Article pubs.acs.org/jpr
Atlantic Salmon Carries a Range of Novel O‑Glycan Structures Differentially Localized on Skin and Intestinal Mucins Chunsheng Jin,† János Tamás Padra,† Kristina Sundell,‡ Henrik Sundh,‡ Niclas G. Karlsson,† and Sara K. Lindén*,† †
Department of Medical Chemistry and Cell Biology, ‡Department of Biological and Environmental Sciences, University of Gothenburg, Gothenburg SE-405 30, Sweden S Supporting Information *
ABSTRACT: Aquaculture is a growing industry, increasing the need for understanding host−pathogen interactions in fish. The skin and mucosal surfaces, covered by a mucus layer composed of mucins, is the first point of contact between fish and pathogens. Highly O-glycosylated mucins have been shown to be an important part of the defense against pathogens, and pathogens bind to host surfaces using lectinlike adhesins. However, knowledge of piscine O-glycosylation is very limited. We characterized mucin O-glycosylation of five freshwater acclimated Atlantic salmon, using mass spectrometry. Of the 109 O-glycans found, most were sialylated and differed in distribution among skin, pyloric ceca, and proximal and distal intestine. Skin O-glycans were shorter (2−6 residues) and less diverse (33 structures) than intestinal O-glycans (2−13 residues, 93 structures). Skin mucins carried Oglycan cores 1, 2, 3, and 5 and three types of sialic acids (Neu5Ac, Neu5Gc, and Kdn) and had sialyl-Tn as the predominant structure. Intestinal mucins carried only cores 1, 2, and 5, Neu5Ac was the only sialic acid present, and sialylated core 5 was the most dominant structure. This structural characterization can be used for identifying structures of putative importance in host− pathogen interactions for further testing in biological assays and disease intervention therapies. KEYWORDS: Atlantic salmon, fish, O-glycan, mucins, skin, gastrointestinal tract, mucus, glycosylation
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molecules.8 In mammals, each mucin can carry on the order of 100 different carbohydrate structures, which provides a vast array of potential binding sites as well as a possible food source for microbes.9 We recently showed that the furunculosiscausing bacterium Aeromonas salmonicida ssp. salmonicida binds differentially to mucins isolated from skin and intestinal regions of the Atlantic salmon, in a sialic acid-dependent manner;10 however, knowledge of fish mucin O-glycosylation is limited. Glycomic studies focused on fish eggs and skin have revealed complicated and species-specific O-glycan profiles. In the Salmonidae family, O-glycans from unfertilized egg polysialoglycoprotein (PSGP) have been characterized in several species.11 These O-glycans comprise polysialic acid [up to 25 α2-8-linked N-acetylneuraminic acid (Neu5Ac) or N-glycolylneuraminic acid (Neu5Gc)] or a single deaminoneuraminic acid (Kdn) linked to the C6 of N-acetylgalactosamine (GalNAc) at the reducing end.11 This polysialic acid chain is further capped with α2-8-linked Kdn. The proximal or penultimate GalNAc residues at the reducing end are sometimes also modified with a single α2-3-linked sialic acid (Neu5Ac, Neu5Gc, or Kdn). There is a species-specific
INTRODUCTION Aquaculture is the fastest growing animal producing sector in the world and surpassed capture fisheries for human consumption in 2014 (OECD/FAO outlook 2014). Farming of Atlantic salmon (Salmo salar L.) is rising in concert, and in 2012, more than 2 million tons of Atlantic salmon were produced.1 In highly intensive aquaculture, rapid spreading of infections between fish is a risk and thus successful prevention and treatment is vital. The adult teleost fish is covered by mucus on all epithelial surfaces facing the external environment, such as the skin, gills, and gastrointestinal tract. The mucus layer forms a protective, physical barrier against the external environment, which contains multitudes of potential harmful components including pathogens.2,3 Continuous replacement of mucus produced by goblet cells on these epithelial surfaces may prevent microbial colonization and avoid erosion of the underlying epithelia. Mucins, the major components of the mucus layer, are extensively O-glycosylated proteins. Orthologues of mammalian mucins have been identified in the genomes of pufferfish (Fugu rubripes)4 and zebrafish (Danio rerio),5,6 and next-generation sequencing of Atlantic salmon skin has revealed sequences with homology to the human MUC2 and MUC5 mucins.7 OGlycans account for 50−80% of the mass of the mucin © XXXX American Chemical Society
Received: March 17, 2015
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DOI: 10.1021/acs.jproteome.5b00232 J. Proteome Res. XXXX, XXX, XXX−XXX
Article
Journal of Proteome Research
Norway) and exposed to a simulated natural photoperiod. Five fish (31.06 ± 0.49 cm length and 280.70 ± 12.78 g weight; mean ± SEM) were randomly netted, anesthetized in metomidate (12.5 mg/L), and killed with a blow to the head. Mucus from the skin was sampled by gentle scraping of the entire fish skin using two glass microscope slides. The dissected intestine was cut open along the mesenteric border, the proximal region was separated from the distal at the ileorectal valve, and the mucus and mucosa were scraped off using microscope slides. The pyloric ceca were dissected out using small scissors and placed in liquid nitrogen for pulverization using a mortar and pestle. All samples were placed in 10 mM sodium dihydrogen phosphate containing 0.1 mM phenylmethanesulphonyl fluoride (PMSF), pH 6.5 (sampling buffer).
structural diversity of polysialic acids in PSGP from salmonid eggs, e.g., being exclusively composed of Neu5Gc in rainbow trout but from mixtures of sialic acid residues in other salmonid species.11 Salmonid egg mucus also contains a Kdn-rich glycoprotein.11 In addition to polysialic acids, galactosylated galactose (di-Gal or Galβ1-4Gal) and modifications of nonreducing-end located GalNAc have been shown to be present. The latter includes fucosylation of terminal GalNAc (Fucα13GalNAc) and extension of GalNAc residues into [Neu5Acα23(GalNAcβ1-4)GalNAcβ1-3] in salmonid egg mucus Oglycans.11 In Atlantic salmon skin, a disialylated structure (diSia or Neu5Acα2-8Neu5Ac) has been detected on N-glycans of two cysteine proteinase inhibitors (salmon kininogen and salarin). However, only simple sialylated core 1 O-glycans were found on these two proteins.12 Consistent with this, an Nglycan with di-Sia motif has been reported on a glycoprotein isolated from rainbow trout (Oncorhynchus mykiss) ovarian fluid.13 Di-Sia, di-Gal, and Fucα1-3GalNAc terminal epitopes were also detected in zebrafish embryos.14,15 A study on rainbow trout skin mucus revealed that the majority of the Oglycans were sialyl Tn (Neu5Acα2-6GalNAcol) and sialylated core 5 [GalNAcα1-3(NeuAcα2-6)GalNAcol].16,17 Neither Neu5Gc nor Kdn was detected on O-glycans from rainbow trout skin mucus,16,17 whereas Kdn-containing O-glycans have been detected in loach (Misgurnus anguillicaudatus) skin mucus.18 Lectin staining has also suggested the presence of terminal GalNAc (determined by soy bean agglutinin, SBA) but the absence of α-Fuc (determined by Ulex europaeus agglutinin, UEA-I) in skin mucus from rainbow trout.19 So far, core 5 Oglycans have thus been detected in both salmonid egg mucus and skin.11,16,17 In contrast to eggs and skin, knowledge of O-glycan profiles in the fish GI tract is mainly based on qualitative histochemical and lectin binding studies. However, studies on the GI tract from four carp species (genus Cyprinus) indicate that the GI Oglycan profiles are more complex and diverse than those of eggs or skin. For example, using the Dolichos biflorus lectin (DBA), high levels of terminal α-GalNAc were detected,20,21 whereas the levels of sialic acid [detected by MAA (Maackia amurensis agglutinin) and SNA (Sambucus nigra bark lectin)] and βGalNAc (detected by RCA, Ricinus communis agglutinin) were moderate.21 Using α-Fuc-specific lectins (such as UEA-I and Lotus tetragonolobus LTA), α1-6- and α1-2-lined fucose were also found.20,21 To our knowledge, currently, less than 10 O-glycans have been characterized from fish mucins, if the degree of polymerization of polysialic acids is not considered. The main objective of the present study was to characterize the Oglycosylation of the mucins from Atlantic salmon skin, pyloric ceca, and proximal and distal intestine.
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Histochemistry
For histological analyses, another cohort of the same stock of Atlantic salmon (n = 4) of similar developmental status was used. Tissue pieces of the skin were excised from the area under the dorsal fin. Several pyloric ceca and parts of the proximal and distal intestinal tubes were excised, immersed in fresh Carnoy’s fixative (60% ethanol, 30% chloroform, and 10% glacial acetic acid), and embedded in wax for histology. Sections (7 μm) were used for Alcian blue−periodic acid−Schiff (AB-PAS) staining after dewaxing. Briefly, sections were pretreated in 99.5% ethanol for 10 min, rinsed in running tap water for 10 min, and immersed in 3% acetic acid (HAc) acid for 2 min. Staining with Alcian blue was carried out with 1% Alcian blue 8GX in 3% HAc (pH 2.5) for 2.5 h. Slides were then immersed in 3% HAc for 1 min and rinsed under running tap water for 9 min. Sections were oxidized in 1% periodic acid for 10 min, rinsed under running tap water for 5 min, immersed in 25% Schiff’s reagent (Sigma-Aldrich, St. Louis, MO), and rinsed under running tap water for 5 min. Slides were treated with 0.5% sodium metabisulfite three times for 1 min, washed with tap water, dehydrated, and mounted. Scoring of the AB-PAS staining was carried out by counting the goblet cells in five microscopic fields of a tissue section and categorizing them as blue-stained (contains acidic mucins) or magenta-stained (contains neutral mucins). Photos were taken with a Nikon Eclipse 90i microscope with a DS-Vi1 camera (Nikon Instruments Europe B.V.). Isolation and Purification of Mucins
The mucins were isolated as previously described:10 the scrapings and pulverized pyloric ceca in sampling buffer were placed into five sample volumes of extraction buffer (6 M guanidine hydrochloride (GuHCl), 5 mM EDTA, 10 mM sodium phosphate buffer, pH 6.5, containing 0.1 mM PMSF), dispersed using a Dounce homogenizer (four strokes with a loose pestle), and stirred slowly at 4 °C, overnight. The insoluble material was removed by centrifugation at 23 000g for 50 min at 4 °C (Beckman JA-30 rotor), and the pellet was reextracted twice with 10 mL of extraction buffer. The supernatants from these three extractions were pooled and contained the GuHCl soluble mucins. Although a small proportion of the mucins remains in the pellet, we have previously shown that soluble mucins are in the majority: 76% of the mucins in the skin were soluble, and both pyloric ceca and proximal intestine contained 97% soluble mucins, whereas distal intestine contained 91%.10 Thus, we focused on soluble mucins for this study. The samples were dialyzed twice against 10 volumes of extraction buffer and filled up to 26 mL with extraction buffer. CsCl was added by gentle stirring, and the
METHODS AND MATERIALS
Fish and Sampling Procedures
Juvenile Atlantic salmon originating from a wild anadromous strain (river Göta älv, southwestern Sweden) were obtained from Långhults Lax AB (Långhult, Sweden). The fish were maintained in 500 L tanks at the Department of Biological and Environmental Sciences. The fish were held in recirculating 10 °C fresh water, supplemented with 10% salt water (yielding a salinity of 2−3 ‰), at a flow rate of 8.5 L/min. The fish were hand-fed ad libitum once daily with a commercial dry pellet (Nutra Olympic 3 mm; Skretting Averøy Ltd., Stavanger, B
DOI: 10.1021/acs.jproteome.5b00232 J. Proteome Res. XXXX, XXX, XXX−XXX
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Journal of Proteome Research
°C overnight. To remove sialidase enzymes after treatment, samples were applied to self-packed porous graphitized carbon in a C18 Ziptip (Millipore) after washing the column with 90% acetonitrile (AcCN) and water containing 0.5% trifluoroacetic acid (TFA). Oligosaccharides were eluted with 40% AcCN with 0.5% TFA. Released O-glycans were analyzed by liquid-chromatography−mass spectrometry (LC−MS) using a 10 cm × 250 μm i.d. column, prepared in-house, containing 5 μm porous graphitized carbon (PGC) particles (Thermo Scientific, Waltham, MA, USA). Glycans were eluted using a linear gradient from 0 to 40% acetonitrile in 10 mM ammonium bicarbonate over 40 min at a flow rate of 10 μL/min. The eluted O-glycans were detected using an LTQ mass spectrometer (Thermo Scientific) in negative-ion mode with an electrospray voltage of 3.5 kV, capillary voltage of −33.0 V, and capillary temperature of 300 °C. Compressed air was used as a sheath gas. In LC−MS, full scans were performed in the mass range of m/z 380−2000. MS/MS was performed at normalized collisional energy of 35% with a minimal signal of 300 counts, isolation width of 2.0 m/z, and activation time of 30 ms The data were processed using Xcalibur software (version 2.0.7, Thermo Scientific). Glycans were annotated from their MS/MS spectra manually and validated by available structures stored in Unicarb-DB database (2014-12 version).23 The annotated structures were submitted to the Unicarb-DB database, and they will be included in the next release. For statistical analysis, the relative amount of glycan was calculated on the basis of the integrated peak areas in the LC−MS chromatograms. The peak area was used as a number proportional to the amount of glycan. The relative amount of each glycan was presented as a percentage of the total area and used for comparison between tissues and fish.
samples were transferred to Quick Seal ultracentrifuge tubes (Beckman Coulter). The tubes were filled with 10 mM NaH2PO4 to give a starting density of 1.35 g/mL and subjected to density gradient centrifugation at 40 000g for 90 h at 15 °C. The fractions were collected from the bottom of the tubes. Density gradient fractions of purified mucins were analyzed for carbohydrates as periodate-oxidizable structures in a microtiterbased assay: fractions diluted 1:100, 1:500, and 1:1000 in 4 M GuHCl were coated onto 96-well plates (PolySorpTM, NUNC A/S, Roskilde, Denmark) and incubated overnight at 4 °C. The rest of the assay was carried out at 23 to 24 °C. After washing three times with washing solution (5 mM Tris-HCl, 0.15 M NaCl, 0.05% Tween-20, 0.02% NaN 3 , pH 7.75), the carbohydrates were oxidized by adding 25 mM sodium metaperiodate in 0.1 M sodium acetate buffer, pH 5.5, for 20 min. The plates were washed again, and the wells were blocked with DELFIA blocking solution (50 mM Tris-HCl, 0.15 M NaCl, 90 mM CaCl2, 4 mM EDTA, 0.02% NaN3, 0.1% BSA, pH 7.75) for 1 h. After further washing steps, the samples were incubated for 1 h with 2.5 mM biotin hydrazide in 0.1 M sodium acetate buffer, pH 5.5, followed by another washing step. Europium-labeled streptavidin was diluted 1:1000 in DELFIA assay buffer (50 mM Tris-HCl, 0.15 M NaCl, 20 mM diethylenetriaminepentaacetic acid, 0.01% Tween-20, 0.02% NaN3, 1.5% BSA, pH 7.75) and added to the wells. After a 1 h incubation, the plates were washed six times and then incubated with DELFIA enhancement solution (0.05 M NaOH, 0.1 M phthalate, 0.1% Triton X-100, 50 mM tri-n-octylphosphine oxide, 15 mM biotin nitrilotriacetic acid) for 5 min on an orbital shaker. Fluorescence (λexcitation = 340 nm and λemission = 615 nm) was measured using a Wallac 1420 VICTOR2 plate reader with the Europium label protocol (PerkinElmer, Waltham, MA, USA). Density measurements were performed using a Carlsberg pipet as a pycnometer: 300 μL of sample was sucked into the pipet and weighed, and density was calculated as g/mL. DNA content was calculated from UV light absorbance at 260 nm. The DNA peak was generally located around fractions 3 to 4 and was baseline-separated from the glycan peak containing the mucins, which occurred around fraction 9−13, at a density of 1.30−1.37 g/mL. Gradient fractions containing mucins were pooled together to obtain one sample for each gradient.
Statistics
ANOVA followed by Dunnet’s posthoc test was used to compare levels of glycans between skin and intestinal sites. Statistical analysis was performed using the GraphPad Prism 6.0 (GraphPad Software Inc.) software package.
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RESULTS
LC−MS/MS Revealed Differences in the O-Glycan Profiles of Mucins from Skin and Intestinal Sites
Characterization of O-Glycan Profiles from Skin and Gastrointestinal Mucins
Mucins from Atlantic salmon skin, pyloric ceca, and proximal and distal intestine that bind differentially to the pathogen A. salmonicida in a sialic acid-dependent manner10 were analyzed to identify the structural composition of these complex glycoconjugates. The O-glycans of skin, pyloric ceca, and proximal and distal intestinal mucins were released and analyzed by LC−MS/MS. O-Glycans were detected as singly and doubly charged ions in negative-ion mode (Figure 1). Overall, more than 100 O-glycans were verified by MS/MS. Among them, 109 O-glycans with distinguishing MS/MS spectra were used for further analysis (Table S1). The major annotated O-glycan structures isolated from skin, pyloric ceca, and proximal and distal intestine are labeled in the base peak chromatograms (Figure 1). The O-glycans were 2−13 residues long, and most O-glycans were sialylated. There were 36 Oglycans detected in skin, 60 in pyloric ceca, 69 in proximal intestine, and 83 in distal intestine (Figure 3A). Thirteen out of 109 (12%) O-glycan structures were detected in all isolated samples. More than two-thirds of the O-glycans (68%) were detected only in the GI tract, and most structures unique for
Fish mucins (approximately 100 μg, from skin, pyloric ceca, and proximal and distal intestine; n = 5 of each region) were dotblotted to PVDF membrane (Immobilon P membranes, Millipore, Billerica, MA) in order to remove the extraction buffer from purified mucins. Dots were visualized by Alcian blue (Sigma-Aldrich) staining. Blue dots were excised and subjected to reductive β-elimination. In brief, excised dots were incubated with 0.5 M NaBH4 and 50 mM NaOH for 16 h at 50 °C. Reactions were quenched with glacial acetic acid, and the samples were desalted and dried as previously described.22 It is worth noting that O-acetylated derivatives of sialic acid tend to lose the ester group during reductive β-elimination. Desialylation was performed by using sialidase A (cleaves all nonreducing terminal branched and unbranched sialic acids, α2-3, -6, -8, and -9 linkages) or S (removes sialic acids with α2,3-linkage, ProZyme, CA). Sialidase A (1 μL of 5 mU) or sialidase S (2 μL of 2 mU) was incubated with an aliquot of released O-glycans in 50 mM sodium phosphate, pH 6.0, at 37 C
DOI: 10.1021/acs.jproteome.5b00232 J. Proteome Res. XXXX, XXX, XXX−XXX
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Journal of Proteome Research
5 structures were the most prominent complex O-linked oligosaccharides on skin mucins, in addition to the abundant sialyl Tn found within the lower molecular mass structures (Figure 1 and Table S1). In addition to a high prevalence of low molecular mass core 5 structures, a low abundance of isomeric core 3 (GlcNAcβ1-3GalNAcα1-) structures was also detected. The simplest core 5 structures, detected as GalNAcα1-3GalNAcol ([M − H]− ions of m/z 425, Figure 2B) and its sialylated version ([M − H]− ions of m/z 716, Figure 2B) were identified by their retention times of 18 and 24 min, respectively. The core 3 equivalent, GlcNAcβ1-3GalNAcol, with or without Neu5Ac was detected at 12 and 18 min (verified by a standard present in porcine gastric mucins), respectively. Core 5 structures, with or without sialic acid, were even more abundant on GI mucins than on skin mucins (Figure 3B and Table S1). Frequently, a further extension of HexNAc was found to be linked to the penultimate GalNAc residue of core 5. For example, the glycan with a composition of Hex1HexNAc3 ([M − H]− ion of m/z 790) was detected in all tissues (Figure 4A). The fragmentation ion at m/z 682 ([M − H]− − C3H8O4) and B2 ion at m/z 567 of this structure suggests a linear core 5.24 The presence of Y2α (m/z 587) and Y2β/B2 (m/z 405) indicated terminal HexNAc as well as Hex (Gal). The intense cross-ring cleavage fragment at m/z 262 implied terminal HexNAc linked to penultimate HexNAc, where terminal Hex was also found to be attached. The absence of fragments indicating C4 and/or C4/C3 substitution indicated that the penultimate HexNAc was substituted on C3 and C6. Additional structures showed elongation of both the C3 and C6 branch of penultimate HexNAc. As an example, a structure with additional Neu5Ac and HexNAc (NeuAc1Hex1HexNAc4, m/z 6422−) is shown in Figure 4B. A fragmentation ion at m/z 901 (Z2α) suggested that HexNAc was linked to terminal Gal residues. Due to the absence, or low abundance, of core 3 type structures of Atlantic salmon mucin low molecular weight oligosaccharides, isomeric core 5 was the core type believed to be extended into other structures (45 out of 109 O-glycans). In addition, cores 1 and 2 were also detected in Atlantic salmon mucins. Fifteen out of 109 O-glycans contained core 1, and 10 of them were sialylated. Forty-one glycans contained core 2. Five core 3 O-glycans were exclusively detected in skin mucin. These core 3 structures were also detected with addition of α2,6-linked NeuAc to C6 of GalNAc aditol or elongated with β1,4-linked Gal to the β1,3-linked N-acetylglucosamine (GlcNAc). In Atlantic salmon skin, sialyl Tn was the predominant structure (61%), and core 5 was the most abundant extension core structure (27%). Only low amounts of cores 1, 2, and 3 were detected (
