©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Novel, Specific O-Glycosylation of Secreted Flavobacterium meningosepticum Proteins
Asp-Ser (*)AND Asp-Thr(*)-Thr CONSENSUS SITES (*)

Thomas H. PlummerJr. (1)(§), Anthony L. Tarentino (1), Charles R. Hauer (1) (2)

From the (1) Division of Molecular Medicine, Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, New York 12201-0509 and (2) The School of Public Health, State University of New York, Albany, New York 12201

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A new type of O-linked oligosaccharide has been discovered on several proteins secreted by the Gram-negative bacterium Flavobacterium meningosepticum, including Endo F (three sites), Endo F (one site), and a P40 protease (one site). The oligosaccharide moiety is covalently attached via a mannose residue to a serine or threonine at consensus sites corresponding to Asp-Ser* or Asp-Thr*-Thr. Preliminary characterization by mass spectroscopy revealed an oligosaccharide of 1244 Da at each of the proposed glycosylation sites. Collision-associated dissociation analysis showed a characteristic daughter ion series of m/z 218, 394, and 556, indicative of a common Flavobacterium oligosaccharide. Compositional analysis demonstrated an unusual profile of monosaccharides, including hexoses, methylated hexoses, and uronic acid derivatives.


INTRODUCTION

Post-translational glycosylation is now widely recognized as an important process for modifying the structure/function of eukaryotic proteins. Glycosylation additions in bacterial systems, however, are much less well understood and thus far restricted mainly to oligosaccharide polymers and lipopolysaccharides and to complex cell wall and cell surface components. Mescher and Strominger in 1976 (1) first reported the presence of a single N-linked and multiple O-linked carbohydrates in the envelope protein of Halobacterium salinarium. More recent studies have reported the presence of N-linked carbohydrate in bacterial cell wall proteins (2, 3) and O-linked glycan in Thr/Pro-rich regions of cellulase complexes of the cellulolytic bacteria Clostridium thermocellum and Bacteriodes cellulosolvens(4) .

In the course of our studies on the hydrolases produced by Flavobacterium meningosepticum, we observed anomalies which suggested a type of glycosylation different from what had been previously observed in prokaryotes. This Gram-negative bacterium secretes into the culture medium at least eight major proteins that can be purified to homogeneity by hydrophobic interaction chromatography in conjunction with high-resolution ion-exchange chromatography (5, 6) . These proteins include two proteases, termed P27 and P40()(7) and four oligosaccharide chain-cleaving enzymes, including one amidase, termed PNGase() F, and three endoglycosidases designated as Endo F, F, and F (5). The genes for all four oligosaccharide chain-cleaving enzymes were cloned and their nucleotide sequences determined (8, 9, 10) . The genes for Endo F and Endo F were expressed in Escherichia coli, and the molecular weights of the cloned enzymes were compared with those of the native enzymes on SDS-polyacrylamide gel electrophoresis. Native Endo F ran slightly larger and native Endo F significantly larger than their cloned enzyme counterparts. Mass spectrometry confirmed that Endo F and Endo F were modified and contained approximately 3731 and 1244 daltons of mass, respectively, not accounted for in the gene structure. Re-examination of Edman sequence data led to the suggestion that Endo F and Endo F were post-translationally modified with carbohydrate during secretion by possible linkage to the hydroxyl of serine (10) . A problem existed in characterizing the oligosaccharide moieties of Endo F and Endo F, since isolation of these enzymes from 12 liters of cultural filtrate yielded only 9 and 15 nmol of protein, respectively. We will demonstrate that the P40 protease is also a glycoprotein containing a carbohydrate moiety O-linked to serine at one unique well defined aspartyl-serine consensus site. This oligosaccharide is easily obtained in amounts of 1 µmol and serves as a model for Endo F and Endo F. In fact, mass spectrometry data strongly suggests that the O-linked carbohydrate on P40, Endo F, and Endo F are identical.

Prokaryotic extracellular glycoproteins like Endo F, Endo F, and P40 that have O-linked oligosaccharides at specific consensus sites have never been described before. This report outlines the isolation of glycopeptides containing these consensus sites and demonstrates the existence of a new type of bacterial glycan that is distinguished by having an unusual array of uronic acids and methylated neutral sugars linked via mannose to serine/threonine. The following paper (11) presents a detailed characterization of this novel glycan.


MATERIALS AND METHODS

Proteins

Endo F and Endo F were isolated essentially as described (5) . The final purification step of Endo F and Endo F was achieved on a high resolution sulfopropyl (Protein-Pak SP8HR, Waters Chromatography Division) column at pH 4.5 (6) . P40 was chromatographed on TSK-phenyl-Toyopearl 650 M(3) and then rechromatographed on TSK-butyl-Toyopearl 650S using the conditions outlined for TSK-butyl-Toyopearl 650 M column chromatography (5) . Pooled fractions were dialyzed at 4 °C against two changes of 10 mM EDTA and one change of 5 mM EDTA followed by one change of distilled water for 4 h before lyophilization.

Tryptic Digestion

Lyophilized P40 (42 mg) was warmed at 37 °C for 45 min in 1.6 ml of 0.4 M Hepes, pH 8.25, containing 8 M urea. The solution was diluted 3-fold with 4.8 ml of 0.4 M Hepes and 315 µl of L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (4.0 mg/ml, 3% w/w) in 2 mM HCl, 100 mM CaCl was added. After 6 h, a second identical aliquot of trypsin was added. The digestion was incubated at 37 °C for 24 h and was terminated by addition of 0.4 ml of 8 M HPO. A precipitate was removed by centrifugation.

Thermolytic Digestion

Lyophilized Endo F (1.23 mg) was suspended in 0.4 ml of 0.2 M Hepes, pH 8.24, containing 5 mM CaCl; and 50 µg of thermolysin, 3% w/w, (Calbiochem) was added. After 6 h, a second identical aliquot of thermolysin was added. The digestion was incubated at 50 °C for 24 h and was terminated by addition of 50 µl of 4.0 M HPO.

Cyanogen Bromide Digestion

To approximately 500 pmol of Endo F in a 500-µl Eppendorf tube was added 100 µl of 70% trifluoroacetic acid and a crystal of CNBr. The sample was flushed with N, capped, and allowed to sit for 24 h at room temperature. The solution was lyophilized to near dryness, redissolved in 500 µl of water, lyophilized again, and then redissolved in 200 µl of water prior to separation on a 1 50-mm ABI RP-300 column.

Glycopeptide Purification

Supernatant (7.5 ml) from the tryptic digestion of P40 was applied to a column of Toyopearl HW-40S (1.5 234 cm, Supelco) equilibrated in 0.1 N acetic acid containing 1% butanol. The flow rate was 11.2 ml/h, and fractions of 2.8 ml were collected. Peptides were detected by absorbance at 230 nm. Carbohydrate was monitored by the phenol-HSO assay (12) . The glycopeptide was shown to be exclusively in the nonretarded position on the column. These fractions were pooled, concentrated by rotary evaporation at 40 °C, and rechromatographed on a column of Bio-Gel P-4 (0.9 91 cm, 200-400 mesh, Bio-Rad) equilibrated in 0.1% trifluoroacetic acid. The flow rate was 12 ml/h, and fractions of 0.8 ml were collected. Glycopeptide was detected by absorbance at 215 nm and by reacting aliquots with fluorescamine (13) . Carbohydrate was monitored as before and pertinent fractions were lyophilized.

The supernatant from the thermolytic digestion of Endo F was handled identically to that of P40 except on a smaller scale. The initial separation was on a TSK HW40S column (0.6 93 cm) equilibrated in 0.1% trifluoroacetic acid. The flow rate was 6.0 ml/h, and fractions of 0.5 ml were collected. Peptides were monitored at 215 nm. The glycopeptide-containing fractions (tubes 28-30) were lyophilized. Ten percent was applied directly to a Bio-Gel P-4 column (0.6 63 cm) and 90% was subjected to alkaline-borohydride oligosaccharide release (see below) prior to chromatography on Bio-Gel P-4.

Oligosaccharide Release

Lyophilized P40 glycopeptide (1 µmol) was dissolved in 450 µl of freshly prepared 1 M NaBH in 50 mM NaOH (14) and incubated at 45 °C. After 16 h, the reaction was terminated by the addition of glacial acetic acid to pH 4.0, and the sample was applied to the aforementioned larger Bio-Gel P-4 column. Uncleaved peptide, released oligosaccharide, and released peptide were detected as before. Endo F glycopeptide was handled similarly, and released oligosaccharide was separated from reduced peptide on a Bio-Gel P-4 column (0.6 63 cm).

Amino Acid Analyses

Peptides were hydrolyzed in constant boiling HCl at 110 °C for 22-24 h and analyzed for amino acids with a Beckman System Gold amino acid analyzer or a Pickering amino acid resolution column and buffers on a Waters 625 LC system.

Edman Degradation

Automated Edman degradation was performed with a model 477A Applied Biosystem pulsed liquid sequenator equipped with a model 120A amino acid analyzer. Samples of 250-2000 pmol were applied in 100-200 µl of 25% acetic or formic acid. Peptide composition determined by amino acid analysis was used to predict the number of Edman cycles to be completed for each peptide.

Carbohydrate Analysis

Suitable aliquots were hydrolyzed either with 2 N trifluoroacetic acid or with 2 N hydrochloric acid in N-flushed, evacuated tubes for varying time intervals. Acids were removed by lyophilization (2 N trifluoroacetic acid) or under vacuum at 40 °C (2 N HCl). Monosaccharide samples were taken up in water (115 µl), made alkaline with 2 N NaOH (10 µl), and aliquots (100 µl) were analyzed by HPAEC/PAD on a CarboPac PA-I column, using the chromatography conditions of Clarke et al.(15) , or on a MA-1 column using conditions recommended by the manufacturer (Dionex) for sugar alcohols. Released, reduced P40 oligosaccharide was chromatographed on a Carbo-Pac PA-100 column using the general procedure of Townsend et al.(16) . Eluant 1 was 500 mM sodium acetate, eluant 2 was water, and eluant 3 was 1 M NaOH. The column was equilibrated in 10 mM sodium acetate, 200 mM sodium hydroxide at a flow rate of 1 ml/min. After sample addition, the concentration of sodium acetate was increased to 400 mM over 60 min using a linear gradient.

Compositional analyses were also accomplished by preparing alditol acetate derivatives of glycosyl components followed by gas chromatography and combined gas chromatography/mass spectrometry (GC/MS) analysis. Trimethylsilyl derivatives of the methyl glycosides were prepared to detect any amino sugars (17, 18) and analyzed by GC and GC/MS (compositional analyses were performed by Dr. Roberta K. Merkle at the University of Georgia, Complex Carbohydrate Research Center).

Mass Spectrometry

Data were acquired with a Finnigan-MAT (San Jose, CA) TSQ-700 triple quadruple mass spectrometer equipped with a Finnigan electrospray ionization (ESI) source. Salt-free samples were infused into the ESI source at a flow rate of 2 µl/min. Collisional activated dissociation (CAD) spectra were obtained on doubly and/or singly charged ions; argon was used as the collision gas. Carboxylic acid groups were converted to the corresponding methyl esters as described previously (19) . Peptide amine terminal NH groups were acetylated with a 3:1 mixture of methanol/acetic anhydride.


RESULTS AND DISCUSSION

The secreted proteins of Flavobacterium meningosepticum are separated initially by hydrophobic interaction chromatography on columns of either Toyopearl TSK-butyl (5) or Toyopearl TSK-phenyl (7). A typical chromatogram of 12 liters of culture medium on a TSK-phenyl column was surveyed for carbohydrate content by the phenol-HSO assay. The only protein peak that was coincident with carbohydrate was that of the protease, P40 (peak C, Ref. 7). The fractions containing Endo F and F, although glycosylated, showed no carbohydrate owing to the lack of sensitivity of this technique at their concentration levels. P40 was rechromatographed on a column of TSK-butyl (not shown), desalted by dialysis against dilute EDTA, and then subjected to total tryptic hydrolysis. The soluble peptides, containing 80% of the original carbohydrate, were subjected to gel filtration on a column of TSK HW-40S (Fig. 1). All of the carbohydrate was recovered in a glycopeptide eluting in the nonretarded fraction of the column. Amino acid analysis of the glycopeptide indicated the presence of six amino acids, with 2 mol of serine/mol of aspartic acid and no apparent amino sugars. The glycopeptide was then subjected to alkaline borohydride to release the oligosaccharide moiety. After overnight incubation at 45 °C excess borohydride was decomposed with glacial acetic acid, and the reaction mixture was separated on a Bio-Gel P-4 column (Fig. 2). All components were monitored by absorbance at 215 nm; the carbohydrate- and peptide-containing fractions were determined with phenol-HSO and fluorescamine, respectively. The peak corresponding to the intact glycopeptide (Fig. 2, solid bar) was greatly decreased following alkaline borohydride treatment and replaced by two lower molecular weight fractions; a moderate-sized peak containing the majority of the carbohydrate (Fig. 2, CHO), and a large fluorescamine-positive, carbohydrate-negative peak containing the peptide. Molecular weight analysis by ESI-MS indicated a mass of 746.6 Da for the deglycosylated peptide. The CAD spectra of the native, acetylated, and methyl ester-converted peptide demonstrated a sequence of S-X-X-D-A-T-K, where X is either leucine or isoleucine. Edman degradation established that the sequence was S-I-L-D-A-T-K. The presence of 1 mol of alanine corresponds to the complete reduction of the dehydroalanine formed by -elimination of the oligosaccharide moiety at the serine residue under the conditions employed. Thus, the P40 glycopeptide corresponds to the sequence S-I-L-D-S*-T-K (), where the starred serine represents the site of attachment to the oligosaccharide.


Figure 1: Gel filtration of tryptic digest of P40 on Toyopearl HW-40S. The column (1.5 234 cm) was equilibrated in 0.1 N acetic acid at a flow rate of 11.2 ml/h, and fractions of 2.8 ml were collected. Peptides were detected by absorbance at 230 nm () and carbohydrate was monitored by the phenol-HSO assay (). The bar marks fractions pooled for further study.




Figure 2: Gel filtration of P40 glycopeptide mixture after release of oligosaccharide chain by 1 M NaBH in 50 mM NaOH at 45 °C for 16 h. The column (0.9 91 cm) was equilibrated in 0.1% trifluoroacetic acid at a flow rate of 9 ml/h, and fractions of 0.8 ml were collected. Peptides were detected by absorbance at 215 nm () and by reaction of aliquots with Fluram (); carbohydrate was monitored by the phenol-HSO assay (). Filled rectangle, position of original glycopeptide before carbohydrate release; open rectangle, fractions pooled for further study.



Preliminary information concerning the Flavobacterium oligosaccharide moiety was obtained as follows. Mild acid hydrolysis of the free oligosaccharide followed by HPAEC chromatography on a PA-1 column indicated the presence of glucose, mannose, and glucuronic acid, plus at least two unknown components in significant amount. Sugar composition determined by GC and GC/MS analysis of the TMS and alditol acetate derivatives indicated the presence of mannitol, presumably the linking sugar of the oligosaccharide, and nearly equimolar amounts of a 2-O-methyl-deoxyhexose (probably rhamnose), 2-O-methyl mannose, glucose, and glucuronic acid, as well as lower amounts of mannose. The presence of mannitol was verified by HPAEC/PAD chromatography using a MA-1 column. No amino sugars were apparent using any of the above techniques.

Mass analysis of the free oligosaccharide demonstrated a molecular weight of 1264 Da for the major component. An intense signal for the M + H ion, compared with Na and K chelated species, indicated the presence of a basic moiety in the oligosaccharide, in spite of the absence of amino sugars in the compositional analysis. The CAD spectrum of this oligosaccharide (Fig. 3A) exhibited a daughter ion series with prominent signals at m/z 218, 394, and 556; this daughter ion pattern was also observed in the CAD spectra of glycopeptides with the 1244-Da pendant group. Also, the m/z 218 signal indicated the possible presence of an N-acetylhexuronic acid, and the m/z 218 - m/z 394 difference of 176 indicated the possible presence of a hexuronic acid or a methyl hexose. To test for the presence of hexuronic acids the released oligosaccharide was treated with methanolic HCl, to convert any carboxylate groups into methyl esters. Molecular mass analysis of the resultant derivative indicated a mass of 1306 Da. This shift of 42 Da corresponds to the presence of three carboxylic acid groups in the P40 oligosaccharide. In Fig. 3B, the CAD spectrum of the methyl ester converted oligosaccharide, the shift of the m/z 218 ion to m/z 232 supports an assignment of N-acetylhexuronic acid, whereas the constant difference of 176 between the m/z 232 and m/z 408 ions argues for the presence of a methyl hexose as an adjacent sugar group.


Figure 3: Comparison of the CAD spectra of (A) the free oligosaccharide from P40 and (B) the corresponding methyl ester derivative.



The reduced P40 oligosaccharide was chromatographed directly on a PA-100 column. Because of the three acidic groups in this oligosaccharide, it was necessary to increase the standard gradient sodium acetate concentration 2-fold in order to elute the oligosaccharide in the middle of the profile. Under these conditions the oligosaccharide was resolved into three glycoforms; a main component representing 83% of the PAD response, with minor components accounting for 14 and 3%, respectively. These findings are consistent with MS analyses that indicated a main component with a mass of 1264 Da, a minor component with a mass of 1104 Da (-160 Da), and a trace component with a mass of 1441 Da (+176 Da).

Evidence has also been obtained that Endo F and Endo F are O-glycosylated like the P40 protease described above. A glycopeptide was isolated from Endo F using the same procedures developed for the P40 protease, albeit on a smaller scale. Intact enzyme was digested at 50 °C by thermolysin, and the glycopeptide was isolated by gel filtration in the nonretarded fractions of a TSK-HW40S column. The glycopeptide was subjected to oligosaccharide release by alkaline-borohydride followed by gel filtration on Bio-Gel P-4 to separate the components. Trifluoroacetic acid hydrolysis of intact glycopeptide followed by HPAEC chromatography on a PA-1 column gave a pattern of unknown and known sugars very similar to that of the P40 oligosaccharide. Edman degradation of the carbohydrate-free peptide established the following sequence for the 10 cycles programmed: L-Q-D-X-T-K-L-P-G-G. The X represents a blank cycle which should contain phenylthiohydantoin-derivative amino-butyric acid, for which there was no standard. The sequence L-Q-D-T-T-K corresponds to residues 46-51 of the Endo F molecule (10) . Mass spectrometry determined a molecular mass of 2431 Da for the glycopeptide. The CAD spectrum of this species was consistent with the peptide L-Q-D-T*-T-K-L-P-G-G-T-G, modified, like the P40 protease, with a pendant moiety of mass 1244 Da. This MS/MS spectrum exhibited the characteristic daughter ion pattern of m/z 218, 394, 556 associated with the putative common Flavobacterium glycoform. The methyl ester converted sample exhibited a mass of 2501 Da, corresponding to the presence of five carboxylic acids (three on the oligosaccharide and two on the peptide chain), i.e. the expected value for this glycopeptide.

The gene for Endo F has been isolated and cloned in E. coli(10) . A comparison of the molecular weights of native versus cloned Endo F showed a molecular weight difference of +3731 for the native enzyme, suggesting potential multiple glycosylation sites. Edman data of two tryptic peptides containing Asp-Ser sequences revealed no phenylthiohydantoin-derivatives for these serines. To account for the mass difference, we proposed (10) that native Endo F was post-translationally modified during secretion by glycosylation with an oligosaccharide moiety at three specific serine residues in an Asp-Ser diad, namely at positions 27-28, 43-44, and 97-98. This proposal was confirmed by the following mass analysis. Cleavage of Endo F with cyanogen bromide followed by reverse phase high performance liquid chromatography separation on a RP-300 (C8) column yielded four distinct fractions. Infusion ESI mass analysis of one of these fractions revealed two nonglycosylated peptides, with respective molecular masses of 9482 (major product) and 9711 Da (minor product). These values coincide with the calculated masses for the projected cleavage products (as homoserine) at amino acid residues 206-288 and 206-290 of Endo F. The three other CNBr fractions were glycosylated peptides with masses of 2327, 5369, and 18,460 Da, respectively. These values each correlate to predicted cleavage residue sites 39-47, 1-38, and 51-205 with the addition of a 1244-Da oligosaccharide moiety (). For example, the CAD spectrum of the 2327-Da peptide is consistent with the glycopeptide sequence, R-W-L-P-D-S*-L-D-Hse. Moreover, each glycopeptide fraction showed the characteristic oligosaccharide daughter ion series of m/z 218, 394 and 556.

represents a synopsis of sequences of F. meningosepticum secreted proteins and their sites of glycosylation. In every instance where there is a DS* sequence, the starred serine is fully substituted by addition of the common Flavobacterium oligosaccharide. We have found only one instance of a D-T*-T sequence (Endo F) and it too is fully substituted. A DT sequence alone, however, is not sufficient to allow for glycosylation, as evidenced by the finding that the single DT sequences in Endo F, Endo F, Endo F, and P40 () do not contain covalently bound carbohydrate. These substitutions should be evident by MS analysis even if on less than 10% of the protein. It is of interest to note that all five nonsubstituted DT sequences are followed by asparagine, aspartic acid, or glutamine. Further sequence studies will reveal whether these residues could be a negative signal for glycosylation.

The function of this novel oligosaccharide will require further study. The only other reported O-linked glycan on prokaryotes are those found in the extracellular cellulase complexes of Clostridium thermocellum and Bacteriodes cellulosolvens(4) . These glycans are linked mainly to threonine via a galactosyl group, and the attachment sites appear to be restricted to threonine/proline enriched areas somewhat reminiscent of O-linked carbohydrates of eukaryotic systems. In contrast, the Flavobacterium oligosaccharide has a completely different glycan structure that is attached randomly at very specific consensus sites. The oligosaccharide moiety may be involved in protein folding/stability as has been demonstrated in mammalian systems. We have successfully used the pMAL amplification system for overproduction of PNGase F() and glycosylasparaginase (20) in E. coli. Neither of these proteins is glycosylated by F. meningosepticum. In contrast, Endo F which is heavily O-glycosylated, is unable to be amplified in E. coli with the pMAL system despite being properly engineered into the vector. Endo F appears to be unstable without its normal carbohydrate complement in this system and is recovered in trace amounts.

We anticipate the production of antibody probes to ascertain whether this oligosaccharide and its linkage sequence is unique to Flavobacterium, or importantly, is an unreported but more common glycoform in other secreted bacterial glycoproteins.

  
Table: Consensus sites for glycosylation of F. meningosepticum proteins



FOOTNOTES

*
This work was supported in part by Grant 30471 from the National Institute of General Medical Sciences, United States Public Health Service, DHHS (to T. H. P. and A. L. T.), by the National Institutes of Health Biomedical Resource Center Program, Grant 1-P41-RR05351-05 to the Complex Carbohydrate Research Center, The University of Georgia, and by National Science Foundation Grant BIR-9217648 (to C. R. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 518-474-4187; Fax: 518-473-2900.

The protease previously termed P45 with an apparent molecular weight of about 45,000 on SDS-PAGE has been renamed P40, because mass spectrometry confirmed a molecular weight of 40,086 daltons. P40 has since been identified as a zinc-aspartyl endoprotease (21) that cleaves appropriate substrates on the amino-terminal side of aspartic acid.

The abbreviations used are: PNGase, peptide-N-(N-acetyl--D-glucosaminyl)asparagine amidase; Endo, endo--N-acetylglucosaminidase; HPAEC/PAD, high performance anion-exchange chromatography/pulsed amperometric detection; GC/MC, gas chromatography/mass spectrometry; ESI, electrospray ionization; CAD, collisional activated dissociation.

Conducted in collaboration with Dr. Chudi Guan, New England Biolabs.


ACKNOWLEDGEMENTS

We thank Arthur W. Phelan and William Heffernan for excellent technical assistance and Dr. Li-Ming Changchien for Edman sequence analysis.


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