Surface-exposed Glycoproteins of Hyperthermophilic Sulfolobus

Article pubs.acs.org/jpr

Surface-exposed Glycoproteins of Hyperthermophilic Sulfolobus solfataricus P2 Show a Common N-Glycosylation Profile Gianna Palmieri,† Marco Balestrieri,† Jasna Peter-Katalinić,‡,§ Gottfried Pohlentz,∥ Mosè Rossi,† Immacolata Fiume,† and Gabriella Pocsfalvi*,† †

Institute of Protein Biochemistry, National Research Council of Italy, Napoli, Italy Institute of Medical Physics and Biophysics, University of Münster, Münster, Germany § Department of Biotechnology, University of Rijeka, Rijeka, Croatia ∥ Institute for Hygiene, University of Münster, Münster, Germany ‡

S Supporting Information *

ABSTRACT: Cell surface proteins of hyperthermophilic Archaea actively participate in intercellular communication, cellular uptake, and energy conversion to sustain survival strategies in extreme habitats. Surface (S)-layer glycoproteins, the major component of the S-layers in many archaeal species and the best-characterized prokaryotic glycoproteins, were shown to have a large structural diversity in their glycan compositions. In spite of this, knowledge on glycosylation of proteins other than S-layer proteins in Archaea is quite limited. Here, the N-glycosylation pattern of cell-surface-exposed proteins of Sulfolobus solfataricus P2 were analyzed by lectin affinity purification, HPAEC-PAD, and multiple mass spectrometry-based techniques. Detailed analysis of SSO1273, one of the most abundant ABC transporters present in the cell surface fraction of S. solfataricus, revealed a novel glycan structure composed of a branched sulfated heptasaccharide, Hex4(GlcNAc)2 plus sulfoquinovose where Hex is D-mannose and D-glucose. Having one monosaccharide unit more than the glycan of the S-layer glycoprotein of S. acidocaldarius, this is the most complex archaeal glycan structure known today. SSO1273 protein is heavily glycosylated and all 20 theoretical N-X-S/T (where X is any amino acid except proline) consensus sequence sites were confirmed. Remarkably, we show that several other proteins in the surface fraction of S. solfataricus are N-glycosylated by the same sulfated oligosaccharide and we identified 56 N-glycosylation sites in this subproteome. KEYWORDS: Archaea, N-glycosylation, Sulfolobus solfataricus, surface layer, SSO1273, ABC transporters

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INTRODUCTION Glycosylation of asparagine residues (N-linked glycosylation) is one of the most ubiquitous forms of posttranslational modification, imparting fundamental structural and functional roles to membrane and secreted proteins.1 A recent highthroughput mapping of N-glycoproteomes of six phylogenetically distant eukaryotic species significantly extended the number of known glycosylation sites, allowing questions relating to the evolution of the N-glycoproteome to be addressed.2 A surprisingly high number of N-glycosylated proteins specific to the phylogenetic class of the model organisms studied was found within the major eukaryotic lineages. Beside this specificity, eukaryotic N-glycoproteins also share several common features, including canonical sequence motifs (N-X-S/T), subcellular localization and a core pentasaccharide glycan structure (Man3GlcNAc2). It has long been known that prokaryotic cells are also capable of synthesizing N-glycosylated proteins. The first noneukaryal Nglycoprotein was purified and characterized from Halobacterium salinarum by Mescher and Strominger in 1976.3 Since then, © 2013 American Chemical Society

several other glycoproteins have been identified, predominantly from the surface (S)-layer and the flagella of halophilic (Hbt. salinarum, Haloferax volcanii and Haloarcula marismortui),4−6 methane producing (Methanococcus voltae, Methanococcus maripaludis and Methanococcus fervidius)7−9 and sulfur-dependent (Sulfolobus acidocaldarius)10,11 Archaea species (Figure 1). On the basis of current data, archaeal N-glycans show an unexpected structural diversity not seen in the other two domains of life. Furthermore, they are usually short in length (less than six monosaccharaides) and often undergo modifications such as methylation and sulfation. For example, a methylated pentaglycan has been purified from two halophilic Archaea (H. volcanii and H. marismortui)6,12 and sulfated sugars have been described for S. acidocaldarius10,11 and Hbt. salinarum.3 Due to the analytical complexities associated with studying structurally diverse N-glycans, there are only 21 wellcharacterized archaeal glycoproteins described in the recently Received: February 7, 2013 Published: April 15, 2013 2779

dx.doi.org/10.1021/pr400123z | J. Proteome Res. 2013, 12, 2779−2790

Journal of Proteome Research

Article

Figure 1. Schematic view of N-glycan structures identified in different archaeal organisms.

concentrations.25 An impaired N-glycosylation pathway results in unstructured S-layer formation26 and reduction in the number of flagella associated with reduced cellular stability and motility.24 Sulfolobus solfataricus P2 was first isolated in Solfatara volcano near Naples, Italy and is widely used as a model organism of aerobic thermoacidophilic crenarchaeon in archaeal research. S. solfataricus is metabolically dependent on sulfur and belongs to the family of Sulfolobales. It grows optimally at 80 °C and at a pH of 3−4. Sulfolobales possess a single membrane and a “quasi-periplasmic space” between the cytoplasmic membrane and the S-layer. Transmission electron microscopy analysis of the three-dimensional structure of S. solfataricus Slayer shows the layer p3 symmetry with a reported lattice constant of around 21 nm.27 Recent studies suggested that the S-layer protein and several membrane-bound multisubunit complex containing solute binding proteins (SBPs) of ABC transporters are N-glycosylated.28−30 Nevertheless, the glycan structure(s) of N-glycoproteins of S. solfataricus has not yet been characterized. In the present study, by analyzing the Nglycosylation pattern, glycan type and glycosylation sites of the cell surface proteins of S. solfataricus, we provide evidence that the chitobiose core specific to S. acidocaldarius is characteristic to several cell surface proteins of this hyperthermoacidophile.

published ProGlycProt database (http://www.proglycprot. org).13 The archaeal N-glycoproteins characterized to date lack a common core N-glycan structure, except the S-layer,11 Cytochrome b558/56610 and flagella14 glycoproteins of S. acidocaldarius, which share a common chitobiose core disaccharide (GlcNAc2) characteristic of eukaryotic N-linked glycans too.11 While the eukaryal and bacterial pathways of N-glycosylation are relatively well-defined, little was known until recently of the corresponding processes in Archaea. Recent studies shed light on salient aspects of sugar transport and archaeal Nglycosylation pathway,9,15−18 such that a more comprehensive picture of the archaeal version of this protein modification is now emerging. Briefly, the process of N-glycosylation starts at the cytoplasmic side of the plasma membrane. Activated sugar precursors are added stepwise onto the lipid carrier dolichol by specific glycosyltransferases to assemble the nascent oligosaccharide chain. The preassembled oligosaccharide is flipped across the cytoplasmic membrane by a putative flippase not yet identified in Archaea. The oligosaccharide is transferred by a single oligosaccharyltransferase, called AglB in Archaea, to the asparagine residue in the canonical glycosylation motif N-X-S/ T (where X is any amino acid except proline) of the protein. Interestingly, apart from the AglB, other archaeal enzymes involved in the N-glycosylation machinery show no homology to each other, indicating a wide diversity in the N-glycosylation process across the Archaea.17 While N-linked glycosylation seems not to be essential for euryarchaeota,19 it is not true for crenarcheota, such as S. solfataricus, in which tunicamycin inhibits cell division by affecting protein glycosylation.20 In addition, N-glycosylation has been shown to be important in motility, cellular adhesion, cell−cell communication, biofilm formation and maintenance of cellular shape, and thus for adaptation to extreme environments.9,21−24 In H. volcanii, for example, impaired or absent N-glycosylation of the S-layer glycoproteins greatly reduces cell proliferation at high salt

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MATERIALS AND METHODS

S. solfataricus Growth Conditions

S. solfataricus P2 (DSM 1617) strain was aerobically grown at 80 °C in glycine-buffered Brock’s medium supplemented with 0.05% yeast extract, 0.2% sucrose and 0.2% tryptone at pH 3.2 to the late exponential phase (OD650 of 1.2). Preparation of Cell Surface Protein Fraction and Glycoprotein Enrichment

Cells were harvested, resuspended in sample buffer (20 mM Tris-HCl pH 6.5, 0.7 mM PMSF and protease inhibitor 2780



Article

Purification of SSO1273 Protein from the Cell Envelope

cocktail) and lysed by eight cycles with a 15 s pulse and 45 s off sonication. Cell debris was removed by low-speed centrifugation at 2000× g at 4 °C for 20 min. Cell membranes were collected by ultracentrifugation at 100000× g at 4 °C for 45 min. The pellet was washed four times in sample buffer and solved in solubilization buffer (sample buffer supplemented with 1% Triton X-100) for 30 min at 37 °C. Insoluble material was removed by ultracentrifugation at 250000× g at 4 °C for 45 min, and the supernatant was dialyzed against 20 mM Tris-HCl pH 6.5, 0.05% Triton X-100. This is the surface membrane protein sample used in all the subsequent study. Protein concentration was determined by the Bradford assay (Bio-Rad). The membrane surface protein sample was enriched in glycoproteins using a ConA-Sepharose 4B affinity media (5 mL, GE Healthcare) according to manufacturer’s instruction. Briefly, the sample (2 mg) was loaded in buffer A (20 mM Tris-HCl pH 6.5, 0.5 M NaCl, 0.05% Triton X-100) and washed five times with the same buffer. Bound glycoproteins were eluted by 200 mM α-methylmannopyranoside prepared in buffer A. After enrichment, the sample was dialyzed against 20 mM Tris-HCl pH 6.5 and stored at −80 °C until use.

The purification of SSO1273 protein was performed by electroelution as described earlier.29 Briefly, surface membrane protein sample (500 μg) was loaded onto 8% SDS-PAGE (20 × 20 cm) from which the spacers between a few lanes had been removed to achieve high loading capacity. Gel electrophoresis was carried out at 14 mA overnight at 4 °C. A vertical strip of the gel, containing the reference well with the molecular weight marker (Bio-Rad, Hercules, CA) and the beginning of the large well, was stained with Coomassie brilliant blue and used as the reference for the horizontal cut of the lane containing the SSO1273 from the unstained portion of the gel. The excised gel band was subjected to electroelution for 2 h at 50 mA at 25 °C in a dialysis membrane tube containing 1 mL of SDS-PAGE running buffer. Detergent was removed by dialysis against 20 mM Tris-HCl, 0.05% sodium deoxycholate pH 6.5, and the purified protein was used for further analysis. Molecular mass of the purified protein was measured by SELDI-TOF MS (BioRad, Hercules, CA) using a NP20 protein-chip (BioRad Hercules, CA) and saturated sinapinic acid in 50% ACN, 0.2% trifluoracetic acid as a matrix in the range of m/z 1000−180000. Three independent measurements were performed.

1DE-SDS-PAGE Analysis and Protein Identification

In-solution Digestion and Mass Spectrometry Analysis of SSO1273 Derived (Glyco)Peptides

Surface protein and surface glycoprotein enriched samples (20 μg) were electrophoretically separated on a precast Novex 4− 12% Bis-Tris NuPAGE gel (Life Technologies, Carlsbad, CA) using MOPS running buffer, according to the manufacturer’s instructions. Each gel lane was cut into 30 slices (2 mm each) washed and in-gel digested according to Shevchenko et al31 using trypsin (Promega, Madison, WI) at 6 ng/μL in 50 mM NH4HCO3 10% ACN for 16 h. Peptides were extracted sequentially using 100 μL 30% ACN 3.5% formic acid (HCOOH), 100 μL 50% ACN 5% HCOOH and 100 μL ACN. The fractions were combined, vacuum-dried and analyzed by nano-RP-HPLC−ESI−MS/MS using the QStar Elite (Applied Biosystems, Foster City, CA) equipped with a nanoflow electrospray ion source and interfaced with an Ultimate 3000 (LCPackings, Sunnyvale, CA) HPLC system. A Trap column PepMap, C18, 5 mm length, 300 Å, (LCPackings, Sunnyvale, CA) at 30 μL/min flow rate was used for 5 min to purify and concentrate the peptides. Peptide separation was performed by using a capillary column, PepMap, C18, 15 cm length, 75 μm ID, 300 Å (LCPackings, Sunnyvale, CA, USA), solvent A: 2% ACN in 0.1% HCOOH and solvent B: 98% ACN in 0.1% HCOOH at a flow rate of 300 nL/min. The following gradient was used: 5−50% B in 30 min, 50−98% B in 6 s. CID experiments were carried out in IDA mode using nitrogen as a collision gas. Two independent nano-HPLC− ESI−MS/MS experiments were performed for each sample. Tandem mass spectra were extracted and peak lists were generated by Analyst QS 2.0 software using the default parameters. Combined peak lists were analyzed using Mascot Server (version 2.2) and X! tandem. Mascot Server was set up to search the S. solfataricus P2 proteome extracted from NCBInr database and containing 7290 entries. Trypsin was specified as the digestion enzyme with a maximum of one missed cleavage site. Scaffold_3.6.2 (Proteome Software Inc.) was used to validate MS/MS based peptide and protein identifications. Protein identifications were accepted if they were greater than 99.0% probability and contained at least two unique identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm.32

SSO1273 (40 μg, 50 μL) was denatured by addition of denaturing buffer (100 mM Tris, 6 M guanidinium hydrochloride, pH 8.6) to a final volume of 250 μL at 56 °C for 60 min. The sample was than buffer exchanged to approximately 480 μL of 10 mM ammonium bicarbonate, pH 7.5 using NAP 5 Sephadex G-25 DNA grade columns (GE Healthcare, Buckingamshire, UK). Enzymatic digestion was performed overnight at 37 °C at a substrate/trypsin/chymotrypsin ratio of 25:1:2.6. The product of proteolysis was vacuum-dried, solubilized in 30% methanol, 5% HCOOH and analyzed in the positive ion mode using two different instruments. NanoESI-MS experiments were carried out on a quadrupole time-of-flight (Q-TOF) mass spectrometer (Micromass, Manchester, U.K.) equipped with a Z-spray source. Typical source parameters were: source temperature 80 °C, desolvation gas (N2) flow rate of 75 L/h, a capillary voltage of 1.1 kV, and a cone voltage of 30 V. For low energy CID experiments, the (glyco)peptide precursor ions were selected in the quadrupole analyzer and fragmented in the collision cell using a collision gas (Ar) pressure of 3.0 × 10−3 Pa and collision energies of 20− 40 eV (Elab). (Glyco)peptide structures were deduced from the resulting fragment ion spectra. RP-nano HPLC−ESI−MS/ MS experiments were carried out in IDA mode using the Ultimate 3000 (LCPackings, Sunnyvale, CA) HPLC online coupled to the Qstar Elite (Applied Biosystems, Foster City, CA) mass spectrometer with the experimental parameters described before. For peptide sequence identification Mascot Server 2.2 (Matrix Science, London, U.K.) was used. Trypsin and chymotrypsin were specified as the digestion enzymes with a maximum of three missed cleavage sites. CAM for cysteine was set as fixed modification. Allowed variable modifications were oxidation of methionine and pyro-glutamic acid formation at the N-terminus. Electroblotting onto PVDF Membrane, PNGase Enzymatic Cleavage and Purification of N-Linked Glycans

Glycoproteins separated by 1D-SDS-PAGE were transferred onto polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA) using a Trans-Blot Cell apparatus (Bio-Rad, 2781



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measurement accuracy was determined to be within ±5 ppm in MS/MS mode in the m/z range 300−1500.

Hercules, CA) at 50 mA constant current at 4 °C overnight. Membrane was stained with Coomassie brilliant blue. PVDF bands were cut, placed into eppendorf tubes, washed once with 200 μL of 1% (v/v) NP40 in 50% methanol for 20 min under agitation, and six times with water. N-Linked glycans were enzymatically released from the protein by addition of 5 μL of 0.5 unit/μL PNGase F (Proteomics grade, Sigma-Aldrich, Milano, Italy) and 10 μL 10 mM phosphate buffer pH 7.6 at 37 °C overnight. Released oligosaccharides were purified on graphitized carbon, Extract Clean Carbo (Grace Division Discovery Sciences) packed on the top of 10 μL ZipTip C18 pipet tips (Millipore, Billerica, MA) according to Wilson et al.33 Eluted glycans were vacuum-dried and 1/10 of the sample was used for HILIC−ESI−MS/MS experiments and the rest for HPAEC-PAD analysis.

Bioinformatics

SignalP 4.1 Server program was used to identified proteins for N-terminal signal peptides indicative for the type II protein secretion system.34 Proteins that were positive at least for one of the three SignalP predictions (Gram-positive prokaryotes, Gram-negative prokaryotes, and eukaryotes) were considered to be potentially exported by Sec or Tat mechanisms. Putative Tat signal peptides were predicted by TatP 1.035 and TATFIND 1.4.36 FlaFIND 1.0 was used to determine the presence of archaeal class III (type IV pilin-like) signal peptides.37 Nonclassical secretion mechanisms were predicted by SecretomeP 2.0.38 N-Linked glycosylation analysis was performed using NetNGlyc 1.0 server.39 ProGlycProt database13 (http://proglycprot.org/index.aspx) was used to consult already characterized prokaryotic N-glycan structures. TransportDB (http://www.membranetransport.org)40 was referred for the classification of archaeal transporters.

Analysis of Oligosaccharide and Monosaccharide Composition of SSO1273 Glycoprotein by HPAEC-PAD

For oligosaccharide mapping, the purified PVDF-membrane released N-linked glycans were separated by HPAEC and analyzed by pulsed amperometric detection (PAD) on a BioLC HPLC system (LCPackings, Sunnyvale, CA) using a CarboPack PA100 (2 × 250 mm) column (LCPackings, Sunnyvale, CA). Oligosaccharides were eluted with a gradient of 0−210 mM sodium acetate in 160 mM NaOH over 27 min at a flow rate of 0.25 mL/min. For monosaccharide analysis, oligosaccharides of the glycoproteins blotted onto PVDF-membrane were acid hydrolyzed. Hydrolysis was performed at 100 °C for 4 h in 400 μL of 2 N TFA or 6 N HCl for the quantitative release of neutral and acidic monosaccharides, respectively. Supernatants were vacuum-dried and dissolved in water before injection. Monosaccharides were separated on a CarboPack PA 100 (2 × 250 mm) column (LCPackings, Sunnyvale, CA) and eluted isocratically using 16 mM NaOH at 0.25 mL/min flow rate. MonoStandards (LCPackingş Sunnyvale, CA) containing fucose, galactosamine, glucosamine, galactose, glucose and mannose were used for calibration.

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RESULTS

Cell Surface Glycoproteins of Sulfolobus solfataricus

Cell surface subproteome of S. solfataricus P2 cells grown in tryptone medium were isolated using differential solubilization and centrifugation of the cell membrane fraction.41 The sample was separated by 1DE-SDS-PAGE (Figure 2) and proteins were in-gel digested and analyzed by nano-LC−ESI−MS/MS for protein identification (Table 1 and Supplementary Table 1A, Supporting Information). Members of the genus Sulfolobus are obligate aerobes and thus contain a membrane associated

Glycan Analysis by Nanoflow HILIC-ESI−MS and HILIC-ESI−MS/MS

Purified glycans released by PNGase F from the blotted SSO1273 glycoprotein were analyzed by hydrophilic-interaction liquid chromatography (HILIC) using an Ultimate 3000 (LCPackings, Sunnyvale, CA) HPLC system online coupled to a QStar Elite (Applied Biosystems) mass spectrometer. Mobile phase A (90% ACN, 0.1% (v/v) HCOOH,) and mobile phase B (10% ACN, 0.1% (v/v) HCOOH) were used. Sample was concentrated on a μ-precolumn TSK Amide-80, 300 μm i.d. × 5 mm length, 3 μm (LCPackings, Sunnyvale, CA) using mobile phase A at 30 μL/min flow rate for 5 min. Glycan separation was performed by using a Nano Series column, 75 μm i.d. × 15 cm packed with TSK Amide-80, 3 μm (LCPackings, Sunnyvale, CA). Glycans were eluted with a gradient of 90−10% mobile phase A over 17 min at 300 nL/min flow rate. MS and MS/MS spectra were acquired in negative ion mode over a m/z range 300−2000. Accurate Mass Measurements for the Determination of Elemental Composition of Unusual Monosaccharide Figure 2. SDS-PAGE image of the cell-surface protein fraction of Sulfolobus solfataricus P2 before (lane 2) and after glycoprotein enrichment using Concanavalin A chromatography (lane 3). Lane 1: molecular weight markers.

Accurate mass measurements were carried out using the QStarElite (Applied Biosystems, Foster City, CA) mass spectrometer in positive ion mode at 15.000 mass resolution. Mass 2782


2783

Transporter DB: ATP dependent ABC family maltose binding protein Transporter DB:ATP dependent ABC family iron binding protein

BLAST: thermopsin [Sulfolobus tokodaii str. 7] CD: glycopeptidase PNGAse famlily CD48 BLAST: sugar transport substrate binding component of an ABC transporter BLAST: DUF1512 superfamily TransporterDB: substrate binding domain of sugar binding ABC transporter BLAST: thermopsin glycosylated S-layer protein, SlaA [Sulfolobus islandicus HVE10/4] CD: substrate-binding domain of an ABC-type nickel/oligopeptide-like import system Peptidase related protein SSO3053 SSO0485

gi|13813642, gi|15897411

SSO2181

SSO1288

SSO2602 SSO0389

SSO0097 SSO3066

SSO2552 SSO0421 SSO2712

SSO1175

SSO2551

SSO2045 SSO1171

SSO2847

SSO1287

SSO1273

gi|13816455, gi|15899758

gi|13815479, gi|15898960

gi|13815906, gi|15899329 gi|13813536, gi|15897321, gi|85719258 gi|13814488, gi|15898131

gi|13813227, gi|15897058 gi|13816474, gi|15899771

gi|13815856, gi|15899286 gi|13813572, gi|15897351 gi|13816032, gi|15899428

gi|13814367, gi|15898028

gi|13815855, gi|15899285

gi|13815329, gi|15898836 gi|13814361, gi|15898024

gi|13816205, gi|15899562

gi|13814487, gi|15898130

gi|13814470, gi|15898115

SSO0999

gi|13814184, gi|15897875

ORF SSO2619

gi|13815927, gi|15899344

protein accession numbers

57

40

33

94

53 51

109 55

82 77 73

231

98

125 122

184

211

419

396

416

Mascot protein score

34932

79955

117402

90994

111417 131899

41577 69287

68597 86578 53094

65063

141801

96439 56820

61090

44777

97897

53249

80367

molecular mass

4

8

4

8

3 6

12 4

4 16 8

25

5

10 14

17

21

41

58

14

peptide matches

11

7

2.1

1.8

1.9 4.1

26.3 5.8

4.9 20.8 12.1

8.8

3.4

5 18.2

7

10.7

12.7

17.6

9.5

sequence cov. % 1DE band

11, 12

5, 6

1

5

2, 3 1, 2, 3, 4, 5

10, 11 14

5, 6 5, 6 5, 6, 7, 8

4, 5, 6, 7

2, 3

2, 3, 4 6, 7, 8, 9

2, 3, 4, 6, 7, 8, 9, 10 6, 7, 8, 9, 12 1, 2, 3, 4, 5, 6 1, 2, 4, 8, 9, 10, 11, 12 6, 7, 8

secretion

NC

Sec

Sec

NC

0 Tat

NC Sec, Type IV

Sec 0 Sec, Type IV

Sec

NC

NC Tat, Type IV

Type IV

NC

Tat

Type IV

Type IV

b

9

14

34

21

46 40

2 13

21 1 15

21

35

37 10

13

11

20

10

18

number of predicted N-glycosylation sitesc

0

0

0

0

0 9

0 1

1 0 1

5

4

4 3

0

3

20

2

3

number of observed N-glycosylation sites

Description of protein function is based on the NCBInr database definition when avilable. Otherwise TransporterDB description, result of BLAST or conserved domain (CD) search are in the descriptipon. bPredicted secretion: SignalP was used to predict N-terminal signal peptides used in Type II Sec and Tat protein secretion. Tat secreted proteins were predicted by TATFIND and TatP programs. FlaFind were used to predict archaeal type IV pilin-like signal peptides (type IV) . Nonclassical (NC) secretion was predicted by SecP progam. cN-glycosylation site (Asn-Xaa-Ser/Thr, where Xaa is any amino acid except Pro) was predicted by NetNGlyc 1.0 Server.

a

Peptidase related protein [Sulfolobus solfataricus P2] Maltose ABC transporter, maltose binding protein ABC transporter

Hypothetical protein SSO1288

Conserved hypothetical protein Hypothetical protein SSO0389

Conserved hypothetical protein Arabinose ABC transporter, arabinose binding protein

Conserved hypothetical protein AAA family ATPase Hypothetical protein SSO2712

Serine protease, subtilase family, putative Hypothetical protein SSO1175

Protease Maltose ABC transporter

Sugar-binding periplasmic protein

Hypothetical protein SSO1287

Conserved hypothetical protein

description

TransporterDB: substrate binding domain of oligopeptide binding ABC transporter TransporterDB: trehalose/maltose ABC domain The substrate-binding domain of an ABC-type transporter BLAST: putative lipopolysaccharide biosynthesis protein [Helicobacter pylori F32] TransporterDB: substrate binding domain of sugar binding ABC transporter thermopsin TransporterDB: substrate binding domain of maltose/maltodextrin ABC transporter serine protease

protein name in NCBInr

Dipeptide ABC transporter, periplasmic dipeptide binding protein (dppA) ABC Transporter

a

Table 1. Surface Proteins of Sulfolobus solfataricus P2 Enriched in N-Glycosylated Proteins by Concanavalin-A Affinity Chromatography

Journal of Proteome Research Article



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aerobic electron transport chain coupled to oxidative phosphorylation. Accordingly, different enzyme subunits of the aerobic respiration chain were the main components identified in the surface fraction (Supplementary Table 1A). Recent immunolabeling data showed that A1AO ATPase, the key enzyme complex of cellular bioenergetics is mainly located in the cytoplasmic membrane of S. solfataricus.15,42,14,41 In fact, besides NADH-dehydrogenase subunit D (SSO0324), the succinate-dehydrogenase flavoprotein subunit (SS2356) and four subunits of A1AO-ATPase/synthase (SSO0563, SSO0564, SSO0559 and SSO0561) were identified. Another characteristic of the cell envelope of S. solfataricus is the presence of ATPbinding cassette (ABC) transporters. Archaeal ABC transporters are ubiquitous proteins that use the energy derived from ATP hydrolysis to import/export various substrates across the cell membrane. A typical ABC transporter system is composed of three domains having different functions and localization: (i) two nucleotide binding transmembrane domains that energize transport via ATP hydrolysis (ATPases), (ii) two membrane spanning domains that act as a membrane channel for the substrate (permeases), and (iii) a substratespecific solute binding protein (SBP) located on the cell surface which recognizes and binds the substrate. There are 12 S. solfataricus SBPs known today: eleven reported in TransportDB (http://www.membranetransport.org)40 and one (SSO1273) has been published recently.43 Four SBPs have been identified here, two involved in the peptide (SSO1273 and SSO2619) and two in the sugar transport30 AraS (SSO3066) and TreS (SSO0999). The analysis of cell surface fraction of S. solfataricus resulted in generally lower sequence coverage than that of another previously studied crenarcheal organism, Aeropyrum pernix.41 This was mainly associated with the high number of putatively N-glycosylated proteins found in this fraction. To check this hypothesis, the sample was enriched in N-linked glycoproteins by concanavalin A affinity chromatography (Figure 2). In this fraction 20 putatively N-glycosylated proteins, all of which contained at least one N-glycosylation consensus, were identified (Table 1 and Supplementary Table 1B, Supporting Information). The fraction was particularly enriched in SBPs (SSO0485, SSO3053, SSO1171, SSO2619, SSO1273, SSO2847 and SSO3066) and proteases (SSO2045, SSO1175, SSO2602 and SSO2551). It was observed that several CID mass spectra did not turn up in a positive protein hit in the usual proteomic workflow. These spectra showed glycan specific fragment ions characteristic for glycopeptides (Figure 7A). This observation prompted us to determine the main N-glycan structure characteristic of this hyperthermophilic microorganism.

Figure 3. MALDI-TOF MS mass spectrum of the purified SSO1273 glycoprotein.

analyses. The HPAEC-PAD chromatogram (Figure 4A) shows a single main oligosaccharide peak eluted at 25.4 min. To

N-Glycosylation of SSO1273 Oligopeptide Binding Protein

Figure 4. Glycan and monosaccharide profiles of SSO1273 protein obtained by high performance anion exchange chromatography with pulsed amperiometric detection. (A) Purified PNGAse digest of PVDF membrane blotted SSO1273 glycoprotein. (B) Monosaccharide profile top trace: separation of a mixture of monosaccharide standard containing fucose, galactosamine, glucosamine, galactose, glucose and mannose, middle curve: four-hour 2 N TFA hydrolysate of PVDF membrane blotted SSO1273 glycoprotein, bottom trace: four-hour 6 N HCl hydrolysate of PVDF membrane blotted SSO1273 glycoprotein.

The cell-surface bound SSO1273 protein has recently been identified as a highly selective oligopeptide binding component of an ABC transporter system.43 SSO1273 has been shown to be glycosylated; however, the structure(s) and heterogeneity of the glycan(s), and the sites of N-glycosylation have not yet been determined. The average molecular mass of the purified protein was measured to be 125157 Da (standard deviation 81 Da) using SELDI-TOFMS (Figure 3). It is 30415 Da higher than the calculated mass of the mature protein (94743 Da). Oligosaccharides were released by PNGase-F from the PVDF membrane blotted protein and purified on graphitized carbon packed on the top of ZipTip C18 pipet tips.33 The N-glycan pattern was determined by HPAEC-PAD and HILIC-ESI−MS

obtain structural data, the oligosaccharides were analyzed by HILIC-ESI−MS and MS/MS in negative ion mode. Note that positive ion mode was also tried but it did not detect the underivatized oligosaccharides. Negative ion ESI−MS has been shown to be useful in the structural analysis of negatively charged sulfated glycans.44 HILIC allowed the separation of the N-glycans released from SSO1273 protein into one major peak fraction (Figure 5A and B), which resulted in a simple ESI−MS spectrum showing a singly charged molecular ion peak at m/z 1297.487 (Figure 5C). This molecular mass is higher than that of the most complex N-glycan of S. acidocaldarius previously described.11 MS/MS taken on m/z 1297.487 molecular ion did 2784



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SSO1273 N-glycoprotein was determined by HPAEC-PAD after acid hydrolysis performed on the membrane. Figure 4B shows the neutral and amino monosaccharide patterns obtained respectively by the TFA and HCl hydrolysis procedures. The presence of D-glucose, D-mannose and D-glucosamine and at least one nonstandard monosaccharide eluted at higher retention time than the standard monosaccharides was detected. The high retention time of the latter may indicate its acidic nature.45 The closely related hyperthermoacidophilic crenarchaeon, S. acidocaldarius is known to contain a sulfonated deoxyhexose, 6-sulfoquinovose in the glycans of Cytochrome b558/5669 and S-layer glycoproteins.11 S. acidocaldarius’ genome encodes a gene cluster important for the assembly of the Nglycans. In this gene cluster the agl3 gene (Saci0423) encoding for UDP-Sulfoquinovose synthase and involved in the biosynthesis of sulfoquinovose has recently been identified.14 In the S. solfataricus genome sulfolipid biosynthesis protein (sqdB) (SSO2583) a sequence homologue of Agl3 could also be identified with 98% sequence identity using BlastP 2.2.27.46 This indicates that S. solfataricus similarly to S. acidocaldarius beside D-glucose, D-mannose and D-glucosamine may harbor 6sulfoquinovose, a monosaccharide that is commonly found in photosynthetic membranes of plants including phototrophic bacteria. To obtain sequence information on the N-glycan of SSO1273, an in-solution digest of SSO1273 glycoprotein was subjected to RP nano-HPLC−ESI−MS/MS and nano-ESI− MS/MS analyses. Based on the RP nano-HPLC−ESI−MS/MS analyses the sequence of 99 unique peptides were identified resulting in 49% protein sequence coverage (Figure 6A and Supplementary Table 2A, Supporting Information). SSO1273 has a 30 amino acid long putative N-terminal signal peptide. The C-terminal portion of the protein is highly hydrophobic and contains a potential glycophosphatidylinositol (GPI) modification site at S878 for anchoring of the protein to the cell membrane.43 The protein sequence contains 20 predicted N-glycosylation consensus sites (Table 1). None of the potential peptides covering the putative N-glycosylation sites were identified by the proteomic workflow suggesting that they are likely to be modified. On the other hand, the analysis led a number of unidentified peptides showing a common fragmentation pattern. These peptides were subjected to nano-ESI−MS/MS analysis in positive ion mode by manual selection of the parent ions using direct infusion of the digest. CID spectra show abundant glycosidic fragment ions together with less abundant y and b type fragments derived from the peptide backbones (an example is shown in Figure 7A). The molecular mass of the neutral N-glycan common to the various glycopeptides was deduced to be 1298.4 Da (with an increment mass of 1280.4 Da), confirming the results obtained by the HILIC-ESI-MS measurements. Based on the manual interpretation of the spectra the N-glycan is Hex4(HexNAc)2 plus IR 226 Da (Figures 6 and 7). The branched heptasaccharide is linked to the asparagine residue by HexNAc−HexNAc at the consensus sequence. Because HPAEC-PAD analysis revealed no GalNAc, the chitobiose core structure (GlcNAc−GlcNAc) characteristic to the closely related S. acidocaldarius could be confirmed for S. solfataricus too. Sugar specific internal fragment ions, B5/Y2/Y2β (m/z 204.14), B5/Y2α (m/z 430.20), B5/Y3 (m/z 592.29), B5/Y4 (m/z 754.38) and B5/Y5 (m/z 916.44) produced by double cleavages were observed in these spectra as a common signature of the glycan. The increment mass of the internal monosaccharide attached to the chitobiose core (IR

Figure 5. Negative ion mode HILIC-ESI−MS and ESI−MS/MS of the PVDF-membrane released oligosaccharide of SSO1273 protein. (A) Total and (B) selected ion (m/z 1297) chromatograms showing a single oligosaccharide peak eluted at 28.4 min. (C) ESI−MS single stage mass spectrum at 28.4 min.

not yield abundant fragment ions in the low energy CID spectrum, therefore the doubly charged molecular ion at m/z 648.19 was selected for analysis. The CID mass spectrum (not shown) of this ion shows abundant glycan characteristic fragment ions but does not allow full sequence determination of the glycan itself. Monosaccharide composition of the purified 2785



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Figure 6. SSO1273 glycoprotein (A) primary sequence with identified peptides and glycopeptides (Supplementary Table 2A and 2B, Supporting Information). Identified peptides are highlighted in yellow, identified glycopeptides are underlined by wavy blue line, N-terminal signal sequence is highlighted in blue, predicted N-glycosylation sites are highlighted in red and potential glycophosphatidylinositol (GPI) modification site at S878 is highlighted in green. (B) Symbolic representation of the structure of the sulfated heptasaccharide, Hex4(HexNAc)2 plus 6-sulfoquinovose. (C) WebLogo representation of the N-glycosylation site consensus sequences of SSO1273 protein.

Surface Proteins of S. solfataricus Are Glycosylated by the Same Type of N-Glycan

226.2 Da) corresponds to the sulfoquinovose (Molecular mass 244.2 Da). Accurate mass measurements carried out on the B5/ Y2α disaccharide fragment ion at m/z 430 bearing the unusual monosaccharide unit confirmed C14H24NO12S (+1.8 ppm error) elemental composition and supported the presence of sulfate group in the glycan (Supplemental Table 4, Supporting Information). The characteristic set of glycan specific ions was used to monitor the presence of N-glycosylated peptides in the tandem mass spectra of SSO1273 protein digests. The subtraction of the glycan residue mass from the measured molecular mass of the modified peptide (Mw − 1 glycan, Supplementary Table 2B, Supporting Information) resulted in the expected molecular mass of the unmodified proteolytic peptide. On the other hand, manual peptide sequencing allowed peptide identification. By this analysis, all 20 theoretical N-X-S/T consensus sequence sites of SSO1273 were confirmed to be decorated by the same type of glycan (Supplementary Table 2B).

HILIC-ESI-MS/MS analyses of the N-linked glycans enzymatically released from the PVDF membrane (Figure 1) showed a single negatively charged N-glycan at 1297 Da molecular mass, similar to SSO1273 (Figure 5C). In addition, it was noticed that the proteomic data obtained on the ConA enriched surface subproteome of S. solfataricus P2 contained numerous unidentified tandem mass spectra. Data analysis revealed that many of these show the same characteristic signature of the B/ Y sugar fragment ions that was observed in the CID spectra of SSO1273 derived glycopeptides. These signature ions were then used to highlight glycopeptides having the same type of Nglycan in the proteomic data sets. This analysis led to the identification of 39 glycopeptides and 36 N-glycosylation sites which have been assigned to 11 distinct proteins (apart of SSO1273) present in the ConA enriched surface protein fraction (Supplementary Table 3, Supporting Information, and Table 1). These include the identification of 9 out of the 40 2786



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Figure 7. (A) nano-ESI−MS/MS spectrum of the doubly charged glycopeptide precursor ion, (M + 2H)2+ at m/z 1101.47 derived from a tryptic/ chymotriptic in-solution digest of SSO1273 protein showing both peptide (bn and yn) and glycan derived (Yn and Bn) fragment ions. (B) Corresponding fragmentation scheme.

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theoretical N-glycosylation sites of S-layer glycoprotein

DISCUSSION

Archaea, a major constituent of our ecosystem possess a characteristic cell envelope which is distinct from that of Bacteria and Eukaryotes. Most of the archaeal species constitute a unique structure with an outer surface-layer over the cytosolic cell membrane that consists of bipolar ether lipids and membrane proteins. Though N-glycosylation of S-layer and cell surface appendages (flagella and pili) related proteins have been investigated for some archaeons (Figure 1), knowledge on

(SSO0389). Surprisingly, several surface-exposed ABC transporters (SSO1273, SSO2619, SSO0999, SSO1171, SSO2712 and SSO3066) and proteases (SSO2045, SSO2551 and SSO1175) undergo the same type of N-glycosylation as the S-layer glycoprotein of S. solfataricus. 2787



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the homologues of oligosaccharyltransferase aglB17and there is no A. pernix glycoprotein characterized today. Further research however is needed in order to conclude whether or not A. pernix lacks N-glycosylation. Interestingly, about 2% of the predicted N-glycosylated protein of S. solfataricus (79 proteins) and S. acidocaldarius (59 proteins) are highly glycosylated, having more than 10 potential N-glycosylation sites. An attempt has been made to annotate the set of highly N-glycosylated proteins of S. solfataricus into functional categories (Supplementary Table 5, Supporting Information). It was noted that thermopsin-like proteases and ABC transporters seem to be the most represented members in this cluster, suggesting that N-glycosylation could play a specific role for these functional classes in S. solfataricus.

the N-glycosylation of other cell surface exposed proteins is limited. To understand both specific and common characteristics of N-glycosylation in this domain of life, it is of importance to experimentally characterize glycosylation sites. Here, by undertaking a global glycoproteomic approach on the cell-surface fraction of S. solfataricus we have found a single prominent glycan structure characteristic of a number of different surface exposed proteins of S. solfataricus (Table 1). The novel N-glycan is composed of a branched sulfated heptasaccharide, Hex4(GlcNAc)2 plus sulfoquinovose where Hex is D-mannose and D-glucose. The chitobiose glycan core and the presence of a sulfated sugar building block are common to another closely related archaeal species, S. acidocaldarius. The presence of other less abundant glycans, structurally different from the one characterized cannot be excluded. Next, we were interested in understanding which mechanisms are used to secrete the identified proteins. In silico analysis of Nterminal signal peptides allowed identification of eighteen secreted proteins in the glycoprotein fraction (Table 1). Using a combination of prediction tools these were predicted to be secreted by the Sec (six proteins) and Tat pathways (three proteins) of the type II secretion system, type IV archaeal secretion (six proteins) and nonclassical mechanisms (five proteins). It suggests that the S. solfataricus uses different secretion mechanisms for the membrane translocation of surface proteins before they are N-glycosylated by Hex4(GlcNAc)2 plus sulfoquinovose oligosaccharide. To shed light on the N-glycosylation frequency in prokaryotes four archaeal (S. solfataricus, S. acidocaldarius, A. pernix and H. volcanii) and a well-studied bacterial (Campylobacter jejuni) proteomes were analyzed using NetNGlyc prediction software.39 Figure 8 shows that a significantly higher

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ASSOCIATED CONTENT

S Supporting Information *

Supplementary Table 1. Protein identified in the cell surface fraction of Sulfolobus Solfataricus P2 (A) before and (B) after ConA lectin affinity purification. Supplementary Table 2. (A) Peptide and (B) Glycopeptide list of SSO1273 protein cleaved by chymotripsin and trypsin (merged results of three independent experiments). Supplementary Table 3. Glycopeptides from the different surface proteins identified in the ConA enriched fraction (merged results of three independent experiments). Supplementary Table 4. Elemental composition analysis of B5/Y2α fragment ion at m/z 430 in nine independent nano-HPLC−ESI−MS/MS experiments. Supplementary Table 5. Predicted highly glycosylated proteins (with more than 10 glycosylation sites) in the proteome of Sulfolobus solfataricus P2 and their functional classes. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Ph: +39 081 6132 585. Fax: +39 081 6132 249. E-mail: g. [email protected]. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS G.P. thanks the CNR SHORT-TERM MOBILITY - 2008 fund for financial support.

Figure 8. Graph showing number of potentially N-glycosylated proteins calculated by NetNGlyc across four archaeal (S. solfataricus, S. acidocaldarius, A. pernix and H. volcanii) and one bacterial (C. jejunii) model organisms. *A selected Human data set containing around 6000 curated human proteins including 189 annotated N-glycosylated protein according to Gunda and Brunak.39

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ABBREVIATIONS MS, mass spectrometry; HPLC, high performance liquid chromatography; HPAEC-PAD, high performance anion exchange chromatography pulsed amperometric detection; SBP, solute binding protein; CID, collision induced dissociation; IDA, information-dependent acquisition; RP, reverse phase; Q-TOF, quadrupole time-of-flight; CAM, carbamidomethylation; GPI, glycophosphatidylinositol; Hex, hexose; GlcNAc, N-acetylglucosamine; HexNAc, N-acetylhexosamine; PNGase F, peptide N-glycosidase F; ConA, Concanavalin-A; Sec-pathway, secretion-pathway; Tat-pathway, twin-arginine translocation; ACN, acetonitrile; HCOOH, formic acid.

percentage of N-glycosylation is predicted in Archaea than in Bacteria in the model organisms studied. A particularly high frequency of N-glycosylation was predicted in the proteomes of the two Sulfolobus species (S. solfataricus (62%) and S. acidocaldarius (59%) comparing them to the hyperthermophile A. pernix (30%) and the halophile H. volcanii (40%) archaea. It is interesting to note that among the more than 50 archaeal genomes screened for the homologues of different agl genes of the archaeal N-glycosylation pathway, A. pernix appears to lack 2788



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