Arnaud Pailot

Katia D'ambrosio

Catherine Corbier

François Talfournier
email: francois.talfournier@maem.uhp-nancy.fr

Guy Branlant
email: guy.branlant@maem.uhp-nancy.fr

Maem

Invariant T244 is essential for efficient acylation step of the nonphosphorylating Glyceraldehyde-3-phosphate Dehydrogenase from Streptococcus mutans

Keywords: ALDH, T244, acylation step, hydride transfer, conformation of nicotinamide, Xray structure

One of the most striking features of several X-ray structures of CoA-independent Aldehyde Dehydrogenases (ALDH) in complex with NAD(P) is the conformational flexibility of the nicotinamide mononucleotide (NMN) moiety. However, the fact that the rate of the acylation step is high in nonphosphorylating Glyceraldehyde-3-phosphate Dehydrogenase (GAPN) from Streptococcus mutans implies an optimal positioning of the nicotinamide ring relative to the hemithioacetal intermediate within the ternary GAPN complex to allow an efficient and stereospecific hydride transfer. Substitutions of invariant T244 for serine and K178 for alanine, result in a drastic decrease of the efficiency of hydride transfer which becomes ratelimiting. The crystal structure of the binary complex T244S GAPN/NADP shows that the absence of the β-CH 3 group leads to a well defined conformation of the NMN part, including the nicotinamide ring, clearly different from that depicted to be suitable for an efficient hydride transfer in the wild type. The ∼ 0.6-unit increase in pK app of the catalytic C302 observed in the ternary complex for both mutated GAPNs is likely due to a slight difference in positioning of the nicotinamide ring relative to C302 with respect to the wild-type ternary complex. Altogether, the data support a critical role of the T244 β-CH 3 group, held in position through hydrogen bond interaction between the T244 β-OH group and the ε-amino group of K178, in permitting the nicotinamide ring to adopt a conformation suitable for an efficient hydride transfer during the acylation step for all the members of the CoA-independent ALDH family.

INTRODUCTION

Nonphosphorylating aldehyde dehydrogenases (ALDH) belong to a family of phylogenetically and structurally-related enzymes that catalyse the NAD(P)-dependent oxidation of a wide variety of aldehydes to their corresponding non-activated or CoAactivated acids. ALDHs are known to participate in many biological functions such as intermediary metabolism, detoxification, osmotic protection and cellular differentiation. They share a similar chemical mechanism involving two major steps: first, an acylation step common to both families and, second, a deacylation step that differs by the nature of the acyl acceptor. The acylation step involves the formation of a thiohemiacetal intermediate via nucleophilic attack of the catalytic C302 1 on the aldehydic function followed by an oxidoreduction process leading to the formation of a thioacylenzyme intermediate and NAD(P)H. The deacylation step includes the nucleophilic attack of a water or coenzyme A (CoA) molecule on the thioacylenzyme intermediate and release of the non-activated or CoAactivated acids. Moreover, previous studies have highlighted major differences in the kinetic mechanism of ALDHs depending upon the nature of the deacylation step. In CoAindependent ALDHs, kinetic data support an ordered sequential mechanism in which NAD(P)H dissociates last [START_REF] Marchal | Role of glutamate-268 in the catalytic mechanism of nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans[END_REF][START_REF] Vallari | Kinetic mechanism of the human cytoplasmic aldehyde dehydrogenase E1[END_REF], whereas CoA-dependent ALDHs exhibit a ping-pong mechanism in which the release of the reduced cofactor occurs prior to the transthioesterification step [START_REF] Shone | Steady-state and pre-steady-state kinetics of coenzyme A linked aldehyde dehydrogenase from Escherichia coli[END_REF][START_REF] Söhling | Purification and characterization of a coenzyme-Adependent succinate-semialdehyde dehydrogenase from Clostridium kluyveri[END_REF]. Mechanistic aspects were studied extensively for CoA-independent ALDHs, and several invariant residues were shown to be critical for the chemical mechanism [START_REF] Farres | Investigation of the active site cysteine residue of rat liver mitochondrial aldehyde dehydrogenase by site-directed mutagenesis[END_REF][START_REF] Vedadi | Involvement of cysteine 289 in the catalytic activity of an NADP(+)-specific fatty aldehyde dehydrogenase from Vibrio harveyi[END_REF][START_REF] Wang | Involvement of glutamate 268 in the active site of human liver mitochondrial (class 2) aldehyde dehydrogenase as probed by site-directed mutagenesis[END_REF][START_REF] Vedadi | Critical glutamic acid residues affecting the mechanism and nucleotide specificity of Vibrio harveyi aldehyde dehydrogenase[END_REF]. In addition, evidence has been provided for the chemical activation of the catalytic C302 upon cofactor binding to nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) from Streptococcus mutans through a local conformational rearrangement which has been shown to be strongly kinetically favoured by substrate binding to the binary complex GAPN-NADP [START_REF] Marchal | Evidence for the chemical activation of essential Cys-302 upon cofactor binding to nonphosphorylating glyceraldehyde 3-phosphate dehydrogenase from Streptococcus mutans[END_REF]. Moreover, enzymatic and structural studies on GAPN and other ALDHs have highlighted the essential roles of (i) an oxyanion hole composed of the side chain of the invariant N169 residue and the main chain nitrogen of C302 that allows an efficient hydride transfer without base catalyst assistance [START_REF] Steinmetz | Structure of mitochondrial aldehyde dehydrogenase: the genetic component of ethanol aversion[END_REF][START_REF] Johansson | Structure of betaine aldehyde dehydrogenase at 2.1 A resolution[END_REF][START_REF] Cobessi | Structural and biochemical investigations of the catalytic mechanism of an NADP-dependent aldehyde dehydrogenase from Streptococcus mutans[END_REF][START_REF] Marchal | Chemical mechanism and substrate binding sites of NADP-dependent aldehyde dehydrogenase from Streptococcus mutans[END_REF] and (ii) the invariant E268 residue in the ratelimiting hydrolysis step through activation and orientation of the attacking water molecule [START_REF] Marchal | Role of glutamate-268 in the catalytic mechanism of nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans[END_REF].

Additionally, numerous crystal structures of CoA-independent ALDHs have already been solved in the presence of NAD(P). One of the most striking features of many ALDH structures is the conformational flexibility of the nicotinamide mononucleotide (NMN) moiety of the cofactor, which is seen as missing or weak electron density [e.g., see 11, 14-15].

EXPERIMENTAL PROCEDURES

Materials -T244S, T244S/C382S, T244V and K178A GAPNs were produced as described previously [START_REF] Marchal | Evidence for the chemical activation of essential Cys-302 upon cofactor binding to nonphosphorylating glyceraldehyde 3-phosphate dehydrogenase from Streptococcus mutans[END_REF]. For purification, cells were harvested by centrifugation, resuspended in 50 mM potassium phosphate buffer, pH 8.2 (buffer A) and disrupted by sonication. The extract was clarified by centrifugation at 15000 g for 90 min, and solid ammonium sulfate was added to either 40% saturation for T244S and T244S/C382S GAPNs or 30% saturation for T244V and K178A, respectively. The precipitate was removed by centrifugation, and the supernatant was applied to a high performance phenyl-Sepharose column using a FPLC system (Amersham Biosciences) previously equilibrated with buffer A containing 1 M ammonium sulfate.

Following equilibration, mutated GAPNs were eluted using a descending gradient (1 to 0 M) of ammonium sulfate at 5 ml.min -1 . Fractions containing mutated GAPNs were pooled, concentrated and dialyzed exhaustively against buffer A and applied to a Q-Sepharose column equilibrated with buffer A. After washing with buffer A, mutated GAPNs were eluted using a gradient (0 to 2 M KCl) at 5 ml.min -1 . At this stage, mutated GAPNs were considered to be pure as judged by 7.5 % SDS/polyacrylamide gel electrophoresis and by electrospray massspectrometry analysis. T244V and K178A GAPNs were isolated as apo forms, whereas 0.8 mole of NADP remained bound per tetramer of T244S and T244S/C382S GAPNs. Enzyme concentrations were determined spectrophotometrically as the apo form by using a molar extinction coefficient of 2.04x10 5 M -1 .cm -1 at 280 nm [START_REF] Marchal | Evidence for the chemical activation of essential Cys-302 upon cofactor binding to nonphosphorylating glyceraldehyde 3-phosphate dehydrogenase from Streptococcus mutans[END_REF] and expressed either as tetramer (molarity, M) or monomer (normality, N) concentration. All other materials were reagentgrade or better and were used without further purification. NADP (Roche) was dissolved in H 2 O and the stock concentration was determined spectrophotometrically by using a molar extinction coefficient of 18000 M -1 .cm -1 at 259 nm. D,L-G3P or D-G3P (Sigma, St. Louis, MO) was obtained by hydrolysing D,L-G3P diethyl acetal or D-G3P diethyl acetal, respectively, according to the manufacturer and its concentration was enzymatically assessed using the wild-type GAPN.

Quantification of NADP content -NADP content of purified GAPNs was enzymatically quantified after addition of 0.2 mM (D,L)-G3P to 5 µN GAPN by following NADPH appearance on a SAFAS flx spectrofluorometer. Fluorescence measurements were carried out in 50 mM potassium phosphate buffer at pH 8.2. The excitation wavelength was set at 358 nm, and spectra were recorded in the range 380-540 nm. Maximum emission intensity was observed at 460 nm.

Steady-State Kinetic Analyses -Initial rate measurements were carried out on a Kontron

Uvikon 933 spectrophotometer by following the appearance of NADPH at 340 nm. Determination of the kinetic parameters were carried out at the optimum pH of 8.2 in 50 mM TES buffer and 5 mM β-mercaptoethanol. The temperature of the solutions was maintained at 25 °C by thermostated sample holders using a circulating water bath for all measurements.

Catalytic constants k cat and K M were determined by measuring steady-state velocity at various (D,L)-G3P and NADP concentrations under conditions where no inhibitory effect was observed either with NADP or (D,L)-G3P. Data were plotted according to Michaelis-Menten equation using least squares regression analysis.

Deuterium isotope effects -D-[1-2 H]G3P was prepared as described previously [START_REF] Corbier | The nicotinamide subsite of glyceraldehyde-3-phosphate dehydrogenase studied by site-directed mutagenesis[END_REF] and its concentration was determined enzymatically. Isotopic effects on T244S and K178A GAPNs were measured in 50 mM TES buffer, pH 8.2, and 5 mM β-mercaptoethanol by direct comparison of the initial velocities measured with D-G3P and D-[1-2 H]G3P at concentrations of 0.1 mM and 0.2 mM for both mutated GAPNs in the presence of NADP at 25°C.

pH-dependence of the steady-state rate constant of K178A and T244S GAPNs -

The pHdependence of the steady-state rate constant k cat , which was shown to be associated with the rate-limiting acylation step (see results), was studied over the pH-range 5.25-8.75 or 5.25-9.00 in a mixed reaction buffer consisting of 120 mM Tris, 30 mM imidazole and 30 mM acetic acid (pH adjusted with NaOH or HCl), adjusted with 3 M NaCl to a constant ionic strength of 0.15 M. The apparent pK a parameters governing the pH-dependence of k cat were extracted from k obs vs pH profiles and analysed as described by Marchal and Branlant [START_REF] Marchal | Evidence for the chemical activation of essential Cys-302 upon cofactor binding to nonphosphorylating glyceraldehyde 3-phosphate dehydrogenase from Streptococcus mutans[END_REF].

Kinetic reactions with 2,2'-Dipyridyl Disulfide (2PDS) -Due to the high reactivity of

cysteine residues in all mutated GAPNs, kinetic measurements were carried out on a SX18MV-R Applied Photophysics stopped-flow apparatus (Leatherhead, UK) at 25°C, under pseudo-first-order conditions in 30 mM acetic acid, 30 mM imidazole, 120 mM Tris buffer at a constant ionic stength of 0.15 M over a pH range of 5.50-8.75 for T244S and T244/C382S GAPNs and 5.75-9.25 for K178A GAPN. The pH range was chosen in function on the pH stability of the mutated GAPNs. One syringe contained apo or apo-like 2 form of GAPN, and the other contained 2PDS. The reaction was monitored at 343 nm with 10 µN GAPN and 200 µM 2PDS and pyridine-2-thione was quantified using an extinction coefficient of 8080 M - 1 .cm -1 . The pseudo-first-order k obs were determined at each pH by fitting the absorbance A at 343 nm versus time t to a monophasic expression (1) :

A 343 = a(1-e -kobs*t ) + c (1)
where k obs is the observed rate constant, a is the burst magnitude and c represents the value of the ordinate intercept, or to a biphasic expression (2) :

A 343 = a 1 (1-e -kobs1*t ) + a 2 (1-e -kobs2 *t ) + c (2)
where k obs1 and k obs2 are the observed rate constants for the fast and the slower phase respectively, a 1 and a 2 are the relative amplitude values for the two phases, and c represents the value of the ordinate intercept.

The second-order rate constants k 2 were determined at each pH value and data were analysed as described by Marchal and Branlant [START_REF] Marchal | Evidence for the chemical activation of essential Cys-302 upon cofactor binding to nonphosphorylating glyceraldehyde 3-phosphate dehydrogenase from Streptococcus mutans[END_REF].

Crystallisation, data collection and processing -Crystals of the T244S GAPN with NADP were obtained using the hanging drop vapour diffusion method at 293 K. 2-6 µl of a 5 mg.ml -1 protein solution in 50 mM imidazole buffer at pH 6.8 and mixed with 1-2 µl of reservoir solution composed of 1.9-2.0 M ammonium sulphate, 0.1 M HEPES buffer pH 7.5, and 2%

PEG 400 [START_REF] Cobessi | Apo and holo crystal structures of an NADP-dependent aldehyde dehydrogenase from Streptococcus mutans[END_REF][START_REF] Cobessi | Structural and biochemical investigations of the catalytic mechanism of an NADP-dependent aldehyde dehydrogenase from Streptococcus mutans[END_REF]. The crystals grew within a few days. A 2.50 Å resolution data set was collected at 100 K at the synchrotron source in Trieste. The crystals belong to the space group P21212, with unit cell dimensions a= 143.7 Å, b= 154.4 Å and c = 115.0 Å. Diffracted data were processed using the HKL crystallographic data reduction package [START_REF] Otwinowski | Processing of X-ray diffraction data collected in oscillation mode[END_REF]. Statistics of the data set are summarized in Table 1.

Phasing, refinement and quality of the model -The structure of the T244S GAPN was solved by difference Fourier techniques, using the wild-type apo-like structure as a starting model (PDB code 2euh) [START_REF] Cobessi | Apo and holo crystal structures of an NADP-dependent aldehyde dehydrogenase from Streptococcus mutans[END_REF].

Several cycles of rigid-body refinement carried out with CNS [START_REF] Brünger | Crystallography & NMR system: a new software suite for macromolecular structure determination[END_REF], dropped the crystallographic R-factor and R-free values to 0.23 and 0.28, respectively. The analysis of the (2Fo -Fc) electron density map at this stage of refinement was very well defined for all the structure, including the mutated residue. The ordered water molecules were added automatically and checked individually. Moreover, seven sulfate ions were added (one in the A and C monomers, two in the B monomer and three in the D monomer) and refined with occupancy factors ranging from 0.5 to 1. After refinement (CNS) and manual rebuilding [START_REF] Roussel | TURBO-FRODO, Silicon Graphics Applications Directory, Silicon Graphics, Mountain View[END_REF] the final crystallographic R-factor and R-free values calculated for the 78424 independent reflections (in the 20.00-2.50 Å resolution range) were 0.176 and 0.227, respectively. The Structural Bioinformatics (accession code XXX). Figure 1A was drawn using MOLMOL [START_REF] Koradi | MOLMOL: a program for display and analysis of macromolecular structures[END_REF] and Figure 1B with BOBSCRIPT [START_REF] Esnouf | Further additions to MolScript version 1.4, including reading and contouring of electron-density maps[END_REF] RESULTS

Rationale for the mutations -As indicated in the Introduction, we have postulated that T244

and its potential interaction with K178 could be critical in the correct positioning of the NMN moiety of the cofactor during acylation, so that hydride transfer can occur with an optimum efficiency. In order to validate this assumption, the roles of residues T244 and K178 have been probed by site-directed mutagenesis. Introduction of a residue such as serine in position 244 would provide experimental support for the role of the Van der Waals interaction between the β-CH 3 of T244 and the nicotinamide ring, whereas T244V and K178A

substitutions would assess the role of the K178/T244 hydrogen bond interaction in the correct positioning of the β-CH 3 of T244 relative to the nicotinamide ring of the cofactor. In the apo and apo-like forms of GAPN, C302 and C382 were previously shown to be reactive towards 2PDS [START_REF] Marchal | Evidence for the chemical activation of essential Cys-302 upon cofactor binding to nonphosphorylating glyceraldehyde 3-phosphate dehydrogenase from Streptococcus mutans[END_REF]. Therefore, to assign pK app and k 2 values to C302 in the apo-like form of T244S GAPN, C382 has been substituted by a serine residue.

Determination of the pK apps and reactivities of the cysteine residues in apo form of K178A GAPN and apo-like forms of K178A, T244S and T244S/C382S GAPNs with 2PDS

-For all mutated GAPNs, the reaction with 2PDS under native conditions obeyed pseudo-first-order kinetics with formation of 2 mol of pyridine-2-thione per subunit as determined from the absorbance at 343 nm except that only 1 mol of 2PDS reacted per subunit of T244S/C382S GAPN. This is in accord with what was described for the wild type in which two residues i.e.

C302 and C382 were shown to be reactive per monomer [START_REF] Marchal | Evidence for the chemical activation of essential Cys-302 upon cofactor binding to nonphosphorylating glyceraldehyde 3-phosphate dehydrogenase from Streptococcus mutans[END_REF]. For the K178A GAPN apo form, the two cysteines exhibited distinct k 2 constants. The pH-k 2 profile for each thiolate showed sigmoidal behavior with pK app values of 8.80 and 9.15 and k 2 values of 3.3×10 5 M -1 .s -1 and 3.05×10 4 M -1 .s -1 , respectively (Figure 2A and Table 2). The high k 2 values indicated that both cysteine residues are accessible within the active site of K178A GAPN, in contrast to what was observed for the wild-type apo form [START_REF] Marchal | Evidence for the chemical activation of essential Cys-302 upon cofactor binding to nonphosphorylating glyceraldehyde 3-phosphate dehydrogenase from Streptococcus mutans[END_REF]. The high accessibility of C302 is probably not catalytically relevant but is rather another example of the great conformational flexibility exhibited by this residue within the active site [START_REF] Marchal | Evidence for the chemical activation of essential Cys-302 upon cofactor binding to nonphosphorylating glyceraldehyde 3-phosphate dehydrogenase from Streptococcus mutans[END_REF][START_REF] Marchal | Characterization of the amino acids involved in substrate specificity of nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans[END_REF]. The apo form of K178A GAPN incubated at 4°C in the presence of saturating concentration of NADP for 5 h at pH 5.5 under conditions where the conformational rearrangement 1 of the active site was shown to occur in the apo wild type, displayed a similar pH-k 2 profile to that described for the apo K178A

GAPN with pK app values of 8.85 and 9.25 and k 2 values of 2.97×10 5 M -1 .s -1 and 2.9×10 4 M -1 .s - 1 , respectively (curves not shown, see Table 2). Since the apo form of T244S GAPN could not be obtained irrespective of the experimental procedure used (data not shown), titration by 2PDS was only performed on the apo-like form of T244S GAPN after incubation for 5h at 4°C and pH 5.5 in the presence of saturating concentration of NADP. A pH-k 2 profile similar to that of apo and apo-like forms of K178A GAPN was obtained with pK app values of 8.5 and 9.0 and k 2 values of 3×10 3 M -1 .s -1 and 9.5×10 4 M -1 .s -1 , respectively (Figure 2B, inset ; Table 2). The pH-k 2 curve of the apo-like form of T244S/C382S GAPN fitted only to a monosigmoidal profile with a pK app value of 9.0 and a k 2 value of 2×10 5 M -1 •s -1 (Figure 2B and Table 2). Such a result strongly suggests that the pK app of 9.0 determined in the T244S GAPN represents that of C302.

Kinetic properties of T244S, T244V and K178A

GAPNs -In a previous study, it was shown that acylation and deacylation steps of wild-type GAPN could be kinetically resolved and that deacylation was rate-limiting under steady-state conditions [START_REF] Marchal | Role of glutamate-268 in the catalytic mechanism of nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans[END_REF]. It was also demonstrated that the rate-determining step within deacylation was associated with hydrolysis, whereas hydride transfer was rate-determining in acylation. Therefore, before interpreting the steady-state kinetic data, we first determined whether the rate-limiting step was still associated with the deacylation step for mutated GAPNs. For that, experiments were carried out at pH 8. These results showed that the rate-limiting step is associated with the acylation step for T244S, T244V and K178A GAPNs in contrast to the wild type (Table 3). More precisely, as shown below, the rate-limiting step in T244S and K178A GAPNs is associated with hydride transfer. Therefore, to analyze a possible effect of the substitutions introduced at positions 178 and 244 in terms of change in the apparent affinity constant for the G3P substrate and in rate constants, only the k ac value and the K M value for G3P of the wild type in the acylation step have been considered. was used as a substrate (Table 4). This strongly suggested that hydride transfer is ratelimiting. Therefore, the hydride rate constant is 2900-fold decreased compared to the wild type. The pH-rate profile was then studied over a pH range of 5-8.75 at saturating concentration of 1.5 mM (D,L)-G3P and at concentration of 30 µM NADP, where NADP does not behave as an inhibitor. As shown in Figure 3, the pH-k cat profile exhibited a sigmoidal curve that can be related to the contribution of one ionizable group of pK app 6.8 that must be deprotonated for acylation, and characterized by a k cat of 0.27 s -1 . Compared with wild type, pK app of 6.8 would correspond to that of catalytic C302. The fact that the pH-k cat profile depends on C302 pK app and not on E268 pK app which was shown to be 7.6 in the wild type [START_REF] Marchal | Evidence for the chemical activation of essential Cys-302 upon cofactor binding to nonphosphorylating glyceraldehyde 3-phosphate dehydrogenase from Streptococcus mutans[END_REF] could be indicative of relative positioning of C302 and E268 in the T244S ternary Michaelis complex, slightly different from that found in the wild-type ternary complex.

T244S GAPN -As shown in

T244V and K178A GAPNs -The kinetic parameters are summarized in Table 3. The T244V substitution led to a drastic 2.5.10 5 -fold decrease of the k cat value. The k cat value of 2.10 -3 s -1

was so low that determination of K M values for NADP and (D,L)-G3P was not possible. The k cat value for the K178A GAPN was strongly reduced by a factor 2.5.10 4 and the K M values for NADP and (D,L)-G3P were 3-fold higher and 40-fold lower, respectively, with respect to the wild type. The large decrease in the (D,L)-G3P K M value remains to be explained. Under steady-state conditions at optimum pH 8.2, a substrate kinetic isotopic effect of 2.1 was observed with D-[1-2 H]G3P (Table 4). Thus, hydride transfer is also rate-limiting and is 2.5.10 4 -fold less efficient than with the wild type. The pH-rate profile was also studied over a pH range of 5-9. k cat values for K178A were determined at saturating concentrations of 0.15 mM (D,L)-G3P and 1 mM NADP, respectively. As shown in Figure 3, the pH-k cat profile exhibited a sigmoidal curve that can also be related to the contribution of one ionizable group of pK app 6.7 and characterized by a k cat value of 0.02 s -1 . Compared to the wild type, pK app of 6.7 would correspond to that of catalytic C302, a value similar to that determined for T244S GAPN.

Structure of the T244S apo-like form -

The (2Fo-Fc) electron density map is well defined for all the structure. Difference maps revealed the presence of NADP in the four monomers.

Three sulphate ions coming from the crystallisation medium have also been identified in monomer D while only two were present in monomer B, and one in monomers A and C. The sulphate anion whose position is conserved in all four monomers occupies the same location as the sulphate anion SO 4 a found in the wild type apostructure (Apo1 and Apo2, [START_REF] Cobessi | Apo and holo crystal structures of an NADP-dependent aldehyde dehydrogenase from Streptococcus mutans[END_REF][START_REF] Cobessi | Structural and biochemical investigations of the catalytic mechanism of an NADP-dependent aldehyde dehydrogenase from Streptococcus mutans[END_REF]), while the other is located on the protein surface.

The least-squares superimpositions involving the 474 Cα atoms of one subunit of the apo-like wild type [START_REF] Cobessi | Apo and holo crystal structures of an NADP-dependent aldehyde dehydrogenase from Streptococcus mutans[END_REF] with each subunit of the T244S GAPN structure result to root-mean square differences less than 0.29 Å, showing that the overall structures are very close. Local differences are limited to solvent-exposed regions, mobile loops and to the active site region.

In the wild-type structure, T244 is located at the end of a β-strand and its β-OH group is hydrogen-bounded to the ε-NH 2 group of K178, a residue located in an α-helix. In the T244S structure, the β-OH group of S244 adopts the same conformation and its interaction with K178 is conserved. The quality of the (2Fo-Fc) electron density map is good enough to model the NMN moiety of the NADP molecule (Figure 1 B). In particular, the pyridinium ring adopts a conformation, clearly different from that depicted for wild-type GAPN/NADP and ALDH2/NAD binary complexes which was described to be suitable for an efficient hydrid transfer [START_REF] Perez-Miller | Coenzyme isomerization is integral to catalysis in aldehyde dehydrogenase[END_REF]. In the T244S GAPN, the pyridinium ring is stabilised through van der Waals interactions with residues C302, P167 and L174. These residues are located after an α-helix, lies at the end of a β-strand and on an α-helix respectively. Moreover, the conformation of the side chain of the catalytic C302 is closer to that found in the wild-type apo form than in the wild-type/NADP complex and the salt-bridge between R459 and E268 remains present 2 , albeit NADP is bound (Figure 1A).

Finally, while the side chain of R301 was shown to bind the sulphate anion, called SO 4 a, in the wild-type apostructures (Apo1 and Apo2, [START_REF] Cobessi | Structural and biochemical investigations of the catalytic mechanism of an NADP-dependent aldehyde dehydrogenase from Streptococcus mutans[END_REF][START_REF] Cobessi | Apo and holo crystal structures of an NADP-dependent aldehyde dehydrogenase from Streptococcus mutans[END_REF]) and shown to contribute to both G3P binding and to be involved in the efficient positioning of G3P relative to C302 within the ternary complex GAPN/NADP/G3P [START_REF] Marchal | Characterization of the amino acids involved in substrate specificity of nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans[END_REF], it is positioned opposite to the SO 4 a anion corresponding to the C-3 phosphate of G3P and interacts with the carbonyl groups of G294 and A293. It is probable that the conformation adopted by the side chain of R301 in the binary complex rather corresponds to an alternative conformation which is not representative of the positioning of its guanidinium group within the competent wild-type ternary complex. Indeed, no significant K M change for G3P accompanies the drastic decrease of the acylation rate observed for the T244S GAPN in contrast to what was observed for the R301L GAPN [START_REF] Marchal | Characterization of the amino acids involved in substrate specificity of nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans[END_REF].

DISCUSSION

A key aspect of the chemical mechanism of the CoA-independent ALDH family is the requirement of a conformational isomerization of the NMN(H) portion of the cofactor after the oxidoreduction process to allow E268 to play its role in the deacylation step [START_REF] D'ambrosio | The first crystal structure of a thioacylenzyme intermediate in the ALDH family : new coenzyme conformation and relevance to catalysis[END_REF]. The fact that the acylation step in GAPN and also in many other CoA-independent ALDHs, is not ratelimiting and is efficient implies several prerequisites for this step. One of those relates to the optimal positioning of the nicotinamide ring towards the hemithioacetal within the ternary group leads to a unique stabilized position of the nicotinamide ring clearly distinct from that hypothesized for the "hydride transfer" conformation in CoA-independent ALDHs including GAPN [START_REF] Steinmetz | Structure of mitochondrial aldehyde dehydrogenase: the genetic component of ethanol aversion[END_REF][START_REF] Cobessi | Apo and holo crystal structures of an NADP-dependent aldehyde dehydrogenase from Streptococcus mutans[END_REF][START_REF] Moore | Sheep liver cytosolic aldehyde dehydrogenase: the structure reveals the basis for the retinal specificity of class 1 aldehyde dehydrogenases[END_REF][START_REF] Perez-Miller | Coenzyme isomerization is integral to catalysis in aldehyde dehydrogenase[END_REF]. The crystal structure presented herein is likely representative of the position occupied by the nicotinamide ring within the T244S GAPN/NADP complex in solution. This is in agreement with the fact that i) the formation of the binary complex T244S GAPN/NADP is not able to promote the chemical activation of C302 (its pK app remains around 9) in contrast to what was observed for the wild type [START_REF] Marchal | Evidence for the chemical activation of essential Cys-302 upon cofactor binding to nonphosphorylating glyceraldehyde 3-phosphate dehydrogenase from Streptococcus mutans[END_REF] and, ii) the salt-bridge between R459 and E268, which is considered as typical of the apo form [START_REF] Cobessi | Structural and biochemical investigations of the catalytic mechanism of an NADP-dependent aldehyde dehydrogenase from Streptococcus mutans[END_REF], is still present in the X-ray binary complex structure.

Determination of the structure of a hemithioacetal intermediate is not possible because of its transient nature. If we assume that the nicotinamide conformation in the T244S hemithioacetal intermediate/NADP complex is the one observed in the crystal structure of the binary complex T244S GAPN-NADP, the poor efficiency of the hydride transfer could be explained by non-optimal positioning of the nicotinamide ring relative to the hemithioacetal intermediate. The 0.6-unit increase in pK app of C302 compared to the wild type, which is similar to the contribution of the positively charged nicotinamide ring in lowering the pK app of C302 in the wild type [START_REF] Marchal | Evidence for the chemical activation of essential Cys-302 upon cofactor binding to nonphosphorylating glyceraldehyde 3-phosphate dehydrogenase from Streptococcus mutans[END_REF], can be also interpreted as the consequence of positioning of the nicotinamide ring relative to C302 slightly different from that in the wild-type acylating ternary complex. Therefore, the role of the β-CH 3 group of T244 in the wild type would be to mainly allow the nicotinamide ring to adopt a conformation suitable for an efficient hydride transfer.

An alternative explanation would relate to the positioning of the C 1 -hydroxylate of the hemithioacetal intermediate relative to the oxyanion hole rendering it unsuitable for hydride transfer. However, this latter assumption is rather unlikely. First, the apparent affinity constant of T244S GAPN for G3P is not significantly changed. Second, whatever the X-ray structures considered including those of the binary complex T244S GAPN/NADP, the apolike wild type, the ternary thioacylenzyme intermediate, the side chain of N169 involved in the oxyanion hole formation, is always similarly positioned in all the structures [START_REF] Cobessi | Apo and holo crystal structures of an NADP-dependent aldehyde dehydrogenase from Streptococcus mutans[END_REF][START_REF] D'ambrosio | The first crystal structure of a thioacylenzyme intermediate in the ALDH family : new coenzyme conformation and relevance to catalysis[END_REF].

Role of the β-OH group of T244 -

As mentioned in the Introduction, the T244 OH group, which is hydrogen bonded to the K178 ε-amino group, can help the β-CH 3 group of T244 to position the nicotinamide ring in a conformation suitable for hydride transfer. The T244V substitution was therefore intended i) to determine the consequence of the loss of the hydrogen bond between the T244 β-OH group and the K178 ε-amino group and, ii) to introduce a Val residue invariant at this position in CoA-dependent ALDHs whose acylation step was shown to be as efficient as in GAPN [START_REF] Stines-Chaumeil | Mechanistic characterization of the MSDH (methylmalonate semialdehyde dehydrogenase) from Bacillus subtilis[END_REF]. Removing the β-OH group in fact alters drastically the catalytic properties of the enzyme. The efficiency of the rate-limiting acylation step is decreased by a factor 2.5×10 5 -fold and 86-fold compared to the wild-type and the T244S GAPNs, respectively. Although it was not possible to determine whether hydride transfer remained rate-determining within the acylation step because of the very low k cat value, the larger decrease of k cat likely originates from the disruption of the hydrogen bond interaction between the residues at position 244 and 178, thus leading to formation of a non efficient T244V ternary complex. However, one can not exclude the possibility that the very low k cat value of the T244V GAPN is also due to additional structural changes induced by the presence of a Val residue in the hydrophilic environment of the GAPN active site.

The substitution of the counterpart residue K178 for alanine also appears to be informative and complementary to the studies done on T244S GAPN. In fact, the K178A and T244S

GAPNs exhibited very similar catalytic properties. First, the rate-limiting step is associated with hydride transfer and a 2.5×10 4 -fold decrease of the k cat value is observed. Second, the pK app values for C302 are equivalent in both ternary complexes i.e. 6.7 vs 6.8. Therefore, it is tempting to interpret these results in a similar way to that developped in the preceding section for the T244S GAPN. So, the disruption of the T244/K178 hydrogen bond interaction by substituting either T244 by a valine residue or K178 by an alanine residue, would alter the positioning of T244, in particular of its β-CH 3 group and thus of the nicotinamide ring that could explain the very poor efficiency of the hydride transfer. 178 is mostly occupied by a methionine residue. Thus, it is tempting to propose that one of the two methyl groups of the β-branched side chain of V244 is positioned like that of β-CH 3 of T244 and therefore fullfils a similar function thanks to the other methyl group of V244 which could interact via hydrophobic interactions with the side chain of residues 178. Such an hypothesis remains to be validated. In addition to T244, the side chain of invariant E399 was shown to play an essential role by anchoring the NMN ribose through hydrogen bonds with the hydroxyl groups of the ribose [START_REF] Ni | Involvement of glutamate 399 and lysine 192 in the mechanism of human liver mitochondrial aldehyde dehydrogenase[END_REF]. Lastly, it was also hypothesized that the negative charge of the tetrahedral oxyanion intermediate participates in the efficient positioning of the positively charged nicotinamide ring within the ternary complex hemithioacetal intermediate/NAD(P) [START_REF] D'ambrosio | The first crystal structure of a thioacylenzyme intermediate in the ALDH family : new coenzyme conformation and relevance to catalysis[END_REF]. Altogether, this likely delineates a pattern of interactions that stabilizes an efficient "hydride transfer" conformation of the NMN moiety of the cofactor, once the transient hemithioacetal intermediate is formed.

Relevance to the two-step catalytic mechanism

Finally, recent structural data on the thioacyl intermediate of GAPN suggested that a low energetic barrier exists between the conformations adopted by the NMN(H) moiety during the two-step catalytic cycle of the CoA-independent ALDHs [START_REF] D'ambrosio | The first crystal structure of a thioacylenzyme intermediate in the ALDH family : new coenzyme conformation and relevance to catalysis[END_REF]. Based on the structure of the crystalline binary complex T244S GAPN/NADP, we propose that the β-CH 3 group of T244 might play a role in the efficiency of the conformational isomerization of the NMNH after hydride transfer as well.

FOOTNOTE

Footnote 1 : The amino acid numbering used for the biochemical and structural data is that defined by Wang and Weiner [START_REF] Wang | Involvement of glutamate 268 in the active site of human liver mitochondrial (class 2) aldehyde dehydrogenase as probed by site-directed mutagenesis[END_REF] and this nomenclature has been used for all biochemical studies done on members of the ALDH family. and the loss of the salt-bridge between E268 and R459 [START_REF] Marchal | Evidence for the chemical activation of essential Cys-302 upon cofactor binding to nonphosphorylating glyceraldehyde 3-phosphate dehydrogenase from Streptococcus mutans[END_REF][START_REF] Cobessi | Apo and holo crystal structures of an NADP-dependent aldehyde dehydrogenase from Streptococcus mutans[END_REF]. K178A (d) (3.0 ± 0.1)×10 5 8.85 ± 0.05 (2.9 ± 0.2)×10 4 9.25 ± 0.05 T244S (d) (3.0 ± 0.3)×10 3 8.5 ± 0.1 (9.5 ± 2)×10 4 9.0 ± 0.2 T244S/C382S (d) (2.0 ± 0.4)×10 5 9.0 ± 0.1

The experimental conditions are described in Experimental procedures. Transients obtained on the apo and apo-like K178A and apo-like T244S GAPN were best described by a double exponential expression reflecting the contribution of C302 and C382 residues and data were analysed according to equation 2. For the apo-like T244S/C382S GAPN, experimental data were best described by a monoexponential expression according to equation 1. The secondorder rate constants at each pH were fitted to a monosigmoidal equation to determine the pK app 's in Sigmaplot (SPSS). The error estimates are those provided by the program. (a) From [START_REF] Marchal | Evidence for the chemical activation of essential Cys-302 upon cofactor binding to nonphosphorylating glyceraldehyde 3-phosphate dehydrogenase from Streptococcus mutans[END_REF]. with either (D,L)-G3P or D-G3P showed that both enantiomeric forms equally behaved as efficient substrates for the wild-type GAPN [START_REF] Marchal | Role of glutamate-268 in the catalytic mechanism of nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans[END_REF]. Therefore, (D,L)-G3P was used as a substrate for mutated GAPN-catalyzed reactions. K M values for NADP and (D,L)-G3P were determined by fitting the experimental data to the Michaelis-Menten equation. (a) Corresponding to rate constant and K M values for the cofactor and the substrate in the acylation step and determined with D-G3P [START_REF] Marchal | Role of glutamate-268 in the catalytic mechanism of nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans[END_REF]. Substrate inhibition was described to be observed for [D-G3P] > 200 µM (see [START_REF] Marchal | Role of glutamate-268 in the catalytic mechanism of nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans[END_REF]). The acylation rate constant was determined at 10°C. Therefore to compare the k cat values of the mutated GAPNs and k ac value of the wildtype GAPN, the reported k ac value of the wild type was extrapolated from the value measured at 10°C, assuming a 2.5-fold increase of the rate constant k ac per 10°C (from [START_REF] Marchal | Role of glutamate-268 in the catalytic mechanism of nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans[END_REF]). (b) The k cat value was too low to permit determination of K M values for NADP and (D,L)-G3P.

(c) Substrate inhibition was observed for [(D,L)-G3P] > 150 µM. (a) The acylation rate constant for the wild type was extrapolated from the value measured at 10°C (from [START_REF] Marchal | Role of glutamate-268 in the catalytic mechanism of nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans[END_REF]).

(b) From Table 3.

(c) The k cat value was to low to allow accurate determination of pK a and substrate isotopic effect.

Figure 1 : A, Superimposition of the active site regions of the apo-like wild-type enzyme (in red) and of the T244S GAPN (in blue). The conformation of the NMN moiety in the wildtype structure is the one described to be suitable for efficient hydride transfer [START_REF] Marchal | Evidence for the chemical activation of essential Cys-302 upon cofactor binding to nonphosphorylating glyceraldehyde 3-phosphate dehydrogenase from Streptococcus mutans[END_REF][START_REF] Perez-Miller | Coenzyme isomerization is integral to catalysis in aldehyde dehydrogenase[END_REF]. B, Stereoview of the cofactor molecule in the (2Fo -Fc) electron density map (contour level: 1 σ). provided pK app values of 8.50 and 9.00 and k 2 values of 3.3×10 3 M -1 .s -1 ( ) and 9.5×10 4 M - 1 .s -1 ( ), respectively. Standard error on each experimental point was less than 5 %.

  2 and 25°C under the following conditions i.e.T244S GAPN (16 and 32 µN), NADP (0.03 mM), (D,L)-G3P (1.5 mM); K178A GAPN (16 and 32 µN), NADP (1 mM), (D,L)-G3P (0.15 mM); T244V GAPN (16 and 32 µN), NADP (1 mM), (D,L)-G3P (0.2 mM). No burst of NADPH formation was observed irrespective of the mutated GAPN and the measured rate constants were similar to the rate values determined under steady-state conditions (see next paragraph).

  acylating complex. Several X-ray structures of binary ALDH/NAD(P) complexes reveal a great conformational flexibility of the NMN portion of the NAD(P) molecule, in particular of the nicotinamide ring [e.g. see 11, 14-15]. However, once the transient hemithioacetal intermediate is formed, the NMN moiety, in particular in GAPN, must be positioned such that an efficient and stereospecific transfer of hydride ion occurs towards the C 4 of the nicotinamide ring. As indicated in the Introduction, the invariant T244 is postulated to play a major role in the positioning of the nicotinamide ring within the ternary complex of CoAindependent ALDHs. The kinetic and structural data reported here on GAPN strongly favour such an assumption, in particular of the critical role of the β-CH 3 group of T244.Role of the β-CH 3 group of T244 -Substitution of T244for serine leads to a change in the nature of the rate-limiting step which becomes associated with the acylation step. Hydride transfer becomes rate-limiting and its efficiency is strongly decreased by a factor of 2900 compared to the wild type. This drastic effect could be the consequence of changes occurring in the location and orientation of the nicotinamide ring relative to the hemithioacetal intermediate within the ternary complex, thereby preventing an efficient hydride transfer. The T244S GAPN structure in the presence of NADP clearly shows that the absence of the β-CH 3

-

  The major feature that has emerged from this study lies in the critical contribution of the β-CH 3 group of invariant T244 in the efficiency of the GAPN acylation step. It likely contributes to formation of a transient hemithioacetal intermediate with an adequate positioning of the nicotinamide ring to allow an efficient hydride transfer. Such a role is expected to be general for all the members of the CoA-independent ALDH family. This is further supported by the presence of an invariant valine residue at position 244 in the CoA-dependent ALDH family. In these ALDHs, position

Footnote 2 :

 2 In contrast to the term holo form, the term apo-like form indicates here an apo form saturated by NADP but in which the cofactor has not yet induced the local conformational rearrangement of the active site, including the decrease of the pK a of C302

Figure 2 :

 2 Figure 2 : pH-dependence of the second-order rate constant k 2 for the reaction of 2PDS on apo K178A and apo-like T244S and T244S/C382S GAPNs.Kinetics were performed at 25°C as indicated under "Experimental conditions". The GAPN and 2PDS concentrations were 10 µN and 200 µM, respectively. Rate constants k obs were determined using nonlinear regression analysis, and second order rate constants k 2 were calculated by dividing the k obs values by the concentration of 2PDS. Transients obtained on the apo K178A and apo-like T244S GAPNs were analysed according to eq. 2 reflecting the contribution of C302 and C382 residues. Transients obtained on the apo-likeT244S/C382S GAPN were analysed according to eq. 1. (A) Apo K178A GAPN : Both data sets were fitted against a single pKa model, which provided pK app values of 8.80 and 9.15 and k 2 values of 3.3×10 5 M -1 .s -1 (•) and 3.05×10 4 M -1 .s -1 ( ), respectively. (B) Main panel : Data sets obtained on the apo-likeT244S/C382S GAPN were fitted against a single pKa model, which provided pK app and k 2 values of 9.0 and 3.3×10 5 M -1 .s -1 , respectively. Inset : Data sets obtained on the apo-like T244S GAPN were fitted against a single pKa model, which

Figure 3 :

 3 Figure 3 : pH dependence of the steady-state rate constant k cat for the K178A and T244S GAPNs.pH-dependence of k cat was studied over the pH-range 5.25-8.75 for T244S GAPN or 5.25-9.00 for K178A GAPN with a mixed reaction buffer consisting of 120 mM Tricine, 30 mM imidazole and 30 mM acetic acid at a constant ionic strength of 0.15 M. The other experimental conditions were as follows : (D,L)-G3P 0.15 mM, NADP 1 mM and K178A GAPN 6.6 µN ; and (D,L)-G3P 1.5 mM, NADP 0.03 mM and T244S GAPN 2 µN.Data for T244S ( ) and K178A (•) GAPNs were best fitted by nonlinear regression analysis against a single pK app model, identified by the best-fit theoretical curves (solid line and dotted line, respectively). Standard error on each experimental point was less than 5 %.

  FIGURES

  Figure 2

Biochemical Journal Immediate Publication. Published on 8 Sep 2006 as manuscript BJ20060843 Copyright 2006 Biochemical Society stereochemical

  quality of the model was assessed by PROCHECK[23]. The most favoured and additionally allowed regions of the Ramachandran plot contained 99.6% of the nonglycine residues. The final model consisted of 14362 non-hydrogen atoms, 1109 water molecules and 7 sulphate ions coming from the crystallization solution. The refined structure presented a good geometry with r.m.s.d. from ideal bond lengths and angles of 0.005 Å and 1.26°, respectively. The average temperature factor (B) for all atoms was 24.6 Å 2 . The statistics for refinement and model geometry are summarized in Table1. Coordinates and structure factors have been deposited in the Protein Data Bank at Research Collaboratory for

Table 3 ,

 3 substituting T244 with serine resulted in a 2-fold decrease in K M for NADP with a value of 10 µM. An inhibitory effect was observed for

[NADP] > 60 µM. The K M value for (D,L)-G3P decreased by a factor 4 and the k cat value drastically decreased by a factor of 2900 relative to the k ac value of the wild type (Table

3 & Table 4

). At optimum pH 8.2, the k cat value was decreased by a factor 2.1 when D-[1-2 H]G3P

Table 2 :

 2 Parameters for the pH-dependence of the second-order rate constants for the

	reaction of 2PDS with K178A, T244S and T244S/C382S GAPNs.
	GAPN	k 2 (M -1 .s -1 )	pK app
	Apo-GAPNs
	Wild-type (a)	1350 ± 28	8.84 ± 0.04
	C382A (a)	225 ± 9	8.49 ± 0.07
	C302A (a)	588 ± 14	9.07 ± 0.02
	K178A	(3.30 ± 0.25)×10 5	8.80 ± 0.07
		(3.05 ± 0.25)×10 4	9.15 ± 0.15
	Holo-GAPN
	Wild-type (a)	> 2×10 5	ND
	Wild-type (b)	182 ± 2	6.1 ± 0.2
	Apo-like-GAPNs (c)

Table 3 :

 3 Kinetic parameters of the acylation step for the T244S, T244V and K178A

	GAPNs,
	GAPN	NADP	(D,L)-G3P	k cat (s -1 )	Rate-limiting step
		K M (µM)	K M (µM)
	Wild-type (a)	25 ± 2 (a)	490 ± 50 (a)	790 ± 20 (a)	Deacylation
	T244S	10 ± 3	120 ± 14	0.27 ± 0.03	Acylation
	T244V	-	-	~ 0.002 (b)	Acylation
	K178A	70 ± 6	12 ± 5 (c)	0.020 ± 0.002	Acylation
	Initial velocities of the reaction catalysed by mutated GAPNs were measured in 50 mM TES
	buffer, pH 8.2 and 5 mM β-mercaptoethanol at 25°C. Previous enzyme activity measurements

Table 4 :

 4 Parameters for the pH-dependence of the k cat for the T244S, T244V

	and K178A GAPNs.
	GAPN	pK app1	pK app2	k cat (s -1 )	Substrate isotopic effect
	Wild type (a)	6.2 ± 0.1	7.5 ± 0.1	790 ± 20 (a)	2.7
	T244S	6.8 ± 0.1	-	0.27 ± 0.03 (b)	2.1
	T244V (c)	-	-	~ 0.002 (b)	-
	K178A	6.7 ± 0.1	-	0.020 ± 0.002 (b)	2.1
	pK app and k cat values were deduced from nonlinear regression analysis of experimental data
	obtained at 25°C against a single pKa model. The substrate isotope effects were determined as
	described under "Experimental Procedures".

Biochemical Journal Immediate Publication. Published on 8 Sep 2006 as manuscript BJ20060843 Copyright 2006 Biochemical Society

Biochemical Journal Immediate Publication. Published on 8 Sep 2006 as manuscript BJ20060843

ACKNOWLEDGEMENTS

We are grateful to S. Boutserin for her very efficient technical help. We also thank Dr. Van Dorsselear and G. Chevreux for mass determination and Dr. S. Sonkaria for careful reading of the manuscript. A. Pailot was fellow of the French Ministère de la Recherche et des Nouvelles Technologies (MRNT). This research was supported by the Centre National de la Recherche Scientifique, the French Ministère de la Recherche et de l'Enseignement Supérieur, the University Henri Poincaré Nancy I, the IFR 111 Bioingénierie and local funds from the Région Lorraine.

TABLES

Copyright 2006 Biochemical Society (b) This pK app value of 6.1 for C302 was determined using iodoacetamide as a kinetic probe (from [START_REF] Marchal | Evidence for the chemical activation of essential Cys-302 upon cofactor binding to nonphosphorylating glyceraldehyde 3-phosphate dehydrogenase from Streptococcus mutans[END_REF]).

(c) Apo-like forms refer to GAPN/NADP binary complexes after incubation at 4°C of the apo forms (or partially apo forms in the case of T244S and T244S/C382S GAPNs) in the presence of saturating concentration of NADP for 5 h at pH 5.50 under conditions where the conformational rearrangement 1 occurred in the wild-type enzyme (from [START_REF] Marchal | Evidence for the chemical activation of essential Cys-302 upon cofactor binding to nonphosphorylating glyceraldehyde 3-phosphate dehydrogenase from Streptococcus mutans[END_REF]) but not in the mutated GAPNs. (d) Although no additional experimental data can be obtained beyond the inflection point of the curves, non-linear regression analysis provides accurate pKa values.


William C Lamanna

Rebecca J Baldwin

Michael Padva

Ina Kalus

Gerdy Ten Dam

Toin H Van Kuppevelt

John T Gallagher

Kurt Von Figura

Thomas Dierks
email: thomas.dierks@uni-bielefeld.de

Catherine L R Merry
email: catherine.merry@manchester.ac.uk

HEPARAN SULPHATE 6-O-ENDOSULPHATASES-DISCRETE IN VIVO ACTIVITIES AND FUNCTIONAL COOPERATIVITY

Heparan sulphate (HS) is essential for normal embryonic development. This requirement is due to the obligatory role for HS in the signalling pathways of many growth factors and morphogens that bind to sulphated domains in the HS polymer chain. The sulphation patterning of HS is determined by a complex interplay of Golgi-located N-and O-sulphotransferases that sulphate the heparan precursor and cell surface endosulphatases that selectively remove 6-O-sulphates (6S) from mature HS chains. In the present study we generated single or dual knock out mice for the two murine endosulphatases mSulf1 and mSulf2. Detailed structural analysis of HS from mSulf1 - /-fibroblasts showed a striking increase in 6-O-sulphation, which was not seen in mSulf2 -/-HS.

Intriguingly, the level of 6-sulphation in the double mSulf1 -/-/2 -/-HS was significantly higher than that in the mSulf1 -/-counterpart. These data imply that mSulf1 and mSulf2 are functionally cooperative. Unlike their avian orthologues, mammalian Sulf activities are not restricted to the highly sulphated S-domains of HS. Mitogenesis assays with FGF2 revealed that Sulf activity dampens the activating potential of newly-synthesised HS suggesting an important role for these enzymes in cell growth regulation in embryonic and adult tissues.

. Sulphate pattern regulation therefore plays a critical role during development and disease pathogenesis. Within a mature HS chain (typically between 50 and 200 saccharides in length) long sequences of repeating GlcA-GlcNAc residues are separated by composite regions consisting of highly sulphated S-domains (predominantly IdoA(2S)-GlcNS sequences) with adjacent transition zones comprised of alternating GlcNS and GlcNAc containing disaccharides [4]. Both the Sdomains and transition zones are further modified by 6-O-sulphation (6S) of glucosamine residues and variations in the degree and patterning of 6S contribute significantly to the fine structural heterogeneity in HS. The structural diversity in HS is non-random, with regulation observed at the level of tissue type and developmental stage [5, 6]. Within the specific domain types loss or gain of 6S alone is sufficient to alter the outcome of the ligand-HS interaction [7].

Dynamic changes in 6S correlate with modulated susceptibility to FGF signal transduction during neural development and tumour transformation [START_REF] Brickman | Structural modification of fibroblast growth factor-binding heparin sulphate at a determinative stage of neural development[END_REF][START_REF] Jayson | Coordinated modulation of the fibroblast growth factor dual receptor mechanism during transformation from human colon adenoma to carcinoma[END_REF].

The regulation of 6S patterning was thought to be solely the result of the action of the HS biosynthetic enzymes; however the identification of the Sulfs, a family of HS endo-6-Osulphatases, has led to a revision of this viewpoint. The first Sulf was discovered in a molecular cloning screen for Sonic hedgehog responsive genes, which led to the characterization of QSulf1, a sulphatase regulating cellular differentiation through modulation of Wnt signalling in developing quail embryos [START_REF] Dhoot | Regulation of Wnt signalling and embryo patterning by an extracellular sulfatase[END_REF]. Orthologues were identified in mammalian species [START_REF] Morimoto-Tomita | Cloning and characterization of two extracellular heparin-degrading endosulfatases in mice and humans[END_REF], however, many of the insights into Sulf activity have come from studies using QSulf1 and its recently characterized isoform QSulf2 [START_REF] Ai | Substrate Specificity and Domain Functions of Extracellular Heparan Sulfate 6-O-Endosulfatases, QSulf1 and QSulf2[END_REF][START_REF] Ai | QSulf1 remodels the 6-O sulfation states of cell surface heparan sulfate proteoglycans to promote Wnt signaling[END_REF][START_REF] Viviano | Domain-specific Modification of Heparan Sulfate by Qsulf1 Modulates the Binding of the Bone Morphogenetic Protein Antagonist Noggin[END_REF][START_REF] Wang | QSulf1, a heparan sulfate 6-O-endosulfatase, inhibits fibroblast growth factor signaling in mesoderm induction and angiogenesis[END_REF]. The unique localisation of the Sulfs at the cell surface and their endolytic activity, rather than the exolytic and therefore catabolic activity of lysosomal sulphatases, carried with it important implications for the novel hydrophilic domain that is inserted into the catalytic domain of the Sulfs. This highly and mainly positively charged domain has recently been characterised as a decisive functional element for both substrate recognition and cell surface localisation [START_REF] Ai | Substrate Specificity and Domain Functions of Extracellular Heparan Sulfate 6-O-Endosulfatases, QSulf1 and QSulf2[END_REF]. The native substrate specificity of the Sulfs has, until now, been largely studied employing HS disaccharide analysis from QSulf over-expressing cellular systems or in vitro analysis [START_REF] Ai | Substrate Specificity and Domain Functions of Extracellular Heparan Sulfate 6-O-Endosulfatases, QSulf1 and QSulf2[END_REF][START_REF] Ai | QSulf1 remodels the 6-O sulfation states of cell surface heparan sulfate proteoglycans to promote Wnt signaling[END_REF]. Results from these experiments indicate a substrate specificity of QSulf1 and QSulf2 which is completely restricted to GlcA/IdoA(2S)-GlcNS(6S) type disccharides from the S-domains of HS [START_REF] Ai | Substrate Specificity and Domain Functions of Extracellular Heparan Sulfate 6-O-Endosulfatases, QSulf1 and QSulf2[END_REF][START_REF] Ai | QSulf1 remodels the 6-O sulfation states of cell surface heparan sulfate proteoglycans to promote Wnt signaling[END_REF][START_REF] Viviano | Domain-specific Modification of Heparan Sulfate by Qsulf1 Modulates the Binding of the Bone Morphogenetic Protein Antagonist Noggin[END_REF].

Attention has now turned to the mammalian Sulf orthologues in mice and humans, as both Sulf1 and 2 isoforms have been shown to be mis-regulated during mammalian tumourigenesis [START_REF] Lai | Loss of HSulf-1 up regulates heparin-binding growth factor signaling in cancer[END_REF][START_REF] Li | Enhanced levels of Hsulf-1 interfere with heparin-binding growth factor signaling in pancreatic cancer[END_REF][START_REF] Morimoto-Tomita | Sulf-2, a proangiogenic heparan sulfate endosulfatase, is up regulated in breast cancer[END_REF]. Investigations into the influence of Sulf activity on HS-binding factors active in tumourigenesis such as VEGF, FGF, HGF and HB-EGF [START_REF] Lai | HSulf-1 modulates HGF-mediated tumor cell invasion and signaling in head and neck squamous carcinoma[END_REF][START_REF] Lai | HSulf-1 Sulfatase promotes apoptosis of hepatocellular cancer cells by decreasing heparin-binding growth factor signaling[END_REF][START_REF] Uchimura | HSulf-2, an extracellular endoglucosamine-6-sulfatase, selectively mobilizes heparin-bound growth factors and chemokines: effects on VEGF, FGF-1, and SDF-1[END_REF] have led to the hypothesis that the modulation of Sulf activity may be a target for therapeutic intervention.

In this study we describe the first characterisation of HS produced by cells with a knock out for mSulf1, mSulf2 or both. We have combined detailed structural analysis of the HS with expression analyses, HS epitope characterisation and growth factor activity studies. We conclude that mSulf1 and mSulf2 show functional cooperativity and that although increased mSulf1 expression can compensate for loss of mSulf2 activity, mSulf2 is unable to fulfil the role of mSulf1. Importantly, the 6-O-desulphation catalysed by the mSulf enzymes was found to be extensive, and more widely distributed throughout the HS chain than observed with the QSulf orthologues, with significant implications for the range of HS-ligand interactions affected.

EXPERIMENTAL PROCEDURES

Materials -Dulbecco's modified Eagle's medium and foetal calf serum were from Life Technologies, Inc. Heparinase I-III (Flavobacterium heparinum) were all from Grampian Enzymes (Orkney, UK). Chondroitinase ABC (Proteus vulgaris) and monoclonal antibodies against diphosphorylated ERK1/2 were from Sigma. The gels used were Sepharose CL-6B and DEAE-Sephacel (GE Healthcare) and Bio-Gels P-10 (fine grade) and P-2 (Bio-Rad). The ProPac PA-1 HPLC column was from Dionex (Camberley, UK). D-[6- 3 as well as into the blunted BseRI site of mSulf2 exon 1 (Figure 1 A/B), thereby disrupting the respective reading frames and generating stop codons in all frames. NotI-linearized constructs were electroporated into 129Sv/Ola embryonic stem cells (cell line D3, kindly provided by Peter Gruss, Goettingen). G418-resistant clones were selected and genotyped for specific recombination by Southern blotting. Positive ES clones were injected into C57BL/6 blastocysts to produce chimeric mice. Male chimeras were mated with C57BL/6 females, which led to germ line transmission of the targeted alleles. From these, heterozygotes were intercrossed to generate wild type and knock out mice. Northern and Southern blotting was performed according to standard methods using PCR-generated probes (primers listed in Supplementary Table I). The Preparation of HS from MEF cultures -Embryos (E12.5) obtained from mating mSulf1 -/-, 2 -/- or mSulf1 -/-/mSulf2 +/-mice were dissected, MEFs isolated and the HS chains radiolabelled and purified as described previously [START_REF] Merry | The molecular phenotype of heparan sulfate in the Hs2st-/-mutant mouse[END_REF]. Primary MEFs from the mSulf1 -/-/2 -/-embryos could not be maintained in culture. We therefore prepared immortalised lines (mSulf1 -/-/2 -/-and wild type control) by stable transfection of MEFs (E12.5) with the pMSSVLT vector containing an SV40 fragment including the large T gene [START_REF] Schuermann | An expression vector system for stable expression of oncogenes[END_REF], prior to isolation of HS chains.

Disaccharide compositional analysis -HS chains (50 Kcpm of 3 H) were digested with a combination of heparinases I, II, and III and the resultant disaccharides identified following SAX-HPLC as described previously [START_REF] Merry | The molecular phenotype of heparan sulfate in the Hs2st-/-mutant mouse[END_REF].

Low pH nitrous acid depolymerisation -100 Kcpm ( 3 H) of HS was taken for low pH nitrous acid depolymerisation [START_REF] Merry | The molecular phenotype of heparan sulfate in the Hs2st-/-mutant mouse[END_REF]. Oligosaccharides produced were separated and the tetrasaccharides purified essentially as described by Viviano et al. [START_REF] Viviano | Domain-specific Modification of Heparan Sulfate by Qsulf1 Modulates the Binding of the Bone Morphogenetic Protein Antagonist Noggin[END_REF]. Recovered tetrasaccharides were then separated by SAX-HPLC as for the heparinase I/II/III generated disaccharides.

Heparinase III digestion -100 Kcpm ( 3 H) of HS was treated with heparinase III essentially as described by Merry et al. [START_REF] Merry | Highly sensitive sequencing of the Sulfated Domains of Heparan Sulphate[END_REF]. Isolated sized fragments were pooled, desalted and further fractionated by SAX-HPLC. Disaccharides were analysed as described for heparinase I/II/III generated disaccharides. For larger oligosaccharides a single ProPac PA-1 SAX column was used with a linear gradient running from 0-0.75 M NaCl over 110min. Peak identification was made by comparison with previously published profiles [START_REF] Merry | Highly sensitive sequencing of the Sulfated Domains of Heparan Sulphate[END_REF].

Real Time PCR -Primary wild type, mSulf1 -/-and mSulf2 -/-MEFs together with immortalised wild type and mSulf1 -/-/2 -/-MEFs were washed in PBS then harvested. RNA was extracted using the RNeasy Protect mini kit (Qiagen, 74124). Synthesis of cDNA from mRNA transcripts was performed using the following method: RNA (5µg, denatured at 65°C for 3 minutes and placed on ice), dNTP (250µM), oligo dT (5µg; Promega, UK), 1X AMV buffer (Promega) and AMV reverse transcriptase (40 units; Promega) in a total volume of 200µl was incubated at 42°C for 1 hour, followed by 98°C for 5 minutes. Real time PCR assays to the Sulf enzymes together with housekeeping genes L19 and EEF1G were designed using the Exiqon Mouse Universal Probe Library system [START_REF] Mouritzen | The Probe Library: Expression profiling of 99% of all human genes using only 90 dual labeled real time PCR probes[END_REF]; (www.universalprobelibrary.com) (Roche, Switzerland), with amplification primers obtained from MWG (Germany). Amplicons were designed to be intron-spanning typically between 60-100 nucleotides in length. Real time PCR reactions were set up using SensiMix (dT) (Quantace) as per instructions. Briefly for a 10µl reaction, 5ng of cDNA, 0.1µM each forward and reverse primer, 0.1µl of the appropriate mouse probe (Universal ProbeLibrary Set, Roche applied science), 5 µl reaction buffer and water up to 10 µl was mixed per well of a 384 well clear optical reaction plate (ABgene). Each primer/cDNA set was set up in triplicate Copyright 2006 Biochemical Society and the whole experiment was repeated three times. For primer/probe combinations see Supplementary Table II. Experiments were performed on an ABI 7900 Real Time Sequence Detection System, using an Epmotion 5070 robot (Eppendorf, Germany) for plate set up. Assays were tested for efficiency using reference RNA and a 10 fold dilution series; only assays with greater than 99% amplification efficiency were used for subsequent work. Data analysis was performed using the Ct method [START_REF] Livak | Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method[END_REF], normalising against L19 and EEF1G allowing the fold change relative to wild type to be calculated. formaldehyde, and transferred to a FACS tube (Falcon, 352054). Control cells were incubated as above omitting the primary antibody step. Cell fluorescence was measured in a Becton Dickinson FACScan. Viable cells were gated using forward and side scatter, and the fluorescence of this population was measured.

FACS analysis

Mitogenesis assay -A simple mitogenesis assay was established using wild type and knock out primary MEFs. Cells were plated at 25,000 cells per well on a 24-well plate in 1ml of serum free DMEM. After incubating for 4 hours at 37°C, HGF or FGF2 was added at 10ng/ml. Cells were subsequently incubated at 37°C for 36 hours before being counted manually under a light microscope in 15 individual fields per well. The average number of cells per well was calculated and the fold increase/decrease was determined by comparing the average number of cells per well in growth factor induced and untreated control cells.

RESULTS AND DISCUSSION

An essential function of HS is to provide a scaffold on the cell surface for mediating the interaction of signalling molecules, whereby the sulphation patterns of HS largely determine ligand binding affinities. The endo-sulphatases Sulf1 and Sulf2 act as key players in sulphation pattern remodelling. To investigate the role of these enzymes in vivo, mSulf1 and mSulf2 knock out mice were generated.

Phenotypic characterisation -mSulf1 and mSulf2 knock out mice were generated by the classical approach, i.e. by insertion of a neomycin resistance cassette into exon 2 of the murine Sulf1 gene and exon 1 of the murine Sulf2 gene, as outlined in Figure 1A/B. Northern analysis of mRNA from knock out mouse embryonic fibroblasts (MEFs) verified the absence of wild type mSulf1 or mSulf2 transcripts, respectively (Figure 1C). By real-time PCR very low amounts of targeted, i.e. non-functional, transcripts could be detected (see below). mSulf1 -/-mice show no obvious phenotype. However, their mortality in the first month of life was slightly, but consistently increased. mSulf2 -/-mice show a small reduction in litter size and body weight.

Their mortality within the first 6 weeks was increased. Death of the mSulf2 -/-mice was usually associated with malformations, in particular of the brain (I. Kalus et al., in preparation).

Survivors appear normal. Double knock out animals were difficult to obtain. After many heterozygous matings we obtained very few fertile mSulf1 -/-/2 -/-animals, which were used for homozygous matings. In this way we recently succeeded to obtain five litters with a 2-fold reduced size (4.4 0.9 animals). These animals typically have a very short life span and a clear reduction in body weight. Double knock out MEFs could be obtained from E12.5 embryos and are described below.

The knock out approach is distinct from those which have previously investigated the function of the Sulf enzymes in that we are interpreting Sulf activity from the effects of its loss, as opposed to the over-expression studies frequently used, or the in vitro recapitulations of activity using isolated enzyme fractions. We believe this strategy is more biologically significant, as the amount and sub-cellular localisation of all the other components of the HS biosynthetic and modification machinery are maintained and thus are only influenced by the loss of Sulf activity.

Therefore the interplay between these factors and the Sulf enzymes is likely to be more representative of their true in vivo activity.

Molecular Phenotype: Effect on HS disaccharide composition and overall sulphation -As stated earlier, HS structure is tissue specific; however, it has previously been demonstrated that the loss/mutation of HS biosynthetic enzymes in a knock out model has a similar effect on all tissues [START_REF] Ledin | Heparan sulfate structure in mice with genetically modified heparan sulfate production[END_REF][START_REF] Merry | The molecular phenotype of heparan sulfate in the Hs2st-/-mutant mouse[END_REF]. We therefore chose MEFs as a convenient source of material for detailed structural analyses. Metabolically 3 H/ 35 S-radiolabelled HS was purified from primary wild type, mSulf1 -/-and mSulf2 -/-MEFs as well as from immortalised mSulf1 -/-/2 -/-and wild type MEFs.

Immortalisation was necessary as it proved impossible to maintain the mSulf1 -/-/2 -/-MEFs in culture for long enough to allow the preparation of radiolabelled HS. HS from all cells was digested with a combination of heparinases I, II and III and the resultant disaccharides resolved on a Bio-Gel P2 column. The disaccharides were then pooled and fractionated by SAX-HPLC (Table 1 and Supplementary Figure 1). Significant increases were observed in all 6S disaccharide species in the mSulf1 -/-HS compared with the wild type HS (Table 1). No overall increase in 6S

was observed in the mSulf2 -/-HS although there was a significant increase in the UA-GlcNAc(6S) disaccharide unit known to be associated with transition zones (see below). The mSulf1 -/-/2 -/-HS showed large increases in all 6S disaccharides. Comparing the relative abundance of 6S disaccharides between single and double knock out cells revealed a greater than two fold increase of S-domain associated disaccharides, UA-GlcNS(6S) and UA(2S)-GlcNS(6S), in the double knock out HS as compared to either single knock out HS, thus indicating cooperativity between the two Sulf activities within the S-domains (Table 1).

Interestingly, the increase in 6S in the mSulf knock out cells was accompanied by small but statistically significant reductions in other sulphation events, particularly in the level of 2S

(Table 1 and Supplementary Figure 1). It is therefore important to apply caution to the interpretation of experiments where loss (or gain) of Sulf activity influences HS-ligand interactions, as these may not necessarily be due to changes in 6S alone. The effect on NS and 2S may be due to the Sulf enzymes influencing the biosynthetic pathway. However, whether this occurs as a result of direct interaction between the Sulfs, the nascent HS chain and/or other processing enzymes in the Golgi remains to be determined. Alternatively, these changes in NS and 2S levels could result from downstream changes in HS biosynthetic enzyme expression levels as a consequence of changes in Sulf activity.

Effect on N-sulphate distribution and transition zones -Previous in vitro analysis using

QSulfs indicated that these enzymes preferentially remove 6S from the tri-sulphated IdoA(2S)-GlcNS(6S) disaccharide, and have no activity outside of the highly sulphated S-domains [START_REF] Viviano | Domain-specific Modification of Heparan Sulfate by Qsulf1 Modulates the Binding of the Bone Morphogenetic Protein Antagonist Noggin[END_REF]. In order to analyse the domain specificity of the mammalian Sulfs, low pH nitrous acid and heparinase III depolymerisation were used to isolate and analyse the respective S-domains, nonsulphated domains and transition zones which make up the HS chain [START_REF] Viviano | Domain-specific Modification of Heparan Sulfate by Qsulf1 Modulates the Binding of the Bone Morphogenetic Protein Antagonist Noggin[END_REF][START_REF] Merry | Highly sensitive sequencing of the Sulfated Domains of Heparan Sulphate[END_REF]. HS from all cell populations was cleaved at GlcNS residues using low pH nitrous acid [START_REF] Shively | Formation of anhydrosugars in the chemical depolymerization of heparin[END_REF]. We compared the profiles of the resulting oligosaccharides on a Bio-Gel P10 column to determine the proportion of GlcNS-containing disaccharides within the chains (Figure 2), and to additionally provide information on domain organization (see Figure 3A). Regions of contiguous N-sulphated disaccharides (S domains) will be depolymerised to disaccharides (dp2; where dp = degree of depolymerisation) with regions of alternating GlcNS and GlcNAc residues (transition zones)

generating tetrasaccharides (dp4). All profiles (Figure 2 left), were characteristic of a typical HS structure with N-sulphated residues occurring predominantly in S-domain clusters or in UA-GlcNS-UA-GlcNAc type transition zones. Minimal differences in overall macromolecular organization were visible between wild type and mSulf knock out cells. However, preparative fractionation using Bio-Gel P-10 and SAX-HPLC of low pH nitrous acid resistant tetrasaccharides did reveal important differences in sulphate composition of the transition zones (Figure 2 right). The transition zone derived tetrasaccharides separate into non-, mono-and disulphated species, with the variation primarily due to the addition of 6S, as these regions typically contain little 2S [START_REF] Maccarana | Domain structure of heparan sulfates from bovine organs[END_REF]. By comparing the contribution of each of these species we could demonstrate that there is a significant increase in 6S in the transition zones of all mSulf knock out HS, predominantly observed as an increase in mono-sulphated tetrasaccharides, while in the mSulf1 -/-/2 -/-HS there was a significant increase in both mono-and di-sulphated species (Figure 2 bottom).

Effect on S domain organization and composition -Heparinase III excises the S-domains in

HS by specific scission of the non-sulphated regions and transition zones (see diagram in Figure 3A) [START_REF] Merry | Highly sensitive sequencing of the Sulfated Domains of Heparan Sulphate[END_REF]. Following heparinase III digestion and subsequent fractionation of the resultant oligosaccharides by Bio-Gel P-10 (Figure 3B) we observed that, similar to the nitrous acid fractionation, the overall chain macromolecular organization was conserved between the HS types (not shown). Each of the size-defined peaks were isolated separately and re-fractionated using SAX-HPLC. For tetrasaccharides and longer oligosaccharides we took advantage of a Copyright 2006 Biochemical Society previous study in which heparinase III digestion was used to isolate S-domains from 3T3 HS prior to sequence analysis [START_REF] Merry | Highly sensitive sequencing of the Sulfated Domains of Heparan Sulphate[END_REF]. By comparing the profiles generated in this study with those from the 3T3 HS we were able to confidently identify many of the oligosaccharides generated.

Where possible, peak assignments were confirmed by isolation and disaccharide compositional analysis.

Disaccharide analysis -The disaccharide peak generated by heparinase III contained three species corresponding to UA-GlcNAc, UA-GlcNS and UA-GlcNAc(6S). These disaccharides are typically restricted to the non-sulphated and transition regions of the HS chain, confirming heparinase III enzyme specificity. In comparison to wild type HS, a significant two fold increase in UA-GlcNAc(6S) residues (p 0.01) was seen in mSulf1 -/-material (Figure 3C, Table 2).

Compensatory decreases in the non 6-O-sulphated disaccharide UA-GlcNAc were observed balancing this increase in UA-GlcNAc(6S). A similar decrease in UA-GlcNAc residues was also seen in mSulf2 -/-material, although only a 40% increase in 6-O-sulphated UA-GlcNAc(6S) residues was apparent. mSulf1 -/-/2 -/-material also showed a significant (60%) increase in UA-GlcNAc(6S) when compared to the wild type control. Thus, as verified by the independent nitrous acid mediated domain analysis (see above), both mSulf1 and to a lesser extent mSulf2 are active 6-O-endosulphatases within the non-sulphated and transition domains.

Tetrasaccharide analysis -Only one major tetrasaccharide species was visible in all MEF HS types following separation by SAX-HPLC (data not shown). This tetrasaccharide corresponds to UA-GlcNS-IdoA(2S)-GlcNAc, confirmed by disaccharide analysis.

Hexasaccharide analysis -Eight major peaks were detected in all heparinase III derived hexasaccharides (dp6s), labelled a-h (Figure 3D). These species fall into two groups, those that are 6S deficient (6a-6c) and those with 6S (6d-6h). Peaks a-d have previously been sequenced [START_REF] Merry | Highly sensitive sequencing of the Sulfated Domains of Heparan Sulphate[END_REF], and are identified as shown in Figure 3D. Hexasaccharides e-h have an unknown sequence, however the peaks form a repeating pattern with a, b and c anticipated to have the same relationship to each other as d, e and f, etc. We could therefore predict the sequences of these species. Compared to wild type, mSulf1 -/-cell extract HS contains fewer lower sulphated sequences (peaks a-c), whereas hexasaccharides containing one or more 6-O-sulphated glucosamine residue are enriched (peaks d-h) (Figure 3D). A 53% decrease in peak a was seen in mSulf1 -/-HS, consistent with a 26% and 27% increase in peaks d and g which are modifications of a, to contain either one or two 6S glucosamine residues, respectively. The mSulf2 -/-profiles indicate little difference in the sulphated regions compared to wild type, where the only consistent difference was an 18% increase in peak d, containing one 6-O-sulphated glucosamine residue. Comparison of wild type and mSulf1 -/-/2 -/-hexasaccharides extended the trend established in the mSulf1 -/-material, showing a marked increase in highly sulphated hexasaccharide species. This data is thus comparable to the compositional disaccharide analysis whereby distinct differences were detected between the various MEF cells in UA-GlcNS(6S) and UA(2S)-GlcNS(6S) residues contained within the S-domains (Table 1).

Octasaccharide analysis -As observed previously, the complexity of the S-domain SAX-HPLC profiles increased with increasing length [START_REF] Merry | Highly sensitive sequencing of the Sulfated Domains of Heparan Sulphate[END_REF]. Numerous species of octasaccharide (dp8) were visible, denoted a-i (Figure 3E). Some of these sequences have been previously characterised [START_REF] Merry | Highly sensitive sequencing of the Sulfated Domains of Heparan Sulphate[END_REF], however oligosaccharide peaks labelled g-i are of unknown composition, and are likely to contain more than one octasaccharide species per peak, an observation supported by subsequent analytical work [START_REF] Robinson | VEGF165-binding sites within heparan sulfate encompass two highly sulfated domains and can be liberated by K5 lyase[END_REF]. mSulf1 -/-material contained clearly reduced levels of species a-c, representing S-domain regions devoid of 6S (Figure 3E). However mSulf1 -/-material was enriched in species which elute at higher salt concentrations from the column, most noticeably in octasaccharides f-i, with a 40% total increase in these saccharides compared to wild type. Again, the trends observed for mSulf1 -/-material was amplified when comparing immortalized wild type and mSulf1 -/-/2 -/-octasaccharides (Figure 3E).

Overall, the analyses of heparinase III generated hexa-and octasaccharides demonstrate that there is a clear increase in 6S in mSulf1 -/-and mSulf1 -/-/2 -/-material observed in all sizes of Sdomain studied. mSulf2 -/-HS however, shows an increase in 6S almost exclusively within the non-sulphated and transition zones. Thus, the mSulf2 knock out primarily becomes manifest outside the S-domains. Importantly, this observation contrasts with previous studies on the avian Sulf1 and Sulf2 orthologs where activity was restricted to within the S-domains. Specific analysis of the longer sulphated S-domains released by heparinase III as hexa-(dp6) and octasaccharides (dp8) was made possible by incorporation of data from previous studies, in which we have sequenced many of these sub-species [START_REF] Merry | Highly sensitive sequencing of the Sulfated Domains of Heparan Sulphate[END_REF]. This demonstrated clear differences in the effect of each mSulf knock out on S-domain sulphation patterning. As shown in Figure 3 D/E, mSulf1 -/-HS contained a greater proportion of dp6s with either one (6d-f) or two (6g-h) 6S groups; a corresponding decrease in the non-6S species (6a-c) was observed. There was also some indication that mSulf1 activity may be favoured on adjacent GlcNS(6S) residues as the increase in species 6g containing this pattern, was greater than that seen in 6d, containing only one GlcNS(6S) residue. mSulf1 -/-/2 -/-HS-derived S-domain oligosaccharides were similar to, but more extreme than those seen in the mSulf1 -/-HS. This detailed domain specific HS analysis provides the molecular basis needed to gain insight into the effect of dynamic changes in 6S on the biological phenotypes of the mSulf knock out mice, which are currently being characterised.

Regulation underlying 6S patterning -We were interested in a possible co-regulation of mSulf1 and mSulf2 at the level of gene expression. Real time PCR analysis identified an upregulation of mSulf1 expression in the mSulf2 -/-MEFs (Figure 4, mSulf1 increased by 4.3 fold in mSulf2 -/-cells), which was confirmed by Northern analysis (Figure 1C). This compensatory increase of mSulf1 in mSulf2 -/-MEFs is in good agreement with our disaccharide data where mSulf2 -/-material showed a surprising decrease in S-domain related 6S disaccharides (UA-GlcNS(6S), UA(2S)-GlcNS(6S)), and where the effect of mSulf1 -/-/2 -/-dual knock out was in excess of that suggested by either single knock out alone.

This differential activity of mSulf1 and mSulf2 in the mSulf knock out MEFs may have important biological implications. Indeed it is important to note that although no mouse 6-Osulphotransferase knock outs have yet been published; in vitro experiments suggest that individual isoforms are non-specific and complementary in their sites of action [START_REF] Do | Overexpression of heparan sulfate 6-O-sulfotransferases in human embryonic kidney 293 cells results in increased N-acetylglucosaminyl 6-O-sulfation[END_REF][START_REF] Smeds | Substrate specificities of mouse heparan sulphate glucosaminyl 6-O-sulphotransferases[END_REF].

Therefore the predominant control of 6S patterning as seen by binding ligands is probably due to mSulf activity. How mSulf activity is dynamically coupled to generate specific sulphation patterns is likely to be critical for understanding how changes in HS structure are generated to direct cell signalling events during development and disease.

mSulf activity alters surface epitopes recognized by HS-specific antibodies -Methods to

quickly and non-destructively analyse cell-surface HS sulphation patterns could significantly improve our ability to screen for sulphation state changes linked with developmental processes and disease. To approach this issue we have recently developed a method that is based on the use of a panel of phage-display derived antibodies which have specificity for a variety of saccharide patterns within HS. The ability of these antibodies to recognise specific native HS structures (some of which have recently been defined) can provide a 'fingerprint' of HS epitope expression on a cell [START_REF] Van Kuppevelt | Generation and application of type-specific anti-heparan sulfate antibodies using 19 Biochemical Journal Immediate Publication. Published on 11 Aug 2006 as manuscript BJ20060848 Copyright 2006 Biochemical Society phage display technology. Further evidence for heparan sulfate heterogeneity in the kidney[END_REF]. When this panel of antibodies was screened against immortalised knock out and wild type cells, we found that for some of the antibodies there was little change in binding, for others the binding was decreased in the knock outs and for one antibody, RB4EA-12, binding was increased, particularly for the mSulf1 -/-/2 -/-line (Figure 5). This differential binding Copyright 2006 Biochemical Society behaviour supports biochemical analyses, as RB4EA-12 has previously been shown to have a specific requirement for 6S [START_REF] Dennissen | Large, tissue-regulated domain diversity of heparan sulfates demonstrated by phage display antibodies[END_REF][START_REF] Jenniskens | Heparan sulfate heterogeneity in skeletal muscle basal lamina: demonstration by phage display-derived antibodies[END_REF]. Furthermore, antibodies EV3C3 and HS4E4 which showed reduced binding to mSulf knock out MEFs are known to be inhibited by 6S [START_REF] Dennissen | Large, tissue-regulated domain diversity of heparan sulfates demonstrated by phage display antibodies[END_REF]. Therefore, in keeping with the documented complexity of Sulf activity on HS-ligand binding, we found that depending on the antibody used, loss of mSulf activity had a negative effect (i.e. less binding), no effect or a positive effect on antibody binding (Figure 5) demonstrating the potential for differential regulation of ligand-binding epitopes within HS by altered mSulf activity. This further indicates that mSulf activity can simultaneously influence multiple ligand-binding events, likely through induced changes in HS structure [START_REF] Raman | Structural insights into biological roles of protein-glycosaminoglycan interaction[END_REF]. In view of the paucity of detailed 3D information on HS motifs, this panel of antibodies provides a useful screening tool for evaluating these changes.

Loss of mSulf activity increases the mitogenic response of MEFs -The antibody study

above suggested that ligand-binding may be affected by mSulf activity. However, the regulatory activity of HS for its many interacting factors is more complex than can be explained by binding affinity alone [START_REF] Merry | Highly sensitive sequencing of the Sulfated Domains of Heparan Sulphate[END_REF] and indeed, in some instances high affinity binding is inhibitory for activity. mSulf activity has previously been linked with the activity of a number of HS-dependent growth factors and morphogens, including the multi-functional FGF2 [START_REF] Wang | QSulf1, a heparan sulfate 6-O-endosulfatase, inhibits fibroblast growth factor signaling in mesoderm induction and angiogenesis[END_REF]. We therefore employed a simple proliferation assay using primary MEFs to ascertain if there was any differential activity between the mSulf mutant lines. MEF cell cultures have previously been shown to be suitable for these studies [START_REF] Merry | The molecular phenotype of heparan sulfate in the Hs2st-/-mutant mouse[END_REF]. Results demonstrate a significant increase in proliferation of both single mSulf knock out cells, particularly for mSulf1 -/-in which FGF2 induced a three-fold increase in proliferation compared to wild type (Figure 6). No mitogenic response was observed with HGF in any of the MEFs tested, in agreement with previous studies (C. Merry, unpublished).

Conclusions

In conclusion, loss of mSulf1 or mSulf2 activity in MEFs is associated with increases in HS 6S.

Notably, the domain-specific pattern of this increase, its influence on other sulphation activity and the potential effect this has on ligand binding patterns (as revealed by antibody reactivities) are different for the two enzymes. Additionally, this study presents strong evidence to suggest that mSulf1 and mSulf2 act cooperatively and are the major regulators of 6S patterns within HS (Figure 7). The focus of future study will be the analysis of the knock out mice themselves, from wild type, mSulf1 -/-and mSulf2 -/-MEFs, using probes for mSulf2, mSulf1 and -actin. Following treatment with low pH nitrous acid, HS was fractionated by Bio-Gel P-10 (left hand panel) to separate size-defined oligosaccharides (dp2, dp4 etc. where dp=degree of depolymerisation e.g. dp2=disaccharide). The proportion of counts in each sized peak was found to be very similar between the samples -dp2=14-15%, dp4 28-30% (primary cells) and dp2=22%, dp4=31-32% (immortalised cells). Tetrasaccharides (from UA-GlcNAc-UA-GlcNS regions of the chains) were further fractionated by SAX-HPLC (right hand panels) into di-, mono-and non-sulphated species. These were then quantified (bottom panels) for the primary Exposure to HGF resulted in no mitogenic response in all MEFs. The addition of 6S to the nascent HS chain within the Golgi is mediated by a family of 6-Osulphotransferases. Our data suggests that these modifications are fairly extensive throughout the chain (leading to ~40% of disaccharides containing 6S groups). It is the activity of the mSulf enzymes that reduces this level to the ~15% which is found in HS prepared from tissues and which finalises the complex sulphate patterning observed on mature HS chains.

TABLES

  H]glucosamine hydrochloride (20-45 Ci mmol -1 ) and Na 2 35 SO 4 (1050-1600 Ci mmol -1 ) were from GE Healthcare and Perkin Elmer Life Sciences, respectively. Recombinant human FGF2, and HGF/SF were from R&D Systems (Abingdon, UK). Generation of knock out mice, genotyping and Northern analysis -By screening a genomic cosmid library of the 129Sv/Ola mouse (German Resource Center for Genome Research, Berlin) with labelled cDNA fragments as probes, cosmids were identified that carried fragments of the mSulf1 or mSulf2 gene. A 3.1 kb HindIII fragment carrying exon 2 of mSulf1 and a 3.8 kb SalI/EcoRI fragment carrying exon 1 of mSulf2 with about equal homology arms of 1.5-2.0 kb on both sides (Figure 1 A/B) were sequenced, subcloned into pBluescriptII SK and used for construction of the targeting vectors. The neomycin resistance cassette, a blunted XhoI/SalI fragment of the pMC1neo vector (Stratagene), was inserted into the BsaAI site of mSulf1 exon 2

mSulf2 5 '

 5 -external probe was a 420 bp EcoRI/HindIII fragment of EST clone IMAGp998G091033Q2 (German Resource Center for Genome Research, Berlin).

Figure 1 .

 1 Figure 1. Targeted disruption of murine Sulf1 and Sulf2. A, B: Schematic representation of the murine Sulf1 (A) and Sulf2 (B) loci in the area of the targeted exon 2 (A) and exon 1 (B), respectively. The three illustrations show the wild type locus (top), the targeting vector (middle) and the recombined allele (bottom). Recombined alleles carry the neomycin resistance cassette inserted into the BsaAI site of mSulf1 exon 2 (A) and the BseRI site of mSulf2 exon 1 (B), respectively. The location of the 5' and 3' external probes used for Southern hybridization and the relevant restriction sites and resulting fragments are indicated. Successful and specific germline targeting was confirmed by Southern blotting of StuI-or SacIdigested genomic DNA using the indicated external probes (see blot insets in A and B, respectively). Results were confirmed by independent external probes detecting the indicated 3.9 kb fragment in BamHI-digested DNA (SULF1, A) and the 5.2 kb fragment in KpnI/XhoIdigested DNA (SULF2, B) (data not shown). C: Northern blot analysis of total RNA (10 µg)

Figure 2

 2 Figure 2 Low pH nitrous acid depolymerisation of cell extract HS.

  MEFs: wild type (black bars), mSulf1 -/-(white bars) and mSulf2 -/-(check bars); and the immortalised MEF lines: wild type (downwards diagonal bars) and mSulf1 -/-/2 -/-(upwards diagonal bars). All profiles are representative of at least three separate fractionations.

Figure 3 .

 3 Figure 3. Sulphated Domain analysis of cell extract HS. HS was digested with heparinase III. A: Site of action of heparinase III (white arrow) and low pH nitrous acid (black arrows). Non-N-sulphated domains (black box), transition zone NS/NAc (white box) and S-domains (striped box) are indicated. B: Typical HS profile following

Figure 4 .

 4 Figure 4. Real-time PCR analysis of the expression of mSulf enzymes in knock out MEFs.An average of the two house-keeping genes L19 and EEF1G was used to normalise the cDNAs of each sample relative to each other. The fold change for mSulf1 -/-and mSulf2 -/-MEFs was calculated relative to the primary MEF wild type control. Values for mSulf1 -/-/2 -/-MEFs were calculated relative to an immortalised wild type control. A fold change greater than 2 was judged to be significant (dotted line). mSulf1 -/-(clear bars), mSulf2 -/-(check bars) and mSulf1 -/-/2 -/- (upwards diagonal bars). mSulf1 expression was reduced 10 fold in the mSulf1 -/-cells, increased 4.3 fold in the mSulf2 -/-cells and decreased 18 fold in the mSulf1 -/-/2 -/-cells. mSulf2 expression was decreased 2.2 fold in the mSulf1 -/-cells, decreased 77 fold in the mSulf2 -/-cells and decreased 19 fold in the mSulf1 -/-/2 -/-cells.

Figure 5 .

 5 Figure 5. HS epitope-specific ScFv antibodies show altered cell binding characteristics for mSulf knock out MEFs. Immortalised wild type (black line), mSulf1 -/-(green line), mSulf2 -/-(blue line) and mSulf1 -/-/2 -/- (purple line) MEFs were incubated with a panel of HS sequence-specific ScFv antibodies. These were then analysed by FACS to demonstrate a shift in binding profile for each of the MEFs with the various antibodies. The profiles shown for three of the antibodies, as indicated, are representative of at least three different experiments. Control profiles (using secondary antibody alone) are shown as the filled red profiles.

Figure 6 . 22 Biochemical

 622 Figure 6. Loss of mSulf activity increases the mitogenic response of MEFs to FGF2.

Figure 7 .

 7 Figure 7. Sulfs are major regulators of 6-O-sulphation within HS.

Figure 3 UA

 3 Figure 3

6 Biochemical Journal Immediate Publication. Published on 11 Aug 2006 as manuscript BJ20060848

10 Biochemical Journal Immediate Publication. Published on 11 Aug 2006 as manuscript BJ20060848

13 Biochemical Journal Immediate Publication. Published on 11 Aug 2006 as manuscript BJ20060848

Table 1 : Disaccharide composition and overall sulphation of knock out HS from cell

 1

	extract fractions. Im. = immortalised
		WT	mSulf1 -/-	mSulf2 -/-	Im. WT	Im.
		(%)	(%)	(%)	(%)	mSulf1 -/-/2 -/-
						(%)
	UA-GlcNAc	49.5	49.6	52.7	45.4	37.5
	UA-GlcNS	21.5	20.0	22.2	23.5	20.3
	UA-GlcNAc(6S)	5.9	11.1	7.7	7.5	12.6
	UA(2S)-GlcNAc	2.7	1.7	1.3	1.4	1.0
	UA-GlcNS(6S)	3.7	5.8	3.1	5.1	13.7
	UA(2S)-GlcNS	10.9	4.5	8.8	10.4	1.8
	UA(2S)-	5.8	7.4	4.2	6.8	13.1
	GlcNS(6S)
	Total NAc	58.1	62.4	61.8	54.2	51.1
	Total NS	41.9	37.6	38.2	45.8	48.9
	Total 2S	19.3	13.5	14.3	18.5	15.9
	Total 6S	15.4	24.2	15.0	19.4	39.3

24 Biochemical Journal Immediate Publication. Published on 11 Aug 2006 as manuscript BJ20060848 Copyright 2006 Biochemical SocietyTable 2 :

 2 Relative amounts of heparinase III generated disaccharides. Im.=immortalised

		WT	mSulf1 -/-	mSulf2 -/-	Im.	Im.
		(%)	(%)	(%)	WT	mSulf1 -/-/2 -/-
					(%)	(%)
	UA-GlcNAc	75.5	69.7	70.5	68.1	66.1
	UA-GlcNS	19.9	21.4	23.0	24.7	22.4
	UA-GlcNAc(6S)	4.7	9.0	6.5	7.2	11.5

Biochemical Journal Immediate Publication. Published on 11 Aug 2006 as manuscript BJ20060848

Biochemical Journal Immediate Publication. Published on 11 Aug 2006 as manuscript BJ20060848 Copyright 2006 Biochemical Society

Acknowledgments -We would like to thank Nicole Tasch, Ellen Eckermann-Felkl, Martina Balleininger and Annegret Schneemann for excellent technical assistance, Peter Gruss for providing embryonic stem cells and Paul Saftig for his advice in knock out mouse generation. This work was supported by the Deutsche Forschungsgemeinschaft, the Fonds der Chemischen Industrie, Cancer Research UK and the British Council UK-Netherlands Partnership Programme in Science.

Abbreviations -The abbreviations used are: HSPG, heparan sulfate proteoglycan; HS, heparan sulfate; FGF, fibroblast growth factor; HGF/SF, hepatocyte growth factor/scatter factor; VEGF, vascular endothelial growth factor; HB-EGF, heparan binding -EGF like growth factor; UA, hexuronic acid; UA, delta 4,5 unsaturated hexuronic acid; GlcA, glucuronic acid; IdoA, iduronic acid; GlcNAc, N-acetylglucosamine; GlcNS, N-sulfoglucosamine;