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J. Biol. Chem., Vol. 278, Issue 34, 32219-32226, August 22, 2003

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The Functional Glycosyltransferase Signature Sequence of the Human 1,3-Glucuronosyltransferase Is a XDD Motif^*

Sandrine Gulberti , Sylvie Fournel-Gigleux  , Guillermo Mulliert ¶, André Aubry ¶, Patrick Netter , Jacques Magdalou  and Mohamed Ouzzine  ||

From the UMR 7561 CNRS-Université Henri Poincaré Nancy 1, Faculté de Médecine, 54505 Vanduvre-lès-Nancy, France and the ^¶UMR 7036 CNRS-Université Henri Poincaré Nancy 1, Faculté des Sciences, 54505 Vanduvre-lès-Nancy, France

Received for publication, August 2, 2002 , and in revised form, June 6, 2003.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The human 1,3-glucuronosyltransferase I (GlcAT-I) is the key enzyme responsible for the completion of glycosaminoglycan-protein linkage tetrasaccharide of proteoglycans (GlcA1,3Gal1,3Gal1,4Xyl1-O-serine). We have investigated the role of aspartate residues Asp¹⁹⁴-Asp¹⁹⁵-Asp¹⁹⁶ corresponding to the glycosyltransferase DXD signature motif, in GlcAT-I function by UDP binding experiments, kinetic analyses, and site-directed mutagenesis. We presented the first evidence that Mn²⁺ is not only essential for GlcAT-I activity but is also required for cosubstrate binding. In agreement, kinetic studies were consistent with a metal-activated enzyme model whereby activation probably occurs via binding of a Mn²⁺·UDP-GlcA complex to the enzyme. Mutational analysis showed that the Asp¹⁹⁴-Asp¹⁹⁵-Asp¹⁹⁶ motif is a major element of the UDP/Mn²⁺ binding site. Furthermore, determination of the individual role of each aspartate showed that substitution of Asp¹⁹⁵ as well as Asp¹⁹⁶ to alanine strongly impaired GlcAT-I activity, whereas Asp¹⁹⁴ replacement produced only a moderate alteration of the enzyme activity. These findings along with molecular modeling and three-dimensional structure comparison of the GlcAT-I catalytic center with that of the Bacillus subtilis glycosyltransferase SpsA provided evidence that the interactions of Asp¹⁹⁵ with the ribose moiety of UDP and of Asp¹⁹⁶ with the metal cation Mn²⁺ were crucial for GlcAT-I function. Altogether, these results indicated that, similarly to the SpsA enzyme, the nucleotide binding site of GlcAT-I contains a XDD motif rather than a DXD motif.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Glycosaminoglycan (GAG)¹ side chains of proteoglycans are ubiquitous components of the extracellular matrix and are widely distributed on the surface of many cell types. They are implicated in numerous biological processes such as regulation of cell growth and proliferation, cytodifferentiation, and tissue morphogenesis (see for review Refs. 1 and 2). The biosynthesis of chondroitin/dermatan sulfate and heparin/heparan sulfate GAG chains is initiated by the formation of a linkage tetrasaccharide GlcA1,3Gal1,3Gal1,4Xyl1-O attached to specific serine residues of core proteins. This typical tetrasaccharide sequence is also found on so-called "part time" proteoglycans (3), such as -thrombomodulin, whose anticoagulant function is regulated by the presence or absence of a chondroitin sulfate chain (4). GAG chains are thereafter built up on this linkage region by the alternate addition of N-acetylhexosamine and glucuronic acid (GlcA) residues (see for review Ref. 5). The assembly process of the GAG-protein attachment region involves the sequential transfer of monosaccharide residues from the corresponding nucleotide sugar starting at the reducing end by xylosyltransferase (6), galactosyltransferase I (7), galactosyltransferase II (8), and glucuronosyltransferase I (GlcAT-I) (9). GlcAT-I is responsible for the completion of the specific GAG-protein attachment tetrasaccharide sequence, thus playing an essential role in the control of further GAG chain elongation. Accordingly, it has been shown that once GlcA is added to the nascent glycopeptidic chain, the tetrasaccharide sequence is efficiently consumed by downstream glycosyltransferases (GTs), suggesting that GlcAT-I may catalyze a rate-limiting step of GAG chain synthesis (10, 11). Like other GTs, GlcAT-I is a type II membrane Golgi-resident protein, with a short N-terminal cytosolic tail, a transmembrane helix, a stem region, and a C-terminal catalytic domain. The enzyme catalyzes the transfer of a GlcA moiety, from UDP--D-glucuronic acid (UDP-GlcA), onto the 3-hydroxyl group of the terminal galactosyl residue, leading to the formation of a 1,3-linkage. On the basis of studies on several nucleotide-sugar transferases that share a similar inverting mechanism, the reaction is believed to be the S_N2 type, whereby a general base would be required for the nucleophilic attack of the acceptor substrate and is often Mn²⁺ ion-dependent.

Sequence alignments of members of the 1,3-glucuronosyl-transferase family have revealed a sequence of three aspartate residues corresponding to the DXD (aspartate-any residue-aspartate) signature motif characteristic for many GTs and other enzymes that use nucleotidic substrates and require a divalent metal ion for activity. In GlcAT-I, this motif corresponds to residues Asp¹⁹⁴, Asp¹⁹⁵, and Asp¹⁹⁶, which lie in a loop located at the junction between the donor and acceptor substrate binding subdomains of the enzyme (12). Several lines of evidence indicate that the highly conserved DXD motif of GTs has a key role in metal-mediated donor substrate binding and phosphate-sugar bond cleavage. However, recent studies support the idea that these motifs are not equivalent in nucleotide binding and catalysis. For example, the DXD motif in GM2 synthase was required for enzymatic activity but was not found critical for UDP binding (13), whereas the presence of an intact DXD sequence was a prerequisite for both donor substrate binding and activity of clostridial cytotoxins (14). In the case of the Bacillus subtilis SpsA protein, a XDD motif instead of the DXD motif has been shown to play a major role in metal-UDP-sugar complex interactions (15).

To gain further insight into the role of GlcAT-I in GAG chain assembly, it is crucial to better understand the structure and function of the protein. Our laboratory has been deeply involved in determining the peptide domains and amino acids that are important for catalysis and membrane organization of UDP-glucuronosyltransferases (16, 17), in particular the human GlcAT-I (18, 19). We have shown that GlcAT-I is organized as a homodimer involving an intermolecular disulfide bond mediated by a cysteine residue located in the stem region (18). Furthermore, we recently identified essential amino acids governing the donor substrate specificity of GlcAT-I (19). In the current study, using site-directed mutagenesis combined with UDP-beads affinity binding experiments and kinetic analyses, we demonstrate that the human GlcAT-I Asp¹⁹⁴-Asp¹⁹⁵-Asp¹⁹⁶ sequence is a major determinant of the UDP-GlcA and metal complex binding site. Furthermore, we showed by mutant expression together with molecular modeling and three-dimensional structure comparison that the two last aspartate residues of this motif, Asp¹⁹⁵ and Asp¹⁹⁶, are crucial for GlcAT-I function and display distinct roles in cosubstrate and divalent metal ion interactions.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—Gal1-S-3Gal (Gal-Gal), UDP-GlcA, UDP, GlcA, D-saccharic acid 1,4-lactone (saccharonolactone), -glucuronidase (bovine liver), Triton X-100, anti-rabbit alkaline phosphatase-conjugated immunoglobulins, and methanol were from Sigma. UDP beads (affinity gel-UDP) were provided by CN Biosciences (Nottingham, United Kingdom). Bacterial and yeast culture media were from Difco BD Biosciences (Le Pont le Chaix, France). Protein assay reagent was obtained from Bio-Rad. The restriction enzymes were provided by New England Biolabs (Hitchin, UK). T4 DNA ligase was from Invitrogen (Cergy Pontoise, France). The Pichia pastoris expression system and competent Escherichia coli cells were purchased from Invitrogen (Groningen, The Netherlands). The QuikChange site-directed mutagenesis kit was from Stratagene (La Jolla, CA). All other reagents were of the best quality available.

Plasmid Construction and Site-directed Mutagenesis—Cloning of the full-length GlcAT-I cDNA and construction of the recombinant pPICZBGlcAT-I vector designed for the heterologous expression of the recombinant enzyme in the methyltrophic yeast P. pastoris was performed as previously described (18). Construction of amino acid-substituted mutants of GlcAT-I was performed using the QuikChange site-directed mutagenesis kit (Stratagene) according to the recommendations of the manufacturer. Mutants were systematically checked by sequencing. The sequence of the sense and antisense mutation primers is indicated in Table I. The various mutants were individually expressed in the yeast P. pastoris as described below.

Heterologous Expression in the Yeast P. pastoris—Each recombinant pPICZ vector was individually transformed into the P. pastoris SMD1168 yeast strain (Invitrogen) using the P. pastoris Easy Comp transformation kit (Invitrogen). Transformants were selected on YPD plates (1% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) dextrose) containing 100 µg/ml of zeocin (Invitrogen). The cells were grown in BMGY medium (1% (w/v) yeast extract, 2% (w/v) peptone, 100 mM potassium phosphate, pH 6.0, 1.34% (w/v) yeast nitrogen base and 1% (v/v) glycerol). Expression was induced by methanol (2%, v/v) for 48 h at 30 °C in a rotary shaker (215 rpm). Yeast cells were harvested by centrifugation at 3,000 x g for 10 min, broken with glass beads, and further submitted to differential centrifugations as previously described (17). The 100,000 x g pellet corresponding to the membrane fraction was resuspended by Dounce homogenization in sucrose-HEPES buffer (0.25 M sucrose, 5 mM HEPES, pH 7.4). Protein concentration was evaluated by the method of Bradford (20) before analysis by SDS-PAGE under reducing conditions (21). Immunoblot analysis was performed using a polyclonal antipeptide antibody and alkaline phosphatase-conjugated anti-rabbit immunoglobulins as secondary antibodies, as previously described (18).

Analysis of GlcAT-I Enzyme Activity—Activity of recombinant human GlcAT-I was evaluated using Gal-Gal as acceptor substrate as previously described (18). Briefly, standard incubations were performed in 100 mM acetate buffer (pH 5.0) with 10 mM MnCl₂, 25–50 µg of membrane protein, 1 mM Gal-Gal, 5 mM saccharonolactone, and 1 mM UDP-GlcA, in a total volume of 100 µl. For the evaluation of the metal ion dependence of GlcAT-I, Mn²⁺ was replaced by various divalent cations (Mg²⁺, Ca²⁺, Co²⁺, Zn²⁺, and Ba²⁺) at different concentrations (0.1, 1, 10, and 50 mM). The mixture was incubated at 37 °C for 60 min and the reaction product was analyzed by high performance liquid chromatography after chromophore labeling by reductive amination by aniline as previously described (22). The labeled water-soluble product was analyzed on a reverse phase C18 column (4.6 x 150 mm, 4 µm, Waters, Milford, MA) at a detection wavelength of 254 nm. The mobile phase was composed of 5% (v/v) acetonitrile and 0.05% (v/v) 1,4-diaminobutane in water adjusted to pH 6.0. Control assays in which either the donor substrate UDP-GlcA or the acceptor substrate Gal-Gal was omitted were simultaneously run under the same conditions. Addition of a GlcA residue on the nonreducing end of Gal-Gal was verified by the susceptibility of the product to hydrolysis by -glucuronidase from bovine liver. A time course of the glucuronosyltransferase reaction showed that the product formation was linear with time over 90 min. Initial rate data were subsequently taken after a 60-min incubation time.

Kinetic Studies of Mn²⁺ Activation—The effects of varying metal and UDP-GlcA concentrations on the initial reaction velocity were determined in the standard assay conditions, i.e. at a saturating concentration of acceptor substrate (Gal-Gal, 1 mM) with an incubation time of 60 min. Activation of wild-type and mutant GlcAT-I by Mn²⁺ was measured at 0, 0.05, 0.1, 0.2, 0.5, and 1 mM MnCl₂, whereas UDP-GlcA concentration varied from 0 to 2.0 mM. To provide further insight into the mechanism underlying Mn²⁺ activation, data were simultaneously fitted to Equation 1, Equation 2, or Equation 3 according to Segel (23) using SigmaPlot 2000 software (SPSS Science, Chicago, IL). Equation 1 corresponds to a general model for metal ion activation in that Mn²⁺ is essential for catalytic activity as well as for formation of the activator-substrate complex, but not for ligand binding to the catalytic site. In this case, Mn²⁺ (A), UDP-GlcA (S), and the Mn²⁺·UDP-GlcA complex (SA) bind to free enzyme (E) and also to the activator-enzyme (EA) complex. Only the activator-enzyme-substrate-activator AE(SA) complex is catalytically active. According to Equation 2, only the Mn²⁺·UDP-GlcA complex (SA) binds to the enzyme. Equation 3 states that Mn²⁺ binds to the enzyme before UDP-GlcA (23).

View larger version (11K): [in this window] [in a new window]   (Eq. 1)

View larger version (10K): [in this window] [in a new window]   (Eq. 2)

View larger version (12K): [in this window] [in a new window]   (Eq. 3)

In the three equations, [A]_t and [S]_t represent the total concentration of Mn²⁺ and UDP-GlcA, respectively, and V_max is the apparent maximal velocity. In Equation 1, K₀ is the equilibrium constant of the metal-UDP-GlcA complex, K_A is the apparent dissociation constant of the Mn²⁺-enzyme complex, K_S is the apparent dissociation constant of the enzyme·Mn²⁺·UDP-GlcA into enzyme and UDP-GlcA, K_EA is the apparent dissociation constant of the enzyme-Mn²⁺ complex, K_SA is the apparent dissociation constant of the enzyme·Mn²⁺·UDP-GlcA complex, is the apparent dissociation constant of the Mn²⁺·enzyme·UDP-GlcA complex into the Mn²⁺·enzyme complex and UDP-GlcA, is the apparent dissociation constant of the Mn²⁺·enzyme·Mn²⁺ complex into the Mn²⁺·enzyme complex and Mn²⁺, and K_SA' is the apparent dissociation constant of the Mn²⁺·enzyme·Mn²⁺·UDP-GlcA complex into the Mn²⁺·enzyme and Mn²⁺·UDP-GlcA complexes. In Equation 2, K₀ is the equilibrium constant of the metal-UDP-GlcA complex and K_SA is the apparent dissociation constant of the enzyme·Mn²⁺·UDP-GlcA complex. In Equation 3, K_A and K_S are the dissociation constants of the enzyme·Mn²⁺ complex and of the enzyme·Mn²⁺·UDP-GlcA complex, respectively.

UDP Beads Binding—Membrane fractions from yeast cells expressing recombinant wild-type and mutant GlcAT-I (100 µg of protein) were treated by 1% Triton X-100 (v/v) for 1 h at 4 °C and submitted to 100,000 x g ultracentrifugation. The supernatants containing solubilized GlcAT-I and mutants were used for UDP binding experiments according to a procedure similar to that published previously (24). Briefly, a 10-µl aliquot of UDP beads (10 µmol of UDP/ml of gel) was incubated at 4 °C for 30 min with the Triton X-100-solubilized material in 100 mM acetate buffer (pH 5.0) containing 0–10 mM Mn²⁺. Incubations were also performed in the presence of UDP, UDP-GlcA, and GlcA used as potential inhibitors of GlcAT-I binding to UDP beads. Following incubation, the beads were harvested by centrifugation, the supernatant was saved and the beads were washed once in acetate buffer solution and then boiled in Laemmli sample buffer. Both the supernatant and the bound material associated with the beads were then analyzed by SDS-PAGE and immunoblotting as described above.

Molecular Modeling—All minimization operations were performed with Amber software version 6 (25), using the GlcAT-I donor substrate complex, Protein Data Band entry 1kws [PDB] . In all cases, the structure of the donor substrate was deleted. To avoid the missing residue regions in this file, all minimizations were done keeping fixed all residues outside a sphere of 10-Å radius from the carbon of residue 195 in the A chain. After addition of hydrogen atoms, 10 different models were minimized: wild-type GlcAT-I and D194A, D195A, D196A, D194E, D195E, D196E, D194A/D196A, D194A/D195A, and D195A/D196A mutants. Energy minimization was carried out for each model using the Sander classic program by means of 100 steps of steepest descent followed by 1000 steps of conjugate gradient relaxation. Superposition of the Protein Data Bank files 1kws [PDB] (GlcAT-I) and 1h7l [PDB] (SpsA) was done using the program topp of the CCP4 Suite (26).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
GlcAT-I Is Preferentially Activated by Manganese Divalent Ions—Coordination with metal divalent ions is a commonly proposed mechanism accounting for the binding of the UDP-sugar cosubstrate by DXD motif-containing GTs. To analyze the specificity of GlcAT-I toward the metal cofactor, we examined the effect of various divalent cations with different sizes and van der Waals volumes (Mg²⁺, Ca²⁺, Mn²⁺, Co²⁺, Zn²⁺, and Ba²⁺) on the enzyme activity. In the absence of metal ions (Fig. 1) or when the cations were chelated by EDTA (data not shown), the recombinant wild-type enzyme was inactive but was strongly activated in the presence of Mn²⁺ (0.1–10 mM), up to a specific activity of 70 pmol·min^–1·mg^–1 protein. Other divalent cations tested were able to stimulate GlcAT-I at various levels only when used at a high concentration (10 mM). Mg²⁺, Zn²⁺, and to a lesser extent Co²⁺, produced a significant activation of GlcAT-I, whereas Ca²⁺ and Ba²⁺ were not able to assist catalysis (Fig. 1). Activation levels produced by Mg²⁺ and Zn²⁺ was about 50% to that produced by Mn²⁺ and 25% in the case of Co²⁺ (Fig. 1). It is noteworthy that no further activation or inhibition of the enzyme was observed when the concentration of Mn²⁺, Mg²⁺, Zn²⁺, or Co²⁺ was increased up to 50 mM (data not shown). Altogether, these results clearly indicated that the metal ion binding site of GlcAT-I can accommodate several divalent metal ions but that Mn²⁺ is the best metal activator of the enzyme.

View larger version (17K): [in this window] [in a new window]   FIG. 1. Activation of recombinant wild-type GlcAT-I by divalent cations. The activity of wild-type GlcAT-I was measured in membrane fractions of recombinant yeast cells in the absence (0 mM) or presence of various divalent ions at 0.1, 1, and 10 mM concentrations, as described under "Experimental Procedures." Values are the mean ± S.E. of three assays.

Kinetic Studies of Manganese Activation of GlcAT-I—To analyze the mechanism underlying Mn²⁺ stimulation of the Glc-AT-I activity, detailed kinetic studies of Mn²⁺ activation were performed. Initial rates of reaction were measured using varying concentrations of UDP-GlcA (0–1 mM) in the presence of different concentrations of Mn²⁺ (0–1 mM) (Fig. 2). Analysis of the results clearly showed that GlcAT-I requires the metal for activity and that the velocity of the enzyme was remarkably stimulated by Mn²⁺ in a range of concentrations up to 1 mM (Fig. 2). In addition, the plots indicated that V_max was approached at the Mn²⁺ concentration from 0.5 to 1 mM. Furthermore, no inhibitory effect at higher Mn²⁺ concentration (up to 50 mM) was observed (results not shown). Visual analysis of the kinetic data treated as Lineweaver-Burk plots (Fig. 2, inset) showed that all the lines intersected in the second quadrant suggesting that the substrate binds weakly to the enzyme in the absence of Mn²⁺. Mathematical treatment of the kinetics was conducted by fitting the data to Equations 1, 2, 3. Fitting the data to Equations 1 and 3 resulted in parameters with standard error values higher than the mean value, except for V_max (data not shown). This poor fit suggested that these models may not account for the activation mechanism of GlcAT-I by Mn²⁺ divalent ions. In contrast, the data were best-fit to Equation 2 (Table II) favoring the model corresponding to a metal-activated enzyme via complex formation between the metal activator and the substrate.

View larger version (22K): [in this window] [in a new window]   FIG. 2. Kinetics of Mn2+ activation of wild-type GlcAT-I. Initial velocity versus total substrate concentration [UDP-GlcA]t = [UDPGlcA] + [Mn2+·UDP-GlcA] was evaluated at the following concentrations of MnCl2: 0 (), 0.05 (x), 0.1 (•), 0.2 (), 0.5 (), and 1 mM (). The experimental data (mean value ± S.E. of three assays) were fitted by a nonlinear curve-fitting program, according to Equation 2. Line-weaver-Burk representation is shown in inset.

View larger version (18K): [in this window] [in a new window]   FIG. 4. Kinetics of Mn2+ activation of GlcAT-I mutants D194A and D194E. Initial velocity versus total substrate concentration [UDP-GlcA]t = [UDP-GlcA] + [Mn2+·UDP-GlcA] was evaluated at the following concentrations of MnCl2: 0 (), 0.1 (•), 0.2 (), 0.5 (), and 1 mM (). The experimental data (mean value ± S.E. of three assays) were fitted by a nonlinear curve-fitting program, according to Equation 2. Panel A is D194A and panel B is D194E. Line-weaver-Burk representation is shown in inset.

View larger version (22K): [in this window] [in a new window]   FIG. 3. Expression and activity of wild-type GlcAT-I and DDD mutants. A, SDS-PAGE and immunoblot analysis. Membrane fraction (10 µg of protein) of nonrecombinant (C), recombinant yeast cells expressing wild-type (WT), and GlcAT-I mutants was loaded on each lane and probed with anti-GlcAT-I antibodies. B, activity of wild-type GlcAT-I and DDD mutants. The activity of wild-type and mutant enzymes was evaluated in the presence of Gal-Gal (1 mM) and UDP-GlcA (1 mM) at 1 mM concentration of Mn2+ as described under "Experimental Procedures." Values are the mean of two experiments in duplicates.

Effect of DDD Mutations on Binding of GlcAT-I to UDP Beads—As a test for the role of the DDD motif in nucleotide sugar binding, we compared the ability of the wild-type and GlcAT-I mutants to bind to UDP beads. As expected, the wild-type enzyme bound efficiently to UDP beads in the presence of Mn²⁺ (Fig. 5A, lanes a and b). In contrast, no binding of the protein was observed in the absence of the cation (lanes c and d), underlining the importance of Mn²⁺ in this process. Furthermore, the binding was prevented by the presence of UDPGlcA (lanes e and f) and of UDP (lanes g and h), but not of GlcA (lanes i and j), indicating that binding of GlcAT-I to UDP beads was the result of a specific recognition of the nucleotide moiety by the enzyme. Analysis of the binding capacity of mutants D194A, D195A, and D196A to UDP beads revealed that Asp to Ala replacement strongly impaired UDP binding. As illustrated in Fig. 5B, for all mutants, about 50% of the protein did not bind to UDP beads and was present in the beads supernatant after centrifugation (compare lanes b, d, and f to a, c, and e). Mutation of both Asp¹⁹⁴ and Asp¹⁹⁶ residues or Asp¹⁹⁴ and Asp¹⁹⁵ or Asp¹⁹⁵ and Asp¹⁹⁶ of the DDD motif abolished the binding of GlcAT-I to UDP beads (lanes g, i, and k), resulting in the presence of the protein in the beads supernatant (lanes h, j, and l). Lowering Mn²⁺ concentration from 10 to 1 mM did not modify the binding of the wild-type or of the mutant proteins to the beads (data not shown). All these data support the idea that the three aspartate residues of the DDD motif are important for efficient metal-dependent binding of the UDP moiety of the donor substrate to the GlcAT-I protein. However, one single mutation in the motif did not completely abrogate UDP binding.

View larger version (23K): [in this window] [in a new window]   FIG. 5. Binding of GlcAT-I and DDD mutants to UDP beads. UDP beads were incubated with Triton X-100-solubilized membrane proteins of yeast cells expressing wild-type and GlcAT-I DDD mutants. A, immunoblot analysis of the binding of wild-type GlcAT-I to UDP beads in the absence of Mn2+ (lanes c and d) or in presence of 10 mM Mn2+ (other lanes). Bound proteins to UDP beads (lanes a, c, e, g, and i) were separated from unbound proteins (lanes b, d, f, h, and j) by centrifugation as described under "Experimental Procedures." UDPGlcA (lanes e and f), UDP (lanes g and h), and GlcA (lanes i and j) were added at 10 mM concentration to the incubation as potential inhibitors of GlcAT-I binding to UDP beads. B, immunoblot analysis of the binding of DDD-alanine mutants to UDP beads in the presence of 10 mM Mn2+. Bound proteins (lanes a, c, e, g, i, and k) were separated from unbound proteins (lanes b, d, f, h, j, and l) by centrifugation of UDP beads as described under "Experimental Procedures."

Molecular Modeling of the DDD Motif—The three-dimensional structural analysis of human GlcAT-I showed that the DDD motif is located in a buried loop of seven residues and interacts through hydrogen bonding with the donor substrate and with the Mn²⁺ ion (27). To further investigate the critical feature of this motif in UDP-sugar interaction, the catalytic center of the enzyme has been studied by molecular modeling (Fig. 6A). This model provides evidence that beside their interactions with UDP-GlcA and the divalent metal ion, the aspartate side chains could also form hydrogen bonds with other amino acid partners of the protein, i.e. Asp¹⁹⁴ with Arg¹⁶¹ and Asn¹⁹⁷, Asp¹⁹⁵ with Arg⁸⁶ and Thr⁸¹, and Asp¹⁹⁶ with Arg³¹⁰. In silico mutations of the DDD motif have been performed and modeling of the aspartate to alanine mutants indicated that none of the D194A, D195A, and D196A single mutations or D194A/D195A, D195A/D196A, or D194A/D196A double mutations induced a major structural modification of the UDP-GlcA binding site. On the other hand, our experimental findings showed that in the case of Asp¹⁹⁵ and Asp¹⁹⁶ and to lesser extent Asp¹⁹⁴, introduction of a lipophilic methyl group by substitution of aspartate by alanine impaired the enzyme activity. Further investigations of the role of DDD residues by three-dimensional structure comparison revealed that Asp⁹⁸ and Asp⁹⁹ residues of the XDD motif of SpsAGT superimpose with Asp¹⁹⁵ and Asp¹⁹⁶ of GlcAT-I (Fig. 6B). This observation suggested that Asp¹⁹⁵ and Asp¹⁹⁶ residues interact with the ribose moiety of UDP-GlcA and with the metal ion as determined for Asp⁹⁸ and Asp⁹⁹ of the SpsA protein. In contrast, Asp¹⁹⁴ exhibited a similar position to the SpsA threonine residue Thr⁹⁷, which probably does not interact with the cosubstrate. Consistently, our data emphasized the critical importance of the structurally conserved Asp¹⁹⁵ and Asp¹⁹⁶ residues in the function of GlcAT-I.

View larger version (31K): [in this window] [in a new window]   FIG. 6. Representation of the UDP-GlcA binding site of GlcAT-I. A, schematic representation of the UDP-GlcA binding site of wild-type GlcAT-I. Monomer A is in dark blue, monomer B in green, the Asp194-Asp195-Asp196 motif is in ball-and-stick, UDP-GlcA and Mn2+ ion are in turquoise. B, superposition of the Asp194-Asp195-Asp196 motif of GlcAT-I and the XDD (Thr97-Asp98-Asp99) motif of the SpsA protein. The UDP-GlcA binding site of GlcAT-I is in bold stick and the UDP-Glc binding site of SpsA is in ball-and-stick. UDP-GlcA is in green and dTDP moiety of the UDP-Glc is in pink. Mn2+ ion (GlcAT-I) is in dark green and Mg2+ ion (SpsA) in magenta. Figures were created using MOLSCRIPT (36).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
GlcAT-I belongs to the wide group of GTs responsible for the biosynthesis of a large variety of complex oligosaccharides. The elucidation of the structure and function of these enzymes is a major challenge because of their increasing implication in several pathologies and their potential as pharmacological targets. For example, inhibition of heparan sulfate synthesis by a xylose-based molecule has recently been shown to be capable of reducing tumor growth and proposed as a novel anti-cancer therapy (28). GTs are usually metal-dependent enzymes containing a DXD motif, a signature common to many members and used as a diagnostic feature (24). An increasing number of studies underlines the important role of this motif in metal-mediated cosubstrate interactions. Here, we showed that the activity of GlcAT-I was strictly dependent on the presence of divalent ions. Activity was undetectable in the absence of metal cations, thus emphasizing the obligatory presence of these cofactors for the function of the enzyme. Among all the cations tested with various sizes and van der Waals volumes, Mn²⁺ exhibited the highest apparent V_max. Compared with Mn²⁺, Mg²⁺ and Zn²⁺ resulted in a 2-fold lower effect, whereas Ca²⁺ and Ba²⁺ ions did not activate the enzyme. This result points out an optimal interaction of Mn²⁺ with the GlcAT-I protein. Structural studies of the SpsA protein in complex with UDP indicated that the two phosphate groups of UDP-sugar were substantially more ordered in the presence of Mn²⁺ than with Mg²⁺, suggesting that manganese is also the optimal metal ion for this enzyme (15). Similarly, several GTs require exogenous divalent cations for their activities and possess specific binding sites for such ions (29, 30). In the case of GlcAT-I, three-dimensional structure analysis suggested that one Mn²⁺ cation per monomer binds to the enzyme (27). Using UDP bead binding experiments, we showed that the presence of Mn²⁺ was required for the binding of UDP to GlcAT-I. Similarly, isothermal titration calorimetry studies performed on 1,3-galactosyl-transferase (31) underlined the requirement of Mn²⁺ in the binding of the donor substrate, UDP-Gal, to the enzyme. Altogether, these data show that divalent ions are essential for GlcAT-I activity as well as for substrate binding, but do not provide information on the activation mechanism. Kinetic analyses combined with site-directed mutagenesis have been successfully used to assess metal effects in enzyme activation (32, 33). We thus initiated kinetic studies of Mn²⁺ activation of wild-type and mutant GlcAT-I. Lineweaver-Burk representation of kinetic data suggested a weak binding of the substrate in the absence of Mn²⁺. Further analysis of the kinetics by a mathematical treatment using rate Equation 1, which describes random binding of the activator, the substrate, and the activator-substrate complex, gave a poor fit. This result is consistent with the absence of inhibition of GlcAT-I by either UDP-GlcA or Mn²⁺ and the presence in the enzyme of probably one Mn²⁺ binding site as suggested by crystallographic data (27). This led us to consider alternative models, model 2 for which the activator-substrate complex is Mn²⁺·UDP-GlcA and model 3 stating that Mn²⁺ binds the enzyme prior to UDPGlcA. Our kinetic analysis favors model 2 whereby activation occurs via binding of a Mn²⁺·UDP-GlcA complex. In a recent report, UDP-glucose-metal complex formation in solution was analyzed by molecular dynamic simulation of UDP-glucose in interaction with Mg²⁺ (34). Consistent with our findings, this study provides evidence for binding between the divalent cation and the donor substrate in solution, leading to a conformation that is energetically favored to bind the nucleotide active site of GTs. Although, our results favor a model where the metal interacts with the nucleotide sugar to form the enzyme substrate, given the complexity of metal enzyme activation mechanism per se, we presume that the GlcAT-1-Mn²⁺ interactions may be more intricate. Thus, direct binding studies of Mn²⁺ to the enzyme by methods such as electron paramagnetic resonance spectroscopy (32) or isothermal titration calorimetry (31) will be a step forward in the understanding of metal activation of GlcAT-I.

Resolution of the crystal structure of the GlcAT-I catalytic domain suggested that the three aspartate residues, Asp¹⁹⁴-Asp¹⁹⁵-Asp¹⁹⁶, are involved in hydrogen bonding with UDP as well as in a direct interaction with Mn²⁺ (12). However, the importance of this motif has not been substantiated experimentally and the relative functional importance of each aspartate has not yet been assigned. As expected, the consensus Asp¹⁹⁴-Asp¹⁹⁵-Asp¹⁹⁶ element was essential for catalysis because the simultaneous conversion of any two of the aspartate residues to alanine destroyed the activity and abolished UDP binding. It is noteworthy that the double mutations did not induce either protein degradation or aggregation, or changes in membrane targeting and homodimerization.² Consequently, the loss of activity induced by the double mutations was probably because of a dramatic change in the interaction with the cosubstrate as demonstrated by UDP bead binding experiments rather than in protein folding. On the other hand, none of the single aspartate mutations resulted in a complete inactivation of the enzyme, although Asp¹⁹⁵ and Asp¹⁹⁶ mutants presented an impaired enzyme activity. A similar observation has been reported by Busch et al. (14) who showed that mutation of the spaced pair of invariant aspartates was required to completely block the glucosyltransferase activity of the clostridial cytotoxins. However, in the case of 1,3-mannosyltransferase (35) or Fringe protein (24), mutation of one of the conserved residues, only, was sufficient to abrogate the activity. In contrast to Asp¹⁹⁵ and Asp¹⁹⁶ mutations, Asp¹⁹⁴ replacement produced only a slight alteration in the enzyme activity but impaired the interactions between GlcAT-I and the metal-UDP-GlcA complex as reflected by an increase in K_SA values for D194A and D194E mutants. One explanation that may account for these observations is that replacement of Asp¹⁹⁴ by alanine prevented on the one hand the hydrogen bond formation between the side chain of the aspartate residue a water molecule and Mn²⁺ cofactor and on the other hand between aspartate residue and GlcA moiety, as suggested from crystallographic data (27). The severely impaired activity produced by mutation of Asp¹⁹⁵ and Asp¹⁹⁶ prompted us to further analyze the effect of these mutations on the structural organization of the active center of the enzyme. Molecular modeling and in silico mutations of Asp¹⁹⁵ and Asp¹⁹⁶ indicated that no major change of the active center organization was induced by these mutations, thus excluding that the loss of activity observed would be because of large structural modification of the active site. The most likely explanation emerges from comparison of the three-dimensional structure of the catalytic center of GlcAT-I with that of SpsA, a GT protein belonging to the same structural superfamily and displaying a similar catalytic machinery (15). Interestingly, Asp¹⁹⁵ and Asp¹⁹⁶ superimpose with Asp⁹⁸ and Asp⁹⁹ of the ⁹⁷XDD⁹⁹ motif of SpsA. Asp⁹⁸ has been proposed to interact with a hydroxyl group of the ribose moiety of UDP and Asp⁹⁹ to coordinate the divalent metal ion. Similar interactions were also suggested for Asp¹⁹⁵ and Asp¹⁹⁶ in GlcAT-I. In contrast, Asp¹⁹⁴ did not superimpose with any carboxylic residue of SpsA, instead it lines with a threonine residue that exhibits a shorter side chain. These observations, in addition to our experimental data, clearly suggest that the structurally conserved Asp¹⁹⁵ and Asp¹⁹⁶ are the major determinants of the Asp¹⁹⁴-Asp¹⁹⁵-Asp¹⁹⁶ motif of GlcAT-I.

In conclusion, we provide the first data demonstrating the crucial role of the Asp¹⁹⁴-Asp¹⁹⁵-Asp¹⁹⁶ motif of human GlcAT-I. We showed by mutational analysis that the last two aspartates of this motif, which are located in a loop at the C-terminal end of the 4 strand of the protein, play a major role in GlcAT-I function, whereas the importance of Asp¹⁹⁴ appears less predominant. From these findings, we can reasonably suggest that GlcAT-I is a XDD motif enzyme, similarly to the SpsA protein, rather than a DXD motif-GT protein.

	FOOTNOTES

 
^* This work was supported in part by grants from Fonds National pour la Science (Action Concertée Incitative "Molécules et Cibles Thérapeutiques" number 0693), Région Lorraine, Communauté Urbaine du Grand Nancy, IFR 111 Bioingénierie, CNRS Program "Protéomique et Génie des Protéines," Ligue Régionale Contre le Cancer, and Contrat de Recherche Clinique CHU Nancy. 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.

To whom correspondence may be addressed: UMR CNRS 7561-Université Henri Poincaré Nancy 1, Faculté de Médecine, BP 184, 54505 Vanduvre-lès-Nancy, France. Tel.: 33-3-83-68-39-72; Fax: 33-3-83-68-39-59; E-mail: sfg{at}medecine.uhp-nancy.fr.

To whom correspondence may be addressed. Tel.: 33-3-83-68-39-72; Fax: 33-3-83-68-39-59; E-mail: ouzzine{at}medecine.uhp-nancy.fr.

¹ The abbreviations used are: GAG, glycosaminoglycan; GT, glycosyltransferase; GlcA, glucuronic acid; GlcAT-I, galactose 1,3-glucuronosyltransferase I; Gal-Gal, galactosyl-1,3-thiogalactose; UDP-GlcA, UDP--D-glucuronic acid.

² S. Gulberti, S. Fournel-Gigleux, G. Mulliert, A. Aubry, P. Netter, J. Magdalou, and M. Ouzzine, unpublished data.

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