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Sulfated and sialylated N-glycans in the echinoderm Holothuria atra reflect its marine habitat and phylogeny

      Among the earliest deuterostomes, the echinoderms are an evolutionary important group of ancient marine animals. Within this phylum, the holothuroids (sea cucumbers) are known to produce a wide range of glycoconjugate biopolymers with apparent benefits to health; therefore, they are of economic and culinary interest throughout the world. Other than their highly modified glycosaminoglycans (e.g. fucosylated chondroitin sulfate and fucoidan), nothing is known about their protein-linked glycosylation. Here we used multistep N-glycan fractionation to efficiently separate anionic and neutral N-glycans before analyzing the N-glycans of the black sea cucumber (Holothuria atra) by MS in combination with enzymatic and chemical treatments. These analyses showed the presence of various fucosylated, phosphorylated, sialylated, and multiply sulfated moieties as modifications of oligomannosidic, hybrid, and complex-type N-glycans. The high degree of sulfation and fucosylation parallels the modifications observed previously on holothuroid glycosaminoglycans. Compatible with its phylogenetic position, H. atra not only expresses vertebrate motifs such as sulfo– and sialyl–Lewis A epitopes but displays a high degree of anionic substitution of its glycans, as observed in other marine invertebrates. Thus, as for other echinoderms, the phylum- and order-specific aspects of this species' N-glycosylation reveal both invertebrate- and vertebrate-like features.

      Introduction

      Sea cucumbers (holothuroids) are a group of organisms living in the benthic zones of seas across the world. As one of the five clades of the Echinodermata (Fig. 1), sea cucumbers are primitive deuterostomes and are thus related to the ancestors of vertebrates. Around 100 of the 1500 extant sea cucumber species are consumed by humans, in part because of their intrinsic nutritional value and the proposed beneficial effects of many of the constituent biopolymers (
      • Correia-da-Silva M.
      • Sousa E.
      • Pinto M.M.M.
      • Kijjoa A.
      Anticancer and cancer preventive compounds from edible marine organisms.
      ). Overexploitation in some regions has led to the spread of sea cucumber fishing throughout the world, with unknown ecological repercussions, as these animals ingest and process detritus from the sea floor and thus play an active role in sea grass and coral reef ecosystems (
      • Gonzalez-Wanguemert M.
      • Aydin M.
      • Conand C.
      Assessment of sea cucumber populations from the Aegean Sea (Turkey): first insights to sustainable management of new fisheries.
      ). There is also interest in these organisms as regeneration models because of their capability for asexual reproduction by fission followed by morphallaxis to produce a smaller but functional complete animal (
      • Dolmatov I.Y.
      Asexual reproduction in holothurians.
      ).
      Figure thumbnail gr1
      Figure 1MALDI-TOF MS analyses of PNGase-F–released neutral and anionic N-glycans after pyridylamination. A–C, total PNGase-F–released N-glycans from neutral and anionic pools were analyzed by MALDI-TOF MS in positive ([M+H]+) and negative modes ([M-H]). The neutral pool (A) contains mainly oligomannosidic-type N-glycans, whereas the anionic pool (B and C) shows a wide range of structures modified with phosphate, sialic acid, and/or sulfate. MALDI-TOF MS analyses were performed with 6-aza-2-thiothymine as matrix, supplemented with ammonium sulfate (A–C), which enhanced the occurrence of [M+H]+ or [M-H] ions but impaired formation of sodiated ions such as [M+Na]+ and [M-Hn+Nan-1], which leads to underestimation of the degree of sulfation of some structures compared with . Asterisks indicate the presence of multiple structural isomers. Example structures are annotated using the Symbol Nomenclature for Glycans: green circles, mannose; yellow circles, galactose; blue circles, glucose; blue squares, GlcNAc; red triangles, fucose; blue diamonds, N-glycolylneuraminic acid; S, sulfate; P phosphate. The right panel shows a simplified phylogenetic tree indicating the different classes of echinoderms compared with chordates (including vertebrates).
      Over the years, various glycosylated biopolymers from sea cucumbers have been analyzed, particularly glycolipids (
      • Yamada K.
      • Matsubara R.
      • Kaneko M.
      • Miyamoto T.
      • Higuchi R.
      Constituents of Holothuroidea: 10: isolation and structure of a biologically active ganglioside molecular species from the sea cucumber Holothuria leucospilota.
      ,
      • Yamada K.
      • Sasaki K.
      • Harada Y.
      • Isobe R.
      • Higuchi R.
      Constituents of Holothuroidea: 12: isolation and structure of glucocerebrosides from the sea cucumber Holothuria pervicax.
      ), fucosylated chondroitin sulfates (
      • Mou J.
      • Wang C.
      • Li W.
      • Yang J.
      Purification, structural characterization and anticoagulant properties of fucosylated chondroitin sulfate isolated from Holothuria mexicana.
      ), and glycosylated terpenoids (
      • Silchenko A.S.
      • Kalinovsky A.I.
      • Avilov S.A.
      • Andryjaschenko P.V.
      • Dmitrenok P.S.
      • Yurchenko E.A.
      • Dolmatov I.Y.
      • Dautov S.S.
      • Stonik V.A.
      • Kalinin V.I.
      Colochiroside E, an unusual non-holostane triterpene sulfated trioside from the sea cucumber Colochirus robustus and evidence of the impossibility of a 7(8)-double bond migration in lanostane derivatives having an 18(16)-lactone.
      ), all of which have been claimed to be bioactive (e.g. anti-inflammatory and anti-tumorigenic (
      • Janakiram N.B.
      • Mohammed A.
      • Rao C.V.
      Sea cucumbers metabolites as potent anti-cancer agents.
      )). On the other hand, as for echinoderms in general, there is no report regarding the standard N-linked oligosaccharides of these species. However, based on O-glycan analyses of the distantly related sea urchins, sialic acids can be expected (
      • Karamanos N.K.
      • Manouras A.
      • Anagnostides S.
      • Makatsori E.
      • Tsegenidis T.
      • Antonopoulos C.A.
      Isolation, biochemical and immunological characterisation of two sea urchin glycoproteins bearing sulphated poly(sialic acid) polysaccharides rich in N-glycolyl neuraminic acid.
      ).
      Here we analyzed the N-glycome of Holothuria atra (commonly called black sea cucumber or lollyfish), which is possibly the most abundant of its genus and widespread in the tropical Indo-Pacific region. The N-glycans were released with PNGase-F and then PNGase-A and subsequently analyzed by HPLC, MS, and enzymatic and chemical treatments (
      • Paschinger K.
      • Wilson I.B.
      Analysis of zwitterionic and anionic N-linked glycans from invertebrates and protists by mass spectrometry.
      ). In addition to a number of common oligomannosidic types, various unusual phosphorylated, fucosylated, sialylated, and multiply sulfated N-glycans were identified that potentially represent phylum- and order-specific aspects of echinoderm glycosylation.

      Results

       Workflow and analytical strategy

      N-glycans of H. atra were released by serial digestion with PNGase-F and then PNGase-A, resulting in free N-glycan pools that were subfractionated on graphitized carbon into neutral and anionic subpools prior to pyridylamination (PA).
      The abbreviations used are: PA
      pyridylamine
      RP
      reverse-phase
      NP
      normal-phase
      HF
      hydrofluoric acid
      g.u.
      glucose unit.
      The initial survey of the complete N-glycome showed some mass shifts of 78 Da between positive and negative MS modes, compatible with the presence of either phosphate or sulfate modifications (Fig. 1). Subsequently, the neutral and anionic PNGase F–released N-glycans were subject to reverse-phase (RP) and normal-phase (NP) HPLC; all fractions were analyzed by MALDI-TOF MS in positive and negative modes (Fig. 2 and Fig. S1). This offline LC-MS approach revealed that the neutral N-glycan pool was relatively simple, containing primarily well-known oligomannosidic structures, whereas the anionic pool contained numerous unusual charged hybrid and complex N-glycans.
      Figure thumbnail gr2
      Figure 2RP Kinetex HPLC of neutral or anionic enriched pools of PNGase-F–released N-glycans from H. atra. A and B, pyridylaminated neutral (A) and anionic (B) pools are annotated with proposed structures as confirmed by MS, MS/MS, and enzymatic and chemical treatments; +nS indicates ambiguity regarding which residues are sulfated. The insets show proposed linkages, also for disialylated antennae, whereas the annotated observed m/z values are either positive-mode [M+H]+ for neutral, sialylated, or phosphorylated glycans or negative-mode [M-H] for monosulfated and [M-Hn+Na(n-1)] for multiply-sulfated glycans. Retention times are given in minutes, and the annotated structures for each fraction are shown in order of abundance, with the most abundant uppermost. Calibration is in terms of glucose units. Antennal fucosylation and multiple sulfation results in earlier RP HPLC elution than core α1,6-fucosylation or monosulfation.

       Oligomannosidic-type N-glycans

      The neutral pool contained a series of Hex4–12HexNAc2 glycans (m/z 1151–2447). These could be assigned as isomers of Glc0–3Man4–9GlcNAc2 because of their retention time on RP/NP HPLC as well as positive MS/MS fragmentation patterns before and after mannosidase digestion. As these common isomers (see Table S1 for a comparison of elution times) were identified previously in other organisms (
      • Hykollari A.
      • Eckmair B.
      • Voglmeir J.
      • Jin C.
      • Yan S.
      • Vanbeselaere J.
      • Razzazi-Fazeli E.
      • Wilson I.B.
      • Paschinger K.
      More than just oligomannose: an N-glycomic comparison of Penicillium species.
      ,
      • Eckmair B.
      • Jin C.
      • Abed-Navandi D.
      • Paschinger K.
      Multi-step fractionation and mass spectrometry reveals zwitterionic and anionic modifications of the N- and O-glycans of a marine snail.
      ), they are not discussed further.
      We also observed unusual N-glycans in both positive and negative modes; they were predicted to be phosphorylated forms of Hex10–12HexNAc2 (m/z 2203, 2365, and 2527; Fig. 3, A and E, and Fig. S2, A–C). The most abundant of these (m/z 2527) was dephosphorylated with either alkaline phosphatase or HF, yielding an MS/MS spectrum and retention time characteristic of the basic endoplasmic reticulum Glc3Man9GlcNAc2 N-glycan precursor (m/z 2447). The thereby predicted P1Glc3Man9GlcNAc2 glycan was also treated with α-mannosidase, resulting in loss of up to five mannose residues regardless of whether the phosphate had been removed, and with endo-α2-mannosidase, which removed a P1Glc3Man1 unit (Fig. 3 and Fig. S2); these data indicated that the phosphate residue is on the triglucosylated A arm. Considering also the MS/MS B1 ions at m/z 241 (P1Hex1) in negative mode, we concluded that the terminal glucose residue is the location of the phosphate modification.
      Figure thumbnail gr3
      Figure 3Analysis of phosphorylated oligomannosidic-type N-glycans. A–H, a phosphorylated oligomannosidic-type N-glycan (P1H12N2; m/z 2527) in the neutral enriched pool (see A and ) was analyzed by MALDI-TOF MS (A–D) and MS/MS (E–H) in positive mode as [M+H]+ in conjunction with enzymatic and chemical treatments. The 80-Da modification was shown to be phosphate because of its sensitivity to alkaline phosphatase treatment (A and B), as also monitored by MS/MS (E and F), whereas the probable position of the phosphate was deduced from α-mannosidase and endo-α-mannosidase digestions (C, D, G, and H) removing, respectively, five mannoses or a P1Glc3Man1 unit. Digestions with phosphatase or endomannosidase (B and D) result in a shift to sodiated adducts (+22 Da), whereas the m/z 2225 species in the mannosidase digest (C) is a contaminant generally found when using the jack bean enzyme preparation. Losses of phosphate (−P) or hexoses (−H1, −H2, etc.), as well as B-ion HexxP fragments (H1P, etc.) are indicated. Based on all of the evidence, including mannosidase treatment and HPLC analysis after hydrofluoric acid treatment (), it is concluded that the underlying backbone of the P1H12N2 glycan (m/z 2527) is a standard Glc3Man9GlcNAc2 structure with the same RP HPLC elution properties as a previously published structure from Pristionchus pacificus (
      • Yan S.
      • Wilson I.B.
      • Paschinger K.
      Comparison of RP-HPLC modes to analyse the N-glycome of the free-living nematode Pristionchus pacificus.
      ).

       Neutral hybrid-type N-glycans

      Within the H. atra glycome, there were five neutral glycans with predicted compositions of Hex4–6HexNAc3Fuc0–2 (m/z 1500–1970). These potentially hybrid structures were analyzed using HPLC, MS, and exoglycosidase treatments to define the nature of their antennae. For instance, a 2D HPLC-purified form of Hex6HexNAc3 (m/z 1678, Fig. 4, A and I) was sensitive to β3-galactosidase (Fig. 4, G and J; loss of one Gal) and α-mannosidase (Fig. 4, H and K; loss of two or three Man) but not to β4-galactosidase (Fig. 4F). This indicated the presence of a type 1 antenna (neo-LacNAc, Galβ3GlcNAcβ-R) on a hybrid backbone, a conclusion confirmed by comparison with a later-eluting isomer with a type 2 antenna (LacNAc, Galβ4GlcNAcβ-R; prepared by in vitro β4-galactosylation; Fig. 4B). The β3-galactosylated hybrid structure appeared to be the basis for a number of sialylated and sulfated glycans, as desulfation of S1Hex6HexNAc3 (m/z 1756) and desialylation of NeuGc1Hex6HexNAc3 (m/z 1985) resulted in a coeluting Gal1Man5GlcNAc3 structure (Fig. 4, C and D).
      Figure thumbnail gr4
      Figure 4HPLC and MALDI-TOF MS analysis of hybrid-type N-glycans. A and E–K, the 19.9-min NP-HPLC fraction containing Gal1Man5GlcNAc3 (m/z 1678 as [M+H]+) was reinjected onto RP HPLC and eluted at 21.9 min (A; 7.2 g.u.). Subsequently, positive MS before (E and I) and after β4-galactosidase (F; no digestion), β3-galactosidase (G and J; loss of one Gal), and jack bean α-mannosidase (H and K; loss of up to three Man) proved the presence of a neo-LacNAc (Galβ3GlcNAc) antenna on a hybrid Man5-type backbone. B–D, in contrast, an isomeric Gal1Man5GlcNAc3 structure (generated by enzymatic remodeling with β4-galactosyltransferase and carrying a LacNAc Galβ4GlcNAc antenna) had a later RP HPLC elution time (B; 23.8 min or 8.2 g.u.), which proves the distinct linkage on the H. atra glycan. Furthermore, treatment of the sulfated S1Gal1Man5GlcNAc3 and sialylated NeuGc1Gal1Man5GlcNAc3 hybrid-type N-glycans with, respectively, methanolysis and sialidase resulted in neo-LacNAc antennae, as judged by coelution with the β3-galactosylated neutral form of Gal1Man5GlcNAc3 (compare C and D with A) as well as subsequent enzymatic digestions (data not shown but similar to E–K). The effects of enzymatic treatments are indicated by red arrows, and digested structures are displayed in gray (C and D).

       Sulfated hybrid-type N-glycans

      Monosulfated fucosylated structures of S1Fuc1–2Gal1Man5GlcNAc3 were analyzed via negative MS and MS/MS (Fig. 5, A, F, H, K, and M), whereby key B ions and neutral losses aided definition of the sulfate and fucose positions. For instance, the two fucosylated isomers of S1Fuc1Gal1Man5GlcNAc3 (m/z 1902, eluting at 4.7 g.u. and 7.8 g.u.) exhibited different negative MS/MS fragmentation patterns (Fig. 5, H and K); the first one with m/z 590 (S1Fuc1Gal1GlcNAc1) and 1603 (loss of GlcNAc1-PA) is concluded to possess an antennal fucose, whereas the second one with m/z 444 (S1Gal1GlcNAc1) and 1457 (loss of Fuc1GlcNAc1-PA) is core α6–fucosylated, as it was released by PNGase-F. The related difucosylated m/z 2048 structure not only presented an m/z 590 B fragment but also an m/z 1603 ion, indicative of loss of Fuc1GlcNAc1-PA (Fig. 5M). Treatment of the antennally fucosylated isomer with either β3/4-galactosidase (also no digestion even after α-fucosidase), α-mannosidase (loss of two or three Man), α3/4-fucosidase and HF (both resulting in loss of one Fuc) (Fig. 5, A–E) aided definition of the A arm as Lewis motifs with sulfated galactose residues. An alternative position for sulfation (rather than on galactose) is concluded for a hybrid-type S1Fuc1Gal1Man3GlcNAc3 glycan, which was β3-galactosidase–sensitive; the m/z 241 and 1213 fragments in particular indicated that the sulfate is, in this case, on mannose (Fig. S3).
      Figure thumbnail gr5
      Figure 5MALDI-TOF MS analysis of monosulfated hybrid-type N-glycans. A–G, S1Fuc1/2Gal1Man5GlcNAc3 glycans (m/z 1902/2048 as [M-H], eluting at 4.7 and 6.8 g.u.; see B) were analyzed by negative MS before (A and F) and after enzymatic or chemical treatments (B–E and G). The hybrid nature and lack of sulfation of fucose were shown by sensitivity to jack bean α-mannosidase (C, removing up to three mannoses) and removal of fucose by either α3/4-fucosidase (D) or HF (E and G) treatments without loss of the negative charge, whereas resistance to β3/4-galactosidase (B) is expected for a Lewis-type antenna regardless of sulfation. H–N, negative and positive MS/MS of these glycans, also compared with a core fucosylated isomer eluting at 7.8 g.u., showed a number of key diagnostic variations. Although the presence of a negative-mode B-fragment ion at m/z 241 (S1Hex1; H, K, and M) is compatible with sulfation of galactose, the position of fucose on either the antenna or core can be deduced on the basis of the sulfated B ions at m/z 444/590 (S1Fuc0/1Gal1GlcNAc1). The neutral losses of either −299 (GlcNAc1-PA, −NPA) or −445 (Fuc1GlcNAc1-PA, −NFPA) are indicative of the absence (H) or presence (K and M) of core fucosylation. On the other hand, positive-mode MS/MS of these glycans and an HF digestion product (I, J, L, and N; m/z 1824, 1678, or 1970 resulting from in-source loss of sulfate) reveals the underlying backbones. Specific diagnostic Y ions at m/z 300/446 (Fuc0/1GlcNAc1-PA) and 1151/1297 (Fuc0/1Man4GlcNAc2-PA) are characteristic of core and hybrid-type structures, whereas m/z 512 B ions are indicative of fucosylated antennae. Highlighted in green and purple are, respectively, fragments aiding definition of the antennae and the core. Asterisks indicate ions [M-179] deriving from 0,2A cross-ring cleavage of the core PA-labeled GlcNAc, as also observed previously in negative-mode MS/MS of sulfated N-glycans from lepidopteran species (
      • Stanton R.
      • Hykollari A.
      • Eckmair B.
      • Malzl D.
      • Dragosits M.
      • Palmberger D.
      • Wang P.
      • Wilson I.B.H.
      • Paschinger K.
      The underestimated N-glycomes of lepidopteran species.
      ). Losses of Fuc (F), Hex (H), HexNAc (N), or HexNAc-PA (NPA) are indicated.
      For sulfated structures in general (
      • Stanton R.
      • Hykollari A.
      • Eckmair B.
      • Malzl D.
      • Dragosits M.
      • Palmberger D.
      • Wang P.
      • Wilson I.B.H.
      • Paschinger K.
      The underestimated N-glycomes of lepidopteran species.
      ), positive mode MS/MS of [M-SO3]+ pseudomolecular ions is useful to define the underlying backbones. Here, positive mode Y-ions at 446 (Fuc1GlcNAc1-PA), 1297/1459 (Fuc1Man4/5GlcNAc2-PA), and m/z 503–1313 (Man0–5GlcNAc2-PA) as well as the B ions at m/z 366/512 (Gal1GlcNAc1Fuc0/1) provided full-sequence coverage of the core and the antennae of the hybrid structures (Fig. 5, I, J, L, and N). Therefore, considering the presence of β3-galactose on nonfucosylated glycans (Fig. 4) and the available LC-ESI-MS data (Fig. S4), the antennal motif for these Lewis-modified glycans is concluded to be (HSO3)4Galβ3(Fucα4)GlcNAc-R, i.e. sulfo–Lewis A.
      For disulfated S2Fuc1–2Gal1Man4–5GlcNAc3 structures (m/z 1842–2150; Fig. 6, A–D), diagnostic negative B ions are consistent with two sulfates substituting either the Gal, GlcNAc, or α3-Man residues on the A arm. Negative-mode MS/MS could indeed distinguish disulfated m/z 2150 isomers (eluting at 42.5 and 48.4 min on NP HPLC); the pattern for the first isomer with m/z 241 (S1Gal1), 854 (S2Fuc1Gal1GlcNAc1Man1), and 1537 (S1Fuc1Man5GlcNAc2-PA) suggests the presence of sulfate on the terminal Gal and lower α3-Man residues (Fig. 6H). For the second one, the occurrence of fragments of m/z 241 (S1Gal1) and 692 (S2Fuc1Gal1GlcNAc1), together with loss of the latter upon HF treatment while retaining both sulfate residues, is compatible with the presence of a disulfo-Lewis motif (Fig. 6, D, E, I, and J); thereby the possibility of sulfation of the fucose is excluded, but sulfation of the Gal and GlcNAc residues is confirmed.
      Figure thumbnail gr6
      Figure 6MALDI-TOF MS analysis of disulfated hybrid N-glycans. A–D and F–I, disulfated hybrid-type S2Fuc1–2Gal1Man4–5GlcNAc3 glycans present in different normal phase fractions () were analyzed by negative MS (A–D), exhibiting some in-source loss of sulfate (indicated by Δ102 and asterisks), and MS/MS (F–I). Monosulfated ([M-H]) and disulfated ([M-2H+Na/K]) B-fragment ions were observed at m/z 241, 282, or 444 (S1Gal0–1GlcNAc0–1), 546/562 (S2Gal1GlcNAc1), 692/708 (S2Fuc1Gal1GlcNAc1), 708/724 (S2Gal1GlcNAc1Man1), and 854/870 (S2Fuc1Gal1GlcNAc1Man1) in addition to Y ions at m/z 1375/1537 (S1Fuc1Man4/5GlcNAc2-PA) aid definition of sulfation of Gal, GlcNAc, or Man. The fragmentation patterns of the two m/z 2150 isomers (H and I) indicated either sulfation on the terminal Gal and lower α3-Man or two sulfate residues on a Lewis motif (blue arrows). E and J, HF treatment of the latter only removed the antennal α4-fucose (whereby the m/z 546 fragment corresponds to a disulfated Hex1HexNAc1). As the 80-Da modification was not lost, it was neither linked to the Lewis-type fucose nor was it an isobaric phosphate. The distinct elution of the disulfated isomers is the result of the spatial arrangement of sulfate residues causing weaker or stronger binding to the HIAX column. Losses of Fuc (F), hexose (H), or reducing terminal Fuc0–1GlcNAc1-PA (−NPA or −FNPA) are indicated.
      For the disulfated S2Fuc2–3Gal2Man5GlcNAc4 structures (m/z 2515 and 2661 as [M-2H+Na]; eluting at 50 min on NP HPLC), two mannoses were removed by α-mannosidase treatment (Fig. S5, A and B), proving their hybrid-type backbones. MS/MS analyses of the parental ion, as well as the [M-SO3] and [M+H-2SO3]+ ions resulting from in-source loss of sulfate, indicated the presence of two sulfo-Lewis–type antennae, presumably β1,2- and β1,4-linked to the α3-linked mannose (Fig. S5, C–E).
      Trisulfated S3Fuc0/2Gal1Man5GlcNAc3 structures eluted rather late on the NP HPLC column (65–68 min) and were best detected when supplementing the matrix with sodium acetate (Fig. S6, A, B, G, and H; m/z 1960 and 2252). Negative- and-positive mode MS/MS of the “real” [M-Hn+Na(n-1)] and “in-source” parent ions yielded fragments (Fig. S6, C–F and I–L) consistent with the three sulfation positions on the A arm (terminal Gal, antennal GlcNAc, and α3-Man), as established for the aforementioned disulfated species.

       Sialylated hybrid-type N-glycans

      The results of offline LC-MS/MS led us to predict a number of sialylated glycans in the H. atra N-glycome (Fig. 2B and Fig. S1B). To resolve some of these, a 2D HPLC approach was applied. NP HPLC–fractionated monosialylated structures (NeuGc1Fuc0/1Gal1Man5GlcNAc3, m/z 1985/2131) were reinjected onto RP HPLC before or after α3-sialidase S treatment, and isomers with different sialylation and sialidase sensitivity were identified (Fig. 7, A, B, E and F). Only rather subtle differences in positive- and negative-mode MS/MS between the monosialylated isomers could be observed, with the main diagnostic sialylated negative/positive B ions at m/z 306/308 (NeuGc1) and 671/673 (NeuGc1Hex1HexNAc1) for NeuGc-modified antennae being shared (Fig. 7, K, M, and R). In case of a sialidase S–resistant isomer, β3-galactosidase treatment resulted in loss of one galactose residue and best revealed a diagnostic m/z 511 NeuGc1GlcNAc1 fragment (Fig. 7, G–J). Thus, the conclusion was that there were two positions for sialylation (“externally” on Gal or “internally” on GlcNAc), and sialidase S only removed the former but not the latter.
      Figure thumbnail gr7
      Figure 7HPLC and MALDI-TOF MS analysis of sialylated hybrid-type N-glycans. A, B, E, and F, NeuGc1Fuc0/1Gal1Man5GlcNAc3 glycans (m/z 1985/2131) eluting on NP HPLC at 28–31 min were reinjected before or after sialidase S treatment onto RP HPLC (A and B), which resolved four structures concluded to display either internal or external sialylation of the antenna (B) and showed that the enzyme specifically removed one NeuGc from the externally sialylated isomers, compatible with the incomplete desialylation observed by MS analysis of the NP 30.1 fraction (E and F). K–M, positive and negative MS/MS of the externally sialylated m/z 2131/1985 glycans showed the occurrence of NeuGc containing B and C fragments at m/z 308/306 (NeuGc1), 673/671 (NeuGc1Gal1GlcNAc1), and 853/851 (NeuGc1Gal1GlcNAc1Man1) as well as Y loss of NeuGc1. G–J, in contrast, internal NeuGc was resistant to sialidases, but the isomer eluting at 24.7 min (A and B) was sensitive to β3-galactosidase (G and H), whose action resulted in a shift in the MS/MS B ions from m/z 673 to m/z 511, indicative of NeuGc linked to GlcNAc (I and J). C and D, in the case of the disialylated hybrid type m/z 2292 glycan containing both the internal and external NeuGc, only the latter was removed by sialidase S treatment, resulting in a shift to a retention time to 21.9 min, i.e. identical to the internally sialylated m/z 1985 isomer. P, Q, and S, MS/MS of such disialylated glycans showed little difference from the externally sialylated glycans, but either low-abundance positive m/z 511 NeuGc1GlcNAc1 or m/z 980 NeuGc2Gal1GlcNAc1 ions were present in addition to disialylated negative B and C ions at m/z 996 and 1788. N, O, and T, negative and positive MS/MS of a sulfo-sialyl–Lewis A–containing structure revealed diagnostic B fragments at m/z 897 (S1NeuGc1Fuc1Gal1GlcNAc1) and 819 (NeuGc1Fuc1Gal1GlcNAc1), whereas negative-mode MS/MS of a sulfated NeuGc containing glycan (T) showed a characteristic ion at m/z 386 (S1NeuGc1) as well as loss of a carboxyl group (−44 Da). L, the m/z 424 fragment ion has been observed in other reports on negative-mode MALDI-TOF MS/MS of neutral and sialylated N-glycans, being annotated as a 1,3A3 ion of the composition Gal-GlcNAc-O–CH = CH-O (
      • Harvey D.J.
      • Jaeken J.
      • Butler M.
      • Armitage A.J.
      • Rudd P.M.
      • Dwek R.A.
      Fragmentation of negative ions from N-linked carbohydrates, part 4: fragmentation of complex glycans lacking substitution on the 6-antenna.
      ). Effects of enzymatic treatments are illustrated by red and blue arrows, and digested structures are displayed in gray (B and D).
      There were also related disialylated NeuGc2Fuc0/1Gal1Man5GlcNAc3 glycans (m/z 2292 and 2438); the former was also rechromatographed before and after α3-sialidase S treatment. Removal of only the terminal NeuGc occurred and resulted in altered retention time (Fig. 7, C and D) and minimal changes in MS/MS fragmentation (Fig. 7, P and R). Negative-mode MS/MS of the m/z 2292 glycan and positive MS/MS of the core-fucosylated m/z 2438 structure resulted in detection of either an m/z 996 C fragment or an m/z 980 B fragment compatible with disialylation of Hex1HexNAc1 (Fig. 7, Q and S). Some sialylated glycans were also sulfated, and MS/MS (Fig. 7, N, O, and T) could show the presence of either a sulfo-sialyl–Lewis–containing motif (B ion at m/z 897, S1NeuGc1Fuc1Gal1GlcNAc1) or a sulfated NeuGc (B ion at m/z 386, S1NeuGc1).

       Complex-type N-glycans

      MS predicted a large number of complex-type N-glycans in H. atra, but the relatively low abundance of these structures “overloaded” with fucose and sulfate residues meant that their analysis was challenging. On RP HPLC, glycans such as S3–4Fuc2–4Hex5–6HexNAc4–5 (m/z 2439–3052) were particularly concentrated in the fraction eluting at 14.5 min (Fig. 2B), whereas on NP HPLC, many eluted after 60 min (Fig. S1B). The RP HPLC fraction was analyzed by negative MS before and after digestion with β3/4-galactosidase and α-fucosidase, which resulted in no loss of the sulfated galactose residues but removal of up to four fucoses (Fig. 8, A–D). MS/MS spectra of such bi- and triantennary complex-type N-glycans (see Fig. 8, E–J, and Fig. S7 for examples) showed the presence of similar B ions (e.g. sulfo–Lewis A at m/z 590) as described above; however, possibly because of their low abundance, no multisulfated fragments were detected, as was the case for di- or trisulfated hybrid glycans, but positive-mode MS/MS facilitated definition of the core and antennal fucose residues. Unlike the hybrid structures, desulfation of multisulfated glycans was inefficient and led to unspecific hydrolysis; thus, an unambiguous definition of all galactose linkages (β3 or β4) was not possible. However, where monosulfated complex glycans were present in certain fractions, loss of galactose residues could be observed upon β3-galactosidase treatment, especially when the glycome pool had been defucosylated previously with HF (Fig. S8).
      Figure thumbnail gr8
      Figure 8MALDI-TOF MS analysis of multisulfated complex-type N-glycans. A and B, the highest degree of sulfation of bi/triantennary N-glycans was observed in early-eluting RP HPLC fractions by negative MS in the presence of sodium acetate buffer, which allows detection of [M-Hn+Nan-1] ions corresponding to multisulfated structures (structures containing two to four sulfates are indicated in bold (m/z 2165–3052), and none were digested with a nonspecific galactosidase). Because of in-source loss of sodiated sulfates (Δ102), pseudomolecular ions (asterisks) with varying sulfated status (S1–S4) were also observed and fragmented in negative mode as [M-(SO3)n] or fragmented in positive mode as [M+H(n-1)-(SO3)n]+. C and D, analysis in the presence of ammonium sulfate (C) results in monosulfated ions, observed in defucosylated form upon fucosidase treatment (D). E–J, example glycans (see also ) were fragmented in negative mode either as in-source pseudomolecular (F and I) or parental ions (G and J), showing the presence of sulfated Lewis motifs as well as some neutral losses from the core, whereas their bi- and triantennary nature as well as core and antennal fucosylation were confirmed by positive B and Y fragments (E and H), such as m/z 446 (Fuc1GlcNAc1-PA), 512 (Fuc1Gal1GlcNAc1), and 1484/1646 (Fuc2Gal1Man2/3GlcNAc3-PA); the two β2/β4-linked antennae on the lower α3-Man are assumed compared with hybrid N-glycans (). ‡ indicates early-eluting reducing-end ManNAc epimers (
      • Ishimizu T.
      • Mitsukami Y.
      • Shinkawa T.
      • Natsuka S.
      • Hase S.
      • Miyagi M.
      • Sakiyama F.
      • Norioka S.
      Presence of asparagine-linked N-acetylglucosamine and chitobiose in Pyrus pyrifolia S-RNases associated with gametophytic self-incompatibility.
      ) of structures predominantly found in later fractions. Losses of Fuc (F), Hex (H), and HexNAc (N) are indicated.

       Core α3-linked N-glycans

      The glycopeptides remaining after PNGase-F digestion were treated with PNGase-A to identify possible core α3-fucosylated N-glycans. This residual pool was also separated in neutral and anionic subpools prior to labeling and injection onto RP HPLC. Although some of the masses in the fractions were the same as those identified previously in the PNGase F digest, two HPLC fractions contained hybrid or complex N-glycans displaying the presence of an additional fucose (i.e. S1Fuc3Gal1Man5GlcNAc3 at m/z 2194 and S1Fuc5Gal3Man3GlcNAc5 at m/z 2893; Fig. 9, A and C). While HF treatment resulted in loss of all fucoses except the core α6-linked one (Fig. 9, B and D), negative-mode MS/MS of the hybrid structure (m/z 2194) showed neutral losses of the difucosylated core as well as the B ions, showing occurrence of a sulfo-Lewis motif (Fig. 9E). On the other hand, positive MS/MS of the corresponding [M-SO3]+ pseudomolecular ion (m/z 2116) yielded a core Y ion at m/z 592 (Fuc2GlcNAc1-PA), which is a further proof of difucosylation of the innermost core GlcNAc (Fig. 9F).
      Figure thumbnail gr9
      Figure 9MALDI-TOF MS analysis of specific PNGase-A–released N-glycans. A and C, larger core α3-fucosylated glycans with compositions of S1Fuc3Gal1Man5GlcNAc3 (A; hybrid-type at m/z 2194) and S1Fuc5Gal3Man3GlcNAc5 (C; complex-type, observed with in-source loss of sulfate at m/z 2893), respectively, eluted at 5.3 and 3.8 g.u. upon RP HPLC of the anionic pool of PNGase-A–released N-glycans. B and D, both fractions were subject to HF treatment, which removed all fucoses (red dashed arrows) except the α6-linked core fucose, indicative that the second core fucose is α3-linked. E and F, furthermore, negative MS/MS of S1Fuc3Gal1Man5GlcNAc3 (E) clearly yielded B ions at m/z 1400 and 1603 (resulting in loss of 591 or 794 Da, i.e. of Fuc2GlcNAc1–2-PA), whereas the positive-mode spectrum of the pseudomolecular [M-SO3]+ ion (F) shows a Y fragment at m/z 592, revealing the presence of an additional core fucose on the reducing terminus, as in many invertebrates (
      • Stanton R.
      • Hykollari A.
      • Eckmair B.
      • Malzl D.
      • Dragosits M.
      • Palmberger D.
      • Wang P.
      • Wilson I.B.H.
      • Paschinger K.
      The underestimated N-glycomes of lepidopteran species.
      ). In comparison, as shown in , M and N, MS/MS of a glycan (S1Fuc2Gal1Man5GlcNAc3) with one core and one antennal fucose shows an m/z 590 negative-mode B ion but lacking the m/z 592 and 1605 difucosylated positive-mode Y ions.

      Discussion

      The N-glycome of H. atra, the first to be described of any sea cucumber, is characterized by 74% of neutral structures (mainly oligomannosidic-type N-glycans) and 26% of anionic structures (1% phosphorylated, 24% sulfated, and 1% sialylated), as judged by RP HPLC fluorescence and MS intensities (Fig. 10 and Table S2). The relatively high amount of sulfated hybrid and complex-type N-glycans were enriched in the anionic pool, whereas isomers with different positions of the fucose (core or antennal), sulfate (four different positions; i.e. either on Gal, GlcNAc, Man, or NeuGc) or sialic acid residues (on Gal or on GlcNAc) could be resolved by NP or RP HPLC (Fig. S1 and Fig. 2B). The enrichment and separation as well as addition of Na+ to enhance sulfate detection by MS proved to be crucial for the in-depth sulfo- and sialoglycomic investigation, as isolation, separation, and detection of anionic glycans remains a challenging task for which special specific protocols involving either fluorescent labeling (
      • Cabrera G.
      • Salazar V.
      • Montesino R.
      • Támbara Y.
      • Struwe W.B.
      • Leon E.
      • Harvey D.J.
      • Lesur A.
      • Rincón M.
      • Domon B.
      • Méndez M.
      • Portela M.
      • González-Hernández A.
      • Triguero A.
      • Durán R.
      • et al.
      Structural characterization and biological implications of sulfated N-glycans in a serine protease from the neotropical moth Hylesia metabus (Cramer [1775]) (Lepidoptera: Saturniidae).
      ,
      • Miyagawa S.
      • Maeda A.
      • Kawamura T.
      • Ueno T.
      • Usui N.
      • Kondo S.
      • Matsumoto S.
      • Okitsu T.
      • Goto M.
      • Nagashima H.
      A comparison of the main structures of N-glycans of porcine islets with those from humans.
      ,
      • Murakami T.
      • Natsuka S.
      • Nakakita S.
      • Hase S.
      Structure determination of a sulfated N-glycans, candidate for a precursor of the selectin ligand in bovine lung.
      ,
      • Yagi H.
      • Takahashi N.
      • Yamaguchi Y.
      • Kimura N.
      • Uchimura K.
      • Kannagi R.
      • Kato K.
      Development of structural analysis of sulfated N-glycans by multidimensional high performance liquid chromatography mapping methods.
      ) or permethylation (
      • Yu S.Y.
      • Wu S.W.
      • Hsiao H.H.
      • Khoo K.H.
      Enabling techniques and strategic workflow for sulfoglycomics based on mass spectrometry mapping and sequencing of permethylated sulfated glycans.
      ) have been employed previously.
      Figure thumbnail gr10
      Figure 10Semiquantitative analysis of the H. atra N-glycome and comparisons within the Deuterostoma. The signal intensities of HPLC and MALDI-TOF peaks containing characterized N-glycans were used to estimate the ratio of each individual class and subclass of N-glycans to provide an overview of their relative abundance. Particular proven epitopes include variable antennal α4 fucosylation, β3 galactosylation, 4-linked sulfation of galactose, α3 sialylation of galactose, α6 sialylation of GlcNAc, sulfation of GlcNAc (putatively 6-linked if otherwise not sialylated), and sulfation of mannose; phosphorylation of triglucosylated glycans and core difucosylation of hybrid/complex glycans were also detected (the latter accounting for some 0.3% of the total N-glycome of H. atra). For a full list of predicted compositions, refer to . The simplified evolutionary tree (left panel, based on Vaughn et al. (
      • Vaughn R.
      • Garnhart N.
      • Garey J.R.
      • Thomas W.K.
      • Livingston B.T.
      Sequencing and analysis of the gastrula transcriptome of the brittle star Ophiocoma wendtii.
      )) exhibits the division between protostomes and deuterostomes in the Animalia (500 million years ago) as well as example resulting species. The depiction of the Deuterostoma (center panel) shows the phyla of the Echinodermata and Chordata. An overall comparison of N-glycomic features (right panel) of H. atra (sea cucumber, this study), Ophiactis savignyi (brittle star, accompanying study (
      • Eckmair B.
      • Jin C.
      • Karlsson N.G.
      • Abed-Navandi D.
      • Wilson I.B.H.
      • Paschinger K.
      Glycosylation at an evolutionary nexus: the brittle star Ophiactis savignyi expresses both vertebrate and invertebrate N-glycomic features.
      )), and Vertebrata (e.g. human and bovine) shows selected similarities (e.g. disialylated motif) and differences (e.g. variation in fucosylation, sulfation, and sialylation levels).
      Overall, our data suggest that at least four sulfates can modify the N-glycans of H. atra, and indeed, most LacNAc-like antennae are not just sulfated but are most commonly fucosylated; sulfated forms of β3-galactose, β-GlcNAc, α3-mannose, and α3-sialic acid residues could be proven by MS/MS. Sulfation of galactose is similar to that in the oyster (
      • Kurz S.
      • Jin C.
      • Hykollari A.
      • Gregorich D.
      • Giomarelli B.
      • Vasta G.R.
      • Wilson I.B.
      • Paschinger K.
      Haemocytes and plasma of the eastern oyster (Crassostrea virginica) display a diverse repertoire of sulphated and blood group A-modified N-glycans.
      ), but the relative dominance of sulfation of α3-mannose is in contrast to insects, in which sulfation of α6-mannose or core fucose is more common (
      • Stanton R.
      • Hykollari A.
      • Eckmair B.
      • Malzl D.
      • Dragosits M.
      • Palmberger D.
      • Wang P.
      • Wilson I.B.H.
      • Paschinger K.
      The underestimated N-glycomes of lepidopteran species.
      ). Unlike the highly sulfated keratan-like N-glycans of unfertilized eggs of a fish, Tribolodon hakonensis (
      • Taguchi T.
      • Iwasaki M.
      • Muto Y.
      • Kitajima K.
      • Inoue S.
      • Khoo K.H.
      • Morris H.R.
      • Dell A.
      • Inoue Y.
      Occurrence and structural analysis of highly sulfated multiantennary N-linked glycan chains derived from a fertilization-associated carbohydrate-rich glycoprotein in unfertilized eggs of Tribolodon hakonensis.
      ), with repetitive sulfated neo-LacNAc motifs, no obvious repeating units were detected in this study. Although the function of glycans in echinoderms is unclear, sulfation is implicated as a critical determinant mediating a diverse range of biological recognition functions on N- and O-glycans (
      • Honke K.
      • Taniguchi N.
      Sulfotransferases and sulfated oligosaccharides.
      ).
      Other hybrid and complex N-glycans in H. atra are sialylated, and some structures are even carrying antennal sialic acid in combination with sulfate and/or fucose modifications. Interestingly, like mammalian fetuin (
      • Green E.D.
      • Adelt G.
      • Baenziger J.U.
      • Wilson S.
      • Van Halbeek H.
      The asparagine-linked oligosaccharides on bovine fetuin. Structural analysis of N-glycanase-released oligosaccharides by 500-megahertz 1H NMR spectroscopy.
      ), sialylation occurs on either antennal galactose or antennal GlcNAc residues; there may, of course, be biosynthetic competition with sulfation for these positions. In the proven β3-galactosylated/α4-fucosylated structures in H. atra, the sialyl–Lewis A element corresponds to the human CA19-9 epitope with roles in cancer (
      • Engle D.D.
      • Tiriac H.
      • Rivera K.D.
      • Pommier A.
      • Whalen S.
      • Oni T.E.
      • Alagesan B.
      • Lee E.J.
      • Yao M.A.
      • Lucito M.S.
      • Spielman B.
      • Da Silva B.
      • Schoepfer C.
      • Wright K.
      • Creighton B.
      • et al.
      The glycan CA19-9 promotes pancreatitis and pancreatic cancer in mice.
      ). Such motifs have a potential role in cell–cell interactions; in the case of echinoderms, it is conceivable that a sialylated glycan could be important for regeneration. Compared with the brittle star described in the accompanying study (
      • Eckmair B.
      • Jin C.
      • Karlsson N.G.
      • Abed-Navandi D.
      • Wilson I.B.H.
      • Paschinger K.
      Glycosylation at an evolutionary nexus: the brittle star Ophiactis savignyi expresses both vertebrate and invertebrate N-glycomic features.
      ), sialylation is less common in H. atra (Fig. 10). Nevertheless, the ability of this species to sialylate N-glycans on two different residues (α2,3 on Gal or α2,6 on GlcNAc) correlates with expansion of the sialyltransferase gene family in echinoderms (
      • Harduin-Lepers A.
      • Mollicone R.
      • Delannoy P.
      • Oriol R.
      The animal sialyltransferases and sialyltransferase-related genes: a phylogenetic approach.
      ). Compared with the evolutionarily more primitive protostome phyla (Fig. 10), nematodes have no sialylation capacity at all, whereas most insect species have single homologs of α2,6-sialyltransferase and CMP-NeuAc synthase (
      • Koles K.
      • Irvine K.D.
      • Panin V.M.
      Functional characterization of Drosophila sialyltransferase.
      ,
      • Mertsalov I.B.
      • Novikov B.N.
      • Scott H.
      • Dangott L.
      • Panin V.M.
      Characterization of Drosophila CMP-sialic acid synthetase activity reveals unusual enzymatic properties.
      ); only for Drosophila are there MS data indicative of sialylation of N-glycans in a nonengineered insect system (
      • Aoki K.
      • Perlman M.
      • Lim J.M.
      • Cantu R.
      • Wells L.
      • Tiemeyer M.
      Dynamic developmental elaboration of N-linked glycan complexity in the Drosophila melanogaster embryo.
      ). However, to date, glucuronic acid and sulfate have been proven to be the most recurrent anionic modifications of invertebrate N-glycans (
      • Paschinger K.
      • Wilson I.B.H.
      Anionic and zwitterionic moieties as widespread glycan modifications in non-vertebrates.
      ).
      All sialylated N-glycans proposed for H. atra contain NeuGc rather than NeuAc, even though both have been reported previously on glycolipids from other sea cucumbers (
      • Yamada K.
      • Matsubara R.
      • Kaneko M.
      • Miyamoto T.
      • Higuchi R.
      Constituents of Holothuroidea: 10: isolation and structure of a biologically active ganglioside molecular species from the sea cucumber Holothuria leucospilota.
      ,
      • Yamada K.
      • Sasaki K.
      • Harada Y.
      • Isobe R.
      • Higuchi R.
      Constituents of Holothuroidea: 12: isolation and structure of glucocerebrosides from the sea cucumber Holothuria pervicax.
      ,
      • Kisa F.
      • Yamada K.
      • Miyamoto T.
      • Inagaki M.
      • Higuchi R.
      Constituents of Holothuroidea: 18: isolation and structure of biologically active disialo- and trisialo-gangliosides from the sea cucumber Cucumaria echinata.
      ). Certainly, the CMP-NeuAc hydroxylase required for NeuGc transfer is known in echinoderms (
      • Schlenzka W.
      • Shaw L.
      • Schauer R.
      Catalytic properties of the CMP-N-acetylneuraminic acid hydroxylase from the starfish Asterias rubens: comparison with the mammalian enzyme.
      ), and NeuGc also occurs in many higher deuterostomes, including cephalochordates, fish, and mice (
      • Guérardel Y.
      • Chang L.Y.
      • Fujita A.
      • Coddeville B.
      • Maes E.
      • Sato C.
      • Harduin-Lepers A.
      • Kubokawa K.
      • Kitajima K.
      Sialome analysis of the cephalochordate Branchiostoma belcheri, a key organism for vertebrate evolution.
      ,
      • Yamakawa N.
      • Vanbeselaere J.
      • Chang L.Y.
      • Yu S.Y.
      • Ducrocq L.
      • Harduin-Lepers A.
      • Kurata J.
      • Aoki-Kinoshita K.F.
      • Sato C.
      • Khoo K.H.
      • Kitajima K.
      • Guerardel Y.
      Systems glycomics of adult zebrafish identifies organ-specific sialylation and glycosylation patterns.
      ,
      • Muchmore E.A.
      • Milewski M.
      • Varki A.
      • Diaz S.
      Biosynthesis of N-glycolyneuraminic acid: the primary site of hydroxylation of N-acetylneuraminic acid is the cytosolic sugar nucleotide pool.
      ), but not humans (
      • Oku H.
      • Hase S.
      Studies on the substrate specificity of neutral α-mannosidase purified from Japanese quail oviduct by using sugar chains from glycoproteins.
      ). However, unlike the brittle star, there is an apparent lack of methylated NeuGc on H. atra N-glycans.
      A rather unusual anionic feature detected in H. atra is phosphorylation of three oligomannosidic-type N-glycans with a triglucosylated A arm (P1Glc3Man7–9GlcNAc2) carrying the phosphate on the terminal glucose; such an N-glycan modification has not been reported previously, in contrast to the “famous” mannose-6-phosphate involved in intracellular cell trafficking via the cognate receptor for lysosomal enzymes (
      • Kollmann K.
      • Pohl S.
      • Marschner K.
      • Encarnação M.
      • Sakwa I.
      • Tiede S.
      • Poorthuis B.J.
      • Lübke T.
      • Müller-Loennies S.
      • Storch S.
      • Braulke T.
      Mannose phosphorylation in health and disease.
      ). The terminal localization of glucose-6-phosphate could have an important role in glycoprotein folding regulation during calnexin/calreticulin cycles in the endoplasmic reticulum (
      • Lamriben L.
      • Graham J.B.
      • Adams B.M.
      • Hebert D.N.
      N-glycan-based ER molecular chaperone and protein quality control system: the calnexin binding cycle.
      ). This phosphorylation position contrasts with the presence of phosphate on antennal GlcNAc residues of the brittle star, as described in the accompanying study (
      • Eckmair B.
      • Jin C.
      • Karlsson N.G.
      • Abed-Navandi D.
      • Wilson I.B.H.
      • Paschinger K.
      Glycosylation at an evolutionary nexus: the brittle star Ophiactis savignyi expresses both vertebrate and invertebrate N-glycomic features.
      ).
      The fucosylation level in H. atra is very high in the hybrid and complex sub-N-glycomes, with many of the sulfated and/or sialylated glycans displaying antennal fucosylation; as the galactose residue on the hybrid glycans is clearly β3-linked, this means that the fucose residue on such antennae is α4-linked, a feature found on complex plant N-glycans as well as some human glycans (
      • Wilson I.B.
      • Zeleny R.
      • Kolarich D.
      • Staudacher E.
      • Stroop C.J.
      • Kamerling J.P.
      • Altmann F.
      Analysis of Asn-linked glycans from vegetable foodstuffs: widespread occurrence of Lewis a, α1,3-fucose and xylose substitutions.
      ,
      • Yamashita K.
      • Tachibana Y.
      • Kobata A.
      Oligosaccharides of human milk: structural studies of two new octasaccharides, difucosyl derivatives of para-lacto-N-hexaose and para-lacto-N-neohexaose.
      ). Furthermore, a small minority of glycans are core α3-fucosylated, a feature known to be common in nematodes, insects, and plants (
      • Paschinger K.
      • Wilson I.B.H.
      Comparisons of N-glycans across invertebrate phyla.
      ), whereas core α6 fucosylation in H. atra is frequent. Thus, there must be at least three fucosyltransferases capable of modifying N-glycans in this echinoderm species.
      Another obvious difference to the brittle star is the relative dominance of hybrid structures compared with complex forms in the sea cucumber. Also, the maximal number of branches appears to be three in H. atra rather than four. This would suggest low processing by Golgi α-mannosidase II but also the presence of GlcNAc transferases I, II, and IV in the sea cucumber; some of the hybrid glycans actually display processing by both GlcNAc transferase I and IV, which results in disubstitution of the α3-mannose (Fig. S5), as observed also in insects or birds, for example (
      • Hykollari A.
      • Malzl D.
      • Stanton R.
      • Eckmair B.
      • Paschinger K.
      Tissue-specific glycosylation in the honeybee: analysis of the N-glycomes of Apis mellifera larvae and venom.
      ,
      • Harvey D.J.
      • Wing D.R.
      • Küster B.
      • Wilson I.B.
      Composition of N-linked carbohydrates from ovalbumin and co-purified glycoproteins.
      ). The high abundance of the same hybrid β3-galactosylated “backbone” in H. atra, regardless of whether the N-glycans are sialylated or sulfated, suggests that these classes of structures are biosynthetically related and not random contaminants from the diet.
      In conclusion, the N-glycome of H. atra contrasts with that of the brittle star, but galactosylation, sialylation, and sulfation of the antennae are common features. The presence of fucose, sulfate, and sialic acid has also been reported in other glycoconjugates of various sea cucumbers, such as glycolipids with a fucose-modified trisialylated glucosyl ceramide, chondroitin sulfate with sulfate-modified difucose branches, and triterpene glycosides, which can also be sulfated (
      • Yamada K.
      • Matsubara R.
      • Kaneko M.
      • Miyamoto T.
      • Higuchi R.
      Constituents of Holothuroidea: 10: isolation and structure of a biologically active ganglioside molecular species from the sea cucumber Holothuria leucospilota.
      ,
      • Yamada K.
      • Sasaki K.
      • Harada Y.
      • Isobe R.
      • Higuchi R.
      Constituents of Holothuroidea: 12: isolation and structure of glucocerebrosides from the sea cucumber Holothuria pervicax.
      ,
      • Mou J.
      • Wang C.
      • Li W.
      • Yang J.
      Purification, structural characterization and anticoagulant properties of fucosylated chondroitin sulfate isolated from Holothuria mexicana.
      ,
      • Silchenko A.S.
      • Kalinovsky A.I.
      • Avilov S.A.
      • Andryjaschenko P.V.
      • Dmitrenok P.S.
      • Yurchenko E.A.
      • Dolmatov I.Y.
      • Dautov S.S.
      • Stonik V.A.
      • Kalinin V.I.
      Colochiroside E, an unusual non-holostane triterpene sulfated trioside from the sea cucumber Colochirus robustus and evidence of the impossibility of a 7(8)-double bond migration in lanostane derivatives having an 18(16)-lactone.
      ). The glycome of H. atra may reflect a high expression level of sulfo- and fucosyltransferases as well as their associated metabolites; thus, if genetic manipulation becomes possible, then it could prove to be a good model to study the regulation, mechanisms, and functions of fucosylation and sulfation. From an evolutionary perspective, the occurrence of β3-galactosylation and core α3-fucosylation on one hand but of sialylation or antennal α4-fucosylation on the other shows that this echinoderm species does present both invertebrate- and vertebrate-like features in its glycome.

      Experimental procedures

       Enzymatic release of N-glycans

      3 g (wet weight) black sea cucumber (H. atra adult form) shredded into 2- to 4-mm cubes were suspended in boiling water and denatured for 5 min prior to addition of 0.1 m ammonium bicarbonate (pH 8.0), 20 mm CaCl2, and 3 mg of thermolysin in a final volume of 15 ml. Proteolysis was allowed to proceed for 2 h at 70 °C, and then the sample was centrifuged to remove residual insoluble material. The resulting glycopeptides were enriched by cation-exchange chromatography (Dowex AG50, Bio-Rad) and gel filtration (Sephadex G25, GE Healthcare), yielding 30 mg of purified glycopeptides. N-glycans were released using peptide:N-glycosidase F (PNGase-F, Roche) in 100 mm ammonium carbonate (pH 8), overnight at 37 °C; the remaining glycopeptides were then digested using peptide:N-glycosidase A (PNGase-A, Roche) in 50 mm ammonium acetate (pH 5) overnight at 37 °C. PNGase-F– and PNGase-A–released N-glycan fractions were further purified by a second round of cation-exchange chromatography (Dowex, Bio-Rad) and separated by a nonporous graphitized carbon column (Supelco) using 40% acetonitrile to elute the neutral glycans, followed by 40% acetonitrile with 0.1% TFA to elute the anionic glycans (
      • Hykollari A.
      • Paschinger K.
      • Eckmair B.
      • Wilson I.B.
      Analysis of invertebrate and protist N-glycans.
      ). All N-glycan fractions were then pyridylaminated as described previously (
      • Hase S.
      • Ibuki T.
      • Ikenaka T.
      Reexamination of the pyridylamination used for fluorescence labeling of oligosaccharides and its application to glycoproteins.
      ). Compared with the RP HPLC fluorescent signal of 10 pmol of a purified PA-labeled N-glycan from a commercial source (30 mV at the detector gain used), the yield of total labeled N-glycans was 7 nmol for the neutral pool and 3 nmol for the acidic pool. Sea cucumbers feed on planktonic algae, amoebae, and small animals; as no pentose-containing glycans were detected, we conclude that no algae were coanalyzed.

       MALDI-TOF MS analysis

      The pyridylaminated N-glycans were fractionated by RP or NP HPLC columns and profiled by MALDI-TOF MS (Autoflex Speed, Bruker Daltonics) in positive- and negative-ion modes using FlexControl 3.4 software. All HPLC peaks were collected, freeze dried, redissolved in 10 μl, and examined by MALDI-TOF MS, using 6-aza-2-thiothymine as matrix (
      • Hykollari A.
      • Balog C.I.
      • Rendić D.
      • Braulke T.
      • Wilson I.B.
      • Paschinger K.
      Mass spectrometric analysis of neutral and anionic N-glycans from a Dictyostelium discoideum model for human congenital disorder of glycosylation CDG IL.
      ). Sample and matrix solutions (1 μl each) were sequentially spotted and dried under a vacuum. To enhance formation of [M+H]+ or [M-Hn+Nan-1] ions, either 1 μl of 20 mm ammonium sulfate or 1 μl of 10 mm sodium acetate was spotted on top of the matrix. MS/MS to confirm the composition of all proposed structures was performed by laser-induced dissociation (the precursor ion selector was generally set to ±0.6%). The detector voltage was generally set at 1977 V for MS and 2133 V for MS/MS; 500–1000 shots from different regions of the sample spots were summed. Spectra were processed with the manufacturer's software (Bruker FlexAnalysis 3.3.80) using the SNAP algorithm with a signal/noise threshold of 6 for MS (unsmoothed) and 3 for MS/MS (four times smoothed). All MS and MS/MS spectra were manually interpreted on the basis of the mass fragmentation pattern and results of chemical and enzymatic treatments; isomeric structures present in different RP HPLC or NP HPLC fractions were defined on the basis of comparisons of the aforementioned parameters. At least four MS/MS fragment ions were used to aid definition of each of the structures, which are depicted according to the Symbol Nomenclature for Glycans (
      • Varki A.
      • Cummings R.D.
      • Aebi M.
      • Packer N.H.
      • Seeberger P.H.
      • Esko J.D.
      • Stanley P.
      • Hart G.
      • Darvill A.
      • Kinoshita T.
      • Prestegard J.J.
      • Schnaar R.L.
      • Freeze H.H.
      • Marth J.D.
      • Bertozzi C.R.
      • et al.
      Symbol nomenclature for graphical representations of glycans.
      ). For further details, refer to the supporting information.

       HPLC purification of N-glycans

      Separation of PA-labeled glycans was carried out on a Shimadzu HPLC system equipped with a fluorescence detector (RF-20AXS) using a Kinetex 5-μm RP column (XB-C18 100A, 250 × 4.6 mm, Phenomenex, Torrance, CA) with a gradient of methanol in 0.1 m ammonium acetate (pH 4) up to 16.5% over 44 min applied at a flow rate of 0.8 ml/min as follows: 0–30 min, 0%–9% methanol; 30–35 min, 9%–12% methanol; 35–40 min, 12%–16.5% methanol; 40–44 min, 16.5% methanol; and 44–50 min, return to 0% methanol. For separation based on size and charge, a HIAX IonPac AS11 NP column (Dionex) was used with 800 mm ammonium acetate (pH 3.85) (buffer A) and 80% (v/v) acetonitrile (buffer B). The following gradient was applied at a flow rate of 1 ml/min: 0–5 min 99% B, 5–50 min 90% B, 50–65 min 80% B, and 65–85 min 75% B. PA-labeled glycans were detected by fluorescence with excitation/emission wavelengths of 320/400 nm. The RP HPLC column was calibrated daily in terms of glucose units using a pyridylaminated dextran hydrolysate, whereas the NP HPLC column was calibrated daily using a mixture of pyridylaminated N-glycans (Man3–9GlcNAc2) derived from white beans; the order of elution of the standards was confirmed by MALDI-TOF MS of collected calibrant fractions (
      • Hykollari A.
      • Paschinger K.
      • Eckmair B.
      • Wilson I.B.
      Analysis of invertebrate and protist N-glycans.
      ).

       Structural elucidation using exoglycosidases and chemical treatment

      The following glycosidases were employed: recombinant Aspergillus niger β3/4-galactosidase (prepared in-house (
      • Dragosits M.
      • Pflügl S.
      • Kurz S.
      • Razzazi-Fazeli E.
      • Wilson I.B.
      • Rendic D.
      Recombinant Aspergillus β-galactosidases as a robust glycomic and biotechnological tool.
      )); Xanthomonas manihotis β3-galactosidase (New England Biolabs); Bacillus fragilis β4-galactosidase (New England Biolabs); bovine kidney α-fucosidase (Sigma-Aldrich); almond α3/4-fucosidase (New England Biolabs); jack bean α-mannosidase (Sigma-Aldrich); purified recombinant Bacteroides xylanisolvens BxGH99 α2-endo-mannosidase, which catalyzes removal of a disaccharide from Glc1Man9GlcNAc2 but not from unglucosylated Man9GlcNAc2 (
      • Thompson A.J.
      • Williams R.J.
      • Hakki Z.
      • Alonzi D.S.
      • Wennekes T.
      • Gloster T.M.
      • Songsrirote K.
      • Thomas-Oates J.E.
      • Wrodnigg T.M.
      • Spreitz J.
      • Stütz A.E.
      • Butters T.D.
      • Williams S.J.
      • Davies G.J.
      Structural and mechanistic insight into N-glycan processing by endo-α-mannosidase.
      ); recombinant Aspergillus saitoi α2-mannosidase (Prozyme); and Streptococcus pneumoniae α3-sialidase S (New England Biolabs). In general, 10% of an HPLC fraction (1 μl) was incubated overnight at 37 °C with 0.8 μl of 100 mm ammonium acetate (pH 5.0) and 0.2 μl of a glycosidase (see above). For removal of phosphate- or α3/4-linked fucose, 30% of an HPLC fraction (3 μl) was dried under a vacuum and incubated overnight on ice with 3 μl of 48% (w/v) hydrofluoric acid (HF) prior to drying again. For removal of sulfate, 30% of an HPLC fraction (3 μl) was dried under a vacuum and incubated for 4 h at 37 °C with 20 μl of 0.05 m methanol-HCl (methanolysis) prior to drying again. Enzymatically or chemically treated N-glycans were generally reanalyzed by MALDI-TOF MS and MS/MS without further purification unless rechromatographed by RP HPLC (see the relevant figure legends). The β4-galactosylated Hex6HexNAc3 standard was generated by treatment of a Man5GlcNAc3 structure with bovine milk galactosyltransferase (Fluka) in the presence of UDP-Gal and Mn(II) ions (
      • Jiménez-Castells C.
      • Stanton R.
      • Yan S.
      • Kosma P.
      • Wilson I.B.
      Development of a multifunctional aminoxy-based fluorescent linker for glycan immobilization and analysis.
      ).

      Author contributions

      J. V., C. J., I. B. H. W., and K. P. formal analysis; J. V., C. J., and B. E. investigation; J. V. methodology; J. V. writing-original draft; I. B. H. W. and K. P. supervision; I. B. H. W. and K. P. funding acquisition; I. B. H. W. and K. P. writing-review and editing.

      Acknowledgments

      We thank Carina Wokurek and Alba Hykollari for help with one of the glycan preparations.

      Supplementary Material

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