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Glycosylation at an evolutionary nexus: the brittle star Ophiactis savignyi expresses both vertebrate and invertebrate N-glycomic features

      Echinoderms are among the most primitive deuterostomes and have been used as model organisms to understand chordate biology because of their close evolutionary relationship to this phylogenetic group. However, there are almost no data available regarding the N-glycomic capacity of echinoderms, which are otherwise known to produce a diverse set of species-specific glycoconjugates, including ones heavily modified by fucose, sulfate, and sialic acid residues. To increase the knowledge of diversity of carbohydrate structures within this phylum, here we conducted an in-depth analysis of N-glycans from a brittle star (Ophiactis savignyi) as an example member of the class Ophiuroidea. To this end, we performed a multi-step N-glycan analysis by HPLC and various exoglyosidase and chemical treatments in combination with MALDI-TOF MS and MS/MS. Using this approach, we found a wealth of hybrid and complex oligosaccharide structures reminiscent of those in higher vertebrates as well as some classical invertebrate glycan structures. 70% of these N-glycans were anionic, carrying either sialic acid, sulfate, or phosphate residues. In terms of glycophylogeny, our data position the brittle star between invertebrates and vertebrates and confirm the high diversity of N-glycosylation in lower organisms.

      Introduction

      The Echinodermata represent the second largest grouping within the deuterostomes, after the Chordata, which also include vertebrates. This lineage has separated from the protostomes around 500–600 million years ago and so is at the nexus between invertebrates and vertebrates (
      • Bottjer D.J.
      • Davidson E.H.
      • Peterson K.J.
      • Cameron R.A.
      Paleogenomics of echinoderms.
      ). Living representatives of the echinoderms include classes of Crinoidea (feather stars), Echinoidea (sea urchins), Holothuroidea (sea cucumbers), Asteroidea (starfishes), and Ophiuroidea (brittle stars). Because of their close relationship to vertebrates, members of this phylum have been intensively studied in terms of the conserved basics of development compared with vertebrates as well as species-specific fertilization and regeneration (
      • McClay D.R.
      Evolutionary crossroads in developmental biology: sea urchins.
      ,
      • Biermann C.H.
      • Marks J.A.
      • Vilela-Silva A.C.
      • Castro M.O.
      • Mourão P.A.
      Carbohydrate-based species recognition in sea urchin fertilization: another avenue for speciation?.
      ).
      The evolutionary position of Ophiuroidea, with some 2000 living species (
      • Stöhr S.
      • O'Hara T.D.
      • Thuy B.
      Global diversity of brittle stars (Echinodermata: Ophiuroidea).
      ), makes this class of organisms an interesting target to study the phylogeny of protein-linked glycans of not only echinoderms but also of vertebrates. To date, a major focus of glycostructural studies of echinoderms has been their O-glycans and glycolipids as well as some proteoglycan-like polymers involved in species-specific induction of the acrosome reaction during fertilization (
      • Yamada K.
      • Hamada A.
      • Kisa F.
      • Miyamoto T.
      • Higuchi R.
      Constituents of Holothuroidea: 13: structure of neuritogenic active ganglioside molecular species from the sea cucumber Stichopus chloronotus.
      ,
      • Inagaki M.
      • Miyamoto T.
      • Isobe R.
      • Higuchi R.
      Biologically active glycosides from Asteroidea: 43: isolation and structure of a new neuritogenic-active ganglioside molecular species from the starfish Linckia laevigata.
      ,
      • Prokazova N.V.
      • Mikhailov A.T.
      • Kocharov S.L.
      • Malchenko L.A.
      • Zvezdina N.D.
      • Buznikov G.
      • Bergelson L.D.
      Unusual gangliosides of eggs and embryos of the sea urchin Strongylocentrotus intermedius: structure and density-dependence of surface localization.
      ,
      • Ijuin T.
      • Kitajima K.
      • Song Y.
      • Kitazume S.
      • Inoue S.
      • Haslam S.M.
      • Morris H.R.
      • Dell A.
      • Inoue Y.
      Isolation and identification of novel sulfated and nonsulfated oligosialyl glycosphingolipids from sea urchin sperm.
      ,
      • Higuchi R.
      • Inagaki M.
      • Yamada K.
      • Miyamoto T.
      Biologically active gangliosides from echinoderms.
      ,
      • Vilela-Silva A.C.
      • Hirohashi N.
      • Mourão P.A.
      The structure of sulfated polysaccharides ensures a carbohydrate-based mechanism for species recognition during sea urchin fertilization.
      ,
      • Pomin V.H.
      Structural and functional insights into sulfated galactans: a systematic review.
      ,
      • Miyata S.
      • Sato C.
      • Kumita H.
      • Toriyama M.
      • Vacquier V.D.
      • Kitajima K.
      Flagellasialin: a novel sulfated a2,9-linked polysialic acid glycoprotein of sea urchin sperm flagella.
      ,
      • Miyata S.
      • Sato C.
      • Kitamura S.
      • Toriyama M.
      • Kitajima K.
      A major flagellum sialoglycoprotein in sea urchin sperm contains a novel polysialic acid, an α2,9-linked poly-N-acetylneuraminic acid chain, capped by an 8-O-sulfated sialic acid residue.
      ,
      • Miyata S.
      • Yamakawa N.
      • Toriyama M.
      • Sato C.
      • Kitajima K.
      Co-expression of two distinct polysialic acids, a2,8- and a2,9-linked polymers of N-acetylneuraminic acid, in distinct glycoproteins and glycolipids in sea urchin sperm.
      ,
      • Kitazume S.
      • Kitajima K.
      • Inoue S.
      • Haslam S.M.
      • Morris H.R.
      • Dell A.
      • Lennarz W.J.
      • Inoue Y.
      The occurrence of novel 9-O-sulfated N-glycolylneuraminic acid-capped a2→5-Oglycolyl-linked oligo/polyNeu5Gc chains in sea urchin egg cell surface glycoprotein: identification of a new chain termination signal for polysialyltransferase.
      ). From these studies, it appears that, in contrast to protostomes, sialic acids are probably a frequent component of echinoderm glycoconjugates and, compared with “higher” vertebrates, occur also in sulfated and methylated forms (
      • Angata T.
      • Varki A.
      Chemical diversity in the sialic acids and related α-keto acids: an evolutionary perspective.
      ). Furthermore, core α1,3/6-difucosylation is a known recurring feature of protostome N-glycomes (
      • Paschinger K.
      • Wilson I.B.H.
      Comparisons of N-glycans across invertebrate phyla.
      ) but is absent from vertebrates and has not been identified in an echinoderm species before. Although there are a number of studies regarding the importance of N-glycan biosynthesis for sea urchin development (
      • Heifetz A.
      • Lennarz W.J.
      Biosynthesis of N-glycosidically linked glycoproteins during gastrulation of sea urchin embryos.
      ,
      • Kabakoff B.
      • Lennarz W.J.
      Inhibition of glycoprotein processing blocks assembly of spicules during development of the sea urchin embryo.
      ), there is little exact information regarding the N-glycosylation capacity of echinoderms. Indeed, in terms of actual structures, there is a single report regarding mass spectrometry analysis of oligomannosidic N-glycans (
      • Şahar U.
      • Deveci R.
      Profiling N-glycans of the egg jelly coat of the sea urchin Paracentrotus lividus by MALDI-TOF mass spectrometry and capillary liquid chromatography electrospray ionization-ion trap tandem mass spectrometry systems.
      ).
      As part of our ongoing efforts to establish a glycophylogeny of animal species, we analyzed the N-glycans of Ophiactis savignyi, an omnivorous small tropical marine organism once claimed to be the most common brittle star on the planet, potentially invasive in some areas and capable of both asexual and sexual reproduction (
      • McGovern T.M.
      Plastic reproductive strategies in a clonal marine invertebrate.
      ). We hypothesized that echinoderm species would display a mixture of invertebrate and vertebrate structural features; indeed, we found many N-glycans with antennal sialic acid or sulfate but also others displaying difucosylation of the core. Furthermore, compared with our other studies of echinoderms (Ref.
      • Vanbeselaere J.
      • Jin C.
      • Eckmair B.
      • Paschinger K.
      • Wilson I.B.H.
      Sulphated and sialylated N-glycans in the echinoderm Holothuria atra reflect its marine habitat and phylogeny.
      and unpublished data), there are also unique motifs that define the individual N-glycome of this species.

      Results

       Overall glycomic approach

      Our initial hypothesis regarding echinoderm N-glycosylation was that there would be a substantial degree of modification of oligosaccharides with anionic moieties as in vertebrates but that there may be vestiges of invertebrate-type N-glycomic features, such as difucosylation of the asparagine-bound chitobiosyl core. Therefore, the glycomic workflow for O. savignyi was aimed not only at separating neutral from anionic N-glycans but also at releasing structures with core α1,3-fucose. Serial use of PNGase F and A followed by solid-phase extraction on nonporous graphitized carbon allowed isolation of three pools of glycans (Fig. S1, enriched in neutral, anionic, and core α1,3-fucosylated structures), all of which were fluorescently labeled and analyzed by off-line HPLC MALDI-TOF MS. The overall predicted N-glycan compositions, based on MS and MS/MS before and after chemical or enzymatic treatments, are summarized in Table S1. Furthermore, O-glycan analysis of the residual glycopeptide fraction was performed by LC-ESI
      The abbreviations used are: ESI
      electrospray-ionisation
      FDL
      fused lobes hexosaminidase
      g.u.
      glucose unit
      HIAX
      hydrophilic interaction anionic exchange
      RP
      reverse phase
      PA
      pyridylamine.
      -MS following reductive β-elimination.

       Neutral N-glycans

      The neutral PNGase F–released pool contains primarily oligomannosidic and hybrid structures (see Fig. 1 for a normal phase profile), and their isomeric status could be evaluated on the basis of RP HPLC elution times (Fig. S2) and MS/MS fragmentation compared with previous studies. Our initial interest was in determining the nature of the potential galactosylation of an example hybrid structure (m/z 1678 as [M+H]+) as well as of a low-abundance biantennary glycan (m/z 1865). Based on use of linkage-specific galactosidases (Fig. 2), β1,3-galactosylation (type 1 chain, Galβ1–3GlcNAcβ1-R), as found in a number of invertebrates, rather than the β1,4-form (type 2 chain, Galβ1–4GlcNAcβ1-R) dominant in mammals, is concluded to be a feature of this echinoderm. Antennal β1,3-galactosylation was also a basic feature of the anionic glycans.
      Figure thumbnail gr1
      Figure 1HIAX-HPLC of the neutral PNGase F pool. Using hydrophilic interaction anionic exchange (HIAX) HPLC, which separates glycans by size and charge, resulted in detection of extended A arm–modified oligomannosidic structures, m/z 2609–3096 (Hex13HexNAc2-Hex16HexNAc2; for MS/MS, see ). The HIAX column was calibrated with a set of PA-labeled N-glycans derived from beans (H3X-H9, i.e. Man3GlcNAc2Xyl1-Man9GlcNAc2). The chromatogram is annotated with glycan structures (each with the positive-mode m/z value for the protonated ion in order of abundance in each fraction, with the most abundant uppermost) according to the symbol nomenclature for glycans. Linkages of example glycans are shown in the inset. For further photographs of the organism, refer to . The simplified evolutionary tree of the Echinodermata and Chordata is 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.
      ).
      Figure thumbnail gr2
      Figure 2Antennal motifs are based on β1,3-galactose as proven by glycosidase digests. A–C, the biantennary glycan (A) m/z 1865 (Hex5HexNAc4Fuc1) was sensitive to a β1,3-specific galactosidase (B) but resistant to a β1,4-specific galactosidase (C). F–H, treatment of the hybrid glycan (F) m/z 1678 (Hex6HexNAc3) with β1,3-galactosidase resulted in loss of one galactose (G), whereas jack bean α-mannosidase slowly removed up to three mannose residues (H); the coeluting Hex7HexNAc2 structure was resistant to galactosidase but sensitive to mannosidase. In the MS/MS spectra of untreated (D and I) and galactosidase-treated glycans (E and J), loss of galactose (Δ162) is indicated by red lines, e.g. shifts in B ions from m/z 366 (Hex1HexNAc1) to m/z 204 (HexNAc1) as well as in Y ions from m/z 1338 (Hex3HexNAc3Fuc1) or m/z 1192 (Hex3HexNAc3) to ones at m/z 1176 (Hex2HexNAc3Fuc1) or m/z 1030 (Hex2HexNAc3).
      In later neutral normal-phase fractions, glucosylated oligomannosidic structures were also found, as were glycans with unexpected Hex13–16HexNAc2 compositions. These were partially susceptible to digestion with jack bean α-mannosidase but also sensitive to a bacterial endo-α-mannosidase, as judged by shifts in the MS and MS/MS spectra (Fig. 3 and Fig. S3). As Hex13HexNAc2 (m/z 2610) lost five hexoses upon either endo-mannosidase or exo-mannosidase digestion, and no glucosidase or galactosidase tested removed the extra hexose residues, the Hex13–16HexNAc2 glycans are predicted to be Glc3Man9GlcNAc2 structures carrying unknown hexose residues on the glucosylated arm.
      Figure thumbnail gr3
      Figure 3Example of enzymatic digests of A arm–elongated N-glycan structures. A–C and G–I, two glycans with m/z 2447 (A, Hex12HexNAc2) and m/z 2610 (G, Hex13HexNAc2) (see MS/MS in D and J and ) are sensitive to B. xylanisolvens endo-α-mannosidase (B and H) cleaving the glucose-substituted α1,2-mannose of the A arm, releasing four to five hexoses, which is reflected by the shift of the diagnostic fragments from m/z 1637/1799 (Hex7–8HexNAc2) to m/z 989 (Hex3HexNAc2), as shown in MS/MS of the digest products in E and K. On the other hand, α-mannosidase removed up to five mannoses from the B and C arm from both structures, resulting in m/z 1637 or m/z 1799 (C and I) with a dominant m/z 665 fragment, as shown in MS/MS (F and L).
      Upon PNGase A treatment of glycopeptides remaining after PNGase F release, a mixture of neutral structures was obtained. Only fucosylated paucimannosidic glycans as well as “residual” oligomannosidic ones were present in this pool. Of the identified structures, two had elution times (8 g.u.), compositions and MS/MS patterns (i.e. a Y ion at m/z 592) indicative of core α1,3/α1,6-difucosylation (Fig. S4). Because of its early elution, the monofucosylated structure at 5 g.u. (m/z 1135, Hex3HexNAc2Fuc1) is also concluded to be core α1,3-fucosylated, which is a feature known from plants and invertebrates but not from vertebrates.

       Separation of anionic N-glycans

      Based on fluorescence intensity of the various N-glycan pools, it was estimated that some 70% of the structures are anionic, and initial mass spectrometric data suggested that both sulfated and sialylated glycans were present (Fig. S1). “Sialylation-friendly” RP HPLC conditions (pH 6) in a first dimension yielded 11 pools that were then subject to charge/size-based separation using a HIAX column; almost 120 2D-HPLC–separated glycan-containing fractions were isolated (Fig. 4 and Fig. S5).
      Figure thumbnail gr4
      Figure 4RP HPLC fractionation of anionic N-glycans. A–K, for the first dimension, anionic enriched N-glycans were separated on a Kinetex C18 RP HPLC (pH 6; calibrated in terms of glucose units). The color code and letters indicate pooled peaks whose second-dimension HIAX-HPLC profiles are shown in . Fractions are annotated with example glycans according to the symbol nomenclature for glycans (where Me and S represent methyl and sulfate, respectively). The illustrated structures are based on a combination of MS/MS analysis, RP and HIAX-HPLC elution times, as well as enzymatic and/or chemical treatments. Because of the separation properties of the RP column used as the first dimension, multiple sulfated structures are shown in A, modified hybrid structures in B, multiple sialylated biantennary structures in C–E, and those with methylated N-glycolylneuraminic acid and multiantennary structures in G–K. The 4 g.u. RP HPLC peak not subject to 2D-HPLC contains a nondigestible glycan not related to other analyzed structures.

       Sulfated and phosphorylated N-glycans

      Although sulfation and phosphorylation both result in an 80-Da modification compared with “parental” neutral N-glycans, their properties differ (
      • Paschinger K.
      • Wilson I.B.
      Analysis of zwitterionic and anionic N-linked glycans from invertebrates and protists by mass spectrometry.
      ); for instance, “in-source” loss in positive mode is a hallmark of sulfate, whereas sensitivity to hydrofluoric acid is a characteristic of phosphate. Therefore, we were able to conclude that both sulfated and phosphorylated N-glycans are synthesized by O. savignyi.
      Based on negative-ion-mode MS/MS and the results of exoglycosidase treatments, three different types of sulfated monosaccharides were observed: core α1,6-fucose, antennal galactose, or antennal GlcNAc. Sulfation of core α1,6-fucose has been found previously in insects, and hallmarks are the m/z 225 sulfated fucose fragment and resistance to bovine fucosidase treatment (Fig. 5, A–E, K, and M). In comparison, an isomeric glycan with sulfation of an antennal GlcNAc was distinguishable on the basis of the m/z 282 B fragment and loss of only one HexNAc upon serial galactosidase/hexosaminidase digestion (Fig. 5, F–J and N). Sulfation of galactose was associated with a dominant m/z 241 fragment (sulfated hexose, Fig. 5L) and galactosidase resistance. Disulfation of type 1 antennae was indicated by the MALDI-TOF MS/MS and LC-ESI-MSn data with, respectively, m/z 546 [M-2H+Na] or 261 [M-2H]2− fragments (Hex1HexNAc1S2, Fig. 5, O–Q) being observed; the galactose is proposed to be 4-sulfated (
      • Minamisawa T.
      • Hirabayashi J.
      Fragmentations of isomeric sulfated monosaccharides using electrospray ion trap mass spectrometry.
      ), whereas the GlcNAc may be 4- or 6-sulfated.
      Figure thumbnail gr5
      Figure 5Analysis of monosulfated and disulfated N-glycans. A–Q, two isomeric sulfated N-glycans (m/z 1943, Hex5HexNAc4Fuc1S1; A and F) separated by 2D-HPLC (G, 26 min; I, 31.2 min; and ) with key diagnostic fragments of either m/z 225 (Fuc1S1) and 727 (HexNAc2Fuc1S1-PA, K) or m/z 282 (HexNAc1S1) and 444 (Hex1HexNAc1S1, N) were subjected to various exoglycosidase digests. The first isomer was sequentially sensitive to β1,3-galactosidase (B), jack bean β-hexosaminidase (D), and jack bean α-mannosidase (E) but resistant to bovine fucosidase (C), confirming the sulfate position on the core fucose. In the case of the second isomer, β1,3-galactosidase removed two galactoses (G) but jack bean β-hexosaminidase only one GlcNAc (H), whereas the A arm–specific FDL removed none (I), in accordance with the key fragment indicative of sulfate linked to an antennal GlcNAc residue. MS/MS analysis of monosulfated N-glycans (K–N) as [M-H] or disulfated N-glycans as [M-SO3] and [M-2H+Na] (O and P) yielded fragments indicative of sulfation of either core fucose (m/z 225/524/727, K and M), terminal galactose (m/z 241, L and O), or subterminal GlcNAc (m/z 282, N and P) or with the B ion at m/z 546, indicative of double sulfate substitution of the Galβ1,3GlcNAc motif (P). Fragment ions at m/z 444 and 606 are indicative of sulfation on Gal1GlcNAc1Man0–1 antennae. For a disulfated hybrid structure, m/z 241, 282, 444, and 546 fragments (Hex1S, HexNAc1S, and Hex1HexNAc1S1–2; O and P) were observed when performing MS/MS on the [M-2H+Na] parent or the [M-SO3] ion, resulting from in-source-loss of one sulfate; it actually appeared that sulfation was most commonly on antennal GlcNAc. LC-ESI-MSn of a disulfated glycan (Q, Hex4HexNAc3Fuc1S2 at m/z 828.97 [M-2H]2−) indicates sulfate on terminal galactose and subterminal GlcNAc, respectively; some of the singly charged fragments were also observed in MALDI-TOF MS/MS, whereas others are doubly charged and are indicated in blue with (2). MS3 of fragment ions at m/z 241 (GalS, inset) resulted in observation of ions at m/z 181, indicative of 4-sulfation of galactose.
      Other glycans with an 80-Da modification were observed in both positive and negative modes and so were concluded to be phosphorylated on HexNAc residues, as judged by the m/z 284 [M+H]+ fragment ions (Fig. 6, A–E). In the case of two hybrid structures, this was verified by sensitivity to jack bean β-hexosaminidase only after hydrofluoric acid treatment; subsequently one or two hexose residues could be removed upon jack bean α-mannosidase treatment (Fig. 6, F–I).
      Figure thumbnail gr6
      Figure 6Identification of phosphorylated N-glycans. A–E, positive-mode MALDI-TOF MS/MS of hybrid and biantennary phosphorylated glycan structures of O. savignyi. The diagnostic fragment ion at m/z 284 is compatible with a phosphate substitution on the terminal GlcNAc. Note that the fragment at m/z 446 (A and B), which refers to the B ion P-GlcNAc-Man, is isobaric to the Y ion for Fuc-GlcNAc-PA (C–E). F–I, the phosphorylated structures at m/z 1272 and 1434 in positive ion mode (m/z 1270/1432 negative ion mode, inset) were sensitive to hydrofluoric acid treatment (G), where the loss of 80 Da demonstrates the presence of a phosphate. After dephosphorylation, jack bean β-hexosaminidase was able to remove the GlcNAc residues (H), in contrast to resistance before treatment (data not shown), proving the position of the phosphate group on the antennal GlcNAc. The susceptibility to jack bean α-mannosidase proved the hybrid nature of the m/z 1272 and 1434 glycans (I).

       Sialylated N-glycans

      A number of glycans contained modifications of either 307 or 321 Da, as defined by results of positive- and negative-mode MS/MS. Relevant B fragments in either positive or negative ion mode indicated the presence of N-glycolylneuraminic acid or its methylated form on terminal HexHexNAc motifs (Fig. 7, A–G, and Fig. S6). As there were multiple isomers for some masses among the 2D-HPLC fractions (e.g. two, three, or even four each of mono- and biantennary structures with m/z 1807, 1821, 2172, or 2333; Fig. S5), a major question was whether there were multiple positions for sialylation. Therefore, selected glycans were incubated with sialidases and/or mild acid.
      Figure thumbnail gr7
      Figure 7MS/MS analysis of sialylated N-glycans. A–D, example positive-ion-mode MALDI-TOF MS/MS of N-glycans modified with either N-glycolylneuraminic acid (A) or its methylated form (C) with the diagnostic B (m/z 673 or 687 for NeuGc1Me0–1Hex1HexNAc1) and Y ions depicted. Negative-ion-mode MS/MS of these glycans resulted in diagnostic fragments at m/z 306 (B) or 320 (D) for NeuGc or NeuGcMe. Because the sialic acids are easily lost in MS/MS experiments, there was no obvious difference in fragmentation pattern between glycans with NeuGc or NeuGcMe on either terminal galactose or internal GlcNAc; however, enzymatic digests enabled these to be distinguished ( and ). E, negative collision-induced dissociation ESI-MS/MS of a disialylated glycan (Hex4HexNAc3Fuc1NeuGc2 at m/z 1056.27 [M-2H]2−) indicates one NeuGc residue linked to subterminal GlcNAc (fragment ions at m/z 509); the fragment ions at m/z 937 are concluded to be due to 0,2X cleavage of NeuGc (red), which is diagnostic for 2,6-linked sialic acid. F and G, positive- and negative-mode MALDI-TOF MS of the Hex4HexNAc3Fuc1NeuGc2 glycan also showing the m/z 978/980 B fragments indicative of the sialyl–Lewis C motif (NeuGc2Gal1GlcNAc1). H–M, the two monosulfated isomers of m/z 2250 as [M-H] eluting at 2D-HIAX (E, 50.5 min; F, 51 min; H and K), with MS/MS in J and M indicating a sulfate linked to antennal GlcNAc (m/z 282) or NeuGc (m/z 386), were subjected to α2,3-specific sialidase S, which removed m/z 307 or 386 (NeuGc or its sulfated version, I and L), resulting in a still-charged structure with a sulfate at m/z 1943 as [M-H] or a neutral glycan at m/z 1865 as [M+H]+. Loss of 45 Da in MS as well as the m/z 341 MS/MS fragment could be a result of decarboxylation of sialic acid (
      • Powell A.K.
      • Harvey D.J.
      Stabilization of sialic acids in N-linked oligosaccharides and gangliosides for analysis by positive ion matrix-assisted laser desorption/ionization mass spectrometry.
      ).
      As shown for the four isomeric monosialylated biantennary Hex5HexNAc4Fuc1NeuGc1 glycans (Fig. 8, 2171 Da), two structures only lost the NeuGc residue upon acid treatment, whereas two others were sensitive to α2,3-specific sialidase S. On the other hand, the sialidase-resistant glycans lost two hexose residues upon β1,3-galactosidase treatment, whereas only one hexose was removed from those sensitive to sialidase S (Fig. 8, B, G, R, and W). Thus, as sialic acids are known to modify either terminal galactose or antennal GlcNAc residues in, e.g., mammalian fetuin, we concluded that this also occurs in the brittle star (see below); indeed some structures with NeuGc on both Gal and GlcNAc of the same antenna were also found (Fig. 7, E–G).
      Figure thumbnail gr8
      Figure 8Structural elucidation of four monosialylated isomeric Hex5HexNAc4Fuc1NeuGc1 N-glycans. A–Z, four 2D-HPLC–separated 2171-Da isomers (H, 33 min; I, 35 min; G, 33 min; H, 34 min; A, F, Q, and V) only showed subtle differences in their MS/MS fragmentation patterns (E, J, U, and Z). In case of isomers with internal NeuGc, β1,3-galactosidase was able to cleave two residues (B and G) compared with one galactose for those with one terminal sialic acid residue (R and W); only after β1,3-galactosidase digest could the key fragment for the NeuGc-HexNAc modification, m/z 511 (insets in B, C, G, and H) be detected. The four isomers also differed in their susceptibility to α2,3-sialidase S, which only cleaves off terminal NeuGc (S and Y), whereas internal NeuGc was removed with acid hydrolysis (D and I). To determine which arms were sialylated, two of the m/z 1338 digest products were reinjected on HPLC (K and L) and coeluted with two standards, A and B, which also showed different key fragment ions (M–P) at m/z 1135 as in standard A and digest product H, 33 min, and m/z 1176 as in standard B and digest product I, 35 min; the other two m/z 2172 structures were subject to lower arm–specific FDL hexosaminidase (T and X), where the respective resistance or sensitivity proves substitution of the lower or upper arm.
      To determine which antennae of the 2171-Da isomers were sialylated, serial chemical or enzymatic treatments were then performed. By comparing the RP HPLC retention time of digestion products with standards, the two isomers with sialylated GlcNAc could be distinguished regarding the NeuGc substitution on either the α1,6 or α1,3 antenna (Fig. 8, A–P). On the other hand, despite similar fragmentation patterns and only slightly different 2D-HPLC elution properties, the two isomers with sialylated galactose were shown to differ in terms of which antenna carried the sialic acid residue by performing β1,3-specific galactosidase treatment followed by incubation with the arm-specific insect FDL hexosaminidase (
      • Léonard R.
      • Rendic D.
      • Rabouille C.
      • Wilson I.B.
      • Préat T.
      • Altmann F.
      The Drosophila fused lobes gene encodes an N-acetylglucosaminidase involved in N-glycan processing.
      ) (Fig. 8, Q–Z).
      A similar approach was employed on two glycans with putative NeuGcMe modifications. In the case of a terminal NeuGcMe residue, sialidase S treatment resulted in loss of 321 Da, but the glycan was resistant to β1,3-galactosidase unless desialylated (Fig. S7, A–C and E); in contrast, the isomeric glycan with later NP-HPLC retention was resistant to sialidase S but sensitive to β1,3-galactosidase and acid treatment (Fig. S7, G–J), consistent with α2,6-sialyl substitution of GlcNAc. Based on treatment of the desialylated/degalactosylated structures with insect FDL, both glycans possessed sialylated lower arm Galβ1,3GlcNAc motifs (Fig. S7, D and K). The MS/MS fragmentation patterns were very similar (Fig. S7, F and L), but the trace ion at m/z 525 (i.e. HexNAcNeuGcMe) verified that the sialidase S–resistant structure was modified by NeuGcMe on the antennal GlcNAc residue.
      Some glycans were predicted to contain both sulfate and sialic acid; in the case of two isomeric structures (Hex5 HexNAc4Fuc1NeuGc1S1) with different 2D-HPLC elution properties, fragments at either m/z 282 or 387 were observed and correlated with loss of either 307 or 387 Da upon sialidase S treatment. This suggested that the sulfate residues were either on an antennal GlcNAc or covalently bound to the terminal sialic acid residue (Fig. 7, H–M).
      As mentioned above, the resistance to hydrofluoric acid of the 80-Da moiety would indicate the occurrence of sulfate, and when treating the complete anionic pool with this reagent, major peaks at, e.g., m/z 1578 and 1943 were still dominant in the overall spectrum. However, the masses of the sialylated glycans shifted by 18 Da (Fig. S8). This change is akin to that upon lactonization and suggested that hydrofluoric acid can also stabilize specific sialic acid linkages, similar to combined 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide/1-hydroxybenzotriazole treatment; the shift in the NeuGc1Hex1HexNAc1 positive-mode fragment from m/z 673 to 655 is also the same (Fig. S8, C–H). Therefore, there were two sets of proof that NeuGc or NeuGcMe on terminal galactose is α2,3-linked. On the other hand, sialic acid linked to GlcNAc is concluded to be α2,6-linked because of the ability to remove it with a nonspecific sialidase, 3-sialylation of GlcNAc being ruled out by the presence of the 3-linked galactose, and LC-ESI-MS3 evidence (Fig. 7E).

       Maximum number of N-glycan antennae

      Not only did the results of hydrofluoric acid treatment of the entire anionic pool indicate lactonization, but we could also detect the presence of trisialylated triantennary structures (m/z 3113 in negative mode, Fig. 9). Upon desialylation of the anionic pool using 2 m acetic acid, we could also observe larger glycans (Hex6–7HexNAc5–6Fuc1) whose monosulfated forms were detected in negative mode (Fig. 9 and Fig. S9, A–D). As MS/MS analysis indicated that these correspond to tri- and tetra-antennary structures, selected RP HPLC fractions containing mono-, bi-, and triantennary glycans were also subjected to mild acid hydrolysis prior to degalactosylation (Fig. S9, E–J) and RP HPLC, as this method is known to distinguish different triantennary isomers (
      • Minamisawa T.
      • Hirabayashi J.
      Fragmentations of isomeric sulfated monosaccharides using electrospray ion trap mass spectrometry.
      ,
      • Léonard R.
      • Rendic D.
      • Rabouille C.
      • Wilson I.B.
      • Préat T.
      • Altmann F.
      The Drosophila fused lobes gene encodes an N-acetylglucosaminidase involved in N-glycan processing.
      ). The underlying structures were compared with asialoagalactoglycans derived from fetuin (Fig. S9, K–M), for which the β1,2-GlcNAc on the α1,6-mannose and the β1,2/β1,4-disubstitution of the α1,3-mannose have been defined previously. The resulting retention time comparisons (Fig. S9N) indicated that the mono-antennary glycans were β1,2-substituted on the α1,3-mannose and the biantennary β1,2-substituted on both α-mannose residues, whereas the triantennary glycans from O. savignyi have the same configuration as those in bovine fetuin.
      Figure thumbnail gr9
      Figure 9Impact of mild acid and hydrofluoric acid treatment on sialic acids. A–C, incubation of an aliquot of the complete anionic N-glycan pool with 2 m acetic acid removed all sialic acids, resulting in residual neutral and sulfated structures. Sulfated tri and tetra-antennary glycans were detected, as verified by the MS/MS analyses shown in the insets. D, an aliquot of the complete anionic enriched N-glycan pool from O. savignyi was incubated with 48% hydrofluoric acid for 48 h at 4 °C, facilitating observation of larger glycan structures. Lactonization (Δ18) of sialylated N-glycans seemingly stabilized the structures, changing the mass difference from Δ307 (N-glycolylneuraminic acid) to Δ289. For MS/MS data, refer to .

       O-glycans

      When performing LC-ESI-MS after reductive β-elimination of residual glycopeptides (Fig. S10 and Table S2), the two major identified O-glycans were predicted to be Galβ1,3GalNAc, the typical core 1 “mucin-type” glycan or T antigen, and Xylα1,3Xylα1,3Glc, a structure known from epidermal growth factor domains (
      • Nishimura H.
      • Kawabata S.
      • Kisiel W.
      • Hase S.
      • Ikenaka T.
      • Takao T.
      • Shimonishi Y.
      • Iwanaga S.
      Identification of a disaccharide (Xyl-Glc) and a trisaccharide (Xyl2-Glc) O-glycosidically linked to a serine residue in the first epidermal growth factor-like domain of human factors VII and IX and protein Z and bovine protein Z.
      ). Core 1 glycans with additional fucose, NeuAc, NeuGc, hexose, HexNAc, or methyl modifications were also identified. Based on MS/MS of the lower abundance O-glycans, also compared with the literature (
      • Liu J.
      • Jin C.
      • Cherian R.M.
      • Karlsson N.G.
      • Holgersson J.
      O-glycan repertoires on a mucin-type reporter protein expressed in CHO cell pools transiently transfected with O-glycan core enzyme cDNAs.
      ,
      • Jin C.
      • Padra J.T.
      • Sundell K.
      • Sundh H.
      • Karlsson N.G.
      • Lindén S.K.
      Atlantic salmon carries a range of novel O-glycan structures differentially localized on skin and intestinal mucins.
      ), it can be concluded that fucosylated core 1 (Fucα1, 2Galβ1,3GalNAc), sialyl Tn (NeuAc/NeuGcα2,6GalNAc), and mono- and disialylated core 1 (Galβ1,3[NeuAc/NeuGcα2,6]GalNAc, NeuAc/NeuGcα2,3Galβ1,3GalNAc, and NeuAcα2, 3Galβ1,3[NeuAcα2,6]GalNAc) structures are also present in O. savignyi. Therefore, it is interesting that NeuAc is well represented in the O-glycome but not detectable in the N-glycome.

      Discussion

      Because of their close relationship to vertebrates at the nexus of protostomes and deuterostomes, echinoderms are historically useful model organisms for studies of development, fertilization, and embryogenesis. As little is known about their N-glycosylation capacity, we conducted an in-depth analysis of N-glycans from O. savignyi. Our initial hypothesis was that the N-glycome of an echinoderm would reflect the evolutionary position of this phylum and would therefore have aspects of both invertebrate and vertebrate glycosylation patterns. Indeed, this presumption, based also on older work, especially on O-glycans and glycolipids, was more than confirmed in the case of the brittle star O. savignyi and a sea cucumber, Holothuria atra (as shown in our companion report). Previously, the occurrence of calcium-binding sulfated N-glycans potentially involved in sea urchin embryonic skeleton formation was postulated (
      • Kabakoff B.
      • Hwang S.P.
      • Lennarz W.J.
      Characterization of post-translational modifications common to three primary mesenchyme cell-specific glycoproteins involved in sea urchin embryonic skeleton formation.
      ,
      • Farach-Carson M.C.
      • Carson D.D.
      • Collier J.L.
      • Lennarz W.J.
      • Park H.R.
      • Wright G.C.
      A calcium-binding, asparagine-linked oligosaccharide is involved in skeleton formation in the sea urchin embryo.
      ), but their exact structures were never defined, whereas the occurrence of classical oligomannosidic structures in echinoderms was confirmed in a mass spectrometry study on egg jelly glycans from the sea urchin Paracentrotus lividus (
      • Şahar U.
      • Deveci R.
      Profiling N-glycans of the egg jelly coat of the sea urchin Paracentrotus lividus by MALDI-TOF mass spectrometry and capillary liquid chromatography electrospray ionization-ion trap tandem mass spectrometry systems.
      ). Therefore, based on our reading of the literature, our two studies are indeed the first in-depth N-glycomic analyses of any echinoderm species. As summarized in Fig. 10A, in the brittle star, we found complex and hybrid N-glycans featuring the type 1 antennal motif, a minor degree of the α1,3/α1,6-difucosylation core modification, glucosylated oligomannosidic structures with an unknown hexose cap, and various anionic modifications, specifically sulfate, phosphate, and sialic acid.
      Figure thumbnail gr10
      Figure 10Summary of glycoepitopes and their abundance in the N-glycome of the brittle star O. savignyi. A, based on fluorescence intensities, 30% of N-glycans are neutral, and 70% carry an anionic moiety. Within these classes, 92% of the neutral N-glycans are classical oligomannosidic Man5–9GlcNAc2 structures, but neutral glycans with extra glucose and unknown hexose residues are also present, in addition to neutral core α1,3-fucosylated, hybrid, and biantennary glycans; antennal galactosylation was solely detected in a β1,3 linkage. Within the acidic N-glycan pool, sialylation (solely N-glycolylneuraminic acid) is the major anionic modification, with up to three sialylated antennae being detected; 20% of the sialylated structures are disialylated (one on Gal, one on GlcNAc) to result in sialyl–Lewis C epitopes (gray box). 5% of sialylated structures are also sulfated, and 20% of NeuGc residues are methylated. Glycans with just sulfation are less abundant, and those displaying phosphorylation account for just 1% of the acidic pool. B, simplified glyco-evolutionary scheme showing the occurrence of selected antennal elements in protostomes and deuterostomes. The phylogeny is adapted from 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.
      ). Depicted are Lewis X; sulfated blood group A; glucuronylated LacdiNAc (also modified by phosphorylcholine); glucuronylated/sulfated type I; sulfated Lewis A; NeuGcMe-modified type I and NeuAc-modified type II antennae from Schistosoma mansoni, Crassostrea virginica, Dirofilaria immitis, Anopheles gambiae, H. atra, O. savignyi, and Homo sapiens (
      • Paschinger K.
      • Wilson I.B.H.
      Comparisons of N-glycans across invertebrate phyla.
      ,
      • Vanbeselaere J.
      • Jin C.
      • Eckmair B.
      • Paschinger K.
      • Wilson I.B.H.
      Sulphated and sialylated N-glycans in the echinoderm Holothuria atra reflect its marine habitat and phylogeny.
      ). Note that anionic N-glycans are unknown in S. mansoni; D. immitis is the only nematode for which glucuronylated N-glycans have been proven.
      Invertebrate characteristics of both the brittle star and sea cucumber glycomes are reflected in the occurrence of some core difucosylated N-glycan structures of the type known from a wide range of insect, mollusk, trematode, and nematode species (
      • Paschinger K.
      • Rendić D.
      • Wilson I.B.
      Revealing the anti-HRP epitope in Drosophila Caenorhabditis.
      ). Modification of antennal GlcNAc residues with β1,3-linked galactose is also shared with oysters and insects (
      • Paschinger K.
      • Wilson I.B.H.
      Comparisons of N-glycans across invertebrate phyla.
      ,
      • Kurz S.
      • Aoki K.
      • Jin C.
      • Karlsson N.G.
      • Tiemeyer M.
      • Wilson I.B.
      • Paschinger K.
      Targeted release and fractionation reveal glucuronylated and sulphated N- and O-glycans in larvae of dipteran insects.
      ,
      • 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.
      ,
      • Stanton R.
      • Hykollari A.
      • Eckmair B.
      • Malzl D.
      • Dragosits M.
      • Palmberger D.
      • Wang P.
      • Wilson I.B.
      • Paschinger K.
      The underestimated N-glycomes of lepidopteran species.
      ), but LacdiNAc (GalNAcβ1,4GlcNAc, found in many invertebrates) was lacking (Fig. 10B). Although β1,3-galactose is also present in mammals, the typical vertebrate modification with β1,4-galactose is absent in the brittle star, as are digalactose motifs, whether these be Galα1,3/4Gal or Galβ1,4Gal, as found in many mammals, birds, and fish (
      • Galili U.
      • Shohet S.B.
      • Kobrin E.
      • Stults C.L.
      • Macher B.A.
      Man, apes, and old world monkeys differ from other mammals in the expression of α-galactosyl epitopes on nucleated cells.
      ,
      • Suzuki N.
      • Su T.H.
      • Wu S.W.
      • Yamamoto K.
      • Khoo K.H.
      • Lee Y.C.
      Structural analysis of N-glycans from gull egg white glycoproteins and egg yolk IgG.
      ,
      • 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.
      ), and histo-blood group motifs. In contrast, the sea cucumber H. atra has sulfo- and sialyl–Lewis A motifs (
      • Vanbeselaere J.
      • Jin C.
      • Eckmair B.
      • Paschinger K.
      • Wilson I.B.H.
      Sulphated and sialylated N-glycans in the echinoderm Holothuria atra reflect its marine habitat and phylogeny.
      ). On the other hand, there are certainly triantennary N-glycans and a trace of tetra-antennary glycans indicative of the presence of a number of branching N-acetylglucosaminyltransferases, including GlcNAc-TIV (
      • Minowa M.T.
      • Oguri S.
      • Yoshida A.
      • Hara T.
      • Iwamatsu A.
      • Ikenaga H.
      • Takeuchi M.
      cDNA cloning and expression of bovine UDP-N-acetylglucosamine: α1,3-d-mannoside β1,4-N-acetylglucosaminyltransferase IV.
      ), an enzyme also predicted to occur in cnidarians and some nematodes as well as insects and vertebrates (Fig. S11). Interestingly, when there is only one Galβ1,3GlcNAc antenna, this is always on the α1,3-mannose, which is an indication that there is no dominant FDL-like hexosaminidase (
      • Léonard R.
      • Rendic D.
      • Rabouille C.
      • Wilson I.B.
      • Préat T.
      • Altmann F.
      The Drosophila fused lobes gene encodes an N-acetylglucosaminidase involved in N-glycan processing.
      ) removing the β1,2-linked GlcNAc from the lower arm, as known from many invertebrates.
      The high degree of “charged” N-glycans in O. savignyi (estimated as 70%, as summarized in Fig. 10A) results in a more “vertebrate-like” characteristic, although we previously found an approximately 50% degree of N-glycan sulfation in oyster plasma (
      • 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.
      ). Indeed, the predominance of sialylation differentiates it from typical invertebrates, but the sialic acid modifications (methylation and sulfation) are different as compared with vertebrates. N-glycolylneuraminic acid is the most abundant sialic acid on N-glycans of the brittle star, with either α2,3-sialylated β1,3-linked galactose residues or α2,6-sialylation of antennal GlcNAc. The latter modification is known from bovine 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.
      ), and corresponding mammalian GlcNAc-6-sialyltransferase activity has been reported (
      • Paulson J.C.
      • Weinstein J.
      • de Souza-e-Silva U.
      Biosynthesis of a disialylated sequence in N-linked oligosaccharides: identification of an N-acetylglucosaminide α(2→6)-sialyltransferase in Golgi apparatus from rat liver.
      ). Recently this “internal” sialylated motif has also been found in the mouse brain and named “sialyl–Lewis C” (
      • Torii T.
      • Yoshimura T.
      • Narumi M.
      • Hitoshi S.
      • Takaki Y.
      • Tsuji S.
      • Ikenaka K.
      Determination of major sialylated N-glycans and identification of branched sialylated N-glycans that dynamically change their content during development in the mouse cerebral cortex.
      ). This epitope, which interacts with Siglec-H, has been found in developing mouse brains and may mediate interactions between dendritic spines and microglia (
      • Handa-Narumi M.
      • Yoshimura T.
      • Konishi H.
      • Fukata Y.
      • Manabe Y.
      • Tanaka K.
      • Bao G.M.
      • Kiyama H.
      • Fukase K.
      • Ikenaka K.
      Branched sialylated N-glycans are accumulated in brain synaptosomes and interact with Siglec-H.
      ). Although glycoproteins and their binding partners still need to be identified in echinoderms, it is known that their neuronal systems display similarities to those in chordates (
      • Zueva O.
      • Khoury M.
      • Heinzeller T.
      • Mashanova D.
      • Mashanov V.
      The complex simplicity of the brittle star nervous system.
      ).
      We also examined O-glycans and found a number of simple structures in the brittle star, some of which are sialylated. The previous literature on echinoderm O-glycans is a bit richer than for N-glycans, and di-, oligo-, or polysialylated structures have been found in sea urchins. Specific examples include mucin-type O-glycans from sperm flagella containing terminal 8-O-sulfated α2,9-linked polyNeu5Ac (
      • Miyata S.
      • Sato C.
      • Kumita H.
      • Toriyama M.
      • Vacquier V.D.
      • Kitajima K.
      Flagellasialin: a novel sulfated a2,9-linked polysialic acid glycoprotein of sea urchin sperm flagella.
      ,
      • Miyata S.
      • Sato C.
      • Kitamura S.
      • Toriyama M.
      • Kitajima K.
      A major flagellum sialoglycoprotein in sea urchin sperm contains a novel polysialic acid, an α2,9-linked poly-N-acetylneuraminic acid chain, capped by an 8-O-sulfated sialic acid residue.
      ) or other sperm and egg O-linked glycoproteins with α2,8-sialic acid and α2,5-sialic acid (
      • Miyata S.
      • Yamakawa N.
      • Toriyama M.
      • Sato C.
      • Kitajima K.
      Co-expression of two distinct polysialic acids, a2,8- and a2,9-linked polymers of N-acetylneuraminic acid, in distinct glycoproteins and glycolipids in sea urchin sperm.
      ,
      • Kitazume S.
      • Kitajima K.
      • Inoue S.
      • Haslam S.M.
      • Morris H.R.
      • Dell A.
      • Lennarz W.J.
      • Inoue Y.
      The occurrence of novel 9-O-sulfated N-glycolylneuraminic acid-capped a2→5-Oglycolyl-linked oligo/polyNeu5Gc chains in sea urchin egg cell surface glycoprotein: identification of a new chain termination signal for polysialyltransferase.
      ,
      • Kitazume-Kawaguchi S.
      • Inoue S.
      • Inoue Y.
      • Lennarz W.J.
      Identification of sulfated oligosialic acid units in the O-linked glycan of the sea urchin egg receptor for sperm.
      ). Also, sialylated glycolipids from various echinoderms have been described (
      • Miyata S.
      • Yamakawa N.
      • Toriyama M.
      • Sato C.
      • Kitajima K.
      Co-expression of two distinct polysialic acids, a2,8- and a2,9-linked polymers of N-acetylneuraminic acid, in distinct glycoproteins and glycolipids in sea urchin sperm.
      ,
      • Hoshi M.
      • Nagai Y.
      Novel sialosphingolipids from spermatozoa of the sea urchin Anthocidaris crassispina.
      ,
      • Inagaki M.
      • Shibai M.
      • Isobe R.
      • Higuchi R.
      Constituents of Ophiuroidea: 1: isolation and structure of three ganglioside molecular species from the brittle star Ophiocoma scolopendrina.
      ,
      • 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.
      ). Therefore, a range of sialyltransferases should be present in echinoderms, and indeed a number of α2,3-, α2,6-, and α2,8-sialyltransferase homologs are encoded by the genome of the sea urchin Strongylocentrotus purpuratus (
      • Harduin-Lepers A.
      • Mollicone R.
      • Delannoy P.
      • Oriol R.
      The animal sialyltransferases and sialyltransferase-related genes: a phylogenetic approach.
      ,
      • Petit D.
      • Teppa E.
      • Mir A.M.
      • Vicogne D.
      • Thisse C.
      • Thisse B.
      • Filloux C.
      • Harduin-Lepers A.
      Integrative view of α2,3-sialyltransferases (ST3Gal) molecular and functional evolution in deuterostomes: significance of lineage-specific losses.
      ,
      • Harduin-Lepers A.
      • Petit D.
      • Mollicone R.
      • Delannoy P.
      • Petit J.M.
      • Oriol R.
      Evolutionary history of the α2,8-sialyltransferase (ST8Sia) gene family: tandem duplications in early deuterostomes explain most of the diversity found in the vertebrate ST8Sia genes.
      ). However, considering the lack of enzymatic characterization of the various homologs in any echinoderm, it is difficult to predict which sialic acid linkages are theoretically possible in each species. Thus, we do not know whether our detection of only monosialylated motifs in the brittle star is either due to lack of α2,5/8/9-sialyltransferases in the brittle star or due to preparative/analytical factors.
      In terms of types of sialic acid, it is remarkable that the NeuGc and its methylated form are the only sialic acid residues on the N-glycans of O. savignyi, whereas O-glycans carry both NeuAc and NeuGc. As CMP-NeuGc biosynthesis requires prior formation of CMP-NeuAc, absence of NeuAc from N-glycans seems curious. However, methylation and sulfation of NeuGc match reports of starfish and sea cucumber O- and lipid-linked glycans (
      • Ijuin T.
      • Kitajima K.
      • Song Y.
      • Kitazume S.
      • Inoue S.
      • Haslam S.M.
      • Morris H.R.
      • Dell A.
      • Inoue Y.
      Isolation and identification of novel sulfated and nonsulfated oligosialyl glycosphingolipids from sea urchin sperm.
      ,
      • Miyata S.
      • Sato C.
      • Kumita H.
      • Toriyama M.
      • Vacquier V.D.
      • Kitajima K.
      Flagellasialin: a novel sulfated a2,9-linked polysialic acid glycoprotein of sea urchin sperm flagella.
      ,
      • Warren L.
      N-glycolyl-8-O-methylneuraminic acid, a new form of sialic acid in the starfish Asterias forbesi.
      ,
      • Klein A.
      • Diaz S.
      • Ferreira I.
      • Lamblin G.
      • Roussel P.
      • Manzi A.E.
      New sialic acids from biological sources identified by a comprehensive and sensitive approach: liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) of SIA quinoxalinones.
      ,
      • Bergwerff A.A.
      • Hulleman S.H.D.
      • Kamerling J.P.
      • Vliegenthart J.F.G.
      • Shaw L.
      • Reuter G.
      • Schauer R.
      Nature and biosynthesis of sialic acids in the starfish Asterias rubens: identification of sialo-oligomers and detection of S-adenosyl-l-methionine: N-acylneuraminate 8-O-methyltransferase and CMP-N-acetylneuraminate monooxygenase activities.
      ), and two starfish enzymes required for generation of methylated NeuGc (CMP-NeuAc hydroxylase and 8-O-methyltransferase) have already been identified (
      • 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.
      ,
      • Kelm A.
      • Shaw L.
      • Schauer R.
      • Reuter G.
      The biosynthesis of 8-O-methylated sialic acids in the starfish Asterias rubens: isolation and characterisation of S-adenosyl-l-methionine:sialate-8-O-methyltransferase.
      ). O-acetylation of sialic acid is known from other echinoderms but is notoriously difficult to detect because of instability, and we did not define any acetylated structures, whereas sulfated sialic acid was found only on a small subset of brittle star N-glycans. As NeuGc and acetylated NeuAc have been reported to be less easily cleaved by bacterial and viral sialidases than NeuAc (
      • Corfield A.P.
      • Veh R.W.
      • Wember M.
      • Michalski J.C.
      • Schauer R.
      The release of N-acetyl- and N-glycolloyl-neuraminic acid from soluble complex carbohydrates and erythrocytes by bacterial, viral and mammalian sialidases.
      ,
      • Corfield A.P.
      • Higa H.
      • Paulson J.C.
      • Schauer R.
      The specificity of viral and bacterial sialidases for α(2–3)- and α(2–6)-linked sialic acids in glycoproteins.
      ,
      • Kleineidam R.G.
      • Furuhata K.
      • Ogura H.
      • Schauer R.
      4-Methylumbelliferyl-α-glycosides of partially O-acetylated N-acetylneuraminic acids as substrates of bacterial and viral sialidases.
      ), it can be speculated that the abundance of NeuGc or its methylated or sulfated forms on the N-glycans of O. savignyi may provide a first line of defense against pathogens.
      In terms of anionic modifications other than sialylation, sulfation and phosphorylation were detected; as mentioned above, there was some sulfation of sialic acid residues, but sulfated galactose, GlcNAc, and fucose were also observed. Here there are parallels to the sulfation of galactose in oysters and of core fucose in insects (
      • Kurz S.
      • Aoki K.
      • Jin C.
      • Karlsson N.G.
      • Tiemeyer M.
      • Wilson I.B.
      • Paschinger K.
      Targeted release and fractionation reveal glucuronylated and sulphated N- and O-glycans in larvae of dipteran insects.
      ,
      • 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.
      ). LC-MS suggested that the galactose residues are 4-O-sulfated, unlike the 3-O-sulfation more common in mammals; nevertheless, 4-O-sulfo-modifications of fucose and GalNAc are known as modifications of sea cucumber chondroitin chains (
      • Santos G.R.
      • Porto A.C.
      • Soares P.A.
      • Vilanova E.
      • Mourão P.A.
      Exploring the structure of fucosylated chondroitin sulfate through bottom-up nuclear magnetic resonance and electrospray ionization-high-resolution mass spectrometry approaches.
      ), and 4-sulfated GalNAc is a component of mammalian chondroitin and dermatan sulfates (
      • Mikami T.
      • Mizumoto S.
      • Kago N.
      • Kitagawa H.
      • Sugahara K.
      Specificities of three distinct human chondroitin/dermatan N-acetylgalactosamine 4-O-sulfotransferases demonstrated using partially desulfated dermatan sulfate as an acceptor: implication of differential roles in dermatan sulfate biosynthesis.
      ). On the other hand, phosphorylation of GlcNAc was found, rather than of mannose, as known from N-glycans of mammalian lysosomal enzymes or slime mold glycoproteins (
      • 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.
      ,
      • Kollmann K.
      • Pohl S.
      • Marschner K.
      • Encarnacao M.
      • Sakwa I.
      • Tiede S.
      • Poorthuis B.J.
      • Lübke T.
      • Müller-Loennies S.
      • Storch S.
      • Braulke T.
      Mannose phosphorylation in health and disease.
      ), but no zwitterionic modifications were detected (
      • Paschinger K.
      • Wilson I.B.
      Analysis of zwitterionic and anionic N-linked glycans from invertebrates and protists by mass spectrometry.
      ).
      Glycosylation is a species-specific process, which is especially reflected in the distinct set of glycans involved in the fertilization process (
      • Mengerink K.J.
      • Vacquier V.D.
      Glycobiology of sperm-egg interactions in deuterostomes.
      ). These differences were also reflected in our studies, as only low amounts of Man1–3GlcNAc2-based paucimannosidic glycans and almost no pseudohybrid glycans (i.e. with an antenna only on α1,6-mannose) were found in O. savignyi. It is noteworthy that the anionic N-glycome of the sea cucumber H. atra is rather biased toward hybrid structures based on Man5GlcNAc2, including sulfated/fucosylated, but more rarely sialylated, HexHexNAc motifs (
      • Vanbeselaere J.
      • Jin C.
      • Eckmair B.
      • Paschinger K.
      • Wilson I.B.H.
      Sulphated and sialylated N-glycans in the echinoderm Holothuria atra reflect its marine habitat and phylogeny.
      ). Additionally, our preliminary data on two sea urchin and two starfish species revealed a distinct set of N-glycans. This highlights that the individual echinoderm species analyzed have a quite different overall N-glycome, even though some motifs overlap. Thus, we conclude that the N-glycome of O. savignyi is unique compared with other species, even compared with that of H. atra. Indicative that there is a push to “final products” of certain biosynthetic pathways is that the hybrid and complex glycans of this species are almost all sulfated and/or sialylated (accounting for 70% of the N-glycome), leaving a neutral subset dominated by oligomannosidic structures, including some with unusual hexose modifications on the “lower arm” triglucose motif. In general, except for the presence of some core difucosylated structures, the O. savignyi N-glycome is probably nearer to those of vertebrates; further comparative studies will certainly show variations and similarities in the glycomotifs associated with emergence of the deuterostome lineage before the Cambrian era.

      Experimental procedures

       Biological material and glycan preparation

      The brittle stars O. savignyi (Müller and Troschel, 1842) were sourced from Haus des Meeres - Aqua Terra Zoo, a public aquarium in Vienna, Austria. There, upon first detection in 2013, they were already living in a total of four coral reef exhibition tanks pertaining to two independent seawater systems. Therefore, only gross reconstruction of origin and date of introduction appears possible; during the last decade, all imports of coral reef materials came from the central Indo-Pacific Ecoregion (Philippines, Indonesia, and northeast Australia) via a Dutch wholesaler (De Jong Marinelife Inc.). The imports of live corals usually comprised small portions of foundation rocks. This suggests that the O. savignyi founder population had hitchhiked in shelters found in crevices of such rocks and that they then populated the open-celled polyurethane foam, which served as the filter of the aquarium system. A regimen of 10 h of dim light/14 h of darkness prevailed in the service room. The inorganic composition of the seawater was routinely analyzed employing inductively coupled plasma - optical emission spectrometry (ICP-OES) and ion chromatography-variable wavelength detection/electrochemical detection (IC-VWD/ECD) methodology (Oceamo Inc., Vienna, Austria; Table S3). Freshly collected animals were put in small Petri dishes for microphotography (Fig. 1 and Fig. S12).
      10 g of starved brittle stars were handpicked from aquarium substrates, washed once with deionized water, and stored at −80 °C. After thawing, the material was heat-inactivated for 10 min in boiling water and lyophilized prior to grinding in liquid nitrogen. The powder was suspended in deionized water, and the pH was adjusted with 100 mm ammonium carbonate buffer to 8.2; CaCl2 was added to a final concentration of 0.5 mm prior to addition of 10 mg of thermolysin (Promega). Proteolysis was allowed to proceed for 2 h at 70 °C and 37 °C overnight for completion. The glycopeptides were purified using standard laboratory protocols (
      • Hykollari A.
      • Paschinger K.
      • Eckmair B.
      • Wilson I.B.
      Analysis of invertebrate and protist N-glycans.
      ) prior to glycan release with PNGase F (Roche) overnight at 37 °C. After cation exchange chromatography on Dowex, the unbound material was subjected to solid-phase extraction on nonporous graphitized carbon (ENVIcarb, Supelco) and eluted with 40% acetonitrile or 40% acetonitrile with 0.1% TFA. These fractions were subjected to a further solid-phase extraction step on C18 reverse-phase resin (LiChroprep, Merck), and the glycans were eluted with water and stepwise increases in methanol (15%, 40%, and 100% (v/v)) concentration. Glycan-containing fractions, as judged by MALDI-TOF MS, were fluorescently labeled with 2-aminopyridine. The remaining glycopeptides bound to the Dowex resin were gel-filtrated (Sephadex G25) prior to incubation with PNGase Ar (recombinant, New England Biolabs) overnight at 37 °C to facilitate release of core α1,3-fucosylated glycans. The glycans released with this enzyme were then no longer bound by Dowex and were subject to the same purification and fluorescent-labeling steps as for the PNGase F released ones. The residual glycopeptides remaining after PNGase Ar digestion were subject to reductive β-elimination. As the animals were starved and no traces of algal glycans (which would be expected to contain pentose residues) were detected, no food contamination of the glycomes occurred.

       N-glycan fractionation

      Complete pyridyl-aminated N-glycomes were fractionated by reverse-phase HPLC (Kinetex 5μ RP column, XB-C18 100Å, 250 × 4.6 mm, Phenomenex®) using two different gradients at either pH 4 or pH 6. With buffer A (0.1 m ammonium acetate (pH 4)) and buffer B (30% methanol), a gradient of up to 55% over 44 min was applied at a flow rate of 0.8 ml/min as follows: 0–30 min, 0–30% B; 30–35 min, 30–40% B; 35–40 min, 40–55% B; 40–44 min, 55% B; 44–50 min, return to 0% B. For higher sialic acid stability, the following gradient was used: buffer C (0.1 m ammonium formate (pH 6)) and buffer B with a flow rate of 0.8 ml/min: 0–5 min, 0–10% B; 5–35 min, 10–40% B; 35–40 min, 40–50% B; 40–45 min, 50–65% B; 45–49 min, hold at 65%; 49–51 min, return to starting conditions. The RP HPLC column was calibrated daily in terms of glucose units using a pyridylaminated dextran hydrolysate. HIAX-HPLC (IonPac AS11 column, Dionex, 4 × 250 mm, combined with a 4 × 50 mm guard column) was used with a two-solvent gradient (buffer D, 0.8 m ammonium acetate (pH 3.85), and buffer E, 80% acetonitrile, LC-MS–grade) at a flow rate of 1 ml/min as follows: 0–5 min, 99% E; 5–50 min, 90% E; 50–65 min, 80% E; 65–85 min, 75% E. The HIAX-HPLC was calibrated using oligomannosidic PA-labeled bean glycans. All manually collected HPLC glycan fractions were analyzed after lyophilization by MALDI-TOF MS and MS/MS.

       Glycan MS

      Monoisotopic MALDI-TOF MS was performed using an Autoflex Speed (Bruker Daltonics) instrument in either positive or negative reflection mode with 6-aza-2-thiothymine or 2,5-dihydroxybenzoic acid as matrix. In general, MS/MS was performed by laser-induced dissociation of [M+H]+ or [M-H] ions (except for derivatized sialylated glycan masses detected as [M+Na]+ with 2,5-dihydroxybenzoic acid as matrix); typically 2000 shots were summed for MS (reflector voltage, lens voltage, and gain of 27 kV, 9 kV, and 2217 V, respectively) and 4000 for MS/MS (reflector voltage, lift voltage, and gain of 27 kV, 19 kV, and 2174 V, respectively). 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 (smoothed four times). Glycan spectra were manually interpreted on the basis of the masses of the predicted component monosaccharides; differences of mass in glycan series; comparison with coeluting structures from other insects, nematodes, and other marine organisms; and fragmentation patterns before and after chemical treatment or exoglycosidase digestion. A list of theoretical m/z values for each glycan composition is presented in Table S1.
      Selected RP HPLC fractions of pyridylaminated N-glycans as well as O-glycans prepared by β-elimination were also analyzed by online LC-MS/MS using a 5-μm porous graphitized carbon column (10 cm × 150 μm, prepared in-house) coupled to an LTQ ion trap mass spectrometer (Thermo Scientific, Waltham, MA). Glycans were eluted using a linear gradient from 0–40% acetonitrile in 10 mm ammonium bicarbonate over 40 min at a flow rate of 10 μl/min. The eluted N-glycans were detected in negative ion mode with an electrospray voltage of 3.5 kV, capillary voltage of −33.0 V, and capillary temperature of 300 °C. Specified ions were isolated for MSn fragmentation by collision-induced dissociation with the collision energy set to 30%. Air was used as a sheath gas, and mass ranges were defined depending on the specific structure to be analyzed. The data were processed using Xcalibur software (version 2.0.7, Thermo Scientific). Glycans were identified from their MS/MS spectra by manual annotation using the fragment nomenclature of Domon and Costello (
      • Domon B.
      • Costello C.E.
      A systematic nomenclature for carbohydrate fragmentations in Fab-MS MS spectra of glycoconjugates.
      ).

       Enzymatic and chemical treatments

      Based on results of HPLC elution and MALDI-TOF MS and MS/MS data, aliquots of the isolated HPLC fractions were subjected to targeted exoglycosidase digestion and chemical treatment. Either α-mannosidases (jack bean from Sigma, Aspergillus saitoi α1,2-specific from Prozyme, Xanthomonas α1,2/3-specific from New England Biolabs, or Xanthomonas α1,6-specific from New England Biolabs), endo-α1,2-mannosidase (recombinant Bacteroides xylanisolvens BxGH99), α-fucosidases (bovine kidney α1,6-specific from Sigma), β-galactosidases (recombinant Aspergillus niger or Aspergillus oryzae, prepared in-house; Xanthomonas β1,3-specific from New England Biolabs; or Bacillus fragilis β1,4-specific from New England Biolabs), β-hexosaminidase (recombinant Apis mellifera FDL, prepared in-house, or native jack bean from Sigma), or α-sialidases (sialidase V, S, or A, Prozyme, from, respectively, Vibrio cholerae, Streptococcus pneumoniae, or Arthrobacter ureafaciens or Neuraminidase, New England Biolabs, from Clostridium perfringens) were used for further treatment of the sample in 50 mm ammonium acetate (pH 5) (pH 6.5 with 5 mm CaCl2 for sialidases and pH 5.5 with 5 mm CaCl2 for β1,3-galactosidase) at 37 °C for 24 or 48 h (37 °C and 1.5 h for FDL). Under these conditions, FDL hexosaminidase is specific for β1,2-GlcNAc linked to the α1,3-mannose residue of the trimannosyl N-glycan core. For attempted digestion of unknown hexoses, β-mannosidase (Helix pomatia from Sigma), α-galactosidase (green coffee bean from Sigma), or α-glucosidases (Trichoplusia ni HighFive cell culture supernatant (
      • Hykollari A.
      • Dragosits M.
      • Rendić D.
      • Wilson I.B.
      • Paschinger K.
      N-glycomic profiling of a glucosidase II mutant of Dictyostelium discoideum by ”off-line” liquid chromatography and mass spectrometry.
      ) or rice glucosidase from Sigma) was used.
      For removal of phosphate groups, selected fractions were dried and incubated for 48 h at 0 °C with 3 μl of 48% (v/v) hydrofluoric acid prior to evaporation in a centrifugal concentrator. The samples were diluted in water and re-evaporated before dissolving again. Under these conditions, N-glycolylneuraminic acids were also stabilized by loss of a water molecule. Otherwise, shrimp alkaline phosphatase (Fermentas) was employed to remove phosphate residues. As appropriate, treated glycans were rechromatographed by RP HPLC (pH 6) to ascertain retention time shifts prior to MALDI-TOF MS; otherwise, an aliquot (generally one-fifth) of any digest was analyzed by MALDI-TOF MS without further purification.

       Linkage-specific sialic acid derivatization and mild acid hydrolysis

      Sialic acid–specific derivatization was performed as described previously (
      • de Haan N.
      • Reiding K.R.
      • Haberger M.
      • Reusch D.
      • Falck D.
      • Wuhrer M.
      Linkage-specific sialic acid derivatization for MALDI-TOF-MS profiling of IgG glycopeptides.
      ). Around one-fifth of a sample was mixed with the carboxylic acid activator 1-ethyl-3-(3-dimethylamino)-propyl)carbodiimide together with the catalyst 1-hydroxybenzo-triazole as follows: a 0.5 m solution of 1-ethyl-3-(3-dimethylamino)-propyl)carbodiimide and 1-hydroxybenzo-triazole in 100% (v/v) ethanol was added to the sample at a 1:1 ratio, and incubation was performed for 1 h at 37 °C. After adding 100% (v/v) MeCN to a final concentration of around 85% (v/v), the reaction mixture was purified using a Hydrophilic Interaction Liquid Chromatography-Solid Phase Extraction (HILIC-SPE) followed by MALDI-TOF analysis using 2,5-dihydroxybenzoic acid as matrix. Because of resistance of some sialic acid linkages toward sialidases, chemical hydrolysis was employed. Around one-fifth of a selected HPLC peak was mixed with 2 m acetic acid and incubated for 2 h at 80 °C (
      • Varki A.
      • Diaz S.
      The release and purification of sialic acids from glycoconjugates: methods to minimize the loss and migration of O-acetyl groups.
      ). To remove the acetic acid, the samples were dried using a SpeedVac and washed twice by adding water and re-evaporation. The desialylated samples were directly analyzed by MALDI-TOF MS.

      Author contributions

      B. E. and C. J. formal analysis; B. E., C. J., and D. A.-N. investigation; B. E. methodology; B. E. writing-original draft; N. G. K. and D. A.-N. resources; N. G. K., I. B. H. W., and K. P. supervision; D. A.-N., I. B. H. W., and K. P. writing-review and editing; I. B. H. W. and K. P. funding acquisition; K. P. conceptualization.

      Supplementary Material

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