0.2 mLỈmin)1 Solvent A was 0.4% acetonitrile, 0.1% formic acid Solvent B was 95% acetonitrile, 0.1% formic acid The following gradient conditions were used: at time t ¼ min, 5% solvent B; t ¼ min, 5% solvent B; gradient to t ¼ 30 min, 50% solvent B; t ¼ 31 min, 100% solvent B; and t ¼ 36 min, 100% solvent B, t ¼ 37 min, 5% solvent B Total run time was 60 Fluorescence was detected at 360 nm ⁄ 425 nm Fractions were collected manually and analysed by MALDITOF-MS FEBS Journal 273 (2006) 347–361 ª 2005 The Authors Journal compilation ª 2005 FEBS M Wuhrer et al S mansoni N-glycans with dimeric LacdiNAc Nano-LC-MS/MS References 2AB-labelled glycans were separated on a PepMap column (75 lm · 150 mm; Dionex ⁄ LC Packings, Amsterdam, the Netherlands) using an Ultimate nano-LC system (Dionex ⁄ LC Packings) equipped with a Switchos guard column system (Pepmap guard column, 300 lm · 10 mm) The system was equilibrated with eluent A (H2O ⁄ acetonitrile 95 : 5, v ⁄ v, 0.1% formic acid) at a flow rate of 150 nLỈmin)1 After injecting the sample, a linear gradient to 50% eluent B (H2O ⁄ acetonitrile : 95, v ⁄ v, containing 0.1% formic acid) in 30 was applied, followed by a final wash with 100% B for The system was directly coupled to an Esquire high capacity trap (HCT) ESI-IT-MS (Bruker) equipped with an online nano-spray source operating in the positive-ion mode For electrospray (900–1200 V), capillaries (360 lm OD, 20 lm ID with 10 lm opening) from New Objective (Cambridge, MA) were used The solvent was evaporated at 165 °C with a nitrogen stream of LỈmin)1 Ions from m ⁄ z 50 to 2000 were registered Automatic fragment ion analysis was enabled, resulting in MS ⁄ MS spectra of the most abundant peaks In order to register predominantly sodium adducts by MS, part of the analyses were performed after addition of 0.8 mm NaOH to solvent A For the analysis of permethylated glycans, the column was conditioned with 15% eluent B After injection, a linear gradient to 70% eluent B 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71–78 14 Dube DH & Bertozzi CR (2005) Glycans in cancer and inflammation – potential for therapeutics and diagnostics Nat Rev Drug Discov 4, 477–488 15 Kannagi R, Izawa M, Koike T, Miyazaki K & Kimura N (2004) Carbohydrate-mediated cell adhesion in cancer metastasis and angiogenesis Cancer Sci 95, 377–384 a-Mannosidase treatment 2AB-labelled glycans (50 pmol to nmol) were treated with a-mannosidase from jack beans (100 mU; Sigma) in 50 lL 50 mm sodium acetate buffer, pH 5.0 for 18 h at 37 °C The reaction mixture was applied to a carbon cartridge (Alltech), and the 2AB-labelled glycans were eluted with mL of 25% acetonitrile, analysed by MALDI-TOF-MS, and subjected to nano-LC-MS ⁄ MS Permethylation and linkage analysis 2AB-labelled glycans were permethylated [42] and analysed by nano-LC-MS ⁄ MS For linkage analysis, permethylated glycans were hydrolyzed (4 m trifluoroacetic acid, h, 100 °C), and partially methylated alditol acetates obtained after sodium borohydride reduction and 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Antibody responses to the fucosylated S mansoni N-glycans with dimeric LacdiNAc 40 41 42 43 LacdiNAc glycan antigen in Schistosoma mansoniinfected mice and expression of the glycan among schistosomes... heterogeneous group of N-glycans expressed by adult worms of the human parasite S mansoni, which feature repeats of LDN units These units can also carry a fucose in the 3-position of N-acetylglucosamine... various monoclonal antibodies [37] In conclusion, the rather minor structural variations found on schistosome glycoconjugates can have a profound effect on their immunological recognition Thus,