Glycan Profiling of Gel Forming Mucus Layer from the Scleractinian

ARTICLE pubs.acs.org/Biomac

Glycan Profiling of Gel Forming Mucus Layer from the Scleractinian Symbiotic Coral Oculina arbuscula Bernadette Coddeville,†,‡ Emmanuel Maes,†,‡ Christine Ferrier-Pag
es,§ and Yann Guerardel*,†,‡ †

Universite de Lille1, Unite de Glycobiologie Structurale et Fonctionnelle, UGSF, F-59650 Villeneuve d'Ascq, France CNRS, UMR 8576, F-59650 Villeneuve d'Ascq, France § Centre Scientifique de Monaco, c/o Musee Oceanographique, Avenue Saint Martin, MC-98000 Monaco ‡

ABSTRACT: The gel forming mucus layer surrounding scleractinian corals play fundamental functions in the maintenance of a favorable microenvironment required for the survival of these organisms. In particular, it harbors a rich partially speciesspecific symbiotic community through yet poorly understood molecular interactions. However, removal or contamination of this community by exogenous bacteria is closely linked to the worldwide bleaching events that are presently devastating coral colonies. The present study investigates the structure of major high molecular weight glycoconjugates that are responsible for both rheological properties of mucus and sugar-protein interactions with microbial communities. We demonstrated that it is composed by two distinct types of sulfated macromolecules: mucin type glycoproteins densely substituted by short unusual O-linked glycans and repetitive polysaccharides.

’ INTRODUCTION The surface of scleractinian corals is covered by a thick layer of mucus, which represents an important fraction of the corals’ energetic demand.1 Mucus was shown to have several functions, from sediment removal2 to nutrition and protection against physical, chemical and biological harm, such as desiccation, variations in temperature and salinity,1 or microbial diseases.3 When released into reef waters, mucus significantly contributes to the functioning of the reef ecosystem because it is of a high nutritional value for many planktonic and benthic organisms4,5 and is degraded and recycled into nutrients by bacteria.6
8 Furthermore, it has been demonstrated that some corals, such as Montastrea cavernosa, Diploria strigosa, Porites astreoides, or Galaxea fascicularis harbor in their tissue and mucus layer, a rich bacterial-cyanobacterial community that may develop symbiotic interactions.9
11 Microorganisms may indeed be involved in a vast array of functions such as nitrogen fixation, food supply, or protection against pathogenic bacteria.10,12 It is noteworthy that the bacterial community harbored by the mucus layer of healthy coral is different from the one in the surrounding water, which clearly demonstrates the impact of the microenvironment of mucus layer on colonization.13 The worldwide coral bleaching events observed over the last two decades was clearly associated to a shift in this microbial community toward dominance of the pathogenic bacteria, in particular, harmful Vibrio species, within the mucus layer.12
14 Despite the fundamental role that this layer plays in coral physiology, and as nutrient and energy carrier, limited information is presently available on the molecular composition of mucus.3,15,16 A recent study however clearly established the presence, within the r 2011 American Chemical Society

mucus isolated from a wide range of coral species, of high molecular mucin-type glycoproteins with physicochemical properties very close to those isolated from higher vertebrates.17 These components were shown to exhibit concentration-dependent gel formation properties. Independent composition analyses of mucus isolated from different coral species showed that it contains different amount of partially species-specific monosaccharides, including arabinose (Ara), xylose (Xyl), fucose (Fuc), mannose (Man), galactose (Gal), N-acetyl-glucosamine (GlcNAc), and N-acetyl-galactosamine (GalNAc) residues, associated to high molecular weight glycoproteins.10,15,18,19 However, the detailed structures of the oligosaccharides bearing these components were only partially established in a single coral species as neutral O-linked glycans that were linked to high molecular mucin-type glycoproteins through a very unusual mannose residue.20 Furthermore, although sulfated components were repetitively observed in several coral species, the structure of these major components have never been established so far. It has been postulated that differences in the mucus composition may control microbial community composition,21 but the exact components that exert the control have not been entirely established. Indeed, the relationship between carbohydrate composition of mucus and bacterial community has so far been mostly examined from the point of view of nutrient supplied in the water column8 but seldom from the aspect of hostsymbionts/pathogen molecular mechanisms. By analogy, mucus Received: December 23, 2010 Revised: March 7, 2011 Published: April 25, 2011 2064

dx.doi.org/10.1021/bm101557v | Biomacromolecules 2011, 12, 2064–2073

Biomacromolecules layers in superior eukaryotes are known to be major players into the regulation of microbes-host interactions through a complex interplay between carbohydrate moiety of host endothelial glycoproteins and microorganisms surface lectins.22
25 In this regard, coevolution of host
pathogen interplays is now recognized as a major driving force toward the building up of the exquisite species-specificity displayed by complex carbohydrate structure.26,27 Consequently, many pathogenic or symbiotic microorganisms developed a rather strict host specificity that is partially controlled by their capacity to interact at the molecular level with the host surface carbohydrate ligands. To our knowledge, a single study established a direct relationship between colonization by pathogenic bacteria and Oculina patagonica mucus layer through a protein
carbohydrate interaction involving D-Gal residues present in the coral mucus layer.28 However, considering the staggering structural diversity that complex glycans exhibit in nature,29 the inhibition of interaction using methyl-β-D-galactopyranoside barely provides any information on the fine structure of the oligosaccharide ligand that is recognized by bacteria. As a consequence, to identify the mosaic of glycan epitopes that may be used by micro-organisms as specific ligands to colonize mucus layer, we propose in the present report to explore the glycan diversity associated to a single model coral species, Oculina arbuscula. By doing so, we hope to provide novel insights into the study of the relationships existing between microbiota and corals by identifying potential molecular targets for micro-organisms.

’ MATERIALS AND METHODS Growing of Corals. Several small colonies of Oculina arbuscula were sampled along the coasts of Carolina (Atlantic Ocean), brought back to the laboratory, and maintained in three open-flow 20 L aquaria at a temperature of 24 °C and light intensity of 150 μmol photons m
2 s
1 (photoperiod: 12 h dark/12 h light). These temperature and light levels are the most appropriate to guarantee long-term maintenance of this coral in culture, with no stress that might induce zooxanthellae expulsion or tissue loss. Seawater renewal rate was 5 L/h and water was sandfiltered so that it contained few zooplankton particles. Light intensity was set to the required level, using metal halide lamps and neutraldensity shade screens, and was measured using a LI-COR data logger (LI-1000) with a spherical quantum sensor. Temperature was monitored daily and was maintained at 24 °C ( 0.5 °C using thermostatregulated aquarium heaters (Visy-Therm, 300W). Colonies were suspended in aquaria, separated from the sediment and the other coral species. Aquaria were regularly cleaned so that no filamentous algae or cyanobacteria could grow on the walls or on the sediment. Collection of Material. Several individual colonies of O. arbuscula were suspended upside down above a beaker and the mucus collected by a gentle rinsing with seawater. The mucus obtained was transferred during 24 h in a Spectra/Por 3 dialysis membrane (Spectrum Laboratories, Inc., Breda, The Netherlands), incubated into 5 L of distilled seawater then milli-Q water for desalting. The mucus was then freeze-dried. Amino Acids and Sugar Analysis. The amino acid composition analysis was performed as previously described30 and the amino acids were analyzed by HPLC (Spectra Physics 8100) in reverse phase with Water PicoTag column (3.9  150 mm). Elution was performed using a binary gradient of sodium acetate 0.1 M of triethylamine and acetonitrile, the flow rate was 1 mL/min during 17 min at 45 °C. The monosaccharide composition analysis was performed according to Zanetta and collaborators31 after methanolysis and analysis of the liberated O-methyl-glycosides as heptafluorobutyrate derivatives. The volatile derivatives were separated by gas
liquid chromatography on

ARTICLE

Carlo Erba GC 8000 Top chromatograph equipped with a 25 m  0.25 mm CP-Sil5 CB low bleed/MS capillary column (Chrompack, Les Ullis, France) coupled to a Finnigan Automass II mass spectrometer. Sulfate Content. The sulfate content was measured by HPAEC. Sulfate contained in the mucus was released by hydrolysis with 1 M HCl (500 μL) for 5 h at 100 °C and then HCl was evaporated under a stream of nitrogen.32 The residue was dissolved in 1 mL of Milli-Q quality water (Millipore Corp., Milford, MA). A total of 30 μL of this solution were directly injected into a Dionex BioLC system equipped with an IonPac AS4A column (250  4 mm), an anion micromembrane suppressor, and a CDM 2 conductivity detector. The column was eluted with 0.04 M NaOH at a flow rate of 2 mL/min, and the separated anions were measured by conductivity detection with a 30 microsiemens sensitivity. The resulting chromatographic data were integrated and plotted with Chromeleon software, version 6.40 (Dionex Corporation). There was a linear relationship between conductivity and sulfate concentration up to 35 μg/mL. A standard curve was constructed with K2SO4, solutions (6.25, 12.5, 25, and 35 μg/mL) to measure the sulfate released from coral mucins. Reduction and Alkylation. A total of 3 mg of sample in 5 mL of 200 mM Tris/HCl, pH 8.0, were denaturated by 6 M guanidine hydrochloride. After addition of 10 mM dithiotreitol, the sample was flushed with nitrogen and incubated at 50 °C for 5 h. Ten mM final iodoacetamide was then added. The reaction was carried out in the dark at room temperature overnight under a N2 atmosphere. After extensive dialysis of the reaction mixtures against water, the sample was freeze-dried. Isolation of Oligosaccharide-Alditols. The mucus was submitted to a reductive β-elimination for 72 h at 37 °C in 100 mM NaOH containing 1 M NaBH4. The reaction was stopped by the addition of Dowex 50  8 (25
50 mesh, Hþ form) at 4 °C until pH 6.5. After filtration on glass wool and evaporation to dryness, boric acid was eliminated by repetitive codistillation as its methyl ester in presence of methanol. The oligosaccharides alditols were submitted to a cationic exchange chromatography on Dowex 50  2 (200
400 mesh, Hþ form) in water to separate acid and neutral oligosaccharides. Oligosaccharide
alditols were desorbed in water and pH was adjusted to 7 with ammonium hydroxide. The fractions containing sugars were purified on Bio-Gel P2 column (Biorad; 15  1 cm) and freeze-dried for further analysis. Mass Spectrometry Analysis. MALDI-TOF. MALDI-TOF mass spectra were acquired on a voyager Elite DE-STR mass spectrometer (Perspective Biosystems, Framingham, MA) in the reflecton positive or negative mode by delayed extraction using an acceleration mode of 20 kV, a pulse delay of 200 ns, and grid voltage of 66%. Samples were prepared by mixing directly on the target 1 μL of oligosaccharide solution (1
5 pmol) with 1 μL of 2,5-dihydroxybenzoic acid matrix solution (10 mg/mL in CH3OH/H2O, 50/50, v/v). Between 50 and 100 scans were averaged for every spectrum. Electrospray Mass Spectrometry (Nano-ESI-MS/MS). All analyses were performed using a Q-STAR pulsar quadrupole time-flight (QTOF) mass spectrometer (Applied Biosystems/MDS Sciex, Toronto, Canada) fitted with a nanoelectrospray ion source (Protana, Odense, Denmark). Glycans dissolved in a solution of 50% methanol and 1% formic acid (1 pmol/μL) for native oligosaccharides and in a solution of 80% methanol and 1% formic acid (0.5 pmol/μL) for methylated oligosaccharides were sprayed from gold-coated “medium length” borosilicate capillaries (Protana). A potential of 800 V or
800 V according to the positive or negative mode was applied to the capillary tip. For the generation of MS/MS data, the precursor ion was selected by the quadrupole, and was subsequently fragmented in the collision cell using nitrogen at a pressure of 5.3  10
5 Torr and appropriate collision energy. The CID spectra were recorded by the orthogonal TOF analyzer over the mass range m/z 50
1000. Methylation. Native oligosaccharide-alditols were permethylated according to Ciucanu and Kerek.33 Briefly, compounds were incubated 2 h in a suspension of 200 mg/mL in NaOH in dry DMSO (100 μL) and 2065

dx.doi.org/10.1021/bm101557v |Biomacromolecules 2011, 12, 2064–2073

Biomacromolecules

ARTICLE

Table 1. Amino Acids Composition of a Total Oculina arbuscula Mucus Extract (Batch 1)a amino acids

Figure 1. Total of 8% PAGE of total mucus extracted from Oculina arbuscula stained with (A) carbohydrate specific PAS and (B) polyanion susceptible Azure A stain; *interface between concentration and separation gels. iodomethane (100 μL). After derivatization, the reaction products were purified on C18 Sep-Pak cartridges (Waters Ltd.) using stepwise elution of 0, 25, 50, and 80% aqueous acetonitrile, 0.1% TFA. The phases containing acetonitrile were pooled and concentrated under nitrogen for further analysis. Linkage Analysis. For linkage analysis, the methylated products were extracted trice in chloroform and washed with water. Lyophilized product was subjected to methanolysis in 500 μL of 0.5 M HCl in anhydrous methanol at 80 °C for 20 h. After drying, products were peracetylated in 200 μL of acetic anhydride and 50 μL of pyridine overnight at room temperature. The reagents were evaporated, and the sample was dissolved in chloroform before analysis in GC/MS. NMR Analysis. 1H and 13C NMR spectra were recorded on a Bruker 9.39 T Avance spectrometer (Centre commun de mesure RMN de Lille) with 1H resonance at 400.33 MHz and 13C at 100.66 MHz. Spectrometer was equipped with a 5 mm Broad Band Inverse probehead with z-gradients. Samples were twice exchanged against 2H2O (99.97% of deuterium atoms, EurisoTop, St Aubin, France) and put in classical 5 mm BB NMR tube. Experiments were achieved using Bruker pulse sequences from their bank pulse programs and parameters; delays and pulses, have been optimized for each experiment. The chemical shifts were calibrated using acetone as internal standard (δ 1H 2.225 and δ 13C 31.55) at 300 K.

’ RESULTS AND DISCUSSION Nature of Glycoconjugate Material. Polyacrylamide gel electrophoresis analysis of total mucus showed two well resolved bands susceptible to carbohydrate specific PAS staining (Figure 1A). Both exhibited a low electrophoretic mobility establishing that they have apparent molecular weights superior to 500 kDa, in agreement with standard molecules. The upper band barely penetrated the separation gel and concentrated at the interface between the concentration and the separation gels, whereas the lower band slightly migrated within the separation gel well above 175 kDa standard molecule. Both bands were also susceptible to Azure A stain (Figure 1B), which established that these components are densely substituted by anionic groups. Staining of gels loaded with identical quantities of total mucus using coomassie brilliant blue staining did not reveal any protein band of lower molecular weight (data not shown). Although these experiments do not rule out the presence of other proteins in mucus, they establish that the two high molecular weight glycoconjugates observed are the two single most abundant components of the mucus.

a

Ser

22.0

Lys

Thr

9.5

Ileu

5.4 3.1

Asp

9.5

Phe

3.0

Gly

8.0

Arg

2.5

Glu

7.8

His

1.7

Ala Val

6.8 6.7

Met Tyr

1.4 0.9

Pro

5.7

Cys

0.6

Leu

5.4

Values are expressed in percent of total amino acids.

Table 2. Monosaccharides and Sulfate Contents of Oculina arbuscula Mucus Extracted from Three Individual Coral Coloniesa batch 1

batch 2

batch 3

Fuc

1.0% (0.3)

1.6% (0.6)

0.9% (0.3)

Man

3.1% (0.90)

2.2% (0.80)

3.5% (1.1)

GlcNAc

4.7% (1)

6% (1)

5% (1)

total

8.8%

9.8%

9.4%

sulfate

3.2%

3.6%

3.5%

a

Values are expressed in percent (weight) of total lyophilized mucus. Ratios of individual monosaccharides are given in parentheses.

Then, the amino acids and carbohydrate contents of the crude material were determined. Analyses revealed the prevalence of hydroxylated amino acids over other amino acids (serine (Ser) þ threonine (Thr) > 30%) as well as high quantity of glycine (Gly) and asparagine (Asp) (Table 1). High ratio of these three amino acids suggests the presence of mucin type glycoproteins. Total carbohydrates (Table 2) represent 8.8% of the crude material and consist of 4.1% neutral sugars and 4.7% hexosamines. Associated monosaccharides exclusively consist of fucose, mannose, and N-acetylglucosamine (Table 2). Surprisingly, for a postulated mucin-type glycoprotein, no N-acetylgalactosamine (GalNAc) residue was observed, whereas it is known as a major component of this type of glycoprotein in vertebrates.34 Although unusual, such a monosaccharide composition is however rather similar to the one observed in other coral species also characterized by the almost absence of GalNAc, in particular Fungia fungites that contains the similar major three monosaccharides Fuc, Man, and GlcNAc.18 In agreement with sensitivity to polyanionic stains of glycoconjugates separated by SDS-PAGE, sulfate was also detected in large amounts within the total crude extracts (about 3%). Altogether, these preliminary analyses established that mucus contains high molecular weight sulphated glycoproteins with physicochemical characteristics similar to those of mucin-type glycoproteins, albeit the lack of GalNAc. Isolation and Studies of Oligosaccharide Moiety. The coral mucus was submitted to alkaline reductive elimination and the glycans released were desalted by ion-exchange and gel filtration chromatography. The yield of saccharides release from the protein backbone was estimated to 50% by comparing monosaccharide content before and after chemical release. Oligosaccharides were first characterized from total mixture by mass 2066

dx.doi.org/10.1021/bm101557v |Biomacromolecules 2011, 12, 2064–2073

Biomacromolecules

ARTICLE

Figure 2. MALDI-TOF mass spectra of native oligosaccharides isolated from Oculina arbuscula coral glycoprotein in positive mode (A) and in negative mode (B); *monosulfated and **disulfated oligosaccharides. Cpd1 was not included in the spectra because of the interfering intense signals originating from the matrix.

Figure 3. Nano-ESI mass spectra of permethylated oligosaccharides isolated from Oculina arbuscula coral glycoprotein in positive mode (A) and in negative mode (B); *monosulfated and **disulfated oligosaccharides; underscored, monohydroxylated oligosaccharides originating from desulfation of their corresponding monosulfated oligosaccharides.

spectrometry using the positive and the negative ion mode, in both native and methylated forms. Then, acidic and neutral oligosaccharides where separated by anion exchange chromatography for further analyses. MALDI-MS spectra of native oligosaccharides and nano-ESI of permethylated oligosaccharides are shown in Figures 2 and 3 and derived compositions are summarized in Table 3.

Monosaccharide compositions of individual molecules deduced from MALDI-MS analysis of native glycans established the presence of several families of short glycans. In particular, experiments ran in positive mode showed seven peaks associated to neutral glycans (cpds 1, 2, 5, 7, 9, 12, 14) and six tentatively attributed to mono- and disulfated glycans (cpds 3, 4, 10, 11, 15, 16; Figure 2A). As previously shown,35,36 sulfated glycans are 2067

dx.doi.org/10.1021/bm101557v |Biomacromolecules 2011, 12, 2064–2073

Biomacromolecules

ARTICLE

Table 3. Assignments of Ions Observed in MALDI Spectra of Native and Permethylated A, Neutral and B, Sulfated Oligosaccharides Isolated from Oculina arbuscula Coral Glycoproteinsa A native cpds

permeth. cpds

þ

assignments

þ

MS

MS

cpds

neutral cpds

390

488

Fuc1HexNAc1

408

534

Hex1HexNAc1 þ ol

2

536

662

Fuc2HexNAc1

5

554 611

708 779

Fuc1HexNAc1Hex1 þ ol Hex1HexNAc2 þ ol

7 9

682

836

Fuc3HexNAc1

12

Fuc2HexNAc1Hex1 þ ol

14

700

1

B native cpds

permeth. cpds

sulfated þ

sulfated


þ

MS

MS

MS

492 510

446 464

576 622

acidic cpds

cpds

desulfated


MS

MSþ

530 576

474 520

Fuc1HexNAc1 þ SO3 Hex1HexNAc1 þ ol þ SO3

3 4

548

618

Fuc1HexNAc1 þ 2SO3

6

566

664

Hex1HexNAc1 þ ol þ 2SO3

8

638

592

750

704

648

Fuc2HexNAc1 þ SO3

10

656

610

796

750

694

Fuc1HexNAc1Hex1 þ ol þ SO3

11

784

694

792

Fuc2HexNAc1 þ 2SO3

13

712

838

Fuc1HexNAc1Hex þ ol þ 2SO3

15

738

878

Fuc3HexNAc1 þ SO3

16

Neutral oligosaccharides are detected as [M þ Na]þ adducts in positive mode, monosulfated oligosaccharides as [M þ 2Na
H]þ in positive mode and [M
H]
in negative mode and disulfated oligosaccharides as [M þ 2Na
H]þ in positive mode and [M þ Na
2H]
in negative mode. In native form, each detected neutral compound potentially originates from genuine neutral O-glycan or from laser induced desulfation of sulfated O-glycan, as demonstrated by the presence of both per-methylated and mono-hydroxylated methylated equivalent compounds.35,36 a

mainly observed as [M þ 2Na
H]þ (M þ 45) adducts, whereas neutral glycans are observed as [M þ Na]þ (M þ 23) adducts in positive mode on the same spectrum. The presence of all monosulfated glycans was confirmed in negative mode by observation of a full set of [M
H]
adducts (Figure 2B). Furthermore, the use of negative mode permitted to identify three additional molecules assigned to disulfated glycans that exhibited [M þ Na
2H]
adducts (cpds 6, 8, 13). Observation of neutral molecules presenting the same compositions that sulfated ones raised the possibility that signal assigned to neutral glycans originated in fact from the partial desulfation of acidic glycans during MALDI-TOF analysis. Further analysis of glycans in mixture following permethylation confirmed the presence of genuine neutral glycans by observing the permethylated equivalents of all neutral compounds as [M þ Na]þ adducts in positive mode, except cpd 14. Furthermore, analysis of permethylated compounds in positive mode showed an additional set of values at m/z 474, 520, 648, 694, which correspond to the monohydroxylated equivalents (M-14 mu) of compounds 3, 4, 10, and 11, respectively. As previously demonstrated, these are the neutral equivalents of permethylated sulfated oligosaccharides in which a
CH3 group has been replaced by a hydroxyl group, originating from an on-target desulfation process.35,36 Furthermore, mass spectrometry analysis of glycans following permethylation permitted to differentiate substitutions by sulfate groups

from eventual phosphate groups (Figure 3). Indeed, although phosphate and sulfate groups cannot be readily differentiated by mass spectrometry of native molecules, phosphate groups are monomethylated by NaOH/ICH3, whereas sulfate groups are not, as previously established37 and further confirmed in the present study by permethylation of phosphorylated commercial monosaccharides (data not shown). Then, the presence of both neutral and sulfated glycans was also confirmed by separation of each family on anionic exchange chromatography and profiling by mass spectrometry (data not shown). Altogether, MS profiling of released glycans in native and permethylated forms permitted to identify 16 signals associated to neutral and sulfated oligosaccharides. Based on calculated monosaccharide composition of individual molecules, two families of oligosaccharides were clearly differentiated. Members of the first family (cpds 2, 4, 7, 8, 9, 11, 14, 15) are composed of variable amounts of HexNAc, Hex, and deHex residues that all include a reduced monosaccharide, as usually observed for O-glycans released by reductive β-elimination (Table 3). The members of the second family (cpds 1, 3, 5, 6, 10, 12, 13, 16) are exclusively composed of HexNAc and deHex residues and surprisingly are never reduced. Together, with monosaccharide identification by gas chromatography, mass spectrometry analyses established the presence of a family of reduced oligosaccharides composed of GlcNAc, Man, and Fuc 2068

dx.doi.org/10.1021/bm101557v |Biomacromolecules 2011, 12, 2064–2073

Biomacromolecules

ARTICLE

Figure 4. MS/MS spectrum of permethylated neutral reduced oligosaccharide (cpd 7) at m/z 708 ([M þ Na]þ), recorded in positive ion mode.

and of a family of nonreduced oligosaccharides composed of GlcNAc and Fuc. Glycan sequences were established by MS/MS fragmentation of individual signals observed in negative and positive modes for sulfated glycans and in positive mode for neutral glycans. Furthermore, both native and permethylated equivalents of each signal were fragmented when possible. These analyses revealed the presence of numerous isobaric structures presenting identical m/z values that could only be discriminated owing to specific fragment ions. Fragmentation analyses demonstrated that all major reduced O-glycans were characterized by the presence of a hexositol residue in reducing position. Observation in positive mode of Y-type [M þ Na]þ fragment ions at m/z 205 or 275 and B-type ions at M-182 or M-252 for native or permethylated glycans established the presence of unsulfated Hex-ol residues, as exemplified by fragmentation of compound 7 (Table 4 A; Figure 4). Similarly, presence of monosulfated Hex-ol residue was typified by the observation of Y-type [M þ Na]þ fragment ion at m/z 285 for native glycans or Y-type [M þ 2Na
H]þ fragment at m/z 363 for permethylated derivatives, as well as B-type ions at M-262 or M-340 for native and permethylated derivatives, respectively. Then, the presence of sulfated Hex-ol was confirmed in negative mode by the observation of Y-type [M
H]
fragment ions at m/z 261 or 317 for native or permethylated glycans (Table 4A). Isobaric structures seemed to differ mainly by the position of the sulfate groups that may substitute either Hex-ol, HexNAc, or deHex residues. For example, ES-MS/ MS analysis in positive mode of permethylated signal at m/z 622.0 from cpd 4 generated two sets of fragments attributable to
[S]Hex-ol and [S]HN residues, which established the presence of two isobaric molecules with HN-[S]Hex-ol and [S]HN-Hexol sequences (Table 4A). Altogether, MS-MS fragmentation patterns established that all reduced oligosaccharides had a similar architecture characterized by a linear (deHex)0
1-HexNAc-Hex-ol sequence or a branched HexNAc-[deHex]0
1-Hexol sequence. Similarly, MS/MS analyses permitted to establish that nonreduced glycans are all based on a similar sequence made of a linear stretch of deHex substituted by a single HexNAc residue in terminal non reducing position. Although these glycans were nor reduced nor tagged in reducing position, thus, at first preventing the determination of their reducing end, their

orientation could be unambiguously established owing to the fragmentation pattern of their permethylated derivatives. Indeed, generation of a single cleavage position between HexNAc and deHex residue in all fragmented oligosaccharides clearly established that the HexNAc bond is exclusively broken through a B/Y type cleavage (Figure 5). This originates from the so-called A-type cleavage of the parent ion that produces in HexNAc containing oligosaccharides a predominant oxonium-type fragment ion widely used for sequencing.38 As for reduced glycans, tandem mass spectrometry permitted one to distinguish numerous isobaric structures exclusively differing by the substitution patterns of their sulfate groups. Indeed, all oligosaccharide cores were observed in either unsulfated (1, 5, 12), mono(3, 10, 16), or even disulfated (6, 13) forms. Furthermore, when present, sulfate groups could substitute either deHex, HexNAc or even both residues at different positions. Altogether, MS/MS analyses permitted to clearly identify 15 nonreduced glycans based on a GlcNAc-(Fuc)1
3 core (Table 4 B) and 12 reduced glycans based on GlcNAc-[Fuc]0
1-Man-ol core sequence, that all originate from the reductive β-elimination of total mucus, all of them being present either as neutral or sulfated glycans. NMR analyses conducted on total coral mucus generated very complex spectra characteristic of glycan mixtures, which confirmed the diversity of glycan motifs observed by mass spectrometry. However, distinctive glycosylation patterns could be inferred from homo- and heteronuclear experiments. In particular, 1H
1H COSY and 1H
13C HSQC-NMR experiments established the presence of a very heterogeneous family of R1
2 substituted fucoses as well as nonreducing terminal fucose residues, owing to the observation of H1/H2/H5 spin systems at 5.33
5.35/3.94
4.02/4.10
4.32 ppm and 5.23
5.30/ 3.8
3.8/4.61 ppm, respectively (Figure 6). The presence of these residues was confirmed by GC/MS analysis of partially methylated and acetylated methyl-glycoside derivatives. Based on their EI-MS fragmentation patterns, two major signals were identified as permethylated and 1,3,4-O-Met-2-O-Ac-fucose, in accordance with the presence of terminal and C2 substituted fucose residues (data not shown). Then, the observation of very deshielded Fuc H3/C3 and H4/C4 set of signals at 4.60/80.2 and 4.90
4.94/78.3
80.2 ppm clearly established that some 2069

dx.doi.org/10.1021/bm101557v |Biomacromolecules 2011, 12, 2064–2073

Biomacromolecules

ARTICLE

Table 4. Fragmentation Ions Generated by MS/MS Analyses of Native and Permethylated (A), Reduced and (B), Nonreduced Oligosaccharides Generated by Reductive β-Elimination of Oculina arbuscula Mucusa A: Reduced Glycans ES/MS-MS fragments cpd

proposed sequences

mode

2

GlcNAc-Man-ol

þ

4

(S)GlcNAc-Man-ol

þ

native B, 226; C, 244; Y, 205; Z, 187

B, 370*; Y, 275




7 8

11

permethylated

97; B, 282*; Y, 181

97; B, 324*; Y, 252 B, 282 ; Y, 363*; sec, 243 97; Y, 317*; Z, 299*

GlcNAc-(S)Man-ol

þ


97; B, 202; Y, 261 ; Z, 243*

Fuc-GlcNAc-Man-ol

þ

B, 372 ; Y, 205, 408

B, 456 ; C, 229; Y, 275, 520; Z, 502

GlcNAc-(Fuc)Man-ol

þ

B, 226 ; Y, 351

Y, 449; sec, 261

(S)(S)GlcNAc-Man-ol




97; B, 384**; Y, 181

(S)GlcNAc-(S)Man-ol




97; B, 282*; 261*

GlcNAc-(S)(S)Man-ol




97; B, 220; C, 202; Y, 363**; Z, 344**

Fuc-GlcNAc-(S)Man-ol

þ [M þ 2Na
H]þ þ [M þ Na]þ


B, 456; Y, 363*, 608; Z, 590*; sec, 268 B, 169, 372; C, 390; Y, 285*, 488*; Z, 470* 97; Y, 261*, 464*; Z, 243*

þ [M þ 2Na
H]þ

Fuc-(S)GlcNAc-Man-ol

B, 544*; Y, 275, 608*; Z, 590*; sec, 544*

þ [M þ Na]þ

B, 169, 452*; C, 470* ; Y, 205, 488*




97; B, 428*; C, 446*; Y, 464*, 181; Z, 163; sec, 282

þ [M þ Na]þ

B, 169, 306*; C, 324*; Y, 488*, 351; Z, 470*




97; B, 282*, 428*; Y, 446*

þ


97; B, 225*; Y, 407*

(S)GlcNAc-[Fuc]Man-ol

Y, 449, 608*; Z, 590*; sec, 261

GlcNAc-[(S)Fuc]Man-ol

B, 317*; Y, 520, 537*

B: Unreduced Glycans ES/MS-MS fragments cpd

proposed sequences

mode

native

permethylated

1

GlcNAc-Fuc

þ

B, 226; C, 244; Y, 187; Z, 169

B, 282; Y, 229

3

(S)GlcNAc-Fuc

þ [M þ 2Na
H]þ

B, 306*; C, 324*; Y, 187

B, 370*; Y, 229




97; B, 282*; Y, 163

97; B, 324*

GlcNAc-(S)Fuc

þ [M þ 2Na
H]þ

B, 226; C, 244

B, 282; Y, 317




97; B, 202; Y, 243*; Z, 225*

97; Y, 271*; Z, 253* B, 282, 456; C, 474; Y, 229, 403

5

GlcNAc-Fuc-Fuc

þ

B, 226, 372; C, 244, 390; Y, 333, 187; Z, 169

6

(S)(S)GlcNAc- Fuc (S)GlcNAc-(S)Fuc





97; B, 384**; Y, 163 97; B, 282* ; Y, 243* ; Z, 225*

GlcNAc-(S)(S)Fuc




97; B, 202; Y, 345**

10

(S)GlcNAc- Fuc-Fuc

þ [M þ 2Na
H]þ

B, 328*, 474*; C, 346*, 492*; Y, 187, 333; Z, 169, 315

þ [M þ Na]þ

B, 306*, 452*; C, 324*, 470*; Y, 187, 333; Z, 169, 315

GlcNAc-Fuc-(S)Fuc




97; B, 282*, 428*; C, 446*; Y, 163, 309

þ [M þ 2Na
H]þ

B, 226, 244; C, 244, 390; Y, 435*, 289*; Z, 417*, 271*

þ [M þ Na]þ

B, 226, 244; C, 244, 390; Y, 413* 97; Y, 243*, 389*; Z, 371* B, 226, 372, 518; C, 390, 536; Y, 479, 333, 187; Z, 315

B, 370*, 544*; C, 562*; Y, 403 97; B, 324*

12

GlcNAc-Fuc-Fuc-Fuc


þ

13

(S)(S)GlcNAc-Fuc-Fuc




97; B, 412**, 586**

(S)GlcNAc-Fuc-(S)Fuc




97; B, 324*; C, 516*; Y, 271*, 445*; Z, 253*

GlcNAc-(S)Fuc-(S)Fuc




(S)GlcNAc-Fuc-Fuc-Fuc

þ [M þ Na]þ

B, 306*, 452*, 598*; C, 324*, 470*, 616*; Z, 333, 479




97; B, 282*, 428*, 574*; C, 446*, 592*

GlcNAc-Fuc-Fuc-(S)Fuc

þ [M þ Na]þ

B, 226, 372, 518; C, 244, 390, 536; Y, 413*, 559*




97; Y, 243*, 389*, 535*; Z, 225*, 371*

16

97; Y, 271*, 445*; Z, 253* B, 456, 630; C, 474, 648; Y, 577, 403

97; C, 516* ; Y, 271*, 533*; Z, 253* 97; B, 324*, 498*, 672*; C, 516*, 690* 97; Y, 271*, 445*, 619*; Z, 253*, 427*

In positive mode, neutral glycans were fragmented from [MþNa]þ adducts, whereas sulfated glycans were fragmented from either [MþNa]þ or [Mþ2Na
H]þ as indicated. In negative mode, all fragments originated from [M-H]
parent ions. * mono-sulfated fragment, ** di-sulfated fragment. a

2070

dx.doi.org/10.1021/bm101557v |Biomacromolecules 2011, 12, 2064–2073

Biomacromolecules

ARTICLE

Figure 5. MS/MS fragmentation pattern of (A) permethylated neutral nonreduced oligosaccharide at m/z 662 ([M þ Na]þ; cpd 5) recorded in positive ion mode and (B) permethylated sulfated oligosaccharide at m/z 704 ([M
H]
; cpd 10) recorded in negative ion mode.

fucose residues were sulfated in C3 and C4 positions. The disparity of NMR signals associated to individual protons was attributed to the heterogeneity of sulfate substitution and monosaccharide positioning within the fucan polysaccharide sequences, as established in mass spectrometry. Finally, several sets of signals associated to β-GlcNAc were identified, which confirmed the identification of this monosaccharide by gas chromatography. Unfortunately, NMR experiments did not permit to unambiguously attribute signals associated to Mannitol residues because most of them are located in the crowded socalled “bulk region” of the spectra. However, GC/MS analysis of partially methylated and acetylated methyl-glycoside derivatives permitted to clearly identify two hexitol residues attributed to the reducing ends of short O-glycans released by reductive βelimination (data not shown). They differed by their substitution patterns, one being detected as monoacetylated in C2 position and the other one as diacetylated in C2 and C6 positions. Altogether, data collected from GC/MS and MS/MS experiments established that the major O-glycans possess either a linear sequence with a 2-linked reducing mannose or a branched structure with a 2,6-linked reducing mannose residue. However, our experiments did not exclude the presence of other minor

branching patterns like trisubstituted mannose residues, as demonstrated by the observation of molecules presenting a disulfated reduced mannose residue (cpd 8.) The fine structural analyses described here have been conducted on the mucus collected from three independent coral colonies, maintained under the same experimental conditions. They showed totally similar results in terms of monosaccharide composition and oligosaccharides sequences, demonstrating that overall glycosylation patterns of single individuals within the same species are similar. The structure of reduced oligosaccharides is highly reminiscent of those previously identified in mucus layer of the coral species Acropora formosa.20 Indeed, all O-linked oligosaccharides isolated from both species are characterized by the very unusual presence of mannose residues in terminal reducing positions instead of the classical GalNAc residue found in the vast majority of animal species, from nematodes to higher vertebrates.34,39,40 In addition, reducing mannose is always substituted by one or several GlcNAc residues in both species. However, contrarily to A. formosa, glycans from Oculina arbuscula contain fucose residues rather than arabinose residues. The present study also established that all O-glycans cores were differentially sulfated on every monosaccharide 2071

dx.doi.org/10.1021/bm101557v |Biomacromolecules 2011, 12, 2064–2073

Biomacromolecules

ARTICLE

Figure 6. 1H/1H COSY NMR analysis of total oligosaccharides isolated from Oculina arbuscula mucus. In upper panel, RFuc H-5/H-6 3JH,H correlations, and in lower panel, RFuc H-1/H-2 3JH,H correlations.

residues, which generates a staggering structural diversity from a very limited set of oligosaccharide sequences. More surprising, mass spectrometry analyses permitted one to identify a large number of nonreduced oligosaccharides in the product of reducing β-elimination. These oligosaccharides were further characterized by a combination of MS/MS and NMR as rather homogeneous polysaccharide with the proposed backbone GlcNAc(β1,2)Fuc(R1,2)Fuc(R1,2)Fuc that were differentially substituted by sulfate groups in C3 and C4 positions. Further reduction in alkali conditions following the purification process of oligosaccharides did not permit the reduction of the Fuc residues in the terminal reducing position of these oligosaccharides (data not shown), strongly suggesting that they do not exhibit any reducing end in native form. Considering that we observed oligosaccharides with reduced sulfated monosaccharides as well as unsulfated nonreduced oligosaccharides, it seemed unlikely that sulfation of reducing monosaccharide may prevent the reduction process. This was definitively ruled out by demonstrating that standard disulfated monosaccharides such as 3,6- and 4,6-disulfated GlcNAc residues were readily reduced in similar experimental conditions than used for coral glycans (data not shown). Altogether, these data support that unreduced oligosaccharides originated from the cleavage of a larger polysaccharide present in β-elimination products. These oligosaccharides may be generated by random in-source fragmentation of the most fragile glycosidic bonds, such as deHex bonds, of a polysaccharide during mass spectrometry analyses. Interestingly, sulfated fucose homopolymers are very common molecules in the extracellular matrix and egg mucus layers from marine invertebrates, including sea cucumbers, star fishes, and sea urchins.41,42 In these organisms, sulfated fucans play important structural roles as protective components

when part of the outer tunics, but may also exert specific biological function in gamete recognition within the egg mucus layers. In particular, they appear as species-specific inducers or the acrosomal reaction of sperm along the fertilization process of sea urchins.43 However, unlike other invertebrates, fucans described so far that are characterized by R1
3 and R1
4 linkages with sulfations in C2 or C4 positions, fucan domain of Oculina arbuscula polysaccharide is linked by R1
2 linkages and sulfated in C3 and C4 positions. Coral polysaccharide also share structural features with highly polymorphic polysaccharides (glyconectins) isolated from sponges surface mucus layer. In particular, the (SO3)GlcNAc-(Fuc)n motif was clearly identified in the sponges species Microciona prolifera and Cliona celata as SO3(3)GlcNAc(R1,3)Fuc-Fuc where they play important roles in cell
cell adhesion through carbohydrate
carbohydrate specific interactions.44
46 Irrespective of their biological origins, polyanionic polysaccharides and high molecular weight glycoproteins, including glycosaminoglycans, fucans, galactans, and mucins, are involved in the building of the extra cellular matrix and mucus layers surrounding eukaryotic cells.47,48 Charge is usually conferred to the glycoconjugates in a very specific manner by a mixture of sialic acids, uronic acids, sulfate, or phosphate groups.32,40,49 In the case of coral mucus, we established that the polyanionic character was essentially the result of a dense sulfation pattern on both glycoproteins and polysaccharide material. In conclusion, the structural analysis of oligosaccharides generated by alkaline hydrolysis of total mucus layer isolated from the scleractinian coral Oculina arbuscula permitted to identify 27 oligosaccharidic compounds, out of which 21 were sulfated. The structural features of these oligosaccharides established the presence of at least two different polymers: one mucin 2072

dx.doi.org/10.1021/bm101557v |Biomacromolecules 2011, 12, 2064–2073

Biomacromolecules type glycoprotein substituted by short unusual O-glycans linked to the protein backbone through mannose residues and a large heteropolysaccharide constituted by fucose and N-acetylglucosamine residues. Irrespective of their origin, most of these compounds were highly sulfated in a seemingly random fashion. Based on this glycan map, we now hope to identify endogenous ligands used by the resident microbiota or competing pathogenic bacteria to interact with the coral surface.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected].

’ ACKNOWLEDGMENT The 400 MHz facility was funded by the Centre Commun de Mesure RMN, Universite de Lille1, Villeneuve d’Ascq, France. The MS facilities were funded by the European community (FEDER), the Region Nord-Pas de Calais (France), the CNRS, and the University of Lille1. The authors thank Colette Brassart for technical help. ’ REFERENCES (1) Brown, B. E.; Bythell, J. C. Mar. Ecol.: Prog. Ser. 2005, 296, 291–309. (2) Hubbard, J. A. E. B.; Pocock, Y. P. Geol. Rundsch. 1972, 61, 598–626. (3) Ritchie, K. B. Mar. Ecol.: Prog. Ser. 2006, 322, 1–14. (4) Wild., C.; Woyt, H.; Huettel, M. Mar. Ecol.: Prog. Ser. 2005, 287, 87–98. (5) Huettel, M.; Wild, C.; Gonelli, S Mar. Ecol.: Prog. Ser. 2006, 307, 69–84. (6) Ferrier-Pag
es, C.; Gattuso, J. P.; Dallot, S.; Jaubert, J. Coral Reef 2000, 19, 103–113. (7) Ferrier-Pag
es, C.; Rottiera, C.; Berauda, E; Levy, O. J. Exp. Mar. Biol. Ecol. 2010, 390, 118–124. (8) Wild, C.; Mayr, C.; Wehrmann, L.; Sch€ottner, S.; Naumann, M.; Hoffmann, F.; Rapp, H. T. Mar. Ecol.: Prog. Ser. 2008, 372, 67–75. (9) Rohwer, F.; Seguritan, V.; Azam, F.; Knowlton, N. Mar. Ecol. Prog. Ser. 2002, 243, 1–10. (10) Tremblay, P.; Weinbauer, M. G.; Rottier, C.; Guerardel, Y.; Nozais, C.; Ferrier-Pages, C. J. Mar. Biol. Assoc. (United Kingdom), 2011, in press. (11) Lesser, M. P.; Falcon, L. I.; Rodriguez-Roman, A.; Enriquez, S.; Hoegh-Guldberg, O.; Iglesias-Prieto, R. Mar. Ecol.: Prog. Ser. 2007, 346, 143–152. (12) Bourne, D.; Iida, Y.; Uthicke, S.; Smith-Keune, C. ISME J. 2008, 4, 350–363. (13) Frias-Lopez, J.; Zerkle, A. L.; Bonheyo, G. T.; Fouke, B. W. Appl. Environ. Microbiol. 2002, 68, 2214–2228. (14) Mao-Jones, J.; Ritchie, K. B.; Jones, L. E.; Ellner, S. P. PLoS Biol. 2010, 8, e1000345. (15) Ducklow, H. W.; Mitchell, R. Limnol. Oceanogr. 1979, 24, 706–714. (16) Coffroth, M. A. Mar. Ecol.: Prog. Ser. 1984, 17, 193–199. (17) Jatkar, A. A.; Brown, B. E.; Bythell, J. C.; Guppy, R.; Morris, N. J.; Pearson, J. P. Biomacromolecules 2010, 11, 883–888. (18) Meikle, P.; Richards, G. N.; Yellowlees, D. Mar. Biol. 1988, 99, 187–193. (19) Wild, C.; Naumann, M.; Niggl, W.; Haas, A. Aquat. Biol. 2010, 10, 41–45. (20) Meikle, P.; Richard, G. N.; Yellowlees, D. J. Biol. Chem. 1987, 262, 16941–16947. (21) Allers, E.; Niesner, C.; Wild, C.; Pernthaler, J. Appl. Environ. Microbiol. 2008, 74, 3274–3278.

ARTICLE

(22) Saeland, E.; de Jong, M. A.; Nabatov, A. A.; Kalay, H.; Geijtenbeek, T. B.; van Kooyk, Y. Mol. Immunol. 2009, 46, 2309–2316. (23) Kobayashi, M.; Lee, H.; Nakayama, J.; Fukuda, M. Glycobiology 2009, 19, 453–61. (24) Scharfman, A.; Degroote, S.; Beau, J.; Lamblin, G; Roussel, P.; Mazurier, J. Glycobiology 1999, 9, 757–764. (25) Couceiro, J. N.; Paulson, J. C.; Baum, L. G. Virus Res. 1993, 29, 155–165. (26) Gagneux, P.; Varki, A. Glycobiology 1999, 9, 747–55. (27) Varki, A.; Freeze, H. H.; Gagneux, P. In Essentials of Glycobiology., 2nd ed.; Varki, A., Cummings, R. D., Esko, J. D., Freeze, H. H., Stanley, P., Bertozzi, C. R., Hart, G. W., Etzler, M. E., Eds.; Cold Spring Harbor: New York, 2009; pp 281
292. (28) Banin, E.; Israely, T.; Fine, M.; Loya, Y.; Rosenberg, E. FEMS Microbiol. Lett. 2001, 199, 33–37. (29) Laine, R. A. Glycobiology 1994, 4, 759–767. (30) Bidlingmeyer, B. A.; Cohen, S. A.; Travin, T. L. J. Chromatogr. 1984, 336, 93–104. (31) Zanetta, J. P.; Timmermann, P.; Leroy, Y. Glycobiology 1999, 9, 255–266. (32) Lo-Guidice, J. M.; Wieruszeski, J. M.; Lemoine, J.; Verbert, A.; Roussel, P.; Lamblin, G. J. Biol. Chem. 1994, 269, 18794–18813. (33) Ciucanu, I.; Kerek, F. Carbohydr. Res. 1984, 131, 209–217. (34) Brockhausen, I.; Schachter, H.; Stanley, P. In Essentials of Glycobiology., 2nd ed.; Varki, A., Cummings, R. D., Esko, J. D., Freeze, H. H., Stanley, P., Bertozzi, C. R., Hart, G. W., Etzler, M. E., Eds.; Cold Spring Harbor: New York, 2009; pp 115
127. (35) Garenaux, E.; Yu, S. Y.; Florea, D.; Strecker, G.; Khoo, K. H.; Guerardel, Y. Glycoconjugate J. 2008, 25, 903–915. (36) Yu, S. Y.; Wu, S. W.; Hsiao, H. H.; Khoo, K. H. Glycobiology 2009, 19, 1136–1149. (37) Moody, S. F.; Handman, E.; McConville, M. J.; Bacic., A. J. Biol. Chem. 1993, 268, 18457–18466. (38) Dell, A. Adv. Carbohydr. Chem. Biochem. 1987, 45, 19–72. (39) Khoo, K. H.; Maizels, R. M.; Page, A. P.; Taylor, G. W.; Rendell, N. B.; Dell, A. Glycobiology 1991, 1, 163–171. (40) Guerardel, Y.; Balanzino, L.; Maes, E.; Leroy, Y.; Coddeville, B.; Oriol, R.; Strecker, G. Biochem. J. 2001, 357, 167–182. (41) Pomin, V. H.; Mour~ao, P. A. Glycobiology. 2008, 18, 1016– 10127. (42) Pomin, V. H. Biopolymers 2009, 91, 601–609. (43) Alves, A. P.; Mulloy, B.; Diniz, J. A.; Mour~ao, P. A. J. Biol. Chem. 1997, 272, 6965–6971. (44) Spillmann, D.; Thomas-Oates, J. E.; van Kuik, J. A.; Vliegenthart, J. F.; Misevic, G.; Burger, M. M.; Finne, J. J. Biol. Chem. 1995, 270, 5089–5097. (45) Misevic, G. N.; Guerardel, Y.; Sumanovski, L. T.; Slomianny, M. C.; Demarty, M.; Ripoll, C.; Karamanos, Y.; Maes, E.; Popescu, O.; Strecker, G. J. Biol. Chem. 2004, 279, 15579–90. (46) Guerardel, Y.; Czeszak, X.; Sumanovski, L. T.; Karamanos, Y.; Popescu, O.; Strecker, G.; Misevic, G. N. J. Biol. Chem. 2004, 279, 15591–15603. (47) Martins, M. F.; Bairos, V. A. Int. Rev. Cytol. 2002, 216, 131–173. (48) Yamaguchi, Y. Cell. Mol. Life Sci. 2000, 57, 276–289. (49) Guerardel, Y.; Kol, O.; Maes, E.; Lefebvre, T.; Boilly, B.; Davril, M.; Strecker, G. Biochem. J. 2000, 352, 449–463.

2073

dx.doi.org/10.1021/bm101557v |Biomacromolecules 2011, 12, 2064–2073