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J. Biol. Chem., Vol. 277, Issue 8, 6230-6239, February 22, 2002

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The Surface Layer (S-layer) Glycoprotein of Geobacillus stearothermophilus NRS 2004/3a

ANALYSIS OF ITS GLYCOSYLATION^*

Christina Schäffer^§, Thomas Wugeditsch, Hanspeter Kählig^¶, Andrea Scheberl, Sonja Zayni, and Paul Messner

From the  Zentrum für Ultrastrukturforschung und Ludwig Boltzmann-Institut für Molekulare Nanotechnologie, Universität für Bodenkultur Wien, A-1180 Wien, Austria and ^¶ the Institut für Organische Chemie, Universität Wien, A-1090 Wien, Austria

Received for publication, September 14, 2001, and in revised form, November 30, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Geobacillus stearothermophilus NRS 2004/3a possesses an oblique surface layer (S-layer) composed of glycoprotein subunits as the outermost component of its cell wall. In addition to the elucidation of the complete S-layer glycan primary structure and the determination of the glycosylation sites, the structural gene sgsE encoding the S-layer protein was isolated by polymerase chain reaction-based techniques. The open reading frame codes for a protein of 903 amino acids, including a leader sequence of 30 amino acids. The mature S-layer protein has a calculated molecular mass of 93,684 Da and an isoelectric point of 6.1. Glycosylation of SgsE was investigated by means of chemical analyses, 600-MHz nuclear magnetic resonance spectroscopy, and matrix-assisted laser desorption ionization-time of flight mass spectrometry. Glycopeptides obtained after Pronase digestion revealed the glycan structure [2)--L-Rhap-(13)--L-Rhap-(12)--L-Rhap-(1]_n = 13-18, with a 2-O-methyl group capping the terminal trisaccharide repeating unit at the non-reducing end of the glycan chains. The glycan chains are bound via the disaccharide core 3)--L-Rhap-(13)--L-Rhap-(1 and the linkage glycose -D-Galp in O-glycosidic linkages to the S-layer protein SgsE at positions threonine 620 and serine 794. This S-layer glycoprotein contains novel linkage regions and is the first one among eubacteria whose glycosylation sites have been characterized.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

For many years, glycosylation of proteins was thought to be limited to eukaryotes. Extensive research in the past two decades, however, has revealed that surface layer (S-layer¹) glycoproteins are among the best-studied prokaryotic glycoproteins. They are widely distributed in the domains Archaea and Bacteria; in Gram-positive eubacteria, they have been demonstrated exclusively within the low G+C phylum (e.g. Bacillus, Geobacillus, Aneurinibacillus, Paenibacillus, Clostridium, Thermoanaerobacter, Desulfotomaculum, and Lactobacillus) (for reviews see Refs. 1, 2).

In general, S-layers are two-dimensional crystalline arrays composed of identical protein or glycoprotein subunits that form the outermost envelope layer of bacterial cells. S-layers have evolved as a consequence of the direct interaction of these cells with different habitats and changing ecological conditions, presumably providing them with a selection advantage (3).

In the past, most efforts on the genetic characterization of S-layers were focused on cloning and sequencing of the corresponding structural genes (for review see Ref. 4). Up to now, more than 40 different S-layer genes have been characterized, including, so far, only seven genes of glycosylated S-layer proteins. Five of them are of archaeal origin; these are the genes encoding the cell surface glycoproteins of Halobacterium halobium (GenBank^TM protein data base accession number P08198), Haloferax volcanii (P25062), Methanothermus fervidus (P27373), Methanothermus sociabilis (P27374), and Haloarcula japonica (BAB39352). The two remaining genes code for the S-layer protein structural genes of the S-layer glycoproteins from the eubacteria Thermoanaerobacter kivui (P22258) and a mutant strain of Geobacillus stearothermophilus (formerly Bacillus stearothermophilus (5)) ATCC 12980 (AAF34763). For these strains, however, no S-layer glycan structures have been elucidated.

Besides the S-layer gene sbsD of G. stearothermophilus ATCC 12980 (AF228338), the S-layer genes of two other, non-glycosylated wild-type strains from the newly reclassified species (5) have been cloned so far. These are the structural S-layer genes sbsA (X71092 (6)) and sbsB (X98095 (7)) from G. stearothermophilus PV72, and sbsC from G. stearothermophilus ATCC 12980 (AF055578 (8)) (for review see Ref. 4). Interestingly, S-layer expression in G. stearothermophilus PV72 can change in response to growth conditions (9-11). It was shown that S-layer variation, i.e. expression of sbsA in the wild-type strain G. stearothermophilus PV72/p6 or expression of sbsB in the variant G. stearothermophilus PV72/p2, observed in response to oxygen limitation in the growth medium, is based on DNA rearrangements between the chromosome and a naturally occurring megaplasmid (12). Under elevated temperatures (67 °C), sbsA transcription is blocked by an insertion element, resulting in an S-layer-deficient mutant (10, 13). Recently, interruption of the S-layer gene by an insertion element leading to an S-layer-deficient variant has also been reported for sbsC of G. stearothermophilus ATCC 12980 (14).

Investigation of the glycosylated S-layer of the wild-type strain G. stearothermophilus NRS 2004/3a started in the early 1980s and was one of the first detailed studies on a eubacterial S-layer glycoprotein (15). After proteolytic digestion of the isolated S-layer glycoprotein (16, 17), glycopeptides bearing rhamnose homopolymers consisting of trisaccharide repeating units were isolated (18). Attempts to determine the linkage of the glycan chains to the S-layer polypeptide led to the proposal of an N-glycosidic linkage between rhamnose and asparagine (19). A similar type of linkage was also proposed for the S-layer glycoprotein of the methanogenic archaeon Methanothrix sochngenii (20).

In this contribution we describe the detailed re-examination of the glycosylation pattern of G. stearothermophilus NRS 2004/3a, including the complete elucidation of the S-layer glycan primary structure and the description of the glycosidic linkage to the S-layer protein. Furthermore, we report on the nucleotide sequence of the structural gene for the S-layer protein, which, following the alphabetical designation of S-layer genes from G. stearothermophilus (for review see Ref. 4), we designate sgsE. Based on the sequence data, the locations of the S-layer glycan chains on the mature glycoprotein were determined. The detailed knowledge of S-layer glycoprotein glycosylation at the molecular level, in combination with the description of the S-layer protein structural gene and the positions of glycosylation on the mature glycoprotein, constitutes the foundations for a future generation of tailor-made S-layer neoglycoconjugates (for review see Ref. 2).

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Bacterial Strain and Growth Conditions-- G. stearothermophilus NRS 2004/3a (5, 15) was continuously grown on modified SVIII medium (0.5% peptone, 0.5% meat extract, 0.3% yeast extract, 0.2% glucose, 0.13% K₂HPO₄, 0.01% MgSO₄, pH 6.9) at 60 °C with an oxygen partial pressure of 10% (v/v) and a dilution rate of 0.2 ml/h in a 15-liter Biostat C fermentor (Braun, Melsungen, Germany). Cells were separated from culture broth by continuous centrifugation (Sepatech 17 RS centrifuge, Heraeus, Vienna, Austria) at 16,000 × g and 4 °C. The biomass was stored at 20 °C.

Analytical and General Methods-- Electron microscopy of freeze-etched preparations of bacterial cells was performed according to published procedures (15). SDS-PAGE was carried out according to a previous study (21). Monosaccharide analysis by HPAEC/PAD on a Carbo-Pac PA-1 column (Dionex, Sunnyvale, CA) and amino acid analysis on a Biotronik LC 3000 amino acid analyzer (Biochrom, Cambridge, UK) were performed according to published procedures (22). In column effluents, carbohydrates were estimated using the thymol reagents (23), and the rhamnose content was determined colorimetrically (24).

N-Terminal sequencing (Edman degradation) of glycopeptides followed standard protocols (25). For deglycosylation of S-layer glycopeptides with trifluoromethanesulfonic acid, the method described by Edge et al. (26) was used. Deglycosylation of S-layer glycoprotein followed essentially the same procedure, except the deglycosylated material was resuspended into 5 M guanidinium hydrochloride (1.5 mg/ml) and extracted for 15 min at 25 °C. The extract was dialyzed at 4 °C, once against 10 mM CaCl₂, three times against distilled water, and lyophilized.

Smith degradation of S-layer glycopeptides was essentially performed as published previously (27), except hydrolysis of the acid-labile polyrhamnan chain was effected with 1 M trifluoroacetic acid for 4 h at 25 °C. Reaction products were recovered after chromatography on a Bio-Gel P-2 column (1.0 × 120 cm, Bio-Rad) with 0.1 mM NaCl as eluent.

DNA Manipulations-- Preparation of chromosomal DNA from G. stearothermophilus NRS 2004/3a was performed using Genomic Tips 100/G from Qiagen. All restriction enzymes required were purchased from Invitrogen. Endonuclease digestion was done according to the manufacturer's instructions, and digests were purified using a QIAquick PCR purification kit (Qiagen). Agarose gel electrophoresis was performed as described by Sambrook et al. (28). For recovery of DNA fragments from agarose gels, the system of Geneclean III from Bio 101, Inc., Vista, CA (Qbiogene) was used.

PCR, Inverse PCR, Oligonucleotides, and DNA Sequencing-- PCR amplifications were carried out using a PCR Sprint thermocycler (Hybaid, Ashford, UK). SgsE oligonucleotide synthesis was done by MWG-Biotech AG (Ebersberg, Germany), SbsC oligonucleotides were kindly provided by Dr. M. Jarosch (Zentrum für Ultrastrukturforschung, Universität für Bodenkultur Wien, Vienna, Austria). The sequences of all oligonucleotides used are listed below in Table I. All amplification reactions were performed in 50-µl reactions using Pwo polymerase (Roche Molecular Biochemicals), and conditions were optimized for each primer pair. DNA sequencing was performed by Agowa (Berlin, Germany).

For PCR amplification of the N-terminal region of sgsE from genomic DNA of G. stearothermophilus NRS 2004/3a, the primer combination sbsC-22/sbsC-23 was used. Based on the sequences of the obtained PCR products, an inverse PCR approach was chosen to amplify unknown downstream and upstream DNA regions (29). For this purpose, ~500 ng of genomic DNA (5 µl) was completely digested with ClaI in a reaction volume of 40 µl and purified using a QIAquick PCR purification kit (Qiagen). The DNA fragments were religated with 3 Weiss units of T4 ligase (New England BioLabs) in a 30-µl reaction volume at 16 °C overnight. DNA was precipitated from the heat-inactivated ligation reaction with ethanol as described by Sambrook et al. (28) and resuspended into 20 µl of distilled water. PCR amplification of ligated fragments with primer pair sbsC-15/sbsC-21, using 20 µmol of each primer, was carried out in a 50-µl reaction volume containing 2 µl of ligation mixture as template, 20 µmol of dATP, dCTP, dGTP, and dTTP, each, and 1 unit of Pwo polymerase in 1× reaction buffer. Based on the combined sequences of the overlapping PCR products from the direct and the inverse PCR reaction, the primer pair sgsE-1/sgsE-2 was used for filling in sequence gaps. The entire sgsE gene was PCR-amplified from genomic DNA of G. stearothermophilus NRS 2004/3a using the primer combination sbsC-22/sgsE-3 and double-strand sequenced.

Sequencing of the N Terminus of the Mature SgsE-- To obtain proteolytic cleavage fragments for sequencing purposes, S-layer glycoprotein and deglycosylated material (8 mg, each) were suspended at a final concentration of 5 mg/ml in 125 mM Tris-HCl buffer, pH 6.8, containing 0.5% SDS, 10% glycerol, and 0.001% bromphenol blue (30), and incubated for 2 min at 100 °C. Proteolytic degradation with V8 protease (Sigma) at a concentration of 30 µg/mg of protein was prolonged for 3 h at 37 °C and stopped by addition of 125 mM Tris-HCl buffer, pH 6.8, containing 2% -mercaptoethanol, 3% SDS, 20% glycerol, and 0.001% bromphenol blue, in a ratio of 1:1 (v/v) and incubated for 2 min at 100 °C. The reaction mixture was separated by SDS-PAGE on a gradient gel (7.5-33% and 3-1.5% cross-linking) at a constant current of 50 mA and 10 °C, using the Protean II electrophoresis apparatus (Bio-Rad). Proteins were transferred to a polyvinylidene difluoride membrane (Bio-Rad) by semidry blotting in a discontinuous buffer system at a constant current of 0.8 mA/cm² (31) using the Multiphor Novablot apparatus (Amersham Biosciences, Inc.). Protein bands were visualized with Coomassie Blue R-250 staining reagent. Bands of interest were excised from the membrane and N-terminally sequenced.

Isolation of S-layer Glycoprotein and S-layer Glycopeptides-- The isolation and purification of S-layer glycoprotein essentially followed published methods (32). For structural investigations of the S-layer glycoprotein glycan, the protein portion of 1.5 g of S-layer glycoprotein was exhaustively degraded with Pronase E (Sigma Chemical Co.) as described previously (25). The degradation products were separated on subsequent chromatography columns (1.5 × 120 cm) of Bio-Gel P-4 (Bio-Rad), Dowex 50W-X8 (H⁺ form, column dimension 1.0 × 5 cm, Bio-Rad), Bio-Gel P-30, and Bio-Gel P-60, all eluted with 0.1 mM NaCl except the cation exchange column, which was eluted with distilled water. For further purification, the appropriate material (45 mg of S-layer glycopeptide mixture) was applied to a chromatofocusing system (Amersham Biosciences, Inc.), run in a linear PBE74 buffer pH gradient between 8.5 and 6.0 as described previously (33). Individual, desalted glycopeptide pools, designated CF 1 through CF 3, were further purified by semipreparative RP(C18)-HPLC (34).

For degradation with papain (Sigma), 250 mg of S-layer glycoprotein was dissolved in 40 ml of 125 mM Tris-HCl buffer, pH 6.8, containing 0.5% SDS, and incubated with 7.5 mg of enzyme (dissolved in 10 ml buffer) at 37 °C. The reaction was stopped after 2 h by heat inactivation of papain. The pool containing the glycopeptide material was separated from peptides by gel permeation chromatography and chromatofocusing as described above. Individual glycopeptide fragments, designated GP 1 through GP 5, were recovered after RP(C18)-HPLC (34) using a modified water/acetonitrile gradient (0-30% in 45 min).

Mass Spectrometry-- MALDI-TOF analysis of 0.8 µl (about 1.8 mg/ml) of glycopeptide samples (pools CF 1-3) was performed using the same volume of matrix solution (2% 2,5-dihydroxybenzoic acid in distilled water containing 30% acetonitrile) according to a procedure described elsewhere (35). Mass spectra were acquired on a Dynamo mass spectrometer (Thermo BioAnalysis, Santa Fe, NM). The instrument was operated in the negative-ion mode with a dynamic extraction setting of 0.3 and calibrated with insulin (from porcine pancreas).

NMR Spectroscopy-- NMR spectra were recorded at 600.13 MHz for ¹H and 150.90 MHz for ¹³C on a Bruker Avance DRX 600 NMR spectrometer using a 5-mm inverse triple-resonance probe (¹H, ¹³C, broad-band) with triple-axis gradient coils.

For the structural assignment of the carbohydrate portion, solutions of the glycopeptide pools (6.9 mg of pool CF 1, 5.3 mg of pool CF 2, and 9.3 mg of pool CF 3) in 0.6 ml of D₂O were measured at 300 K using gradient selected DQF-COSY, TOCSY, NOESY, gradient selected HSQC and HMBC, as well as gradient-selected HSQC-TOCSY and HSQC-NOESY experiments with parameters given elsewhere (33, 36).

For the analysis of the peptide portion, most of the experiments were additionally performed on solutions of the individual CF pools in 0.6 ml of 90% H₂O/10% D₂O using pulsed-field gradients for coherence selection and solvent suppression as described in a previous study (36).

All processing and analyses were done off-line on Silicon Graphics workstations using the Bruker software XWIN-NMR 2.6 and the program AZARA 2.0 (provided by W. Boucher and the Department of Biochemistry, University of Cambridge, UK).

-Elimination-- Glycan chains were released from S-layer glycopeptides under standard -elimination conditions (37). Glycopeptides (1.0 mg/ml) were treated with 0.1 M NaOH, containing 1 M NaBH₄, and 5 mCi of NaB[³H]₄ (348 mCi/mmol, PerkinElmer Life Sciences, Boston, MA) for 40 h at 45 °C. The cooled solution was acidified to pH 6.0 by addition of 50% acetic acid and subjected to gel filtration on a Bio-Gel P-4 column (1.0 × 120 cm) using 0.1 mM NaCl as eluent. Fractions (1.5 ml) were collected and analyzed for incorporated radioactivity.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

General Description of the Organism-- As demonstrated previously by freeze etching, the cell surface of the aerobic, thermophilic, Gram-positive organism G. stearothermophilus NRS 2004/3a is completely covered with an oblique S-layer lattice (a = 11.6 nm, b = 9.4 nm,  ~ 78°) (17). The overall degree of glycosylation of the S-layer protein is ~1.7% (w/w). In SDS-PAGE, the S-layer glycoprotein is separated into four bands with apparent molecular masses of 93, 119, 147, and 170 kDa, respectively (Fig. 1). The three high molecular mass bands give a positive PAS staining reaction. The 119- and the 147-kDa bands show almost equal intensity, both in the protein and carbohydrate staining reaction, whereas the 170-kDa band appears rather weak (Fig. 1, lanes 2 and 4). Upon chemical deglycosylation of the S-layer glycoprotein, a single 93-kDa band remains visible on the gel; thus, this band represents the non-glycosylated protomeric unit of the S-layer glycoprotein (Fig. 1, lane 3). These findings are consistent with the previous description of the S-layer glycoprotein of this organism (17).

View larger version (18K): [in this window] [in a new window]   Fig. 1.   SDS-PAGE analysis of the S-layer glycoprotein of G. stearothermophilus NRS 2004/3a (10% gel). Lane 1, molecular mass standards, myosin (200 kDa), -galactosidase (116.3 kDa), phosphorylase b (97.4 kDa), bovine serum albumin (66.3 kDa); lane 2, S-layer glycoprotein (Coomassie Blue staining); lane 3, deglycosylated S-layer protein (Coomassie Blue staining); lane 4, S-layer glycoprotein (periodic acid-Schiff staining). The amounts of total protein loaded per gel lane were 10, 20, 15, and 50 µg, respectively.

Isolation and Molecular Characterization of sgsE-- Based on the observed conservation of the N termini of the S-layer proteins SbsA and SbsC from G. stearothermophilus strains PV72/p6 and ATCC 12980, respectively (8), sequence similarity was also expected for the N terminus of SgsE from G. stearothermophilus NRS 2004/3a. Thus, the sgsE gene was isolated using a combination of inverse and direct PCR approaches, starting with primers specific for the N terminus of sbsC (Table I, Fig. 2). Amplification of a 3300-nt product allowed the determination of the entire nucleotide sequence of sgsE. The sequence has been deposited at GenBank^TM under the accession number AF328862.

View larger version (11K): [in this window] [in a new window]   Fig. 2.   Schematic representation of the identification of sgsE. A, genomic DNA fragments from G. stearothermophilus NRS 2004/3a used for isolation of sgsE. The full bar indicates the entire gene sequence encompassing the coding region; primers used for amplification in direct and inverse PCR and for sequencing are indicated by short arrows, obtained sequences are represented by large arrows. a, primer pair sbsC-22/sbsC-23 was used to amplify the N terminus of sgsE. Sequencing with primers sbsC-22 and sbsC-23 revealed the sequences S1(+) and S1(). b, after digestion of genomic DNA with ClaI and religation, inverse PCR with primer pair sbsC-15/sbsC-21 generated a 3.6-kb fragment. Upon sequencing with the same primers, the sequences S2(+) and S2() were obtained. c, the sequence gap of the inverse PCR product was filled using the sequencing primers sgsE-1 and sgsE-2, yielding sequences S3() and S3(+), respectively. The entire sgsE was PCR-amplified from genomic DNA using primer combination sbsC-22 and sgsE-3 and double-strand-sequenced. B, glycosylation sites of sgsE. Glycosylation sites on the translated SgsE precursor at positions Thr-620 and Ser-794 are given by capital letters T and S, respectively. The curved line symbolizes the S-layer glycan chains. The full bar represents the mature S-layer protein; the open bar comprises the signal peptide.

The entire sgsE sequence revealed one ORF extending 2709 nt, predicted to encode a protein of 903 amino acids with a calculated molecular mass of 96,652 Da. The ORF starts with an ATG at nucleotide position 1, preceded by a typical Shine-Dalgarno sequence 11 nt upstream the start codon. 25 nt downstream of the TAA stop codon, a putative -independent transcriptional termination signal was identified. The terminator consists of a palindromic stem loop sequence of 41 nt with a perfect stem of 17 nt.

Description of SgsE-- N-terminal sequencing of a V8 protease cleavage fragment of deglycosylated SgsE as well as of the intact glycoprotein revealed the amino acid sequence ATDVATVVSQAKAQM. This sequence was identified on SgsE at position 31-45, indicating that the first 30 amino acids constitute a signal sequence, which is cleaved at the Ala-Ala motif during biosynthetic protein processing. The signal sequence consists of an N-terminal hydrophilic domain (MDKKK) followed by a hydrophobic domain (AVKLATASAVAASAFVAANP) and the cleavage site domain (38). The entire signal peptide has a rather basic pI value of 10.0.

The overall amino acid composition of mature SgsE is within the typical data reported for S-layer proteins of Gram-positive bacteria (3), exhibiting a high content of hydrophobic and acidic amino acids. Among the basic amino acids, lysine preponderates and there is no cysteine present. Mature SgsE has a calculated theoretical molecular mass of 93,684 Da and a pI of 6.1. Secondary structure predictions using the program nnPredict (39) revealed that 65% of the N-terminal part between amino acid 1 and amino acid 286 are -helical, whereas the middle and the C-terminal part of SgsE is mainly organized as loops and -sheets and contains only four strong predictions for -helices. Analysis of the amino acid distribution of SgsE revealed an increased occurrence of arginine, lysine, and tyrosine at the N-terminal region (amino acids 31-270). The N terminus shows contents of arginine, lysine, and tyrosine of 3.3, 11.7, and 7.5 mol%, respectively, whereas these contents decrease to 1.1, 8.5, and 2.8 mol% for the rest (amino acids 271-903) of SgsE. The preponderance of positively charged amino acid residues was also determined at the N termini of SbsA and SbsC (8).

Comparison of SgsE with S-layer Proteins of Other G. stearothermophilus Strains-- A homology search using the Align program revealed that the amino acid sequence of SgsE shows highest identities with the G. stearothermophilus S-layer protein precursors SbsD (AF228338; 93.9% identity), SbsC (AF055578; 40.9% identity), SbsA (X71092; 32.6% identity), and SbsB (X98095; 21.6% identity) (Table II). Less, but distinct identities are given with several other cell wall proteins, such as the S-layer protein (AJ249446) and a crystal protein (AF242295) of Bacillus thuringiensis, the S-layer protein of Bacillus firmus (AF242295), an outer cell wall precursor protein of Bacillus brevis (M14238), and the S-layer protein of Clostridium thermocellum (U79117). Comparing the amino acid sequences of the G. stearothermophilus S-layer proteins SbsA-D with SgsE, it becomes evident that they share similarities with respect to the following features: (i) They are synthesized with a typical N-terminal signal sequence consisting of 30 (SbsA, SbsC, SbsD, and SgsE) and 31 (SbsB) amino acids, respectively; (ii) the signal peptide cleavage site is between the Ala-Ala motif, consistent with the proposed recognition sequence for signal peptidases (40); (iii) all G. stearothermophilus S-layer proteins exhibit a weakly acidic isoelectric point.

Within the N-terminal regions of the S-layer proteins highest sequence identities are found. Identity values are over 82.5%, when comparing amino acids 1-270 of SbsA (82.5%), SbsC (98.1%), and SbsD (96.7%) with SgsE (Table II). In contrast, the N terminus of SbsB does not reveal significant sequence homology; instead, it is the only one among the compared S-layer proteins to possess an S-layer homology domain (41) between amino acids 31 and 168 (42).

S-layer Glycoforms-- In the present paper, the structure of the S-layer glycoprotein of G. stearothermophilus NRS 2004/3a was elucidated, focusing on the description of the linkage region to the S-layer polypeptide. The glycopeptide mixture derived upon degradation of S-layer glycoprotein with Pronase was pre-purified using a combination of Bio-Gel P-4, Dowex 50W-X8, Bio-Gel P-30, and P-60 columns. Subsequently, the mixture was separated in the applied chromatofocusing gradient into three pools, designated CF 1-3, which eluted at pH values in the range of 8.3-8.2 (CF 1, CF 2) and 7.0-6.9 (CF 3), respectively. The pools were further purified by RP(C18)-HPLC, yielding 7.0 mg (CF 1), 5.5 mg (CF 2), and 9.5 mg (CF 3), respectively, of glycopeptides. This material was used for detailed investigations of the S-layer protein glycosylation. The amino acid sequence of the peptide portions was determined by Edman degradation to be predominantly Ser-Ala with trace amounts of Thr-Ser-Asp in pool CF 1; pool CF 2 contained a mixture of the peptide species Ser-Ala and Thr-Ser-Asp; in pool CF 3 the peptide Thr-Ser-Asp preponderated. This result is in agreement with the elution behavior of CF 1 and CF 2 from the chromatofocusing column, where both pools almost coeluted. Carbohydrate analysis by HPAEC/PAD revealed galactose and rhamnose, in slightly varying molar proportions of 1:49 (CF 1), 1:47 (CF 2), and 1:37 (CF 3), as constituents of the S-layer glycan, indicative of variations in S-layer glycan chain length. On the basis of the known trisaccharide repeating unit structure, [2)--L-Rhap-(13)--L-Rhap-(12)--L-Rhap-(1] (Ref. 18; see below), an average chain length of 15 repeating units was calculated from HPAEC/PAD data. For obtaining accurate information on chain length variation, the glycopeptide pools were subjected to MALDI-TOF analysis. The mass spectrum of each pool revealed individual peaks, originating from a major constituent and smaller amounts of other components, always with mass differences exactly coinciding with the mass of one trisaccharide repeat (Table III). The overall degree of polymerization of trisaccharide repeats varies between n = 13 and n = 18 in the glycopeptide pools CF 1-3. The MALDI-TOF spectrum of CF 2 revealed predominant peaks in the range of n = 14-16 (Fig. 3). This result concurred with NMR analyses of the three CF pools, demonstrating that in each pool two major glycopeptide species in varying molar ratios were present.

View this table: [in this window] [in a new window]   Table III MALDI-TOF MS analysis of glycopeptide pools CF 1-3 showing repeating unit-wise variations in S-layer glycan chain length Values for the peptide Ser-Ala (P1, Fig. 3) and Thr-Ser-Asp (P2, Fig. 3) correspond to [M  H] molecular ions.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Negative-ion MALDI-TOF mass spectrum of glycopeptide pool CF 2, showing variation in chain length of the S-layer glycoprotein glycan of G. stearothermophilus NRS 2004/3a occurring on the Ser and Thr glycosylation sites. The number of trisaccharide repeats present on the different glycoforms is given in parentheses. P1 and P2 designate the peptide species Ser-Ala and Thr-Ser-Asp, respectively.

Glycan Structure and Glycosidic Linkage-- For complete characterization of the glycosylation of the S-layer glycoprotein from G. stearothermophilus NRS 2004/3a, the repeating unit structure of the S-layer glycan was re-investigated by straightforward one- and two-dimensional ¹H and ¹³C NMR spectroscopy. The previously determined polyrhamnan repeating unit structure (18) was confirmed by HMBC cross-peaks via the glycosidic linkages in all glycopeptide pools (Fig. 4). As termination points at the non-reducing end of the glycan chains 2-O-methyl groups on all -1,3-linked L-rhamnose units B' could be identified, especially by heteronuclear three-bond correlations between the methyl carbon (_C = 59.42) and H-2 of -L-Rhap B' (_H = 3.700) (Table IV). Additional evidence for the 2-O-methyl--L-Rhap units was derived from the occurrence of 2-O-methylglyceraldehyde-hydrate, obtained after Smith degradation of the glycopeptides (Table V). Due to this observation, the sequence of the differently linked rhamnose residues could be determined unambiguously. The chemical shifts of the anomeric protons and carbons from the L-rhamnose units B, C, and A of the central repeats were readily distinguishable from small signals of other rhamnoses, which represent the core units D and E of this S-layer glycoprotein (Fig. 4). The core is completed by a galactose unit F forming an O-glycosidic linkage either to threonine or serine residues of the polypeptide. The anomeric configuration of this Galp-linkage glycose was found to be  according to the measured coupling constants between H-1 and H-2 (J_H, H ~ 7.5 Hz) and between H-1 and C-1 (J_C, H ~ 162 Hz), respectively (Table IV). All glycosidic linkage types within the S-layer glycan and between the glycan and the polypeptide were determined unambiguously performing two-dimensional NOESY and HMBC NMR experiments. In Fig. 3, an example for the derived linkage information in one single plot is presented, showing a section of an HMBC spectrum with cross-peaks from the anomeric protons to the corresponding ring carbons and the -carbons of the amino acids, respectively. Because the components of the trisaccharide repeating unit are, on average, 15 times more abundant than the terminal residues, and in pool CF 2 the serine- and the threonine-bound glycopeptides were present, the observed cross-peaks to the  carbons of Ser and Thr were very weak. The entire connectivity information between the glycan and the peptides extracted from heteronuclear long range correlations and two-dimensional NOESY spectra is summarized in Table VI. In addition, to elucidate the amino acid sequences of the glycopeptides, DQF-COSY, TOCSY, and NOESY spectra were recorded in distilled water to get cross-peaks involving the amide protons.

View larger version (27K): [in this window] [in a new window]   Fig. 4.   Section of a gradient-enhanced HMBC spectrum of pool CF 2 showing the glycosidic linkages and the cross-peaks between galactose H1/Ser C and galactose H1/Thr C, respectively. A-F represent single sugar units, lowercase S and T denote the linkage to the amino acids Ser J and Thr G, respectively. For details see Fig. 5.

Based on the determined O-glycosidic linkage types, which are -D-Gal-Ser and -D-Gal-Thr, and 2-O-methylation occurring on the terminal rhamnose residue at the non-reducing end of the glycan chain, in combination with peptide sequences, the core saccharide was additionally calculated from the MALDI-TOF MS data (Table III). In concordance with the NMR results, the disaccharide core 2)--L-Rhap-(13)--L-Rhap-(1 was confirmed to be present in all glycopeptide species (Fig. 5, Table III).

View larger version (8K): [in this window] [in a new window]   Fig. 5.   Schematic drawing of the complete structure of the S-layer glycoprotein of G. stearothermophilus NRS 2004/3a. The full curved line symbolizes SgsE; glycosylation sites are the amino acids Thr-620 and Ser-794. Differences in the glycosylation pattern of individual S-layer protomers are indicated by the dotted lines.

Combining results from NMR analyses with the MALDI-TOF MS evidence, the S-layer glycan chains of G. stearothermophilus NRS 2004/3a are composed of [2)--L-Rhap-(13)--L-Rhap-(12)--L-Rhap-(1] trisaccharide repeating units, with a polymerization degree ranging between 13 and 18 units, and with a 2-O-methyl group capping the 1,3-linked rhamnose of the terminal repeat. The glycan chains are linked via a 3)--L-Rhap-(13)--L-Rhap-(1 disaccharide core to the novel linkage units -D-Galp-O-Thr/Ser of the S-layer protein (Fig. 5).

To confirm the presence of O-glycosidic linkages, which have been demonstrated by NMR analysis (this study), -elimination was carried out on S-layer glycopeptides using NaB[³H]₄ in 0.1 M NaOH. After separation of the reaction mixture on a Bio-Gel P-4 column, equimolar amounts of incorporated radioactivity were found in the glycan and peptide portions (not shown). The presence of the ³H label in the carbohydrate fraction indicated that the glycan chains were reduced and present in their alditol forms. Thus, the S-layer glycans could be completely released under the applied conditions, demonstrating their O-glycosidic linkage.

Glycosylation Sites-- For identification of glycosylation sites on mature SgsE, the purified S-layer glycoprotein was partially degraded with papain, aiming at the maintenance of an extended peptide portion on the produced S-layer glycopeptides. Upon separation of the degradation mixture on gel permeation columns and chromatofocusing, three glycopeptide pools, eluting from the ion exchanger at pH intervals of 8.3-7.8, 7.5-7.2, and 7.1-6.8, respectively, were isolated. Final separation of these pools by RP(C18)-HPLC revealed one glycopeptide species, each, to be present in pools 1 and 2 (GP 1 and GP 2), whereas pool 3 split into three species, designated GP 3 through GP 5 (Table VII). Compositional analyses by HPAEC/PAD and amino acid analyses revealed identity in glycose residues, in concordance with the polyrhamnan S-layer glycan of G. stearothermophilus NRS 2004/3a, but differences in amino acids. The glycopeptides as well as the corresponding, chemically deglycosylated fragments were subjected to Edman degradation. The amino acid sequences were determined to be ADLKTTSDNF (GP 1), LTSADV (GP 2), TSAD (GP 3), TLTSAD (GP 4), and TSADVIRV (GP 5) for the deglycosylated fragments (Table VII). Alignment of these sequences with the amino acid sequence of SgsE revealed that GP 1 (decapeptide 1) and GP 2 through GP 5, together constituting a second decapeptide (decapeptide 2), both occurred only once throughout the entire protein. By comparing sequence data of the deglycosylated and intact fragments, which showed a blank signal at the position of the corresponding glycosylated amino acid during sequencing (Table VII), it became evident that Thr-620 of decapeptide 1 was glycosylated, whereas in decapeptide 2 Ser-794 served as a glycosylated amino acid (Fig. 5). From these data we conclude that two O-glycosylation sites are present in SgsE, both located in the C-terminal region of the S-layer protein.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In the course of the investigation of S-layer glycoproteins from different organisms of the domain Bacteria (for a recent review see Ref. 43), G. stearothermophilus NRS 2004/3a has been chosen as a candidate for the design of S-layer neoglycoproteins by means of "genetic carbohydrate engineering" (for review see Ref. 2).

In the present paper, we have elucidated the complete primary structure of the S-layer glycoprotein glycan of G. stearothermophilus NRS 2004/3a. This included the re-examination of the repeating unit trisaccharide constituting the glycan chains (18), the establishment of the sequence of the glycose residues, and focused on the characterization of the core region and the determination of the type of glycosidic linkage between the glycan and the S-layer polypeptide. In addition, the structural gene sgsE (AF328862) has been sequenced and the glycosylation sites on the mature glycoprotein have been determined. G. stearothermophilus NRS 2004/3a is the first eubacterium whose glycosylation sites on the S-layer protein have been shown. Besides the S-layer proteins of this organism (SgsE) and of a temperature-derived variant of G. stearothermophilus ATCC 12980 (SbsD), none of the S-layer proteins investigated thus far from that species possess a glycosylated S-layer.

Based on the structural data of the glycan (Fig. 5), the S-layer glycoprotein of G. stearothermophilus NRS 2004/3a can be classified as a type I S-layer glycoprotein (43). This implies that the S-layer glycoprotein possesses a tripartite architecture, reminiscent of LPS of Gram-negative eubacteria (22, 43). The elongated polyrhamnan chain is composed of identical trisaccharide repeating units, and the terminal repeating unit of each glycan chain is capped with a 2-O-methyl group. This is the first report of a 2-O-methyl capping group occurring on an S-layer glycoprotein glycan. Besides that, 2-O-methyl-rhamnose has been reported as part of the repeating unit of the lipopolysaccharide from Thiocapsa roseopersicina (44) and as a spore-specific constituent of Bacillus cereus (45). The core region of the S-layer glycan form G. stearothermophilus NRS 2004/3a is composed of a 2)--L-Rha-(13)--L-Rha-(1 disaccharide. Interestingly, all core oligosaccharides of S-layer glycans elucidated so far contain -rhamnose residues, either in the L- or in the less common D-configuration. This includes the 3)--L-Rha-(13)--L-Rha-(13)--L-Rha-(13) core oligosaccharides found in Thermoanaerobacter thermohydrosulfuricus L111-69 and Paenibacillus alvei CCM 2051 (32), and the variable cores of Aneurinibacillus thermoaerophilus GS4-97 (34) and A. thermoaerophilus DSM 10155 (46), which are -[-D-Rha-(13)]_n = 0-3- and -[-L-Rha-(13)]_n = 1-2-, respectively. The polyrhamnan S-layer glycans of G. stearothermophilus NRS 2004/3a are bound via -D-galactose units to distinct Thr and Ser residues of mature SgsE. This is the first description of these types of glycosidic linkages in bacterial glycoproteins (for review see Ref. 43). In contrast, all linkage units of bacterial non-S-layer glycoproteins to either threonine or serine investigated so far contain the galactose residue in -configuration. Thus, the occurrence of a -D-galactose unit in the S-layer glycan linkage of G. stearothermophilus NRS 2004/3a emphasizes the preference of -linkages in eubacterial S-layer glycoproteins (for review see Ref. 47). This finding might be explained by the specificities of glycosyl transferases involved in the biosynthesis of the S-layer glycoproteins.

In a biochemical approach to elucidate the biosynthesis pathway of the S-layer glycoproteins of Paenibacillus alvei CCM 2051 (48) and Thermoanaerobacterium thermosaccharolyticum E207-71 (22), nucleoside diphosphate-activated oligosaccharides have been isolated, with saccharide moieties as large as a single repeating unit of the respective S-layer glycan. This is different to the well-known biosynthesis pathway of LPS O-antigens, in which exclusively nucleotide-activated monosaccharides participate. Recently, the synthesis of nucleotide-activated oligosaccharides via a glycosidase-catalyzed transglycosylation reaction from Bacillus circulans has been reported (49), potentially indicating a more widespread occurrence of such compounds in the bacterial metabolism. We conclude that S-layer glycan chains are polymerized repeating unit-wise in the cytoplasm. Because MALDI-TOF analyses of S-layer glycopeptide samples from G. stearothermophilus NRS 2004/3a showed distinct glycopeptide species differing in single repeats (33), there is strong evidence that this concept is also valid for this organism. As has already been discussed for 3-O-methylated S-layer glycans (25, 34), 2-O-methylation of the terminal rhamnose residue might function as a termination signal for chain elongation (for reviews see Ref. 43). Simultaneously with the biosynthesis of the S-layer glycan chain, the core saccharide might be synthesized either on a separate nucleotide activator or cotranslationally by direct transfer of activated glycose units to the nascent S-layer polypeptide. The occurrence of two different types of O-glycosidic linkages at defined positions of SgsE brings up the interesting question as to how the discrimination between these sites is regulated on the enzymatic level.

Only recently, it has been demonstrated with A. thermoaerophilus DSM 10155 that the genes encoding enzymes for the biosynthesis of nucleotide sugar precursors are clustered within the corresponding gene cluster for S-layer glycan biosynthesis (50). First attempts to localize the genes involved in the biosynthesis of nucleotide-activated -L-rhamnose of G. stearothermophilus NRS 2004/3a revealed that the S-layer glycan biosynthesis cluster is located in close vicinity downstream of sgsE.²

The identification of the glycosylation sites on SgsE is a prerequisite for unraveling the S-layer glycoprotein biosynthesis of G. stearothermophilus NRS 2004/3a. We have unambiguously identified amino acids Thr-620 and Ser-794 to be O-glycosylated. On the SDS-PAGE gel, the S-layer glycoprotein separated into four bands with apparent molecular masses of 93, 119, 147, and 170 kDa, with the latter three bands staining with PAS reagent (Fig. 1). Because a distinct 93-kDa band corresponding to mature SgsE was obtained upon chemical deglycosylation, the three glycoprotein bands originate from a single protein species. The value of the deglycosylated S-layer protein is in excellent agreement with the molecular mass of 93,684 kDa, as calculated from the amino acid sequence of SgsE. Based on the identification of two glycosylation sites and one type of glycan chain (Fig. 5), one could expect two glycoprotein bands to appear on the gel. For the observed banding pattern there is currently no conclusive explanation. Assuming a random but site-specific glycosylation of SgsE, the occurrence of a third glycosylation site could cause a banding pattern similar to that observed. However, we have no experimental evidence for an additional glycosylation site on SgsE. A putative third glycosylation site could, if present at all, occur only in very small amounts, as inferred from the low intensity of the 170-kDa band on the gel in comparison to the predominant 119- and 147-kDa bands. Thus, from our data, the existence of a third glycoform cannot be ruled out completely. A final interpretation of the SDS-PAGE banding pattern of the S-layer glycoprotein of G. stearothermophilus NRS 2004/3a, however, will only be possible after purification and separate analysis of each S-layer glycoprotein species.

Comparing recent data base analyses of O-glycosylation sites in proteins (51) with the glycosylation sequences found in SgsE, it becomes evident that the amino acid preferences described for the well investigated mucin-type glycosylation, such as a proline residue at +3 and/or 1 positions favoring glycosylation, cannot be confirmed for S-layer protein glycosylation. Interestingly, besides the two O-glycosylation sites, eight sequons for potential N-glycosylation are distributed throughout mature SgsE. Detailed analysis of the S-layer glycoprotein of G. stearothermophilus NRS 2004/3a revealed that these sites are not glycosylated (this study). In a previous study, an N-glycosidic linkage had been proposed; this assumption had been based on the finding that the glycan chains could only be released by hydrazinolysis but not under rather mild -elimination conditions (pH 9.0/4 °C/45 h (16, 19)). However, combined results of this study (NMR analyses, -elimination reaction) unambiguously demonstrated the exclusive presence of O-glycans. Thus, our previous interpretation has to be revised, implying that eubacterial S-layer glycoproteins seem to possess exclusively O-glycans, so far (for review see Ref. 47). In contrast, in the domain Archaea, O- as well as N-glycans have been observed on S-layer proteins (for reviews see Refs. 52, 53).

The genes coding for the S-layer proteins from five strains of Geobacillus (formerly Bacillus) stearothermohilus strains, which are termed sbsA-D and sgsE, have now been characterized (Table II). SgsE is the first eubacterial S-layer glycoprotein from a wild-type strain whose glycosylation sites have been determined. Comparable analyses have been performed only with the S-layer glycoprotein of the archaeon Halobacterium halobium (53). However, the sequence of the S-layer protein SbsD from a glycosylated, temperature-derived mutant of G. stearothermophilus ATCC 12980 has been deposited at GenBank^TM as the first S-layer glycoprotein sequence (AF228338); but no details about its glycosylation have been published so far. Because there is high overall identity (93.9%) in amino acid sequence between SbsD and SgsE (this study), we performed an alignment of the regions comprising the glycosylation sites of SgsE (Fig. 6). This alignment revealed identical sequences in the vicinity of these sites. Sequence identity is given between amino acids 619 and 654, containing glycosylated threonine at position 620 of SgsE, and between amino acids 780 and 812, containing a glycosylated serine at position 794 of SgsE. Based on this finding, glycosylation of SbsD might occur at sites identical to those identified on SgsE. In this region there is no homology of these S-layer glycoproteins to SbsA-C.

View larger version (26K): [in this window] [in a new window]   Fig. 6.   Sequence alignment of the S-layer proteins SgsE (AF328862; this study) and SbsD (AF228338) between amino acids 595 and 825. The glycosylation sites of SgsE are marked in black. Sequence identities on both proteins in vicinity of these sites are indicated by gray shaded boxes.

Glycosylation of SgsE at the C-terminal region (Thr-620 and Ser-794) might be seen in the context with anchoring the S-layer glycoprotein to the bacterial cell wall. The positively charged amino acids contained in the N-terminal region of SgsE could possibly interact with peptidoglycan-associated secondary cell wall polymers via direct electrostatic interactions or hydrogen bonds, and, thus, the N terminus might mediate attachment to the cell wall (54, 55). The S-layer glycan chains are outwardly orientated and might create a hydrophilic coat around the bacterial cell, comparable to the LPS O-antigens of Gram-negative bacteria.

	ACKNOWLEDGEMENTS

We thank Dr. F. Altmann for performing the MALDI-TOF MS experiments, Dr. K. Vorauer for N-terminal sequence analysis, A. Warter for assistance with the NMR experiments, and R. Christian for critical reading of the manuscript.

	FOOTNOTES

^* This work was supported by the Austrian Science Fund, Projects P12966-MOB and P14209-MOB (to P. M.), and the Hochschuljubiläumsstiftung der Stadt Wien, Project H-96/2000 (to C. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ To whom correspondence should be addressed: Zentrum für Ultrastrukturforschung und Ludwig Boltzmann-Institut für Molekulare Nanotechnologie, Universität für Bodenkultur Wien, Gregor-Mendel-Strasse 33, A-1180 Wien, Austria. Tel.: 43-1-47654 (Ext. 2203); Fax: 43-1-478-9112; E-mail: crs@edv1.boku.ac.at.

Published, JBC Papers in Press, December 10, 2001, DOI 10.1074/jbc.M108873200

² R. Novotny, C. Schäffer, and P. Messner, unpublished data.

	ABBREVIATIONS

The abbreviations used are: S-layer, surface layer; DQF-COSY, double-quantum filtered correlation spectroscopy; HMBC, heteronuclear multiple bond correlation spectroscopy; HPAEC/PAD, high performance anion-exchange chromatography/pulsed amperometric detection; HSQC, heteronuclear single quantum coherence; LPS, lipopolysaccharide; NOESY, nuclear Overhauser enhancement spectroscopy; MALDI-TOF, matrix assisted laser desorption ionization time-of-fight; MS, mass spectrometry; nt, nucleotide; ORF, open reading frame; PAS, periodic acid-Schiff; sgsE, S-layer structural gene of Geobacillus stearothermophilus NRS 2004/3a; TOCSY, total correlation spectroscopy; RP-HPLC, reverse phase-high performance liquid chromatography.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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