Departments of Microbiology and Immunology1 and Medicine2, University of Western Ontario, London, Ontario N6A 5C1, Canada
Author for correspondence: Miguel A. Valvano. Tel: +1 519 661 3996. Fax: +1 519 661 3499. e-mail: mvalvano{at}uwo.ca
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ABSTRACT |
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Keywords: undecaprenol, N-acetylglucosamine, O-antigen biosynthesis, membrane protein, phosphodiester bond
Abbreviations: GlcNAc, N-acetylglucosamine; GPT, UDP-GlcNAc:dolichol phosphate GlcNAc-1-phosphate transferase; TMHMM, Transmembrane Hidden Markov Model; Und-P, undecaprenyl phosphate
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INTRODUCTION |
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The biochemical reactions to synthesize the O repeat occur in the cytoplasm/plasma membrane interface. They involve activated nucleotide sugar precursors that are available in the cytoplasm and membrane-associated glycosyltransferases that catalyse the formation of O-glycosidic linkages. The synthesis of the O repeating subunit begins with the transfer of a sugar-1-phosphate residue to undecaprenyl phosphate (Und-P) forming an Und-P-P-linked saccharide intermediate. This intermediate serves as an acceptor for the glycosyltransferase-dependent additions of the subsequent sugars to complete the synthesis of the O repeat. Various studies have shown that in many E. coli O antigens, the initiating enzyme is a tunicamycin-sensitive UDP-GlcNAc:Und-P GlcNAc-1-P transferase (Alexander & Valvano, 1994 ; Klena & Schnaitman, 1993
; Rick & Silver, 1996
). This enzyme also initiates the synthesis of enterobacterial common antigen (Rick & Silver, 1996
) as well as O antigens in Shigella flexneri (Yao & Valvano, 1994
), Klebsiella pneumoniae (Clarke et al., 1995
) and some of the Salmonella enterica serogroups (Rick & Silver, 1996
). Genetic and biochemical evidence supports that wecA (formerly rfe) is the structural gene encoding the UDP-GlcNAc:Und-P GlcNAc-1-P transferase (Rick & Silver, 1996
). The presence of alternating hydrophobic and hydrophilic domains in the amino acid sequence of E. coli WecA suggests that the protein spans the plasma membrane several times. In a previous study, we have shown that WecA is only present in a fraction containing plasma membrane markers, confirming that it is an integral membrane protein (Amer & Valvano, 2000
).
Significant sequence similarity occurs between E. coli WecA and other bacterial polyisoprenyl phosphate:N-acetylhexosamine-1-P transferases such as MraY, WbcO, RgpG and WbpL (Amer & Valvano, 2001 ; Anderson et al., 2000
; Dal Nogare & Lehrman, 1988
; Lehrman, 1994
). This protein family also includes the eukaryotic UDP-GlcNAc:dolichol phosphate GlcNAc-1-P transferases (GPTs), which catalyse the first step in the biosynthesis of the glycan moiety of glycoproteins (Burda & Aebi, 1999
). The hamster GPT has been characterized (Dan & Lehrman, 1997
; Dan et al., 1996
; Datta & Lehrman, 1993
; Zhu & Lehrman, 1990
). A set of conserved sequences found in eukaryotic GPTs and in prokaryotic enzymes suggested a functional conservation of this enzyme family (Dal Nogare & Lehrman, 1988
), although eukaryotic GPTs function with dolichol-P as an acceptor instead of Und-P (Rush et al., 1997
). Limited information is available on structural motifs and specific amino acids of WecA that may be important for substrate recognition and/or catalysis (Anderson et al., 2000
). We have recently identified conserved amino acid residues in a predicted large cytosolic region of WecA, which may be implicated in the recognition of UDP-GlcNAc (Amer & Valvano, 2001
). In this study, we report the identification of two other regions of the E. coli WecA protein containing aspartic acids that are highly conserved within prokaryotic and eukaryotic members of this family and which are also part of predicted cytosolic loops. We provide evidence suggesting that the conserved aspartic acid residues may be required for the catalysis of the phosphodiester bond between Und-P and GlcNAc 1-phosphate, probably via ionic interactions with divalent metal cations.
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METHODS |
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In vitro transferase and binding assays.
pAA26, encoding the wecAFLAG gene, and the plasmids encoding the mutated wecA derivatives were transformed by electroporation (Dower et al., 1988 ) into E. coli strain MV501, which carries a wecA::Tn10 insertion (Alexander & Valvano, 1994
). Membranes (containing enzymes and endogenous Und-P) were isolated from these transformants as described by Osborn et al. (1972)
, following induction with 0·02% arabinose for 3 h. The reaction mixture for the transferase assay contained the membrane fraction (20 µg total protein), 96 pmol radiolabelled UDP-N-acetyl-[14C]glucosamine (Amersham Pharmacia Biotech) in 250 µl buffer (5 mM Tris-acetate, pH 8·5, 0·1 mM EDTA, 1 mM MgCl2). After 30 min incubation at 37 °C, the lipid-associated material was extracted twice with 250 µl 1-butanol. The combined 1-butanol extracts were washed once with 500 µl distilled water and the radioactive counts of the 1-butanol fraction were determined with a Beckman liquid scintillation counter. Radioactive counts were normalized by determining the amount of parental or mutated WecA proteins in the same membrane fractions used for the enzymic assay. Detection and quantification of WecA proteins was carried out by immunoblot analysis with anti-FLAG antibodies (see below), following densitometry. We also ensured that equal amounts of protein were loaded in the gels and small differences in loading were further corrected by reprobing the blots with anti-OmpA antibodies (see below). Enzyme activity was finally expressed as pmol incorporated GlcNAc (mg membrane protein)-1. Under the conditions used, the enzyme activity of membrane extracts containing the parental WecAFLAG was linear between 10 and 40 µg membrane protein. For comparisons between parental and mutated forms of WecA, enzyme activity was also expressed as the percentage of WecAFLAG activity at 1 mM MgCl2 concentration.
Binding assays were performed as described by Dal Nogare & Lehrman (1988) . This assay is based on the ability of the UDP-GlcNAc analogue tunicamycin to bind and inhibit the enzymic activity of the parental WecA (Alexander & Valvano, 1994
; Barr et al., 1989
) following incubation with membranes containing the WecA mutants. Thus, the level of transferase activity in this assay is a function of the residual concentration of tunicamycin. Forty micrograms of membranes from MV501 cells transformed with plasmids encoding the various wecA mutants was mixed with transferase buffer containing 0·225 µg tunicamycin ml-1 in a total volume of 200 µl and incubated for 10 min at room temperature. The presence of residual tunicamycin failing to bind to the mutant proteins was determined by the standard transfer assay after the addition of 20 µg MV501(pAA26) membranes and radiolabelled UDP-N-acetyl-[14C]glucosamine. We determined that under our experimental conditions 0·225 µg tunicamycin ml-1 are sufficient to inhibit the WecAFLAG parental activity of the membranes from MV501(pAA26). Enzyme activity was expressed as a percentage of parental WecA activity assayed under the same conditions in the absence of tunicamycin.
Immunoblot analysis.
Aliquots of membrane preparations were also used for immunoblot analysis. Two micrograms of membranes were mixed with the appropriate amount of loading sample buffer (50 mM Tris/HCl, pH 6·8, 2% SDS, 10% glycerol and 0·1% bromophenol blue) and incubated at 45 °C for 30 min before loading onto a 10% SDS-polyacrylamide gel. We demonstrated in a previous report that these conditions are optimal to detect WecA, since complete denaturation by boiling prevents its detection by immunoblotting (Amer & Valvano, 2000 ). The transfer of protein to nitrocellulose membranes was performed according to standard procedures. Membranes were blocked overnight with 5% skim milk dissolved in TBS (50 mM Tris, 150 mM sodium chloride, pH 7·6), incubated for 2 h with the FLAG M2 mAb at a concentration of 7 µg ml-1 and washed several times with TBS. This was followed by a 2 h incubation period with horseradish-peroxidase-linked sheep anti-mouse IgG (Amersham Pharmacia Biotechnology) at a dilution of 1:3000. Detection by chemiluminescence was performed using the BM Chemiluminescence Blotting Substrate (Roche Diagnostics), as recommended by the manufacturer. Loading was normalized by determining the relative amounts of OmpA in the membrane preparations using OmpA-specific rabbit antibodies. Nitrocellulose membranes previously reacted with FLAG M2 were incubated at 50 °C for 15 min in stripping buffer (Tris/HCl, pH 6·8, 2% SDS, 2% 2-mercaptoethanol). This was followed by several washes with TBS, and overnight blocking and development as described above, except that the anti-OmpA primary antibody was used at a dilution of 1:10000 and the horseradish-peroxidase-linked sheep anti-rabbit IgG secondary antibody (Amersham Pharmacia Biotechnology) was at a dilution of 1:3000.
LPS analysis.
LPS was extracted and analysed by SDS-PAGE followed by silver staining as described by Marolda et al. (1990) . For determination of the incorporation of a terminal GlcNAc into the lipid A-core oligosaccharide, LPS from E. coli CLM20 containing pMF21 (encoding the O antigen translocase Wzx) and the various plasmids encoding parental and mutated WecA constructs was analysed by immunoblotting using digoxigenin-labelled wheat germ agglutinin as described by Feldman et al. (1999)
.
Fractionation of membranes.
Membranes were prepared and analysed by sucrose gradient fractionations as described previously (Amer & Valvano, 2000 , 2001
)
Amino acid sequence alignments.
BLAST version 2 (Altschul et al., 1997 ) was used to search the database of non-redundant sequences with WecA as a query. Amino acid sequence alignments of WecA homologues were performed using CLUSTAL W (Thompson et al., 1994
). Transmembrane helices were predicted using the Dense Sequence Alignment method (Cserzo et al., 1997
) and the Transmembrane Hidden Markov Model (TMHMM) method (Sonnhammer et al., 1998
).
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RESULTS |
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The in vitro binding activity of mutant proteins was also investigated using a tunicamycin competition assay. Membranes containing WecADD90/91GG bound tunicamycin at levels comparable to WecAFLAG (Table 3). The lack of in vivo complementation and the low level of in vitro transferase activity of WecADD90/91GG (Table 3
), together with a normal substrate-binding activity in vitro suggests that these residues may be involved in interactions with the Und-P-P-GlcNAc reaction product. In contrast, WecADD156/159GG and WecAD156G did not bind tunicamycin, while WecAD159G showed reduced binding activity (Table 3
). Since tunicamycin resembles the structure of the transition compound including UDP-GlcNAc and the polyisoprenyl phosphate (Elbein, 1987
), it was hypothesized that these amino acids could be involved in interactions with the substrates Und-P and UDP-GlcNAc. Alternatively, the loss of negative charges caused by the replacement of aspartic acids with glycine could potentially affect the local conformation of the catalytic site of the enzyme, and this change could be reflected in altered binding and transfer activities. But the conservative substitution of these aspartic acid residues with glutamic acid (DD90/91EE, DD156/159EE and D159E mutants) did not correct the defect (Table 3
), suggesting that these aspartic acid residues, especially D156, may indeed be required for catalytic activity.
We utilized an independent assay to verify these results. During wzy-dependent polysaccharide chain biosynthesis, the transfer of the monosaccharide components of the O repeat occurs on the cytosolic side of the plasma membrane. This step is followed by the translocation or flipping of the Und-P-P-saccharide from the cytoplasmic to the periplasmic surface of the plasma membrane, where the saccharide portion is ligated to preformed lipid A-core oligosaccharide. In a previous study, we have shown that translocation of GlcNAc-P-P-Und and ligation of this sugar to the lipid A-core oligosaccharide can be detected by an immunoblot assay using a digoxigenin-labelled wheat germ agglutinin, and that both WecA and Wzx are required for this process (Feldman et al., 1999 ). Thus, detection of a terminal GlcNAc with this assay represents the sum of activities involving the formation of Und-P-P-GlcNAc, its translocation across the plasma membrane and the ligation of GlcNAc to lipid A-core oligosaccharide. We used the wheat-germ-agglutinin-binding assay to examine whether replacement of D90 and D91 interferes with this process. Fig. 5a
, lanes 4 and 5, shows that wheat germ agglutinin did not bind to the lipid A-core oligosaccharide from cells expressing either WecADD90/91GG or WecADD90/91EE. In keeping with these observations, the lipid A-core oligosaccharide of these cells has the same migration pattern as that of cells containing the control pBAD vector (Fig. 5b
, lane 4). In contrast, cells expressing WecAFLAG displayed a positive reaction with wheat germ agglutinin and a slower migration of lipid A-core oligosaccharide in the gel (Figs 5a
and b
, lanes 2, respectively), denoting the addition of a terminal GlcNAc. Cells expressing WecADD156/159GG, WecADD156/159EE and WecAD156G did not have a terminal GlcNAc attached to lipid A-core oligosaccharide, in contrast to cells expressing WecAD159G (Fig. 5a
and b
, lanes 69), supporting the data obtained from the in vitro transferase assay (Table 3
).
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DISCUSSION |
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The D90/91 and D156/159 mutants were not equivalent with respect to their binding activity as measured by a tunicamycin-binding competition assay. WecADD90/91GG displayed a binding activity similar to the parental WecAFLAG, while WecADD156/159GG showed significantly decreased binding (11% of the parental WecAFLAG activity). A mutant WecA with a single substitution of the nearby D159 had an intermediate level of binding activity relative to that of WecAFLAG, but this mutant protein could complement the synthesis of O antigen in vivo, despite a level of transferase activity in vitro reaching 19% of the WecAFLAG activity. In contrast, the transferase activity of DD90/91GG and DD90/91EE mutants was reduced to 30% of the parental WecAFLAG activity. It has been proposed that tunicamycin acts as an inhibitor by resembling a compound similar to UDP-GlcNAc and the isoprenoid phosphate (Elbein, 1987 ). Therefore, it is conceivable that the lack of binding activity in the DD156/159GG mutant identifies part of the enzymes catalytic site. The functional characterization of mutants with substitutions in D90 and D91 suggest that this region may interact with the reaction product, Und-P-P-GlcNAc. This hypothesis is based on the fact that mutants in these residues had a higher level of transferase activity than the D156 and D159 mutants, but they displayed normal binding activity. We propose that this site may be involved in the release of Und-P-P-GlcNAc, but more conclusive evidence using purified protein and reaction products is required to support this model. There is a precedent for a similar function in Salmonella enterica WbaP (formerly RfbP). This protein is a UDP-galactose:Und-P Gal-1-P transferase that mediates the initiation of O antigen biosynthesis in the major Salmonella serogroups (Wang et al., 1996
). WbaP and WecA do not have any significant similarity in their primary amino acid sequence, nor in their predicted topology. But certain mutations causing amino acid substitutions in WbaP result in the lack of O antigen LPS on the bacterial cell surface while the galactosyltransferase in vitro activity is only slightly reduced (Wang et al., 1996
). These investigators suggested that the mutated residues reveal a function involved in the release of the newly formed polyisoprenol-P-P-linked saccharide to the component or components involved in its membrane translocation (Wang et al., 1996
). Our results showing that WecADD90/91GG cannot function in an in vivo translocation assay that measures the ligation of GlcNAc to the lipid A-core oligosaccharide in a Wzx (O antigen translocase)- and WecA-dependent fashion (Feldman et al., 1999
), suggest a similar role for D90 and D91.
Structural information obtained from several different enzymes catalysing the formation of phosphodiester bonds suggests a mechanism of phosphoryl transfer that involves metal ions, such as Mg2+ or Mn2+ (Davies et al., 2000 ; Shemyakin et al., 1978
), which interact with carboxylates within the protein (Allingham et al., 1999
). We have shown in this study that a metal ion, such as Mg2+ or Mn2+, is required for WecA enzyme activity and we also demonstrated that the levels of enzyme activity vary as a function of the concentration of Mg2+. Conceivably, the aspartic acid residues identified as important for WecA catalytic activity, D90, D91 and D156, and to some extent D159, may also function in divalent metal ion binding to promote substrate binding and the formation of the phosphodiester bond between GlcNAc-1-P and Und-P. Our data, showing that the DD90/91EE and DD156/159EE mutants required an increased concentration of Mg2+ for reaching half-maximal activity, support this conclusion since these findings are consistent with the idea that introduction of glutamic acid residues at these positions lowers the affinity of the protein for Mg2+. Similar behaviour has been reported for an aspartic mutant in the catalytic subunit of an E. coli ATP-dependent efflux pump (Zhou & Rosen, 1999
). Furthermore, the proposed model involving metal ion interactions with the aspartic acids is in part supported by the observation that D90 and D156 are components of two short domains, I-G-A-L-D90 and F-N-M-V-D156, respectively, which both possesses the features ascribed to the Walker B-motif (van der Wolk et al., 1995
). This motif is composed of a stretch of hydrophobic (h) amino acids with the sequence h-X-h-h-D (X=any amino acid), from which the terminal aspartic acid was shown to be required for the coordination of the Mg2+ ion in enzymes possessing nucleotide-binding sites (van der Wolk et al., 1995
). Both h-X-h-h-D motifs are highly conserved in the prokaryotic and eukaryotic WecA homologues examined in this study (Fig. 1
).
In conclusion, our experiments have identified functionally important aspartic acid residues in two regions of the WecA protein that are presumably exposed to the cytosol. Our data demonstrate that both of these regions are important for catalytic activity and they may be part of the catalytic site of the enzyme. The formation of a phosphodiester bond between a sugar phosphate and a polyisoprenyl phosphate lipid carrier is a conserved reaction in prokaryotic and eukaryotic cells. Therefore, it is not surprising that the critical aspartic acid residues identified in this study, as well as the associated Walker B-motifs, are highly conserved in eukaryotic and prokaryotic homologues of this protein family, attesting to their essential functional roles. The purification of WecA, currently under way in our laboratory, will permit us to determine more precisely its enzymic properties, including the demonstration of direct metal-ion-binding, a necessary step to gain an increasing understanding of this enzyme.
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ACKNOWLEDGEMENTS |
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Received 3 August 2001;
revised 1 October 2001;
accepted 11 October 2001.