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J. Biol. Chem., Vol. 279, Issue 21, 22558-22570, May 21, 2004

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A Novel Secreted Endoglycosidase from Enterococcus faecalis with Activity on Human Immunoglobulin G and Ribonuclease B^*

Mattias Collin and Vincent A. Fischetti

From the Laboratory of Bacterial Pathogenesis, The Rockefeller University, New York, New York 10021

Received for publication, February 26, 2004

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The human pathogen Enterococcus faecalis can degrade the N-linked glycans of human RNase B to acquire nutrients, but no gene or protein has been associated with this activity. We identified an 88-kDa secreted protein, endoglycosidase (Endo) E, which is most likely responsible for this activity. EndoE, encoded by ndoE, consists of an -domain with a family 18 glycosyl hydrolase motif and a -domain similar to family 20 glycosyl hydrolases. Phylogenetic analysis of EndoE indicates that the -domain is related to human chitobiases, and the -domain is related to bacterial and human hexosaminidases. Recombinant expression of full-length EndoE or EndoE, site-directed mutagenesis of the catalytic residues, mass spectroscopy, and homology modeling shows that EndoE hydrolyzes the glycan on human RNase B, whereas EndoE hydrolyzes the conserved glycan on IgG. Denaturation experiments indicate that the chitinase activity on RNase B is not dependent on the tertiary structure, although it is on IgG. The ndoE gene and secreted EndoE are present in most E. faecalis but not in Enterococcus faecium isolates. Correspondingly, E. faecalis, but not E. faecium, degrades the glycan on RNase B during growth. Thus, we have identified a secreted enzyme from E. faecalis, EndoE, which by two distinct activities hydrolyzes the glycans on RNase B and IgG. Both activities could be important for the molecular pathogenesis and persistence of E. faecalis during human infections.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Enterococcus faecalis is a human commensal bacterium that inhabits the mucosal surfaces primarily in the gastrointestinal tract. Nevertheless, E. faecalis is a major health problem in humans because it can cause a wide variety of diseases by infecting the urinary tract, bloodstream, and endocardium (1). It is one of the leading causes of nosocomial infections and can cause bacteremia with substantial mortality (2). Therapy of infections is severely complicated by the intrinsic low and the high levels of acquired antibiotic resistance. The complete genomic sequence of the vancomycin-resistant E. faecalis isolate, V583, was recently published, demonstrating the importance of genetic mobile elements in development of resistance, and provides an excellent resource for identifying genes with potential roles in virulence and persistence (3).

Little is known about how E. faecalis obtains nutrients and persists in vivo, but it has been shown that it can release high mannose-type N-linked glycan from host glycoproteins that could support growth in vitro (4). This ability is associated with an endo--N-acetylglucosaminidase activity detected in several enterococcal species, including E. faecalis, but not in the closely related Enterococcus faecium (5). No specific gene or protein has been linked to this activity.

Glycosyl hydrolases can be found in all types of organisms, from bacteria to mammals, and are classified by their enzymatic activities and by their primary and structural similarities (6-8). An excellent up-to-date on-line resource describing the known glycosyl hydrolases and their classification can be found at afmb.cnrs-mrs.fr/CAZY/index.html. Glycosyl hydrolases of family 18 (FGH18)¹ include enzymes that hydrolyze GlcNAc polymers with varying specificity. The FGH18 chitinases degrade chitin, a polymer of GlcNAc, whereas other FGH18s, like the endo--N-acetylglucosaminidases, very specifically hydrolyze the chitin core of various asparagine (N)-linked glycans on glycoproteins. These include EndoF_1-3 and EndoH from Flavobacterium meningosepticum that are widely used in glycobiology research to characterize glycans on glycoproteins (9). The catalytic activities of FGH18s are dependent on a glutamic acid residue, serving as a proton donor, and a well defined 8-amino acid motif has been established for this family (10). The family 20 glycosyl hydrolases (FGH20s) consists of hexosaminidases from bacteria to mammals, including human lysosomal hexosaminidases that are important for degradation of glycoconjugates. Hexosaminidases (EC 3.2.1.52 [EC] ) hydrolyze -glycosidically linked N-acetylglucosamine or N-acetylgalactosamine in glycans. FGH20s are also dependent on a Glu residue as proton donor (11, 12), but there are no well defined enzymatic motifs. Rather, inclusion into this family is based on overall primary and structural similarities (8).

Endo--N-acetylglucosaminidases have been identified in a number of Gram-positive pathogens. For instance, a secreted Streptococcus pneumoniae enzyme, belonging to FGH20, has activity on human glycoproteins and is suggested to be involved in virulence (13). Furthermore, Streptococcus oralis can hydrolyze and metabolize glycans from mucins and ₁-acid glycoprotein (14-16). The important human pathogen Streptococcus pyogenes, which is closely related to E. faecalis, has recently been shown to secrete an endo--N-acetylglucosaminidase (FGH18), EndoS, that exclusively hydrolyzes the complex-type biantennary glycan on the heavy () chain of IgG (17). This activity alters the function of opsonizing IgG through impaired binding to Fc receptors and decreased activation of the classical pathway of complement and thereby increases bacterial survival in human blood (18). Proteolytic and glycolytic modulation of the host immune system seems to be a common theme in the pathogenesis of S. pyogenes as well as other bacterial pathogens (19). Therefore, we asked if E. faecalis might have an enzymatic approach similar to the other Gram-positive pathogens to modulate host immune defense and/or release glycan from glycoproteins for nutritional purposes.

In this study, we utilized the amino acid sequence of the endoglycosidase EndoS from S. pyogenes to probe available bacterial genomic sequences. We identified a novel gene, ndoE, and a corresponding secreted protein, EndoE, in E. faecalis that has glycosyl hydrolase activity on both human RNase B and IgG.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Bacteria, Plasmids, and Culture Conditions—Bacteria and plasmids used in this study are described in Table I. Enterococcal strains were cultured in Todd-Hewitt broth (Difco) at 37 °C without aeration. Escherichia coli strains were cultured in Luria-Bertani broth (Sigma) at 37 °C with aeration. 1.5% (w/v) agar (Difco) was used to solidify media when needed, and 50 µg/ml carbenicillin (Sigma) was added to E. coli cultures harboring pGEX-5X-3 constructs. For analysis of RNase B hydrolysis during growth, enterococci were grown at 37 °C for 24 h in 0.5x chemically defined medium originally developed for S. pyogenes (20) with 0.5x PBS and 0.5 mg/ml human RNase B (Sigma). Culture supernatants were analyzed on 15% SDS-PAGE.

Site-directed Mutagenesis—Mutation of Glu-186 and Glu-662 of EndoE into Gln was performed using QuickChange II Site-directed Mutagenesis kit according to the manufacturer's instructions (Stratagene, La Jolla, CA). The mutagenic oligonucleotide primers (mutations underlined) used were 5'-TGG-GTT-AGA-TAT-TGA-CAT-GCA-AAC-TCG-TCC-AAG-TGA-AAA-AG-3' for E186Q and 5'-TTC-AAT-TTTGGT-GGC-GAT-CAG-TAT-GCA-AAT-GAT-GTC-GAC-3' for E662Q in combination with the antisense of the above sequences and the plasmid pGEXndoE. Mutations were verified by sequencing pGEXndoE(E186Q) and pGEXndoE(E186Q). Recombinant EndoE(E186Q) and EndoE (E662Q) were expressed and purified as described above.

SDS-PAGE and Lectin Blot Analysis—For analysis of glycosidase activity, 10 µg of human IgG or RNase B (Sigma) was incubated with 1 µg of purified EndoE, EndoE, and EndoS or 1 unit of N-glycosidase F (PNGaseF) (Sigma) in a total volume of 20 µl of PBS for 16 h at 37 °C. IgG samples were separated on 10% SDS-PAGE and RNase B samples on 15% SDS-PAGE followed by staining with Coomassie Blue. For lectin blot analysis, proteins were blotted to Immobilon-P PVDF membrane (Millipore) according to Towbin et al. (22). Membranes were blocked with Tris-buffered saline with 0.1% Tween 20 (Sigma) and incubated with 0.5 µg/ml biotinylated GNL lectin (Vector Laboratories, Burlingame, CA). After washing in TBST, membranes were incubated with 1 µg/ml peroxidase-labeled streptavidin (Vector Laboratories). After washing, membranes were developed using SuperSignal West Pico Chemiluminescent Substrate (Pierce) and exposed on Blue Bio Film (Denville Scientific, Metuchen, NJ).

For estimation of the rate of hydrolysis for EndoE, 1 nM IgG-Fc or RNase B was incubated with dilutions (125 ng to 8 µg) of EndoE for 20 h at 37 °C. Subsequently, samples were analyzed by 15% SDS-PAGE and spot densitometry using an Alpha Imager 2200 Documentation and Analysis System (Alpha Innotech Corp., San Leandro, CA).

Denaturation of IgG and RNase B—For analysis of EndoE on partially or fully denatured substrate proteins, 10 µg of IgG in 10 µl of PBS was incubated at 37, 50, 60, 70, 80, and 90 °C for 30 min followed by equilibration at 37 °C. Subsequently, 1 µg of EndoE in 10 µl of PBS was added, and samples were incubated at 37 °C for 2 h. Samples were analyzed on 10% SDS-PAGE. RNase B (10 µg) was denatured by incubation in 20 µl of PBS for 2 min at 90 °C in the presence of 100 mM -mercaptoethanol and 0.5% SDS as described by Chu (23). After cooling to 37 °C, 1 µg of EndoE was added, and samples were incubated at 37 °C for 2 h, followed by analysis on 15% SDS-PAGE.

PCR Analysis of Enterococcal Strains—To investigate the presence of the ndoE gene in enterococcal isolates, single colonies from enterococci grown on Todd-Hewitt agar were boiled in 1x PCR buffer followed by centrifugation at 10,000 x g for 5 min. Supernatants (1 µl) were used as template in a standard PCR using AmpliTaq (Applied Biosystems, Foster City, CA) with an annealing temperature of 48 °C and oligonucleotide primers 5'-ATG-AAC-GGA-GTG-CAG-AAA-GGA-3' and 5'-TTT-AAC-CAC-CAT-ATA-CTC-ACG-ATA-3'. These primers generated a 2505-bp product covering the whole ndoE gene from HER1044. Samples were separated on 1% TAE-agarose gel and stained with ethidium bromide.

Generation of Antibodies and Western Blots—For generation of polyclonal mouse antiserum, 200 µg of EndoE was incubated with 50 µl of 2% Alu-Gel-S suspension (Serva, Heidelberg, Germany) for 20 min at room temperature, followed by centrifugation at 10,000 x g for 10 min and resuspension of pellet in 200 µl of sterile PBS. At day 1, 100 µl of the suspension were injected subcutaneously in BALB/C mice (Charles River Laboratories, Wilmington, MA). At day 14 mice were boosted with an additional 100 µl of suspension. At day 24, mice were sacrificed and bled followed by serum preparation using standard protocols. Reactivity against purified EndoE was tested by Western blots (data not shown). For Western blot analysis of EndoE secretion from enterococcal isolates, cultures were centrifuged at 10,000 x g for 10 min followed by precipitation of proteins in supernatants by using a final concentration of 5% trichloroacetic acid and centrifugation at 10,000 x g for 20 min at 4 °C. Samples were separated on 10% SDS-PAGE and electroblotted to PVDF membranes. Membranes were blocked in PBS with 0.1% Tween 20 (PBST) and 5% (w/v) skim milk (PBSTS), followed by incubation with 1:5,000 of EndoE antiserum in PBSTS, washing in PBST, incubation with 1:2,000 of goat anti-rabbit IgG conjugated to horseradish-peroxidase (Bio-Rad), and development using chemiluminescence as described above.

Sequence Alignments, Phylogenetic Analysis, and Homology Modeling—All protein alignments and phylogenetic analyses were performed using the MacVector^TM 7.1.1 sequence analysis software suite (Accelrys Inc., San Diego, CA). Protein sequences were aligned using the ClustalW algorithm. Alignments were used for reconstruction of phylogenetic trees by using the neighbor-joining method, ignoring gaps and with uncorrected p values. The trees were validated using bootstrap analysis (1,000 repetitions), and consensus trees were generated.

A structure model of the FGH18 domain of EndoE was constructed using Geno3D automated homology modeling (geno3d-pbil.ibcp.fr) (24). This method involves a PSI-BLAST (25) search, selection of Protein Data Bank entries as templates, and calculation of agreement of secondary structure (26); distances and dihedral angle restraints are calculated from the alignment and applied onto the query sequence followed by analysis using the CNS software (27). A model of amino acids 119-250 from EndoE was generated by using a chitinase from the fungus Coccidioides immitis (Protein Data Bank 1D2K [PDB] ) (28) as a template. Models were edited, and figures were created by using 3D-Mol (component of Vector NTI, Informax, Frederick, MD).

Mass Spectroscopy—For mass analysis, 500 µg of RNase B or human IgG Fc fragments (Sigma) were incubated with 1 µg of EndoE, 1 µg of EndoS, or 10 units of PNGaseF (Sigma) in PBS for 16 h. Samples were desalted on a HiTrap Desalting column (Amersham Biosciences). IgG-Fc samples were reduced with a final concentration of 20 mM dithiothreitol. Samples were analyzed on reducing and non-reducing SDS-PAGE to verify cleavage and reduction of IgG-Fc. 1 µl of samples was spotted onto a sample plate. 1 µl of saturated solution of -cyano-4-hydroxycinnamic acid (Fisher) in 50% acetonitrile (Burdick and Jackson, Muskegon, MI) and 0.1% trifluoroacetic acid (Sigma) was deposited on top, and the mixture was allowed to air-dry. MALDI-TOF mass spectra were acquired on a PerSeptive Biosystems Voyager-DE RP equipped with a nitrogen laser (337 nm). Mass spectra were obtained in positive ion mode with an acceleration voltage of 25 kV, grid voltage of 88.0%, and delay time of 300 ns and averaged over 256 laser shots. Spectra were calibrated with RNase B or bovine serum albumin.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Identification of a Putative Endoglycosidase in E. faecalis—The amino acid sequence of the IgG-hydrolyzing endoglycosidase EndoS (GenBank^TM accession number AAK00850 [GenBank] from S. pyogenes strain AP1 (17) was used as a probe to search the bacterial genomes by using the comprehensive microbial resource Blast at www.tigr.org. This resulted in one significant hit (p value of 4.0 x 10^-7) in the recently published genome sequence of E. faecalis V583, a vancomycin-resistant isolate harboring the vanB resistance gene (3, 29). The corresponding open reading frame encodes an 835-amino acid protein annotated as a -N-acetylglucosaminidase (GenBank^TM accession number NP_813917 [GenBank] ). Oligonucleotide primers constructed from the corresponding DNA sequence were used to amplify and clone a homolog from the E. faecalis strain HER1044. Sequencing of this gene revealed an open reading frame encoding an 838-amino acid protein that is 99% identical to the predicted -N-acetylglucosaminidase from V583. The sequences of the protein (denoted EndoE for endoglycosidase of Enterococcus), and the corresponding gene ndoE, have been deposited in GenBank^TM under accession number AY376354 [GenBank] . When the EndoE sequence was analyzed using the SignalP web server for predicting signal sequence cleavage sites at www.cbs.dtu.dk/services/SignalP/(30), it was predicted that the first 55 amino acids constitute a signal peptide with a signal peptidase cleavage site. After removal of the signal peptide, the mature EndoE protein has a predicted size of 88 kDa.

EndoE Belongs to Both Family 18 and 20 of Glycosyl Hydrolases by Similarity—To further analyze the primary sequence of EndoE, a conserved domain search was performed at the NCBI website at www.ncbi.nlm.nih.gov/blast/. This revealed that the carboxyl-terminal portion of EndoE contains a conserved N-acetyl--hexosaminidase region belonging to FGH20s according to Henrissat (7). The amino-terminal region contains no putative conserved domains, but when EndoE was analyzed for protein patterns present in the Prosite data base at us.expasy.org/cgi-bin/scanprosite, a perfect FGH18 motif (7) was identified. Moreover, when the EndoE sequence was aligned with that of EndoS (the IgG glycan hydrolase from S. pyogenes used to identify EndoE), it was apparent that the similarity was confined to the amino-terminal regions of both proteins including the conserved FGH18 motif (Fig. 1A). The carboxyl-terminal domain instead is highly similar to another FGH20 endo--N-acetylglucosaminidase from S. pneumoniae (Fig. 1B). This pneumococcal enzyme has activity on human glycoproteins and has a predicted role in virulence (13). Based on these similarities, we proposed a two-domain model for EndoE composed of a 391-amino acid FGH18 -domain and a 392-amino acid FGH20 -domain (Fig. 1C), and we hypothesized that the two domains might provide two distinct glycosyl hydrolase activities on host glycoproteins.

View larger version (41K): [in this window] [in a new window]   FIG. 1. Alignment of EndoE with FGH18 and FGH20 homologs and a map of EndoE. EndoE was aligned with EndoS, an FGH18 from S. pyogenes (A), or an FGH20 hydrolase from S. pneumoniae (B). Asterisks indicate amino acid identities, and dots indicate similarities. The conserved FGH18 motif is shown in boldface letters. C, a schematic map of EndoE from E. faecalis HER1044. Ss indicates the predicted signal sequence. Numbers above the sequence refer to amino acids of the EndoE sequence submitted to GenBankTM (accession number AY376354 [GenBank] ). A putative FGH18 -domain, which contains an FGH18 motif, and a putative FGH20 -domain are indicated.

View larger version (22K): [in this window] [in a new window]   FIG. 2. Phylogenetic analysis of EndoE and FGH18 and FGH20 homologs. The EndoE -domain was aligned with 11 FGH20 homologs from different species (A), and the EndoE -domain was aligned with 11 FGH18s (B). Phylogenetic trees were constructed using the neighbor-joining method, validated by bootstrap analysis (1,000 repetitions), and resulting consensus trees are shown. Numbers above branching points indicate the percentages of resampling trees supporting the consensus tree. Species names are shown with protein accession numbers in parentheses.

The Family 18 -Domain of EndoE Is Responsible for the Glycosidase Activity on Human Ribonuclease B—It has been described previously that E. faecalis produces an endo--N-acetylglucosaminidase activity that hydrolyzes human ribonuclease B (RNase B) (4), although no corresponding gene or protein has been identified. RNase B is a glycoprotein with a single N-glycosylation site predominantly occupied by high mannose-type glycans (37). Therefore, we investigated if recombinantly expressed purified EndoE had activity on RNase B. Both full-length EndoE and the -domain were expressed fused to GST. After affinity purification, and factor Xa removal of the GST fusion partner, homogenous preparations of 88-kDa full-length EndoE and 44-kDa EndoE were obtained (data not shown). These preparations were incubated with purified human RNase B for 16 h followed by analysis on 15% SDS-PAGE. As controls, RNase B was incubated with purified recombinant EndoS from S. pyogenes (17), purified PNGaseF, or buffer alone. PNGaseF from F. meningosepticum is a peptide-N-(N-acetyl--glucosaminyl) asparagine amidase that hydrolyzes at the base of the pentasaccharide core of N-linked glycan leaving no remaining carbohydrates attached to the asparagine (38). This revealed that both EndoE and EndoE have activity on RNase B as indicated by a shift in the apparent size of RNase B from the fully glycosylated 15.9-kDa form to the deglycosylated 14.7-kDa form to the same extent as when incubated with PNGaseF (Fig. 3A). In contrast, the negative control EndoS has no activity on RNase B, because no shift as compared with RNase B incubated with buffer could be observed (Fig. 3A, EndoS and PBS). This indicates that deletion of the -domain of EndoE does not influence this activity, suggesting that the FGH18 -domain is sufficient for hydrolyzing the N-linked high mannose glycans on RNase B. The single high mannose glycan on RNase B occurs in several different forms with varying numbers of mannose residues (5-9) resulting in five RNase B glycoforms (Man₅-Man₉) (Fig. 3B). To determine in detail where in the RNase B glycan structure EndoE hydrolyzes, RNase B incubated with EndoE was analyzed by mass spectroscopy (MALDI-TOF). Analysis of RNase B incubated with buffer alone clearly shows the mass distribution of the five different glycoforms but also a minor peak at the position of completely unglycosylated RNase B (Fig. 3C, RNaseB). In contrast, EndoE hydrolysis shifts the size of RNase B into a single peak of 13,888 Da (Fig. 3C, RNaseB+EndoE) with no apparent intermediates. This is 205 Da larger than the completely unglycosylated form of RNase (Fig. 3C, RNaseA), suggesting that one monosaccharide is still attached to the protein backbone. Even though one should be cautious with quantitative conclusions from this type of mass spectroscopy data, the fact that the smaller glycoforms of RNase B seems to be shifted to a larger extent than the larger ones could reflect a preference of EndoE for the smaller glycoforms of RNase B.

View larger version (14K): [in this window] [in a new window]   FIG. 3. Analysis of glycosyl hydrolase activity on human RNase B. A, purified RNase B was incubated with EndoE and EndoE and separated on 15% SDS-PAGE as indicated. Incubation with EndoS was used as a negative control, PNGaseF as a positive control, and PBS to assess the integrity of the RNase B preparation as indicated. Arrows to the right indicate the position of intact, fully glycosylated RNase B and RNase B without glycans, respectively. B, schematic representation of the high mannose glycan on RNase B. Boxed area indicates the smallest Man5 glycoform, whereas the presence or absence of the terminal man-nose residues outside the box accounts for the larger glycoforms Man6-Man9. C, MALDI-TOF analysis of RNase B incubated with EndoE or intact RNase B and RNase A as controls. Numbers above individual peaks indicate the mass in daltons.

Taken together, these results suggest that the -domain of EndoE is responsible for the previously observed activity of E. faecalis that deglycosylates RNase B (4) and that the high mannose glycan is hydrolyzed in the chitobiose core leaving one N-acetylglucosamine on the protein backbone.

Full-length EndoE but Not EndoE Has Glycosidase Activity on Human IgG—EndoS, an N-acetylglucosaminidase from S. pyogenes, has a specific activity on human IgG; it hydrolyzes the conserved complex-type biantennary N-linked glycan on the -chain reducing its size by 3 kDa (17). Because EndoE was identified by its similarity to EndoS and is similar to human glycosyl hydrolases, we investigated whether EndoE has activity on human IgG. Purified IgG was incubated with EndoE and EndoE followed by analysis on SDS-PAGE and by lectin blot analysis using Galanthus nivalis (GNL) lectin. GNL specifically binds to mannose residues on the N-linked glycan on IgG (Fig. 4A) and has been used previously to analyze endoglycosidase activity on IgG (17). Separation on SDS-PAGE revealed that EndoE activity shifts the IgG -chain 3 kDa, comparable with what can be seen when incubated with EndoS (Fig. 4B, Stain, EndoE and EndoS) (17). In contrast, EndoE did not shift the -chain as compared with IgG incubated with buffer alone. The 44-kDa protein in this lane marked with an asterisk represents EndoE and not hydrolyzed IgG (Fig. 4B, Stain, EndoE and PBS). PNGaseF, which completely removes the glycan on the -chain, lead to a size shift of 4 kDa (Fig. 4B, Stain, PNGaseF). It should be noted that the limited resolution of the SDS-PAGE only gives an indication of glycan hydrolysis that needs to be confirmed by other methods. Therefore, to verify that the size shifts correspond to hydrolysis of the glycan on the -chain, samples were analyzed by lectin blot using GNL. This clearly showed that IgG incubated with EndoE did not react with GNL (Fig. 4B, Blot, EndoE and EndoE). The reduction in signal was comparable with what could be seen in IgG samples incubated with EndoS or PNGaseF (Fig. 4B, Blot, EndoS and PNGaseF). In contrast, IgG incubated with EndoE was not affected in its reactivity with GNL compared with IgG incubated with buffer alone (Fig. 4B, Blot, EndoE and PBS).

View larger version (25K): [in this window] [in a new window]   FIG. 4. Analysis of glycosyl hydrolase activity on human IgG. A, schematic structure of the fully substituted complex biantennary type oligosaccharide attached to asparagine 297 of the IgG -chain. GNL indicates the binding sites for the G. nivalis lectin. EndoE indicates the proposed hydrolysis site for the EndoE FGH20 domain, and EndoS indicates the hydrolysis site for the S. pyogenes FGH18 IgG-glycan hydrolase. B, purified IgG was incubated with EndoE and EndoE and separated on 10% SDS-PAGE and stained, or electroblotted, onto PVDF membranes and analyzed with GNL lectin. Incubations with EndoS and PNGaseF were used as positive controls, and PBS was used to assess the integrity of the IgG preparation as indicated. Asterisks indicate the positions of EndoE, EndoE, and EndoS, respectively. C, IgG-Fc incubated with EndoE, EndoS, PNGaseF or buffer alone, followed by reduction and analysis on 15% non-reducing SDS-PAGE. D, MALDI-TOF analysis of IgG-Fc incubated with EndoE, EndoS, and PNGaseF or intact IgG-Fc as control. Numbers above individual peaks indicate the mass in Dalton.

To analyze the glycan hydrolysis in more detail, human IgG Fc fragments were incubated with EndoE, EndoS, and PNGaseF and analyzed by mass spectroscopy (MALDI-TOF). The rationale for using IgG-Fc is that we could not obtain satisfactory resolution of the mass spectra within the size range of intact IgG. Prior to MALDI-TOF analysis, samples were reduced to separate the -chains held together by disulfide bonds, desalted, and analyzed by non-reducing SDS-PAGE. This revealed that EndoE, EndoS, and PNGaseF shifted the size of IgG-Fc in a manner comparable with what could be seen with intact IgG, although hydrolysis was not complete even after 20 h of incubation (Fig. 4C). MALDI-TOF spectra of samples showed that both EndoE and EndoS shifts IgG-Fc to single peaks of 25,307 and 25,286 Da, respectively, corresponding to a protein backbone with one N-acetylglucosamine and a core fucose (Fig. 4D, IgG-Fc+EndoE, EndoS). In contrast, PNGaseF shifts IgG-Fc to a single peak of 24,936 Da representing a protein backbone with no remaining carbohydrates complying with the described activity for PNGaseF (Fig. 4D, IgGFc+PNGaseF). Taken together, these data indicate that EndoE hydrolyzes the conserved glycan on IgG in the chitobiose core, leaving one N-acetylglucosamine and a core fucose on the protein backbone, and that the -domain is necessary for this activity.

Homology Modeling of the FGH18 Domain of EndoE—The structures of a number of glycosyl hydrolases have been solved, and despite a wide variety of folds, their overall topology can be divided into three main groups where the active site is within a pocket, a cleft, or a tunnel, respectively (6). In an attempt to predict the topology of EndoE by using homology modeling, it was possible to create a model of amino acids 119-250 covering the predicted FGH18 active site using a chitinase from the pathogenic fungus C. immitis (28) as a template. A space-filled model of this part of EndoE represents the putative active site forming a tunnel with the catalytic Glu-186 residue at the orifice (Fig. 5A). Furthermore, when analyzing the model in detail, the Glu-186 residue is held in close proximity to Asp-184 (Fig. 5B). In the chitinase from C. immitis, the corresponding Asp residue is important for catalytic activity by electrostatically stabilizing the reaction transition state (39). All known FGH18s hydrolyze the glycosidic bonds by an overall retention with the nucleophilic catalytic base close to the carbohydrate anomeric carbon with an average distance between the catalytic residues (Glu and Asp) of 5.5 Å (40). In the generated model, the calculated distance between Glu-186 and Asp-184 is 4.1Å (Fig. 5B).

View larger version (52K): [in this window] [in a new window]   FIG. 5. Homology modeling of EndoE A, space-fill homology model of amino acids 119 -250 of EndoE. The putative catalytic Glu-186 residue is shown in yellow. B, close-up of Glu-186 and the surrounding amino acids with calculated distances in Å.

Site-directed Mutagenesis of FGH18 in EndoE—The glutamic acid in the FGH18 motif has been shown to be essential for enzymatic activity (10), and our homology modeling also indicated that Glu-186 is part of the FGH18 active site. Therefore, we investigated if a site-directed mutation in the -do-main of Glu-186 (Fig. 1) into glutamine affected the enzymatic activity of EndoE and on IgG and RNase B. Purified EndoE, EndoE, or EndoE(E186Q) was incubated with RNase B followed by analysis of size shifts on SDS-PAGE. This revealed that EndoE(E186Q) could not hydrolyze the glycans on RNase B, whereas EndoE and EndoE hydrolyzed the glycans to the same extent (Fig. 6A), confirming that the EndoE FGH18 -do-main is responsible for the hydrolysis of RNase B and that Glu-186 is essential for this activity.

View larger version (33K): [in this window] [in a new window]   FIG. 6. Analysis of EndoE(E186Q) activity on RNase B and IgG. A, purified RNase B was incubated with EndoE, EndoE, or EndoE(E186Q) and separated on 15% SDS-PAGE as indicated. Arrows to the right indicate the position of intact, fully glycosylated RNase B and RNase B without glycans, respectively. B, purified IgG was incubated with EndoE, EndoE, or EndoE(E186Q) and separated on 10% SDS-PAGE and stained, or electroblotted onto PVDF membranes and analyzed with GNL lectin. Asterisks indicate the positions of EndoE, EndoE, and EndoE(E186Q), respectively.

Identification of the FGH20 Active Site in Endo by Mutagenesis—Our findings indicated that the FGH20 -domain of EndoE is responsible for the hydrolysis of the IgG glycan. Therefore, we attempted to identify the catalytic FGH20 residue. As discussed previously there is no well defined enzymatic motif for FGH20, but it has been shown by site-directed mutagenesis that a Glu residue is essential for activity of a -N-acetylhexosaminidase from Streptomyces plicatus (12). This is also the case for human -hexosaminidase B (HexB), where an aspartic acid preceding the Glu residue also is important for activity (11). We aligned the 15 amino acids surrounding the Asp and Glu active site residues in hexosaminidases from S. plicatus and HexB with EndoE. This identified the sequence GGDE that is conserved in all three proteins, where Glu represents position 662 of the EndoE sequence (Fig. 7A). Therefore, we hypothesized that Glu-662 is the proton donor essential for the FGH20 activity on IgG. To address this, we mutated Glu-662 in EndoE into Gln and analyzed the activity on RNase B and IgG. IgG or RNase B was incubated with EndoE or EndoE(E622Q) followed by SDS-PAGE analysis and GNL lectin blot of the IgG samples. This revealed that EndoE(E662Q) hydrolyzes the glycans on RNase B to the same extent as full-length EndoE (Fig. 7B), confirming that the EndoE FGH18 -domain is not important for the hydrolysis of RNase B glycans.

View larger version (21K): [in this window] [in a new window]   FIG. 7. Effect of site-directed mutagenesis of Glu-662 to Gln on RNase B and IgG hydrolysis. A, alignment of EndoE with the regions containing the catalytic residues in hexosaminidases from S. plicatus and humans (HexB). Asterisks indicate amino acid identities, and dots indicate similarities. B, purified RNase B was incubated with EndoE, EndoE(E662Q), or PBS and separated on 15% SDS-PAGE as indicated. Arrows to the right indicate the position of intact, fully glycosylated RNase B and RNase B without glycans, respectively. C, purified IgG was incubated with EndoE or EndoE(E662Q) and separated on 10% SDS-PAGE and stained, or electroblotted onto PVDF membranes and analyzed with GNL.

Effect of Denaturation of RNase B and IgG on EndoE Activity—Glycosidases with activity on glycoproteins, such as the commonly used EndoF_1-3, EndoH, and PNGaseF, require or are enhanced when the substrate glycoprotein is denatured (9). Therefore, we investigated whether denaturation of RNase B or IgG had any effects on EndoE activity. IgG was denatured by increasing temperature followed by 2 h of incubation at 37 °C with full-length EndoE and analysis by SDS-PAGE. The SDS-PAGE analysis revealed that the apparent molecular mass of the IgG -chain was shifted, as compared with incubation with buffer alone, when preincubated at 37-70 °C, whereas incubation at 80-90 °C abolished the activity of EndoE (Fig. 8A). This indicates that EndoE, as EndoS, does not have activity on denatured IgG, unlike many other endoglycosidases.

View larger version (51K): [in this window] [in a new window]   FIG. 8. EndoE activity on denatured IgG and RNase B. A, purified IgG was preincubated at temperatures of 37-90 °C as indicated prior to incubation with EndoE (+) or buffer (-) for 2 h at 37 °C followed by separation on 10% SDS-PAGE. B, RNase B was denatured by incubation at 90 °C under reducing condition in the presence of SDS prior to incubation with EndoE for 2 h at 37 °C. Samples were analyzed on 15% SDS-PAGE. + and -below the gel indicate if RNase B was denatured or not and if EndoE was added or not, respectively. Arrows to the right indicate the positions of intact and deglycosylated RNase B.

Distribution of the ndoE Gene and Secretion of EndoE among Enterococcal Isolates—In order to investigate the presence of the ndoE gene in other enterococcal isolates, PCR experiments using oligonucleotide primers based on the sequence of ndoE from HER1044 were performed on chromosomal DNA from several E. faecalis and E. faecium isolates. This revealed that a 2,500-bp product could be amplified in 11/11 of the E. faecalis strains, but no product could be generated in 3/3 E. faecium strains (data not shown). This suggests that the ndoE gene is harbored by most E. faecalis isolates, including vancomycin-resistant strains, but is not present in E. faecium. This is in concordance with previous studies (5) describing that an N-acetylglucosaminidase activity hydrolyzing the glycans on RNase B could be detected in several enterococcal species, including E. faecalis, but not in E. faecium.

To investigate whether EndoE secretion correlates with the presence of the ndoE gene as determined by PCR, Western blot analysis on culture supernatants from all isolates using polyclonal mouse antiserum against recombinantly expressed full-length EndoE was performed. This revealed that all E. faecalis strains secrete an 88-kDa protein that reacts with the antiserum (Fig. 9A). The varying signal strengths between strains could represent production levels of EndoE but could also be explained by different reactivity of the antiserum because of sequence variations. In contrast, no signal could be detected in the E. faecium strains. This indicates that E. faecalis secretes EndoE as a single stable product during growth in vitro. Moreover, the temporal production of EndoE was investigated by Western blot analysis of culture supernatants withdrawn during growth of E. faecalis strain HER1044. As shown in Fig. 9B, EndoE secretion starts in mid-exponential growth phase, continues to accumulate during stationary phase, and is present as a stable 88-kDa protein after 24 h of growth. In summary, these data indicate that all tested E. faecalis isolates harboring the ndoE gene also secrete an EndoE protein during growth, and this protein is not degraded after prolonged stationary phase incubation in vitro.

View larger version (20K): [in this window] [in a new window]   FIG. 9. Analysis of EndoE secretion in enterococcal isolates and analysis of the temporal production of EndoE. A, Western blot analysis using EndoE antiserum on precipitated culture supernatants separated on 10% SDS-PAGE and blotted to PVDF membranes. E. faecalis and E. faecium strains are as indicated above the blot. B, optical density of E. faecalis HER1044 measured during growth in Todd-Hewitt broth. Samples were withdrawn at indicated time points; culture supernatants were precipitated and separated on 10% SDS-PAGE followed by Western blot analysis using EndoE antiserum.

View larger version (25K): [in this window] [in a new window]   FIG. 10. Hydrolysis of RNase B during growth of enterococci. E. faecalis and E. faecium strains were cultured in the presence of human RNase B followed by analysis of culture supernatants on 15% SDS-PAGE. Strains are indicated above the gel, and arrows to the right indicate the positions of intact and deglycosylated RNase B.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Phylogenetic analysis of EndoE indicates that it is more closely related to mammalian FGH18s (chitobiases) than to other bacterial FGH18s (chitinases and N-acetylglucosaminidases) including EndoS from S. pyogenes. The mammalian chitobiases are lysosomal glycosidases involved in the degradation of N-linked glycoproteins (36). Phylogenetic analysis of EndoE suggests that it is closely related to FGH20s (hexosaminidases) from both bacteria and mammals. The hexosaminidases HexA and HexB from humans are lysosomal proteins involved in degradation of glycoproteins, glycolipids, and proteoglycans. These enzymes are crucial for the degradation of gangliosides, essential outer-membrane lipids, and mutations in the corresponding genes leads to Sandhoff or Tay-Sachs disease with accumulation of gangliosides in neuronal lysosomes resulting in severe neurodegeneration (41-43).

Taken together, EndoE contains two distinct domains, which are related to human lysosomal hexosaminidase and chitobiase, respectively, which are important for turnover of glycoproteins and glycolipids, but with distinct functions. Human lysosomal hexosaminidases are primarily active on N-linked glycans of high mannose type with little activity on complex-type N-linked glycans (44), whereas human chitobiase has activity at the reducing end of complex-type N-linked glycans after prior removal of the glycan from the asparagine by a -D-N-acetylglucosamine asparagine hydrolase (45). EndoE represents the first example of a bacterial enzyme combining a hexosaminidase domain and chitobiase domain closely related to human enzymes, which could have disease implications when secreted by the human pathogen E. faecalis.

Because it is known that E. faecalis can release high man-nose glycans from glycoproteins (4), we hypothesized that EndoE might be responsible for this activity. We could show that recombinantly expressed EndoE hydrolyzes the high mannose glycan on human RNase B leaving one N-acetylglucosamine on the protein backbone. Furthermore, we could show that the -domain was sufficient for this activity. This suggests that we have identified the enzyme responsible for the previously reported E. faecalis activity on high mannose N-linked glycans, and site-directed mutagenesis confirmed that the activity is dependent on the FGH18 active site in the -domain. We could also show that all tested E. faecalis strains, but not E. faecium, hydrolyze the RNase B glycan during growth, confirming the results from earlier studies (5). This was further supported by our finding that the ndoE gene and secreted EndoE are present in all E. faecalis isolates and not in E. faecium isolates. We cannot exclude however that there may be other enzymes secreted during growth of E. faecalis that could contribute to glycan hydrolysis. An ndoE deletion mutant would certainly be of interest, because this might further verify the contribution of EndoE to glycan hydrolysis in vitro and also enable in vivo virulence and persistence studies. However, despite several attempts we have not been able to generate such mutants in the E. faecalis strains we are studying for yet unknown reasons. This might be clarified, however, by our ongoing efforts in finding a successful mutagenesis strategy, which would allow us to investigate the properties of EndoE-negative strains in infection models in the future.

Besides the FGH18 activity on human RNase B, full-length EndoE, but not EndoE, has FGH20 activity hydrolyzing the N-linked glycan on IgG. This activity is not dependent on the family 18 active site as indicated by site-directed mutagenesis (E186Q). Instead, we identified Glu-662 in the -domain as being essential for IgG glycan hydrolase activity by using site-directed mutagenesis (E662Q).

Most interesting, EndoE, as well as EndoS, does not require prior hydrolysis by an -D-N-acetylglucosamine asparagine hydrolase that is required for activity of the related human chitobiases (45), in order to hydrolyze the complex-type glycan on the -chain. Furthermore, EndoE does not have activity on denatured IgG in contrast to many other glycan hydrolases that have enhanced activity or require denaturation of the substrate glycoprotein (9). This suggests that the N-linked glycan on denatured IgG is not sufficient for activity but rather that EndoE is dependent on the tertiary structure of glycosylated IgG. This resembles the activity of the S. pyogenes FGH18 IgG glycan hydrolase EndoS (21), but interestingly the EndoE activity on the IgG glycan is mediated by the FGH20 -domain that is not very similar to EndoS. EndoS does not contain any domain similar to FGH20s, indicating that S. pyogenes and E. faecalis use glycosyl hydrolases belonging to different families to achieve the same goal, hydrolysis of the conserved glycan on IgG.

The conserved IgG glycans attached to asparagine 297 are located in the cavity between the two -chains and are thought to stabilize the molecule (46, 47) (Fig. 3). The glycan is important for several IgG functions including binding to Fc receptors, activation of complement, and clearance of immune complexes from circulation (48-51). Furthermore, defects in IgG glycosylation have been associated with several autoimmune disorders such as Crohn's disease, rheumatoid arthritis, and systemic lupus erythematosus (52-54). It was recently shown that hydrolysis of this glycan by the S. pyogenes endoglycosidase EndoS increased the streptococcal survival in blood containing type-specific opsonizing antibodies. The impairment was mainly because of reduced binding of hydrolyzed IgG to Fc receptors and reduced activation of the classical pathway of complement (18). EndoE activity might also have effects on enterococcal survival within the host, especially during septicemia, because antibody titers against this commensal organism seems to be common in the normal population (55). Furthermore, it has been shown that neutrophil-mediated killing of E. faecalis is enhanced by specific antibodies to the organism (56), suggesting that modulation of IgG functionality might be an efficient bacterial strategy to persist in the bloodstream.

It is fascinating that E. faecalis has acquired two enzymatic domains related to human enzymes and incorporated them into a secreted protein for its own purpose, release of N-linked glycans from host glycoproteins for nutritional purposes or possibly also immunomodulation. These activities might be important for the virulence and persistence of E. faecalis during human infections.

	FOOTNOTES

 
^* This work was supported in part by United States Public Health Service Grant AI11822 (to V. A. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by a fellowship from the Wenner-Gren Foundations, Sweden. To whom correspondence may be addressed. Tel.: 212-327-8170; Fax: 212-327-7584; E-mail: collinm{at}mail.rockefeller.edu.

To whom correspondence may be addressed. Tel.: 212-327-8166; Fax: 212-327-7584; E-mail: vaf{at}rockefeller.edu.

¹ The abbreviations used are: FGH18 and -20, family 18 or 20 glycosyl hydrolases; Endo, endoglycosidase; GlcNAc, N-acetylglucosamine; GNL, G. nivalis lectin; Hex, hexosaminidase; PNGaseF, N-glycosidase F; PBS, phosphate-buffered saline; GST, glutathione S-transferase; PVDF, polyvinylidene difluoride; MALDI-TOF, matrix-assisted laser desorption ionization/time of flight.

	ACKNOWLEDGMENTS

 
We thank Dr. H.-W. Ackermann at Laval University, Quebec, Canada, and Dr. A. Tomasz, Rockefeller University, for providing enterococcal isolates; Dr. R. Schuch and Dr. N. Tapinos for helpful suggestions on the manuscript; and Dr. A. Fiser for sharing insights on homology modeling. We also thank Michele Kirshner at The Rockefeller University Protein Resource Center for MALDI-TOF analyses.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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