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.

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 (1). 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 (2). 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 (3).
Over the years, various glycosylated biopolymers from sea cucumbers have been analyzed, particularly glycolipids (4,5), fucosylated chondroitin sulfates (6), and glycosylated terpenoids (7), all of which have been claimed to be bioactive (e.g. anti-inflammatory and anti-tumorigenic (8)). 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 (9).
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 (10). 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.
known oligomannosidic structures, whereas the anionic pool contained numerous unusual charged hybrid and complex N-glycans.

Oligomannosidic-type N-glycans
The neutral pool contained a series of Hex 4 -12 HexNAc 2 glycans (m/z 1151-2447). These could be assigned as isomers of Glc 0 -3 Man 4 -9 GlcNAc 2 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 (11,12), 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 Hex 10 -12 HexNAc 2 (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 Glc 3 Man 9 GlcNAc 2 N-glycan precursor (m/z 2447). The thereby predicted P 1 Glc 3 Man 9 GlcNAc 2 glycan was also treated with ␣-mannosidase, resulting in loss of up to five mannose residues regardless of whether the phosphate had been removed, and with endo-␣2-mannosidase, which removed a P 1 Glc 3 Man 1 unit ( Fig. 3 and Fig. S2); these data indicated that the phosphate residue is on the triglucosylated A arm. Considering also the MS/MS B 1 ions at m/z 241 (P 1 Hex 1 ) in negative mode, we concluded that the terminal glucose residue is the location of the phosphate modification.

Neutral hybrid-type N-glycans
Within the H. atra glycome, there were five neutral glycans with predicted compositions of Hex 4 -6 HexNAc 3 Fuc 0 -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 Hex 6 HexNAc 3 (m/z 1678, Fig. 4, A and I) was sensitive to ␤3-galactosidase (Fig. 4, G and J; loss of one Gal) and ␣-mannosidase (Fig. 4, H and K; loss of two or three Man) but not to ␤4-galactosidase (Fig. 4F). This indicated the presence of a type 1 antenna (neo-LacNAc, Gal␤3GlcNAc␤-R) on a hybrid backbone, a conclusion confirmed by comparison with a later-eluting isomer with a type 2 antenna (LacNAc, Gal␤4GlcNAc␤-R; prepared by in vitro ␤4-galactosylation; Fig. 4B). The ␤3-galactosylated hybrid structure appeared to be the basis for a number of sialylated and sulfated glycans, as desulfation of S 1 Hex 6 HexNAc 3 (m/z 1756) and desialylation of NeuGc 1 Hex 6 HexNAc 3 (m/z 1985) resulted in a coeluting Gal 1 Man 5 GlcNAc 3 structure (Fig. 4, C and D).   Fig. 2. 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).

Sea cucumber N-glycome
two fucosylated isomers of S 1 Fuc 1 Gal 1 Man 5 GlcNAc 3 (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 (S 1 Fuc 1 Gal 1 GlcNAc 1 ) and 1603 (loss of GlcNAc 1 -PA) is concluded to possess an antennal fucose, whereas the second one with m/z 444 (S 1 Gal 1 GlcNAc 1 ) and 1457 (loss of Fuc 1 GlcNAc 1 -PA) is core ␣6 -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 Fuc 1 GlcNAc 1 -PA (Fig. 5M). Treatment of the antennally fucosylated isomer with either ␤3/4-galactosidase (also no digestion even after ␣-fucosidase), ␣-mannosidase (loss of two or three Man), ␣3/4-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 S 1 Fuc 1 Gal 1 Man 3 -GlcNAc 3 glycan, which was ␤3-galactosidase-sensitive; the  (Fig. 6H). For the second one, the occurrence of fragments of m/z 241 (S 1 Gal 1 ) and 692 (S 2 Fuc 1 Gal 1 GlcNAc 1 ), 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. Trisulfated S 3 Fuc 0/2 Gal 1 Man 5 GlcNAc 3 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

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 (NeuGc 1 Fuc 0/1 Gal 1 Man 5 GlcNAc 3 , m/z 1985/2131) were reinjected onto RP HPLC before or after ␣3-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  (Fig. S2), it is concluded that the underlying backbone of the P 1 H 12 N 2 glycan (m/z 2527) is a standard Glc 3 Man 9 GlcNAc 2 structure with the same RP HPLC elution properties as a previously published structure from Pristionchus pacificus (50).

Sea cucumber N-glycome
monosialylated isomers could be observed, with the main diagnostic sialylated negative/positive B ions at m/z 306/308 (NeuGc 1 ) and 671/673 (NeuGc 1 Hex 1 HexNAc 1 ) for NeuGcmodified antennae being shared (Fig. 7, K, M, and R). In case of a sialidase S-resistant isomer, ␤3-galactosidase treatment resulted in loss of one galactose residue and best revealed a diagnostic m/z 511 NeuGc 1 GlcNAc 1 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.

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 ). Subsequently, positive MS before (E and I) and after ␤4-galactosidase (F; no digestion), ␤3-galactosidase (G and J; loss of one Gal), and jack bean ␣-mannosidase (H and K; loss of up to three Man) proved the presence of a neo-LacNAc (Gal␤3GlcNAc) antenna on a hybrid Man5-type backbone. B-D, in contrast, an isomeric Gal 1 Man 5 GlcNAc 3 structure (generated by enzymatic remodeling with ␤4-galactosyltransferase and carrying a LacNAc Gal␤4GlcNAc 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 S 1 Gal 1 Man 5 GlcNAc 3 and sialylated NeuGc 1 Gal 1 Man 5 GlcNAc 3 hybrid-type N-glycans with, respectively, methanolysis and sialidase resulted in neo-LacNAc antennae, as judged by coelution with the ␤3-galactosylated neutral form of Gal 1 Man 5 GlcNAc 3 (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).

Sea cucumber N-glycome
S 3-4 Fuc 2-4 Hex 5-6 HexNAc 4 -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 ␤3/4-galactosidase and ␣-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 biand 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 (␤3 or ␤4) was not possible. However, where monosulfated complex glycans were present in certain fractions, loss of galactose residues could be observed upon ␤3-galactosidase treatment, especially when the glycome pool had been defucosylated previously with HF (Fig. S8).

Core ␣3-linked N-glycans
The glycopeptides remaining after PNGase-F digestion were treated with PNGase-A to identify possible core ␣3-fucosylated N-glycans. This residual pool was also separated in neutral and

Sea cucumber N-glycome
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. S 1 Fuc 3 Gal 1 Man 5 GlcNAc 3 at m/z 2194 and S 1 Fuc 5 Gal 3 Man 3 GlcNAc 5 at m/z 2893; Fig. 9, A and C). While HF treatment resulted in loss of all fucoses except the core ␣6-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-SO 3 ] ϩ pseudomolecular ion (m/z 2116) yielded a core Y ion at m/z 592 (Fuc 2 GlcNAc 1 -PA), which is a further proof of difucosylation of the innermost core GlcNAc (Fig. 9F).

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 ϩ 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 (14 -17) or permethylation (18) have been employed previously.
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 ␤3-galactose, ␤-GlcNAc, ␣3-mannose, and ␣3-sialic acid residues could be proven by MS/MS. Sulfation of galactose is similar to that in the oyster (19), but the relative dominance of sulfation of ␣3-mannose is in contrast to insects, in which sulfation of ␣6-mannose or core fucose is more common (13). Unlike the highly sulfated keratan-like N-glycans of unfertilized eggs of a fish, Tribolodon hakonensis (20), with repetitive sulfated neo-LacNAc motifs, no obvious repeating

Sea cucumber N-glycome
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 (21).
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 (22), 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 ␤3-galactosylated/␣4-fucosylated structures in H. atra, the sialyl-Lewis A element corresponds to the human CA19-9 epitope with roles in cancer (23). 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 (24), sialylation is less common in H. atra (Fig. 10). Nevertheless, the ability of this species to sialylate N-glycans on two different residues (␣2,3 on Gal or ␣2,6 on GlcNAc) correlates with expansion of the sialyltransferase gene family in echinoderms (25). 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 ␣2,6-sialyltransferase and CMP-NeuAc synthase (26,27); only for Drosophila are there MS data indicative of sialylation of N-glycans in a nonengineered insect system (28). However, to date, glucuronic acid and sulfate have been proven to be the most recurrent anionic modifications of invertebrate N-glycans (29).
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 (4,5,30). Certainly, the CMP-NeuAc hydroxylase required for NeuGc transfer is known in echinoderms (31), and NeuGc also occurs in many higher deuterostomes, including cephalochordates, fish, and mice (32-34), but not humans (35). 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 (P 1 Glc 3 Man 7-9 GlcNAc 2 ) 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 (36).
The terminal localization of glucose-6-phosphate could have an important role in glycoprotein folding regulation during calnexin/calreticulin cycles in the endoplasmic reticulum (37). This phosphorylation position contrasts with the presence of phosphate on antennal GlcNAc residues of the brittle star, as described in the accompanying study (24).
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 ␤3-linked, this means that the fucose residue on such antennae is ␣4-linked, a feature found on complex plant N-glycans as well as some human glycans (38,39). Furthermore, a small minority of glycans are core ␣3-fucosylated, a feature known to be common in nematodes, insects, and plants (40), whereas core ␣6 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 ␣-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 ␣3-mannose (Fig. S5), as observed also in insects or birds, for example (41,42). The high abundance of the same hybrid ␤3-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 (4 -7). 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 per-  N-glycans. A, B, E, and F, NeuGc 1 Fuc 0/1 Gal 1 Man 5 GlcNAc 3 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 (Fig. 2B) 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.

Sea cucumber N-glycome
spective, the occurrence of ␤3-galactosylation and core ␣3-fucosylation on one hand but of sialylation or antennal ␣4-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 CaCl 2 , and 3 mg of thermo-lysin 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 G 2 5, 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:Nglycosidase 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

Sea cucumber N-glycome
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 (43). All N-glycan fractions were then pyridylaminated as described previously (44). 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 pentosecontaining 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 l, and examined by MALDI-TOF MS, using 6-aza-2-thiothymine as matrix (45). Sample and matrix solutions (1 l each) were sequentially spotted and dried under a vacuum. To enhance formation of [MϩH] ϩ or [M-H n ϩNa n-1 ] Ϫ ions, either 1 l of 20 mM ammonium sulfate or 1 l 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 Ϯ0.6%). 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 (46). For further details, refer to the supporting information.

Sea cucumber N-glycome
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 (Man 3-9 GlcNAc 2 ) derived from white beans; the order of elution of the standards was confirmed by MALDI-TOF MS of collected calibrant fractions (43).

Structural elucidation using exoglycosidases and chemical treatment
The following glycosidases were employed: recombinant Aspergillus niger ␤3/4-galactosidase (prepared in-house (47)); Xanthomonas manihotis ␤3-galactosidase (New England Biolabs); Bacillus fragilis ␤4-galactosidase (New England Biolabs); bovine kidney ␣-fucosidase (Sigma-Aldrich); almond ␣3/4-fucosidase (New England Biolabs); jack bean ␣-mannosidase (Sigma-Aldrich); purified recombinant Bacteroides xylanisolvens BxGH99 ␣2-endo-mannosidase, which catalyzes removal of a disaccharide from Glc 1 Man 9 GlcNAc 2 but not from unglucosylated Man 9 GlcNAc 2 (48); recombinant Aspergillus saitoi ␣2-mannosidase (Prozyme); and Streptococcus pneumoniae ␣3-sialidase S (New England Biolabs). In general, 10% of an HPLC fraction (1 l) was incubated overnight at 37°C with 0.8 l of 100 mM ammonium acetate (pH 5.0) and 0.2 l of a glycosidase (see above). For removal of phosphate-or ␣3/4-linked fucose, 30% of an HPLC fraction (3 l) was dried under a vacuum and incubated overnight on ice with 3 l of 48% (w/v) hydrofluoric acid (HF) prior to drying again. For removal of sulfate, 30% of an HPLC fraction (3 l) was dried under a vacuum and incubated for 4 h at 37°C with 20 l 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 ␤4-galactosylated Hex 6 HexNAc 3 standard was generated by treatment of a Man 5 GlcNAc 3 structure with bovine milk galactosyltransferase (Fluka) in the presence of UDP-Gal and Mn(II) ions (49). 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 ␣4 fucosylation, ␤3 galactosylation, 4-linked sulfation of galactose, ␣3 sialylation of galactose, ␣6 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 Table S2. The simplified evolutionary tree (left panel, based on Vaughn et al. (53)) 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 (24)), 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).