Glycosylation Signatures in Drosophila: Fishing with Lectins

Gianni Vandenborre,†,‡ Els J. M. Van Damme,‡ Bart Ghesquiere,§,| Gerben Menschaert,⊥

Mohamad Hamshou,†,‡ Rameshwaram Nagender Rao,†,‡ Kris Gevaert,§,| and Guy Smagghe*,†

Laboratory of Agrozoology, Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University,Coupure links 653, B-9000 Ghent, Belgium, Laboratory of Biochemistry and Glycobiology, Department ofMolecular Biotechnology, Faculty of Bioscience Engineering, Ghent University, Coupure links 653, B-9000

Ghent, Belgium, Department of Medical Protein Research, VIB, B-9000 Ghent, Belgium, Department ofBiochemistry, Ghent University, B-9000 Ghent, Belgium, and Laboratory for Bioinformatics and Computational

Genomics, Faculty of Bioscience Engineering, Ghent University, Coupure links 653, B-9000 Ghent, Belgium

Received February 26, 2010

Glycosylation is a co- and/or post-translational protein modification that generates enormous structuraldiversity among glycoproteins. In this study, immobilized lectins were used to capture glycoproteinswith different glycan profiles from Drosophila melanogaster extracts. On the basis of previous resultsfrom glycan array analyses, the snowdrop (Galanthus nivalis) agglutinin (GNA), the tobacco (Nicotianatabacum) lectin (Nictaba) and the Rhizoctoni solani agglutinin (RSA) were used to select for a broadrange of N- and O-glycan structures. After different lectin affinity chromatographies, the glycoproteomeof Drosophila was analyzed using LC-MS/MS and glycoprotein abundances were calculated by differentlabel-free methods. Bioinformatics tools were used to annotate the identified glycoproteins and theglycoproteins were classified according to their molecular function or their involvement in a biologicalprocess. Subsequent enrichment analysis (using the DAVID database) was employed to find biologicalprocesses or molecular functions in Drosophila in which a particular glycan signature is overrepresented.The results presented here clearly demonstrate that next to the presence of high-mannose and pauci-mannose N-glycans, Drosophila is capable of synthesizing glycoproteins carrying extended hybrid andcomplex N-linked glycans. Furthermore, it was demonstrated that a specific glycosylation signaturecan be associated with a functionally related group of glycoproteins in Drosophila, both in terms ofbiological process and molecular function.

Keywords: Drosophila melanogaster glycan profiles • glycoproteins • lectins

1. Introduction

Many proteins produced by multicellular organisms carrycarbohydrate structures called glycans that mediate importantroles in several biological processes. The two major forms ofthis protein modification are N-glycans and O-glycans, referringto the type of glycosidic linkage of this carbohydrate structureto the amino acids Asn and Ser/Thr, respectively. The repertoireof proteins carrying glycans is very diverse, including enzymesinvolved in a wide variety of metabolic processes and cellularfunctions. During protein biosynthesis, these glycan structurescan be involved in protein folding, quality control, or targeting.1

As constituents of the cell membrane and extracellular matrix,

glycans can regulate intercellular recognition and are importantfor a plethora of biological signaling processes.2-9

In recent years, glycan research has focused on the develop-ment of highly sensitive and rapid mass spectrometric screen-ing strategies for defining a multitude of glycosylated proteinsin Caenorhabditis elegans,10,11 the fruit fly Drosophila mela-nogaster,12 mammalian cells,13 or specific tissues in knockoutmice.14,15 Despite the availability of the complete Drosophilagenome sequence and a broad range of efficient technical toolsfor molecular, genetic, and cellular studies, relatively little isknown about the variety in protein-linked glycan structures inDrosophila. Studies of N-linked glycans in Drosophila havedocumented the abundance of the high-mannose oligosaccha-rides and core fucosylated pauci-mannose glycans.12,16,17 In-terestingly, the N-glycan profile of the fly changes as develop-ment proceeds, suggesting specific regulation of the glycosylationmachinery and roles for certain glycan structures duringdifferent stages of development.17-19 Recent studies in Droso-phila have also identified proteins modified by mucin typeO-linked glycosylation.20,21 Although many types of O-glyco-sylation are known to exist, the mucin type O-glycosylationwhich involves the addition of N-acetylgalactosamine (GalNAc)

* To whom correspondence should be addressed. E-mail: guy.smagghe@ugent.be.

† Department of Crop Protection, Faculty of Bioscience Engineering,Ghent University.

‡ Department of Molecular Biotechnology, Faculty of Bioscience Engi-neering, Ghent University.

§ Department of Medical Protein Research, VIB.| Department of Biochemistry, Ghent University.⊥ Laboratory for Bioinformatics and Computational Genomics, Faculty

of Bioscience Engineering, Ghent University.

10.1021/pr1001753 2010 American Chemical Society Journal of Proteome Research 2010, 9, 3235–3242 3235Published on Web 04/13/2010

to Ser/Thr, often extended with galactose (Gal), is the mostabundant O-glycan structure in Drosophila.21

Lectins are a diverse group of proteins from nonimmuneorigin that specifically bind with different carbohydrate struc-tures. Although lectins have been used in glycoprotein analysisfor decades, it is only recently that they are being applied toglycoproteomics. Lectins can interact with a specific structuralmotif in a particular glycan, and depending on the specificityof the lectin, a selection for different glycan structures can bemade. Nowadays, the use of lectin affinity chromatography isan established method to selectively enrich certain glycancontaining proteins.20,22,23 Previously, lectin chromatographywith immobilized concanavalin A (ConA) and the elderberry(Sambucus nigra) lectin SNA resulted in the enrichment ofhigh-mannose and complex N-glycosylated proteins fromhuman serum and urine, respectively.22,24 In this study, asimilar approach was utilized to explore the full diversity ofglycan structures in Drosophila by enrichment of glycoproteinscontaining both N- or O-glycan structures using the snowdrop(Galanthus nivalis) agglutinin (GNA), the tobacco (Nicotianatabacum) lectin (Nictaba), and the Rhizoctonia solani agglutinin(RSA) (Figure 1). Glycan array analyses revealed that GNA hasa high specificity for oligomannosidic structures and high-mannose N-glycans,25 while Nictaba recognizes high-mannoseand more processed N-glycans with terminal Gal, N-acetylglu-cosamine (GlcNAc), and GalNAc structures.26 A detailed glycanarray analysis suggests that the binding site of Nictaba is mostcomplementary to the Man3GlcNAc2 core of N-glycans (Figure1). Finally, RSA was employed to broaden the range of capturedglycoproteins, displaying a high affinity for certain mucin typeO-linked glycans along with terminal Gal/GalNAc residues oncomplex N-glycans.27

After purification of Drosophila glycoproteins based on theirinteraction with immobilized lectins, their identity was deter-mined using LC-MS/MS and the corresponding protein abun-dance was estimated using established label-free methods.28

To subdivide the identified proteins into functional groups, thePANTHER (Protein ANalysis THrough Evolutionary Relation-

ships) database was used.29 Additionally, gene annotationenrichment analysis was performed using the DAVID (Databasefor Annotation, Visualization and Integrated Discovery) re-sources to extract biologically valuable information from dif-ferent glycan-associated proteomes.30 For the first time, acomparative analysis was made between several identifiedDrosophila glycoproteins and the available information onglycosylation in Drosophila was employed to evaluate the useof lectins to select for subsets of glycosylated proteins.

2. Materials and Methods

2.1. Insects and Lectins. D. melanogaster was maintainedon a corn meal-based diet31 under standard conditions of23-25 °C, 65-70% relative humidity, and a 16:8 (light/dark)photoperiod. GNA was purified from the bulbs of snowdrop(G. nivalis) using a combination of affinity chromatography andion exchange chromatography as described previously.32 Nic-taba was purified from the tobacco plant (N. tabacum cvSamsun NN) after exposure to the plant hormone jasmonic acidmethyl ester.33 RSA was obtained from sclerotes of the plantpathogenic fungus R. solani using several rounds of affinitychromatography and anion exchange chromatography.34 Thecarbohydrate binding specificity of GNA, Nictaba, and RSA wasstudied using glycan array screening provided by the Consor-tium for Functional Glycomics (www.functionalglycomics.org).

2.2. Affinity Purification of Drosophila Glycoproteins. Ho-mogenates of D. melanogaster were made in liquid nitrogenusing a chilled mortar and pestle. The extraction buffer (0.1 Mphosphate buffer (pH 7.6) containing 2.1% of the EDTA-freeHalt protease inhibitor cocktail (Pierce, Rockford, IL)) wasadded at a ratio of 2 mL/g of tissue. The extract was homog-enized at 4 °C using a glass and Teflon homogenizer (10 strokesat 2000 rpm). Afterward, the homogenates were transferred intoplastic tubes, sonicated for 4 min in an ice water bath, frozenat -80 °C overnight, and thawed at 4 °C the next day. Finally,the homogenates were centrifuged at 9500g for 1 h at 4 °C, andthe supernatants were collected.

Before loading the extract onto the GNA-Sepharose, Nictaba-Sepharose, or RSA-Sepharose columns, the extract was adjustedto 0.2 M NaCl. After washing the columns with 0.2 M NaCl,the lectin-binding proteins were eluted with 20 mM unbuffered1,3-propanediamine. Peak fractions were pooled, adjusted to0.2 M NaCl and pH 7.6, and rechromatographed on the samelectin column. The eluate and flow through after lectin chro-matography were analyzed by SDS-PAGE. Protein concentra-tions were determined with the Bradford method using bovineserum albumin (Sigma-Aldrich) as standard.

2.3. Preparation of Peptides for LC-MS/MS Analysis. Puri-fied proteins (approximately 300 µg) from each lectin affinitycolumn were completely dried and redissolved in freshlyprepared 50 mM ammonium bicarbonate buffer (pH 7.8). Priorto digestion, protein mixtures were boiled for 10 min at 95 °Cfollowed by cooling down on ice for 15 min. Sequencing-gradetrypsin (Promega, Benelux, Leiden, The Netherlands) wasadded in a 1:100 (trypsin/substrate) ratio (w/w) and digestionwas allowed overnight at 37 °C. The sample was acidified with10% acetic acid and loaded for RP-HPLC separation on a 2.1mm internal diameter ×150 mm 300SB-C18 column (Zorbax,Agilent technologies, Waldbronn, Germany) using an Agilent1100 Series HPLC system. Following a 10 min wash with 10mM ammonium acetate (pH 5.5) in water/acetonitrile (98/2(v/v), both solvents were “Baker HPLC analyzed” (MallinckrodtBaker B.V., Deventer, The Netherlands)), a linear gradient to

Figure 1. Schematic diagram of glycoprotein purification usinglectin chromatography and mass spectrometry analysis. A seriesof lectin affinity chromatography steps were employed forenrichment of Drosophila proteins with different glycosylatedmodifications. Glycan specificity of each lectin employed isillustrated in boxes.

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3236 Journal of Proteome Research • Vol. 9, No. 6, 2010

10 mM ammonium acetate (pH 5.5) in water/acetonitrile (30/70, v/v) was applied over 100 min at a constant flow rate of 80µL/min. Eluting peptides were collected in 48 fractions between20 and 68 min. To reduce the number of LC-MS/MS runs,fractions separated by 16 min were pooled and vacuum-driedfor further LC-MS/MS analysis. In this way, 16 (and not 48)LC-MS/MS runs were performed to analyze all peptide fractions.

2.4. Mass Spectrometric Analysis. The fractions were redis-solved in 80 µL of 2.5% acetonitrile (HPLC solvent A). Eightmicroliters of this peptide mixture was applied for nanoLC-MS/MS analysis on an Ultimate (Dionex, Amsterdam, TheNetherlands) in-line connected to an Esquire HCT massspectrometer (Bruker, Bremen, Germany). The sample was firsttrapped on a trapping column (PepMap C18 column, 0.3 mmi.d. × 5 mm, Dionex (Amsterdam, The Netherlands)). Afterback-flushing from the trapping column, the sample wasloaded on a 75 µm i.d. × 150 mm reverse-phase column(PepMap C18, Dionex (Amsterdam, The Netherlands)) Thepeptides were eluted with a linear gradient of 3% solvent B(0.1% formic acid in water/acetonitrile (3/7, v/v)) increase perminute at a constant flow rate of 0.2 µL/min. Using data-dependent acquisition, multiply charged ions with intensitiesabove threshold (adjusted for each sequence according to thenoise level) were selected for fragmentation. During MS/MSanalysis, a MS/MS fragmentation amplitude of 0.7 V and a scantime of 40 ms were used.

2.5. Protein Identification and Bioinformatics. The frag-mentation spectra were converted to mgf files using theAutomation Engine software (version 3.2, Bruker) and weresearched using the MASCOT database search engine (version2.2.0, Matrix Science, http://www.matrixscience.com) againstthe Flybase database (release FB2010_01). Peptide mass toler-ance was set at 0.5 Da and peptide fragment mass tolerance at0.5 Da, with the ESI-IT as selected instrument for peptidefragmentation rules. Peptide charge was set to 1+, 2+, 3+.Variable modifications were set to methionine oxidation, pyro-glutamate formation of amino terminal glutamine, acetylationof the N-terminus, deamidation of glutamine or asparagines.The enzyme was set on trypsin. Only peptides that were rankedone and scored above the threshold score set at 95% confidencewere withheld. The peptide-identification results were madepublicly accessible at the proteomics identification (PRIDE)database (experiment accession number 12011) (http://ww-w.ebi.ac.uk/pride).

Several label-free methods were employed to quantify pep-tide and protein amount.35,36 First, the emPAI index wascalculated to estimate the abundance of the glycoproteinsbased on the number of identified tryptically cleaved peptides(Supporting Information Tables S1-S3).28 Second, a spectralcount method was applied to quantify the proteins based onthe number of identified MS/MS spectra (Supporting Informa-tion Tables S1-S3). For this purpose, a clustering step wasintroduced to group highly similar fragmentation spectra.37 Thecluster parameters and score calculations are outlined in theSupporting Information. In addition, the occurrence of poten-tial N-glycosylation sites present on the glycoproteins wasdetermined by counting the number of consensus sequencesAsn-X-Ser/Thr (where X is any amino acid except proline).

To subdivide the identified glycoproteins into functionallyrelated subfamilies, the PANTHER database (http://www.pan-therdb.org) was used.29,38 Enrichment of annotation terms forthe glycoproteins captured with the different lectins wereanalyzed using the Database for Annotation, Visualization and

Integrated Discovery (DAVID) (http://david.abcc.ncifcrf.gov/).30 Fold enrichment analysis was performed between theidentified glycoproteins and a complete adult Drosophilaproteome data set provided by the DAVID resources (basedon the Flybase database). Statistically overrepresented annota-tion terms were detected using the Benjamini statistics formultiple comparison corrections to calculate the false discoveryrate (FDR).

3. Results and Discussion

3.1. Lectin Affinity Chromatography. To study the diversityin glycoproteins present in Drosophila, lectin chromatographywas used to capture different sets of proteins depending ontheir glycan profile (Supporting Information Figure S1). Iden-tification of the Drosophila glycoproteins that specifically bindto GNA, Nictaba, or RSA was achieved by in-solution digestionof purified proteins using trypsin and subsequent LC-MS/MSanalysis of the resulting peptide mixture using an electrosprayionization-ion trap mass spectrometer. Using the Mascot searchalgorithm and the Flybase database, 138, 185, and 148 glyco-proteins from Drosophila that bound to GNA, Nictaba, and RSA,respectively, were identified (Supporting Information TablesS1-S3). Putative N-glycosylation sites were present on 92.0%,86.5%, and 81.8% of the glycoproteins recognized by GNA,Nictaba, and RSA, respectively, indicating that at least 8.0%,13.5%, and 18.2% of the glycoproteins were captured exclusivelythrough interaction with O-glycans on the GNA-, Nictaba-, andRSA-column, respectively (Table 1). Since the available datafrom Drosophila embryos, larvae, adults, and cultured cellsindicated that especially high-mannose and pauci-mannoseN-glycans are dominant,18,39 it was expected that GNA shouldcapture the majority of N-glycosylated glycoproteins. However,comparison of the different glycoproteomes obtained afterselective enrichment on the different lectin columns revealedthat many proteins were only recognized by Nictaba and RSA(and not by GNA), indicating that complex N-glycans and

Table 1. Number of Putative N-Glycosylated DrosophilaProteins Achieved from ESI-MS Analysis of the Peptides byDifferent Lectin-Affinity Chromatography

lectin

no. ofpeptides

identified

no. ofproteins

identified

putativeN-glycosylated

proteins

GNA 553 138 124Nictaba 635 185 160RSA 754 148 121

Figure 2. Venn diagram of the identified Drosophila proteinscaptured by GNA (n ) 138), Nictaba (n ) 185), or RSA (n ) 148)illustrating the number of lectin-specific glycoproteins or glyco-sylated proteins recognized by more than one lectin.

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O-glycans were more abundant than expected. Identificationof the different glycoproteins indicated that 69, 73, and 71 ofthe proteins were unique for GNA, Nictaba, and RSA, respec-tively (Figure 2). In addition, many glycoproteins were identi-fied that were trapped by more than one lectin column. Forexample, 22 proteins were captured by GNA, Nictaba, as wellas RSA (Figure 2), suggesting that multiple glycan structuresare present on these proteins. Taking into account the resultsof the glycan array analyses of the three different lectins,25,26

it can be expected that GNA selects for high-mannose glyco-sylated proteins, whereas Nictaba targets both glycoproteinscarrying high-mannose as well as more complex glycans. Sinceboth GNA and Nictaba will target high-mannose N-glycans, itis not surprising that 63 common proteins are retrieved fromboth lectin columns (Figure 2). Glycan array analysis for RSAlectin revealed specificity toward N-glycans with terminalgalactose sugars along with the mucin-type O-glycans. Acomparative analysis of the proteins retained on the RSA andNictaba columns yielded 71 glycoproteins that were recognizedby both lectins (Figure 2). Only 6 fly proteins were found tobind to both GNA and RSA (Figure 2).

To study the relative abundances of the identified proteinsin the different protein mixtures, the individual exponentialmodified protein abundance index (emPAI) and an abundanceindex based on spectral counting were calculated (SupportingInformation Tables S1-S3). Although independently calculated,both abundance indexes were correlated with each other forthe majority of the glycoproteins. Interestingly, the presenceof a higher number of putative N-glycosylation sites is notcorrelated with a higher abundance index. Another interestingobservation is the occurrence of several glycoproteins withrelatively high abundance indexes like arginine kinase, actin5C, or aconitase that were captured by both Nictaba and RSA,while these proteins were not identified in the protein mixtureretained on the GNA column. The ATP synthase �-subunit isan example of a protein present in the mixtures derived fromall three lectin columns with a relatively high emPAI in theglycoproteome captured by especially RSA, but also afterenrichment by GNA and Nictaba affinity chromatography.These results offer a remarkably rich source of information thatwill allow more detailed questions to be asked about thespecific roles of glycans in Drosophila.

3.2. Functional Distribution of Glycosylated Proteins. Us-ing the PANTHER database, the putatively glycosylated proteinsresolved from Drosophila were subdivided into different func-tional protein families according to their involvement in aspecific biological process or PANTHER protein class (Sup-porting information Figures S2 and S3). Hereby, it was clearthat the captured glycoproteins for all three lectins are involvedin a broad range of biological processes such as metabolism,cell communication, developmental processes, immune sys-tem, cell adhesion, cell cycle, or transport (Supporting informa-tion Figure S2). However, differences were detected in therelative amount of glycoproteins associated with a certainbiological process between glycoprotein fractions obtainedfrom different lectin columns. For example, the group ofglycoproteins with a metabolic function accounted up to 26.7%,34.8%, and 40.5% in GNA, Nictaba, and RSA captured proteins,respectively. Glycoproteins important for cell adhesion weremore abundant in the glycoprotein fraction retained on a GNAcolumn compared to the glycoproteins bound on Nictaba whileabsent in the RSA binding glycoproteins.

Next to classifying the glycoproteins into subfamilies basedon their involvement in a biological process, the PANTHERclassification database was also used to assign the proteins intogroups according to their known or predicted molecularfunction (Supporting information Figure S3). For the GNA-binding proteins, the largest groups were hydrolases (23.8%),proteases (13.4%), proteins associated with nucleic acid binding(8.5%), receptor proteins (6.7%), defense/immunity proteins(6.1%), proteins important for cell adhesion (5.5%), or signaling(5.5%). Proteins with other molecular functions represented lessthan 5% of the total protein. The Nictaba-binding glycoproteinswere classified as nucleic acid binding (14.1%), hydrolases(14.1%), oxidoreductases (12.2%), transferases (9.8%), cytosk-eletal proteins (5.4%), or enzyme modulators (5.4%). Classifica-tion of the RSA binding proteins according to molecularfunction revealed nucleic acid binding proteins (14.7%), trans-ferases (11.5%), hydrolases (10.9%), oxidoreductases (9.6%),chaperones (8.3%), and enzyme modulators (5.8%) as the majorcategories (>5%). Interestingly, hydrolases, proteases, andreceptor proteins were more abundant in the GNA bindingproteins compared to the glycoproteins retained on Nictabaand RSA. In contrast, oxidoreductases and transferases aremore specific for Nictaba and RSA suggesting the presence ofespecially complex N-glycans with terminal Gal/GalNAc onthese enzymes. It is striking that the classification of glycopro-teins bound to Nictaba and RSA is very similar.

3.3. Enrichment Analysis for Lectin CapturedGlycoproteins. For a detailed study of the different glycopro-teomes, the DAVID resources were used to highlight the mostoverrepresented (enriched) GO annotation terms (>2-foldenrichment compared to a Drosophila background; p < 0.05;FDR < 0.05) for biological processes in the different proteinlists. This analysis resulted in only three general gene ontologyterms associated with cell adhesion for the GNA-bindingglycoproteins (Table 2). When enrichment analysis searchesbased on ontology terms related to molecular function for theGNA binding glycoproteins was complete, galactose-specificC-type lectin activity and aminopeptidase activity were en-riched most (>15-fold) (Table 3). Both molecular activities areassociated with membrane proteins which is also in agreementwith the enriched terms for biological processes. Also hydrolaseactivity and ribosomal proteins are annotation terms that areoverrepresented (>5-fold) in the protein fraction retained onthe GNA column.

For the Nictaba bound glycoproteins, 28 ontology termentries were identified as significantly enriched biologicalprocesses (p < 0.05; FDR < 0.05) (Table 2). Pyruvate metabolism,acetyl-CoA metabolism, tricarboxylic acid cycle, and glycolysisare >10-fold enriched compared to the general Drosophilabackground. Moreover, almost all enriched ontology termsbelong to the general cellular respiration activity. When thesame analyses were performed to search for enriched annota-tions for molecular function in the glycoprotein fraction fromthe Nictaba column, enzymes with oxidoreductase activity,coenzyme binding, hydrolase activity, and pyrophosphataseactivity were found (Table 3) which is in agreement with theannotation terms found for biological processes (Table 2). Alsoribosomal proteins were found to be enriched (>5-fold).

When the same enrichment analysis for biological processeswas performed on the list of proteins that bound to RSA, 40term entries were significantly enriched (p < 0.05; FDR < 0.05)(Table 2). Similar to Nictaba, most of the annotation terms wereassociated with cellular respiration. The enrichment analysis

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Table 2. Enrichment Analysis Using DAVID for the Annotation Terms Associated with Biological Processes for GNA-, Nictaba-, andRSA-Captured Glycoproteins

term fold enrichmenta p-valueb

Glycoproteome with GNA-Specific Glycansc

1 cell-cell adhesion 8.5 4.50 × 10-4

2 cell adhesion 4.3 1.40 × 10-2

3 biological adhesion 4.1 1.50 × 10-2

Glycoproteome with Nictaba-Specific Glycansc

1 pyruvate metabolic process 20.8 8.00 × 10-4

2 acetyl-CoA metabolic process 17.7 5.50 × 10-10

3 acetyl-CoA catabolic process 17.3 3.50 × 10-9

4 aerobic respiration 17.3 3.50 × 10-9

5 cellular respiration 17.3 3.50 × 10-9

6 tricarboxylic acid cycle 17.3 3.50 × 10-9

7 coenzyme catabolic process 16.5 3.00 × 10-9

8 cofactor catabolic process 16.1 3.70 × 10-9

9 glycolysis 10.1 1.30 × 10-3

10 alcohol catabolic process 9.5 1.50 × 10-4

11 glucose catabolic process 9.0 8.10 × 10-4

12 hexose catabolic process 9.0 8.10 × 10-4

13 monosaccharide catabolic process 9.0 8.10 × 10-4

14 glucose metabolic process 8.7 8.50 × 10-5

15 hexose metabolic process 6.8 2.20 × 10-4

16 monosaccharide metabolic process 6.4 3.40 × 10-4

17 coenzyme metabolic process 6.0 7.40 × 10-9

18 cellular carbohydrate metabolic process 5.5 2.80 × 10-10

19 cofactor metabolic process 5.5 2.90 × 10-8

20 monocarboxylic acid metabolic process 5.4 1.40 × 10-3

21 cellular catabolic process 4.8 3.00 × 10-9

22 catabolic process 4.4 7.00 × 10-9

23 carbohydrate metabolic process 4.1 4.30 × 10-10

24 generation of precursor metabolites and energy 3.7 7.50 × 10-8

25 organic acid metabolic process 3.7 2.20 × 10-5

26 carboxylic acid metabolic process 3.7 2.20 × 10-5

27 biosynthetic process 2.1 2.60 × 10-4

28 cellular biosynthetic process 2.1 5.20 × 10-4

Glycoproteome with RSA-Specific Glycansc

1 pyruvate metabolic process 26.5 1.80 × 10-4

2 glycolysis 21.0 1.60 × 10-10

3 acetyl-CoA metabolic process 21.0 1.60 × 10-10

4 cellular respiration 20.4 1.80 × 10-9

5 tricarboxylic acid cycle 20.4 1.80 × 10-9

6 aerobic respiration 20.4 1.80 × 10-9

7 acetyl-CoA catabolic process 20.4 1.80 × 10-9

8 translational elongation 19.9 7.40 × 10-4

9 coenzyme catabolic process 19.4 2.60 × 10-9

10 alcohol catabolic process 19.3 1.20 × 10-12

11 cofactor catabolic process 18.9 3.30 × 10-9

12 response to unfolded protein 18.5 1.50 × 10-4

13 response to protein stimulus 18.5 1.50 × 10-4

14 hexose catabolic process 17.8 2.30 × 10-10

15 monosaccharide catabolic process 17.8 2.30 × 10-10

16 glucose catabolic process 17.8 2.30 × 10-10

17 glucose metabolic process 15.0 2.00 × 10-10

18 cellular carbohydrate catabolic process 13.2 4.60 × 10-9

19 response to heat 12.7 8.00 × 10-6

20 response to temperature stimulus 11.4 1.90 × 10-5

21 carbohydrate catabolic process 11.0 4.40 × 10-8

22 hexose metabolic process 10.8 1.20 × 10-8

23 monosaccharide metabolic process 10.2 2.40 × 10-8

24 protein folding 7.8 5.40 × 10-8

25 cellular catabolic process 7.3 6.00 × 10-17

26 cellular carbohydrate metabolic process 6.7 1.00 × 10-12

27 catabolic process 6.5 1.50 × 10-15

28 cellular macromolecule catabolic process 6.5 7.30 × 10-7

29 alcohol metabolic process 6.5 7.30 × 10-7

30 coenzyme metabolic process 6.3 1.30 × 10-7

31 response to abiotic stimulus 6.1 1.40 × 10-5

32 cofactor metabolic process 5.8 4.20 × 10-7

33 macromolecule catabolic process 5.3 1.20 × 10-5

34 carbohydrate metabolic process 4.4 1.60 × 10-9

35 response to stress 4.0 3.40 × 10-4

36 carboxylic acid metabolic process 3.7 2.40 × 10-4

37 organic acid metabolic process 3.7 2.40 × 10-4

38 generation of precursor metabolites and energy 3.4 7.90 × 10-5

39 cellular biosynthetic process 2.2 6.60 × 10-4

40 biosynthetic process 2.1 2.10 × 10-3

a Enrichment factor (fold change >2; FDR < 0.05). b Benjamini p-value. c Enrichment analysis for annotation terms using the DAVID resources.

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for molecular function among the RSA-binding proteins alsorevealed annotation terms similar to those for glycoproteinsbinding to Nictaba (Table 3).

3.4. Interesting Drosophila Glycoproteins. One interestingexample of a glycoprotein identified in the present study is theretinoid- and fatty acid-binding glycoprotein (RFABG), a majorcomponent of the retinal extracellular matrix, which bindsexogenous retinol and palmitic acid, and exhibits endogenousbinding of both fatty acids and retinoids.40 In this report,RFABG was shown to be the second most abundant protein inthe RSA captured glycoproteins (using both the emPAI indexand the abundance index based on spectral counting). Incontrast, RFABG was more than 15 times less abundant afterrunning on a Nictaba column while it was not captured at all

by GNA. These results suggest the presence of especiallyO-glycans or more complex N-glycans with terminal GalNAcresidues, while the high-mannose N-glycans are absent. Be-cause 18 putative N-glycosylation sites were found in theprotein sequence, probably the complex N-glycans are the mostprominent carbohydrate modifications on RFABG. Unfortu-nately, no data are available in the literature to verify ourobservation concerning the glycosylation pattern of RFABG.

The N-glycan profile for the glycoprotein chaoptin wasrecently studied in detail.41 This photoreceptor protein is amember of the leucine-rich repeat family. Although high-mannose type glycans, pauci-mannose, and complex typeglycans were detected on the chaoptin polypeptide, the high-mannose and pauci-mannose were the most abundant N-

Table 3. Enrichment analysis using DAVID for the annotation terms associated with molecular function for GNA-, Nictaba- andRSA-captured glycoproteins

term fold enrichmenta p-valueb

Glycoproteome with GNA-Specific Glycansc

1 galactose binding 34.7 3.30 × 10-3

2 alanine aminopeptidase activity 16.7 5.90 × 10-3

3 membrane alanyl aminopeptidase activity 16.7 5.90 × 10-3

4 aminopeptidase activity 12.4 5.60 × 10-3

5 exopeptidase activity 9.3 6.00 × 10-5

6 hydrolase activity, acting on glycosyl bonds 7.7 5.30 × 10-3

7 structural constituent of ribosome 6.7 7.50 × 10-5

8 structural molecule activity 3.9 2.80 × 10-5

9 peptidase activity 3.1 6.00 × 10-4

10 hydrolase activity 2.2 7.00 × 10-5

Glycoproteome with Nictaba-Specific Glycansc

1 oxidoreductase activity, acting on the aldehyde or oxo group ofdonors

16.1 6.70 × 10-4

2 oxidoreductase activity, acting on the CH-OH group of donors,NAD or NADP as acceptor

9.7 1.90 × 10-5

3 cofactor binding 8.1 1.10 × 10-5

4 coenzyme binding 8.1 7.20 × 10-5

5 oxidoreductase activity, acting on CH-OH group of donors 6.4 4.80 × 10-4

6 structural constituent of ribosome 5.1 1.50 × 10-4

7 structural molecule activity 3.4 1.60 × 10-6

8 nucleoside-triphosphatase activity 2.9 7.50 × 10-4

9 pyrophosphatase activity 2.8 9.40 × 10-4

10 hydrolase activity, acting on acid anhydrides, inphosphorus-containing anhydrides

2.8 1.20 × 10-3

11 hydrolase activity, acting on acid anhydrides 2.8 1.20 × 10-3

12 oxidoreductase activity 2.6 2.00 × 10-4

13 Purine nucleotide binding 2.5 6.70 × 10-5

14 nucleotide binding 2.3 5.90 × 10-5

15 Purine ribonucleotide binding 2.3 5.80 × 10-4

16 ribonucleotide binding 2.3 5.80 × 10-4

Glycoproteome with RSA-Specific Glycansc

1 oxidoreductase activity, acting on the aldehyde or oxo group ofdonors

19.9 3.00 × 10-4

2 unfolded protein binding 9.2 3.40 × 10-3

3 structural constituent of ribosome 6.6 3.10 × 10-6

4 structural molecule activity 4.3 1.60 × 10-9

5 nucleoside-triphosphatase activity 3.3 3.80 × 10-4

6 pyrophosphatase activity 3.2 4.50 × 10-4

7 hydrolase activity, acting on acid anhydrides, inphosphorus-containing anhydrides

3.2 5.40 × 10-4

8 hydrolase activity, acting on acid anhydrides 3.2 5.10 × 10-4

9 oxidoreductase activity 2.8 5.50 × 10-4

10 purine nucleotide binding 2.7 9.00 × 10-5

11 purine ribonucleotide binding 2.6 2.20 × 10-4

12 ribonucleotide binding 2.6 2.20 × 10-4

13 nucleotide binding 2.3 1.00 × 10-3

a Enrichment factor (fold change >2; FDR < 0.05). b Benjamini p-value. c Enrichment analysis for annotation terms using the DAVID resources.

research articles Vandenborre et al.

3240 Journal of Proteome Research • Vol. 9, No. 6, 2010

glycan-types on this glycoprotein which explains its highabundant ranking in the glycoproteins captured by GNA. Asexpected, chaoptin was also captured by Nictaba, but itsabundance was 2.3 times less than on the GNA-column. Incontrast, no chaoptin was bound to RSA, supporting therecently published N-glycan profile data on chaoptin.41

Laminin is a large heterotrimeric glycoprotein which is aprominent component of the basement membrane and isinvolved in important biological roles, including tissue devel-opment and cell differentiation, survival, adhesion, and migra-tion. Laminin is a heavily glycosylated molecule and it has beenreported that between 13% and 30% of the total molecularweight of laminin is due to N-linked glycans.42 In our study,two subunits of laminin were captured by both GNA andNictaba, whereas these glycoproteins were not present amongthe RSA binding proteins. Both lamininA and B were presentamong the 10 most abundant GNA-binding proteins. Theseobservations enable us to predict that the laminin subunits aremainly decorated with high-mannose N-glycan structures.Genetic studies have shown that null mutations in the Droso-phila laminin R3,5 chain lead to embryonic lethality with visibledefects in mesodermally derived tissues leading to dissociatedcell groups in the various organs.43 In the light of these findings,it is interesting to mention an earlier hypothesis about thepossible role of lectin-type interaction between heterophilic celladhesion molecules that involve oligomannosidic glycans onthese molecules.44

The protein called NUCB1 was one of the most abundantproteins among the RSA-binding glycoproteins, while thisprotein was not captured by the other lectins. Moreover, noputative N-glycosylation site was found in the NUCB1 polypep-tide (Supporting Information Table S3), suggesting that bindingto RSA is the result of interactions with putative O-glycanstructures on NUCB1. NUCB1 is associated with the Golgiapparatus, but its exact role remains unknown in Drosophila.45

In agreement with its RSA specific interaction, it was shownthat Drosophila generates the mucin-type T-antigen (Gal�1-3GalNAcR-Ser/Thr) as predominant O-glycan structure onglycoproteins.21 Mutations in one member of the enzymesresponsible for mucin-type O-glycosylation (pgant35A) waseven shown to abrogate enzymatic activity with death occurringthroughout development,19 illustrating the crucial role for thistype of O-glycans during development of Drosophila.

4. Conclusion

In conclusion, the data presented in this paper illustrate amethodology to select for specific glycoproteins based on theuse of several lectins with different carbohydrate specificities.Comparing the glycoproteins captured by GNA, Nictaba, andRSA allowed assigning glycan profiles of the Drosophila proteinsto high-mannose or pauci-mannose N-glycans, complex N-glycans, or O-glycosylated proteins. Moreover, the resultspresented here demonstrate that Drosophila is capable ofexpressing extended hybrid and complex N-linked glycans, inaddition to the expected family of abundant high-mannose andpaucimannose oligosaccharides. A recent publication cor-roborates the existence of complex glycans in Drosophila.39

Furthermore, it was demonstrated that a specific glycosylationsignature can be associated with a functionally related groupof glycoproteins in Drosophila, both in terms of biologicalprocess and molecular function. However, it is worth empha-sizing that, although the large number of identified glycopro-teins from Drosophila is unprecedented, this is not an exhaus-

tive database of glycosylated proteins expressed in Drosophila.Low-abundance glycopeptides might have fallen below thedetection threshold of the MS or generated MS/MS fragmenta-tion patterns of insufficient robustness, and hence could notbe identified. In addition, although GNA, Nictaba, and RSA areperhaps among the most suitable lectins to recover the largestproportion of N- and O-glycosylated proteins, there will beother glycoproteins with glycan structures that do not bindefficiently to any of the lectins utilized in our study.

Acknowledgment. This research was supported by theResearch Council of Ghent University (project BOF10/GOA/003), the Fund for Scientific Research-Flanders (3G016306,G. Smagghe, and E. J. M. Van Damme). B.G. is aPostdoctoral Research Fellow of the Fund for ScientificResearch - Flanders (Belgium). The UGent/VIB labacknowledges support by research grants from the Fund forScientific Research - Flanders (Belgium) (project G015605,G007706, G004207), the Concerted Research Actions fromthe Ghent University and the Inter University AttractionPoles (project BOF07/GOA/012 and IUAP06). We also wantto thank the Consortium for Functional Glycomics forglycan array analyses.

Supporting Information Available: The cluster pa-rameters, emPAI calculation, and spectral count score calcula-tions. Table S1: annotation of the identified proteins bound tothe GNA column. List is characterized by accession number,two abundance indexes (emPAI and spectral count) and thenumber of putative N-glycosylation sites. Table S2: annotationof the identified proteins bound to the Nictaba column. List ischaracterized by accession number, two abundance indexes(emPAI and spectral count) and the number of putativeN-glycosylation sites. Table S3: annotation of the identifiedproteins bound to the RSA column. List is characterized byaccession number, two abundance indexes (emPAI and spectralcount) and the number of putative N-glycosylation sites. FigureS1: Coomassie stained SDS-PAGE analysis of eluate and flowthrough after GNA, Nictaba, and RSA affinity chromatography.Lanes 1 and 9 were loaded with a protein marker (PagerulerPrestained Protein Ladder, Fermentas), lane 2 was loaded withthe Drosophila extract, lanes 3-5 with the peak fraction of theeluate after GNA, Nictaba, and RSA chromatography, respec-tively, and lanes 6-8 with the flow through after GNA, Nictaba,and RSA chromatography, respectively. Figure S2: functionalclassification according to biological process of the glycopro-teins of Drosophila captured by GNA (A), Nictaba (B), or RSA(C) using PANTHER. The numbers on the figure refer to thepercentage of identified proteins within each category. FigureS3: functional classification of Drosophila glycoproteins ac-cording to PANTHER. Proteins were captured by GNA (A),Nictaba (B), or RSA (C). The numbers on the figure refer to thepercentage of identified proteins within each category. Thismaterial is available free of charge via the Internet at http://pubs.acs.org.

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