Isomeric Separation and Recognition of Anionic and Zwitterionic N-glycans from Royal Jelly Glycoproteins
2018; Elsevier BV; Volume: 17; Issue: 11 Linguagem: Inglês
10.1074/mcp.ra117.000462
ISSN1535-9484
AutoresAlba Hykollari, Daniel Malzl, Barbara Eckmair, Jorick Vanbeselaere, Patrick Scheidl, Chunsheng Jin, Niclas G. Karlsson, Iain B. H. Wilson, Katharina Paschinger,
Tópico(s)Magnetic and Electromagnetic Effects
ResumoRoyal jelly has received attention because of its necessity for the development of queen honeybees as well as claims of benefits on human health; this product of the hypopharyngeal glands of worker bees contains a large number of proteins, some of which have been claimed to have various biological effects only in their glycosylated state. However, although there have been glycomic and glycoproteomic analyses in the past, none of the glycan structures previously defined would appear to have potential to trigger specific biological functions. In the current study, whole royal jelly as well as single protein bands were subject to off-line LC-MALDI-TOF MS glycomic analyses, complemented by permethylation, Western blotting and arraying data. Similarly to recent in-depth studies on other insect species, previously overlooked glucuronic acid termini, sulfation of mannose residues and core β-mannosylation of the N-glycans were found; additionally, a relatively rare zwitterionic modification with phosphoethanolamine is present, in contrast to the phosphorylcholine occurring in lepidopteran species. Indicative of tissue-specific remodelling of glycans in the Golgi apparatus of hypopharyngeal gland cells, only a low amount of fucosylated or paucimannosidic glycans were detected as compared with other insect samples or even bee venom. The unusual modifications of hybrid and multiantennary structures defined here may not only have a physiological role in honeybee development, but represent epitopes recognized by pentraxins with roles in animal innate immunity. Royal jelly has received attention because of its necessity for the development of queen honeybees as well as claims of benefits on human health; this product of the hypopharyngeal glands of worker bees contains a large number of proteins, some of which have been claimed to have various biological effects only in their glycosylated state. However, although there have been glycomic and glycoproteomic analyses in the past, none of the glycan structures previously defined would appear to have potential to trigger specific biological functions. In the current study, whole royal jelly as well as single protein bands were subject to off-line LC-MALDI-TOF MS glycomic analyses, complemented by permethylation, Western blotting and arraying data. Similarly to recent in-depth studies on other insect species, previously overlooked glucuronic acid termini, sulfation of mannose residues and core β-mannosylation of the N-glycans were found; additionally, a relatively rare zwitterionic modification with phosphoethanolamine is present, in contrast to the phosphorylcholine occurring in lepidopteran species. Indicative of tissue-specific remodelling of glycans in the Golgi apparatus of hypopharyngeal gland cells, only a low amount of fucosylated or paucimannosidic glycans were detected as compared with other insect samples or even bee venom. The unusual modifications of hybrid and multiantennary structures defined here may not only have a physiological role in honeybee development, but represent epitopes recognized by pentraxins with roles in animal innate immunity. Dedicated to Dr. Hubert Paschinger (1941–2011), chemist, lover of art and music and avid beekeeper. Royal jelly is a product of the hypopharyngeal and mandibular glands of worker honeybees (1Fratini F. Cilia G. Mancini S. Felicioli A. Royal Jelly: An ancient remedy with remarkable antibacterial properties.Microbiol. Res. 2016; 192: 130-141Crossref PubMed Scopus (150) Google Scholar); its natural role is as the food source for worker and drone larvae for the first 3 days but induces queen development when fed beyond that time and remains, thereafter, the food for a queen's entire life. The observed fertility and longer lifespan of queens, as compared with worker bees, has been correlated with this special diet. However, royal jelly has attracted especial attention because of its putative effects on human health. Indeed, it has been used since ancient times as a food supplement or cosmetic and is considered to have a range of antibiotic, anti-inflammatory and other health-relevant effects (2McCleskey C.S. Melampy R.M. Bactericidal Properties of Royal Jelly of the Honeybee.J. Economic Entomol. 1939; 32: 581-587Crossref Google Scholar), although these are controversial and allergic reactions are also known (3Paola F. Pantalea D.D. Gianfranco C. Antonio F. Angelo V. Eustachio N. Elisabetta D.L. Oral allergy syndrome in a child provoked by royal jelly.Case Rep. Med. 2014; 2014: 941248Crossref PubMed Scopus (14) Google Scholar). Royal jelly contains a wide range of compounds including carbohydrates, lipids, vitamins, and proteins, many of which are post-translationally modified (4Zhang L. Fang Y. Li R. Feng M. Han B. Zhou T. Li J. Towards posttranslational modification proteome of royal jelly.J. Proteomics. 2012; 75: 5327-5341Crossref PubMed Scopus (46) Google Scholar, 5Zhang L. Han B. Li R. Lu X. Nie A. Guo L. Fang Y. Feng M. Li J. Comprehensive identification of novel proteins and N-glycosylation sites in royal jelly.BMC Genomics. 2014; 15: 135Crossref PubMed Scopus (37) Google Scholar). Because of their multiple and variable monosaccharide units and their size (typically 2000 Da), N-glycans represent the most diverse and complex set of protein modifications known (6Paschinger K. Wilson I.B.H. Comparative Glycobiology.in: Taniguchi N. Glycoscience: Biology and Medicine. Springer, Japan2015: 795-805Crossref Scopus (3) Google Scholar) and modulate a wide range of biological processes. Based on proteomic and glycoproteomic studies of royal jelly, up to 100 different proteins have been identified including the "major royal jelly proteins" (MRJPs) 1The abbreviations used are:MRJPmajor royal jelly proteins. 1The abbreviations used are:MRJPmajor royal jelly proteins.. Of these, MRJP1 (also known as apisin or royalactin) has a controversial role in queen development (7Kamakura M. Royalactin induces queen differentiation in honeybees.Nature. 2011; 473: 478-483Crossref PubMed Scopus (433) Google Scholar, 8Buttstedt A. Ihling C.H. Pietzsch M. Moritz R.F. Royalactin is not a royal making of a queen.Nature. 2016; 537: E10-E12Crossref PubMed Scopus (59) Google Scholar), but is reported to extend life-span in other invertebrates (9Detienne G. De Haes W. Ernst U.R. Schoofs L. Temmerman L. Royalactin extends lifespan of Caenorhabditis elegans through epidermal growth factor signaling.Exp. Gerontol. 2014; 60: 129-135Crossref PubMed Scopus (26) Google Scholar). Furthermore, MRJP1 and MRJP2 (apalbumin2) have been shown to have antihypertensive and antibiotic activity in their glycosylated state (10Feng M. Fang Y. Han B. Xu X. Fan P. Hao Y. Qi Y. Hu H. Huo X. Meng L. Wu B. Li J. In-depth N-glycosylation reveals species-specific modifications and functions of the royal jelly protein from western (Apis mellifera) and eastern honeybees (Apis cerana).J. Proteome Res. 2015; 14: 5327-5340Crossref PubMed Scopus (36) Google Scholar). Although the sites of glycosylation on MRJPs have been analyzed, there is less information regarding the structures of the glycans on the specific glycoproteins. Although mass spectrometric analyses (11Bíliková K. Mirgorodskaya E. Bukovská G. Gobom J. Lehrach H. Šimúth J. Towards functional proteomics of minority component of honeybee royal jelly: the effect of post-translational modifications on the antimicrobial activity of apalbumin2.Proteomics. 2009; 9: 2131-2138Crossref PubMed Scopus (51) Google Scholar) indicated potentially complex N-glycosylation of MRJP2, HPLC and NMR data showed the presence of oligomannosidic and hybrid glycans (12Kimura Y. Washino N. Yonekura M. N-linked sugar chains of 350-kDa royal jelly glycoprotein.Biosci. Biotechnol. Biochem. 1995; 59: 507-509Crossref PubMed Scopus (40) Google Scholar, 13Kimura Y. Kajiyama S. Kanaeda J. Izukawa T. Yonekura M. N-linked sugar chain of 55-kDa royal jelly glycoprotein.Biosci. Biotechnol. Biochem. 1996; 60: 2099-2102Crossref PubMed Scopus (36) Google Scholar) on monomeric and oligomeric forms of MRJP1. There have also been a series of glycan analyses on royal jelly, culminating in a report that Galβ1,3GalNAcβ1,4GlcNAc units are detectable on N-glycans and that there is a relevant galactosyltransferase activity (14Kimura Y. Miyagi C. Kimura M. Nitoda T. Kawai N. Sugimoto H. Structural features of N-glycans linked to royal jelly glycoproteins: structures of high-mannose type, hybrid type, and biantennary type glycans.Biosci. Biotechnol. Biochem. 2000; 64: 2109-2120Crossref PubMed Scopus (51) Google Scholar, 15Kimura Y. Tsumura K. Kimura M. Okihara K. Sugimoto H. Yamada H. First evidence for occurrence of Galβ1-3GlcNAcβ1–4Man unit in N-glycans of insect glycoprotein: β1–3Gal and β1–4GlcNAc transferases are involved in N-glycan processing of royal jelly glycoproteins.Biosci Biotechnol Biochem. 2003; 67: 1852-1856Crossref PubMed Scopus (9) Google Scholar, 16Kimura Y. Nagai H. Miyamoto M. Kimura M. Yonekura M. Identification of a Royal Jelly Glycoprotein That Carries Unique Complex-Type N-Glycans Harboring the T-Antigen (Galβ1–3GalNAc) Unit.Biosci. Biotechnol. Biochem. 2010; 74: 2148-2150Crossref PubMed Scopus (9) Google Scholar, 17Ichimiya T. Maeda M. Sakamura S. Kanazawa M. Nishihara S. Kimura Y. Identification of β1,3-galactosyltransferases responsible for biosynthesis of insect complex-type N-glycans containing a T-antigen unit in the honeybee.Glycoconj. J. 2015; 32: 141-151Crossref PubMed Scopus (10) Google Scholar). However, none of the hybrid or biantennary glycans described were especially unusual and so it was concluded that a role for these glycans in the biological activity of royal jelly is unlikely. major royal jelly proteins. major royal jelly proteins. Our recent data have proven that insect glycosylation is more complicated than previously thought and that a range of anionic and zwitterionic modifications is present on dipteran and lepidopteran N-glycans (18Kurz S. Aoki K. Jin C. Karlsson N.G. Tiemeyer M. Wilson I.B.H. Paschinger K. Targetted release and fractionation reveal glucuronylated and sulphated N- and O-glycans in larvae of dipteran insects.J. Proteomics. 2015; 126: 172-188Crossref PubMed Scopus (54) Google Scholar, 19Stanton R. Hykollari A. Eckmair B. Malzl D. Dragosits M. Palmberger D. Wang P. Wilson I.B.H. Paschinger K. The underestimated N-glycomes of lepidopteran species.Biochim. Biophys. Acta. 2017; 1861: 699-714Crossref PubMed Scopus (37) Google Scholar). Therefore, we have reappraised the N-glycome of royal jelly using an off-line LC-MALDI-TOF MS workflow; we have also examined the glycosylation of individual major royal jelly proteins and have immobilized N-glycan pools in probably the first study to ever test interactions of natural insect glycans in an array format. A significant proportion of structures was thereby found to be of either the hybrid or complex type with up to three antennae. About 4% of the total N-glycans carry novel combinations of glucuronic acid, sulfate, β-mannose and phosphoethanolamine, whereby these epitopes have potential for physiological activity in both insects and humans. Royal jelly (Wald und Wiese, Vienna, Austria; ca. 9 g wet weight per glycan preparation) was suspended in water and heat-treated before proteolysis with thermolysin (Promega, Madison, WI) (20Hykollari A. Paschinger K. Eckmair B. Wilson I.B.H. Analysis of Invertebrate and Protist N-Glycans.Methods Mol. Biol. 2017; 1503: 167-184Crossref PubMed Scopus (18) Google Scholar) followed by cation exchange (Dowex AG50, Bio-Rad; elution with 0.5 m ammonium acetate, pH 6) and gel filtration (Sephadex G25; GE Healthcare) chromatography of the proteolysate. Thereafter, N-glycans were released from glycopeptides using peptide/N-glycosidase F (PNGase F, 3 U; Roche) at pH 8 as previously described (20Hykollari A. Paschinger K. Eckmair B. Wilson I.B.H. Analysis of Invertebrate and Protist N-Glycans.Methods Mol. Biol. 2017; 1503: 167-184Crossref PubMed Scopus (18) Google Scholar), with a subsequent digestion of the remaining glycopeptides using native PNGase A (0.15 mU; Roche) after adjusting to pH 5; the result was a combined PNGase F/A digest. Alternatively, in a second preparation, the PNGase F-released glycans were isolated from the glycopeptides by Dowex AG50 chromatography and the latter were treated with recombinant PNGase Ar (15 U; New England Biolabs, Ipswich); in this case, the pools of PNGase F- and PNGase Ar-released glycans were separately analyzed. After another round of cation-exchange chromatography (Dowex AG50; flow-through), the glycans were subject to solid-phase extraction on nonporous graphitised carbon (SupelClean ENVICarb; Sigma-Aldrich, Merck) as described (20Hykollari A. Paschinger K. Eckmair B. Wilson I.B.H. Analysis of Invertebrate and Protist N-Glycans.Methods Mol. Biol. 2017; 1503: 167-184Crossref PubMed Scopus (18) Google Scholar); the "neutral" and "anionic-enriched" fractions were then eluted with 40% (v/v) acetonitrile and 40% (v/v) acetonitrile containing 0.1% trifluoroacetic acid (v/v) respectively. The pools of glycans were subject to MALDI-TOF MS before fluorescent labeling by reductive amination using 2-aminopyridine (PA; Sigma-Aldrich) (20Hykollari A. Paschinger K. Eckmair B. Wilson I.B.H. Analysis of Invertebrate and Protist N-Glycans.Methods Mol. Biol. 2017; 1503: 167-184Crossref PubMed Scopus (18) Google Scholar). Similar results were obtained for two different lots of royal jelly from the same supplier. For the workflow in schematic form, refer to the Scheme in the supplement. The neutral and anionic N-glycans (70% of each pool from one PNGase F-released preparation) were separately permethylated as previously reported (21Khoo K.-H. Yu S.-Y. Mass Spectrometric Analysis of Sulfated N- and O-Glycans.Methods Enzymol. 2010; 478: 3-26Crossref PubMed Scopus (32) Google Scholar) with slight modifications. Briefly, the samples were dried in a glass tube before permethylation with 0.2 ml of a slurry of ground NaOH pellets in dimethyl sulfoxide and 0.1 ml of ICH3. The reaction mixture was shaken for 4 h at 4 °C and then quenched on ice with 0.2 ml of cold water, followed by neutralization with 30% aqueous acetic acid, and then applied to a pre-equilibrated C18 solid phase extraction column. Hydrophilic salts and contaminants were stepwise washed off with 3 ml each of water, 2.5 and 10% (v/v) acetonitrile. Subsequently, permethylated N-glycans were eluted serially with 3 ml each of 25 and 50% (v/v) acetonitrile. Complete pyridylaminated N-glycomes (10% of the neutral and 90% of the anionic pools) were fractionated by reversed-phase HPLC (Ascentis Express RP-amide; 150 × 4.6 mm, 2.7 μm; Sigma-Aldrich) and a gradient of 30% (v/v) methanol (buffer B) in 100 mm ammonium acetate, pH 4 (buffer A) was applied at a flow rate of 0.8 ml/min as follows: 0–4 min, 0% B; 4–14 min, 0–5% B; 14–24 min, 5–15% B; 24–34 min, 15–35% B; 34–35 min, return to starting conditions (20Hykollari A. Paschinger K. Eckmair B. Wilson I.B.H. Analysis of Invertebrate and Protist N-Glycans.Methods Mol. Biol. 2017; 1503: 167-184Crossref PubMed Scopus (18) Google Scholar). The RP-HPLC column was calibrated daily in terms of glucose units using a pyridylaminated dextran hydrolysate and the degree of polymerization of single standards was verified by MALDI-TOF MS. Selected RP-HPLC fractions were subject to HIAX-HPLC as a second dimension as described (20Hykollari A. Paschinger K. Eckmair B. Wilson I.B.H. Analysis of Invertebrate and Protist N-Glycans.Methods Mol. Biol. 2017; 1503: 167-184Crossref PubMed Scopus (18) Google Scholar) with an IonPac AS11 column (Dionex, Sunnyvale; 4 × 250 mm, combined with a 4 × 50 mm guard column). A two solvent gradient with buffer A (0.8 m ammonium acetate, pH 3) and buffer B (80% acetonitrile; LC-MS grade) 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; 65–85 min, 75% B. Detection for both columns was by fluorescence (Shimadzu RF-20A XS detector; excitation/emission at 320/400 nm). All manually collected HPLC glycan fractions were lyophilized, redissolved in water and analyzed by MALDI-TOF MS and MS/MS. Monoisotopic MALDI-TOF MS was performed using an Autoflex Speed (Bruker Daltonics, Bremen, Germany) instrument in either positive or negative reflectron modes with 6-aza-2-thiothymine (ATT; Alfa-Aesar, Thermo Scientific) as matrix. MS/MS was in general performed by laser-induced dissociation of the [M+H]+ or [M-H]− pseudomolecular ions (except for permethylated structures analyzed as [M+Na]+ with 2,5-dihydroxybenzoic acid as matrix); typically 2000 shots were summed for MS (reflector voltage, lens voltage and gain respectively 27 kV, 9 kV and 2217 V) and 4000 for MS/MS (reflector voltage, lift voltage and gain respectively 27 kV, 19 kV and 2174 V). 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). Glycan spectra were manually interpreted based on the masses of the predicted component monosaccharides, differences of mass in glycan series, fragmentation patterns, comparison with coeluting structures from other insects and nematodes and chemical treatments or exoglycosidase digestions (see also pages S2-S4 of the Supplement). Negative-ion mode LC-MSn of a 2D-HPLC-enriched glycan was performed as previously described, using a 5 μm porous graphitized carbon column (10 cm × 150 μm) coupled to a Thermo Scientific LTQ ion trap mass spectrometer (22Eckmair B. Jin C. Abed-Navandi D. Paschinger K. Multi-step fractionation and mass spectrometry reveals zwitterionic and anionic modifications of the N- and O-glycans of a marine snail.Mol. Cell. Proteomics. 2016; 15: 573-597Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar); refer also to page S5 of the Supplement for further details. A list of theoretical m/z values for each glycan composition is presented in the supplemental Table S1; mzXML files of raw MS/MS data are available as supplementary information. Glycans were treated, before re-analysis by MALDI-TOF MS, with α-fucosidase (bovine kidney from Sigma-Aldrich), α-mannosidases (jack bean from Sigma-Aldrich, Aspergillus α1,2-specific from Prozyme, Hayward, CA, and Xanthomonas α1,2/3-specific from New England Biolabs), β-galactosidase (β1,3-specific from New England Biolabs), β-glucuronidases (E. coli from Megazyme, Bray, Ireland, and Helix pomatia from Sigma-Aldrich; desalted and concentrated before use), β-N-acetylhexosaminidases (jack bean from Sigma-Aldrich, Xanthomonas β1,2-specific N-acetylglucosaminidase from New England Biolabs, Streptomyces β1,3/4-specific N-acetylhexosaminidase (chitinase) from New England Biolabs or in-house produced recombinant forms of Caenorhabditis elegans HEX-4 specific for β1,4-linked GalNAc residues or Apis mellifera FDL specific for the product of GlcNAc-transferase I (23Dragosits M. Yan S. Razzazi-Fazeli E. Wilson I.B.H. Rendić D. Enzymatic properties and subtle differences in the substrate specificity of phylogenetically distinct invertebrate N-glycan processing hexosaminidases.Glycobiology. 2015; 25: 448-464Crossref PubMed Scopus (23) Google Scholar)) in 50 mm ammonium acetate, pH 5, at 37 °C overnight (except for pH 6.5 in the case of HEX-4, pH 7 in the case of E. coli β-glucuronidase or an incubation time of only 3 h in the case of FDL); these incubations were performed in PCR tubes with a final volume of 3 μl (for further details about conditions and specificities, refer to the supplement). Hydrofluoric acid was used for removal of phosphoethanolamine or α1,3-linked fucose (20Hykollari A. Paschinger K. Eckmair B. Wilson I.B.H. Analysis of Invertebrate and Protist N-Glycans.Methods Mol. Biol. 2017; 1503: 167-184Crossref PubMed Scopus (18) Google Scholar). As appropriate, treated glycans were re-chromatographed by RP-HPLC to ascertain retention time shifts before MALDI-TOF-MS; otherwise, an aliquot (generally one-fifth) of any digest was analyzed by MALDI-TOF-MS without further purification. Before SDS-PAGE, resuspended royal jelly was precipitated (mixed with a 5-fold volume excess of methanol), incubated at −80 °C for one hour, centrifuged at 4 °C, 21,000 × g and dissolved in a reducing sample buffer. After electrophoresis (10 μg/lane) and blotting to a nitrocellulose membrane, the following reagents for detection of glycan epitopes were employed: anti-horseradish peroxidase (Sigma-Aldrich; 1:10,000 diluted in Tris buffered saline with 0.05% Tween and 0.5% BSA, to detect core α1,3-fucose (24Paschinger K. Rendić D. Wilson I.B.H. Revealing the anti-HRP epitope in Drosophila and Caenorhabditis.Glycoconj. J. 2009; 26: 385-395Crossref PubMed Scopus (59) Google Scholar)) and serum amyloid P protein (Fitzgerald, Acton, MA; 1:200, to detect phosphoethanolamine (25Mikolajek H. Kolstoe S.E. Pye V.E. Mangione P. Pepys M.B. Wood S.P. Structural basis of ligand specificity in the human pentraxins, C-reactive protein and serum amyloid P component.J. Mol. Recognit. 2011; 24: 371-377Crossref PubMed Scopus (28) Google Scholar)) as well as C-reactive protein (MP Biochemicals, Santa Ana; 1:200, which binds preferentially to phosphorylcholine (25Mikolajek H. Kolstoe S.E. Pye V.E. Mangione P. Pepys M.B. Wood S.P. Structural basis of ligand specificity in the human pentraxins, C-reactive protein and serum amyloid P component.J. Mol. Recognit. 2011; 24: 371-377Crossref PubMed Scopus (28) Google Scholar)) followed by the relevant peroxidase-conjugated secondary antibodies and development with SigmaFAST 3,3′-diaminobenzidine tetrahydrochloride (19Stanton R. Hykollari A. Eckmair B. Malzl D. Dragosits M. Palmberger D. Wang P. Wilson I.B.H. Paschinger K. The underestimated N-glycomes of lepidopteran species.Biochim. Biophys. Acta. 2017; 1861: 699-714Crossref PubMed Scopus (37) Google Scholar). Other glycan determinants were detected with biotinylated forms of Aleuria aurantia, wheat germ and peanut agglutinins (Vector Labs, Burlingame, CA; 1:1000) followed by phosphatase-conjugated anti-biotin (Sigma-Aldrich; 1:10,000) and development with SigmaFAST BCIP/NBT (26Paschinger K. Gonzalez-Sapienza G.G. Wilson I.B.H. Mass spectrometric analysis of the immunodominant glycan epitope of Echinococcus granulosus antigen Ag5.Int. J. Parasitol. 2012; 42: 279-285Crossref PubMed Scopus (35) Google Scholar). After performing SDS-PAGE (10 μg/lane) and staining with Coomassie Blue, protein bands were excised and washed/destained (twice with 50% acetonitrile in water and successively once with 1:1 0.1 m ammonium bicarbonate/acetonitrile and 100% acetonitrile only) before drying (26Paschinger K. Gonzalez-Sapienza G.G. Wilson I.B.H. Mass spectrometric analysis of the immunodominant glycan epitope of Echinococcus granulosus antigen Ag5.Int. J. Parasitol. 2012; 42: 279-285Crossref PubMed Scopus (35) Google Scholar). The proteins in the gel pieces were reduced for one hour at 56 °C with 10 mm dithiothreitol and alkylated for 45 min with 55 mm iodoacetamide in the dark. After a second round of washing (as above), the gel pieces were dried, covered with a 1:2 mixture of ammonium bicarbonate/trypsin (100 ng/μl) and incubated at 37 °C overnight. The (glyco)peptides were extracted with a mixture of acetonitrile/water/trifluoroacetic acid (660/330/1 (v/v/v)), dried and dissolved in water before MALDI-TOF MS using either α-cyanocinnamic acid (ACH) or 6-aza-thiothymine (ATT) as matrices, using similar mass spectrometer settings as above. Tryptic peptide fingerprint data were analyzed using the Mascot webserver (version 2.6.0) and the Swissprot database (release 2017_06) as described on page S5 of the Supplement. For protein-specific glycan analysis, the (glyco)peptides were first heat-treated to inactivate trypsin and 90% of the samples were subject to PNGase Ar treatment (2.5 U; New England Biolabs) in 20 mm ammonium acetate, pH 5 overnight at 37 °C. Whereas the deglycosylated peptides were analyzed by MALDI-TOF MS, the released N-glycans were purified using two different columns packed respectively with Lichroprep C18/Dowex AG50 and nonporous graphitized carbon/Lichroprep C18. The C18/AG50 column was washed with 2% acetic acid and 60% isopropanol; after equilibration of the column with 2% acetic acid, the N-glycans were acidified with 10% acetic acid before application. The N-glycans were eluted (three column volumes of 2% acetic acid) before use of the carbon/C18 column, which was pre-washed with 100% acetonitrile and equilibrated with water. The N-glycans (combined neutral and anionic pools) were then eluted with 40% acetonitrile containing 0.1% trifluoro-acetic acid. After drying, the glycans were fluorescently labeled by reductive amination using 2-aminopyridine and then analyzed with MALDI-TOF MS and HPLC as above. Free N-glycans of the neutral pool were modified reductively with 2-amino-N-(2-amino-ethyl)-benzamide (AEAB; excitation/emission of 330/420 nm) as described by Song (27Song X. Xia B. Stowell S.R. Lasanajak Y. Smith D.F. Cummings R.D. Novel fluorescent glycan microarray strategy reveals ligands for galectins.Chem. Biol. 2009; 16: 36-47Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). Briefly, the dried N-glycan pool was dissolved in 0.35 m AEAB and 1 m NaCNBH3 and incubated for 2 h at 65 °C. To remove the excess AEAB, the labeled glycans were precipitated three times with 100% acetonitrile followed by solid phase extraction on LC-NH2 (Supelco, Sigma-Aldrich) normal phase material. Successful derivatization of the N-glycans was determined by MALDI-TOF MS and HPLC (also to normalize the concentration of glycans based on fluorescence intensity). The glycan pool was mixed 1:1 with spotting buffer (300 mm sodium phosphate pH 7.5, 0.005% Tween-20) then spotted (n = 10) by noncontact printing (Flexarrayer S1; Scienion, Berlin, Germany) onto NHS-derivatised Nexterion H glass slides (Schott, Jena, Germany). After 16 h of hybridization, slides were blocked (50 mm ethanolamine in 50 mm sodium borate, pH 9.0) for 1 h at RT, washed (TBS + 0.05% Tween-20, TBS, and H2O) and dried (28Jiménez-Castells C. Stanton R. Yan S. Kosma P. Wilson I.B.H. Development of a multifunctional aminoxy-based fluorescent linker for glycan immobilization and analysis.Glycobiology. 2016; 26: 1297-1307PubMed Google Scholar). The slides were incubated with (1) biotinylated forms of peanut agglutinin, wheat germ agglutinin or concanavalin A (VectorLabs; 10 μg/ml or 5 μg/ml in TBS + 0.05% Tween-20 + 1% BSA, i.e. TTBSA) followed by incubation with anti-biotin FITC conjugate (Sigma-Aldrich) (28Jiménez-Castells C. Stanton R. Yan S. Kosma P. Wilson I.B.H. Development of a multifunctional aminoxy-based fluorescent linker for glycan immobilization and analysis.Glycobiology. 2016; 26: 1297-1307PubMed Google Scholar), (2) serum amyloid protein (amyloid P component from human serum, SAP; Fitzgerald, diluted 1:200 in TTBSA) followed by incubation with anti-amyloid P IgG from rabbit (Calbiochem, Merck; in TTBSA) and finally anti-rabbit IgG AlexaFluor-647 conjugate (Invitrogen, Carlsbad, CA; in TTBSA), or (3) anti-L2/HNK-1 (clone 412; diluted 1:1000 in TTBSA) followed by incubation with anti-mouse IgG AlexaFluor-647 conjugate (Invitrogen; in TTBSA). Slides were scanned with an Agilent G2565AA Microarray Scanner (multiple photomultiplier tube (PMT) gain values from 10–100%) and raw fluorescence values (green for FITC and red for AlexaFluor-647) were used to calculate (in Excel) the mean and standard deviation from all ten spots. The negative controls (either only TBS-Tween, no primary lectin or pentraxin for respectively "background" and "no lectin/pentraxin" controls) show fluorescence because of either the AEAB label itself or nonspecific binding of the fluorescent secondary antibodies. Galactosidase (recombinant Aspergillus niger β1,3/4-specific) and glucuronidase (Helix pomatia β-specific) treatments of AEAB-labeled glycans were respectively performed in solution before printing or directly on the printed slides before probing with lectins or antibodies. For further details, refer to pages S6 and S7 of the Supplement. Analysis of glycan-based post-translational modifications of proteins remains a challenge and experience has shown that a thorough analysis of unknown invertebrate glycomes requires multiple fractionation steps in order to prevent suppression effects and to enable separation of isomeric/isobaric structures of the same or similar mass (29Paschinger K. Wilson I.B.H. Analysis of zwitterionic and anionic N-linked glycans from invertebrates and protists by mass spectrometry.Glycoconj. J. 2016; 33: 273-283Crossref PubMed Scopus (19) Google Scholar). Thus, we first separated the free N-glycans of honeybee royal jelly into "neutral" and "anionic" pools, before fluorescent labeling, fractionation on an RP-amide HPLC column (calibrated in terms of glucose units) and MALDI-TOF-MS/MS in combination with chemical and enzymatic treatments (20Hykollari A. Paschinger K. Eckmair B. Wilson I.B.H. Analysis of Invertebrate and Protist N-Glycans.Methods Mol. Biol. 2017; 1503: 167-184Crossref PubMed Scopus (18) Google Scholar). Based on the fluorescence intensities of the two glycan pools, it is estimated that at least 3% of the N-glycans are "anionically" modified. The initial screen, before HPLC, of the pyridylaminated pools by MALDI-TOF MS indicated that the neutral pool contained, in accordance with previous studies (14Kimura Y. Miyagi C. Kimura M. Nitoda T. Kawai N. Sugimoto H. Structural features of N-glycans linked to royal jelly glycoproteins: structures of high-mannose type, hybrid type, and biantennary type glycans.Biosci. Biotechnol. Biochem. 2000; 64: 2109-2120Crossref PubMed Scopus (51) Google Scholar), a range of oligomannosidic and potentially hybrid or c
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