Arne Homann

Rebekka Biedendieck † ‡

Sven Götze

Dieter Jahn

Jürgen Seibel
email: j.seibel@tu-bs.de

Insights into polymer versus oligosaccharide synthesis -Mutagenesis and mechanistic studies of a novel levansucrase from Bacillus megaterium

Keywords: Ls, levansucrase, Ps, polysaccharides, Os, oligosaccharides levansucrase, Bacillus megaterium, fructo-oligosaccharides, site-directed mutagenesis

A novel levansucrase was identified in the supernatant of a cell culture of Bacillus megaterium DSM319. In order to test for the contribution of specific amino acid residues to levansucrase catalysis the wild-type enzyme along with 16 variants based on sequence alignments and structural information (see below) were heterologously produced in Escherichia coli. The purified enzymes were characterised kinetically and the product spectrum of each variant was determined. Comparison of the X-ray structures of the levansucrases from Gram positive Bacillus subtilis and Gram negative Gluconacetobacter diazotrophicus in conjunction with the corresponding product spectra identified crucial amino acid residues responsible for product specifity and catalysis. Highly conserved regions like the previously described RDP and DXXER motifs were identified as being important. Two crucial structural differences localised at the amino acid residues Arg 370 and Asn 252 were of high relevance in polymer versus oligosaccharide synthesis.

INTRODUCTION

Global regulatory responses allow Bacilli to adapt to changes in environmental conditions [START_REF] Priest | Extracellular enzyme synthesis in the genus Bacillus[END_REF].

These responses often include changes in the transcription rates of large sets of target genes and thus, the control of a wide variety of cellular processes in these Gram positive bacteria.

One of these processes is the synthesis of degradative and polymer building enzymes. The secretion of these enzymes enables the cell to build and utilise highly polymeric nutrients like polysaccharides (Ps), nucleic acids, peptides, and lipids. Ps, e.g. inulin and non digestable oligosaccharides (Os) are known to have human health benefit. They are used by the food industry as nutrition supplements [2; 3] and their potential for immune stimulation is currently under investigation [START_REF] Bornet | Nutritional aspects of short-chain fructooligosaccharides: natural occurrence, chemistry, physiology and health implications[END_REF].

Levansucrases form Ps by transfer of fructosyl units to Os. This class of enzymes (E.C. 2.4.1.10) is common in eukaryotes and prokaryotes, e.g. plants, fungi (Aspergillus niger [START_REF] Hidaka | A fructooligosaccharide-producing enzyme from Aspergillus niger ATCC 20611[END_REF],

Saccharomyces cerevisiae [START_REF] Straathoff | Invertase-catalysed fructosyl transfer in water-organic solvent systems[END_REF]), and bacteria (Lactobacilli sp. [START_REF] Van Hijum | Structure-function relationships of glucansucrase and fructansucrase enzymes from lactic acid bacteria[END_REF], Bacillus subtilis [START_REF] Chambert | Kinetic studies of levansucrase of Bacillus subtilis[END_REF]). In these organisms they build up an extracellular energy reserve of fructans. These exopolysaccharides function as stabilizers of cell membranes protecting them against water stress caused by drought or temperature shift [START_REF] Vijn | Fructan: more than a reserve carbohydrate?[END_REF]. Furthermore, it is postulated that fructans may be important for survival of bacteria, for phytopathogenesis and symbiosis [START_REF] Cote | Metabolism in microorganisms, part I: levan and levansucrase[END_REF].

Fructosyl transfering enzymes are divided into 2 classes based on the products formed.

Inulosucrases (E.C.2.4.1.9) mainly form β(2→1) bonds producing the polysaccharide inulin while the second class, levansucrases (E.C. 2.4.1.10), form levan which is β(2→6) linked.

The Os and Ps are composed of a head group of α-D-glucopyranoside to which the fructosyl units are coupled.

One of the best characterised members of Gram positive Bacilli, B. subtilis, secretes the Ls, SacB, into its environment if high amounts of sucrose are available [START_REF] Steinmetz | Carbohydrate Catabolism: Pathways, Enzymes, Genetic Regulation, and Evolution. Bacillus subtilis and Other Gram-Positive Bacteria[END_REF]. Ls catalyses the hydrolysis of sucrose as well as transglycosylation reactions with a growing Os chains as acceptor. B. subtilis Ls synthesises dominantly high molecular levan with a molecular size of up to 3 x 10 6 Da. In contrast, the Ls from the Gram-negative Gluconacetobacter diazotrophicus mainly synthesises short chain Os and low amounts of levan [START_REF] Hernandez | Isolation and enzymic properties of levansucrase secreted by Acetobacter diazotrophicus SRT4, a bacterium associated with sugar cane[END_REF]. Until now, there has been no sufficient explanation for the formation of these different transfructosylation products.

In this study, a novel Ls from B. megaterium which is within the same taxonomic group as B. subtilis [13; 14] was isolated and its product spectra and reaction kinetics were characterised.

Based on amino acid sequence alignments and structural comparisons with enzymes of the glycoside hydrolyse family 68 [START_REF] Coutinho | Carbohydrate-active enzymes: an integrated database approach[END_REF], 16 mutants have been designed by site-directed mutagenesis. A comparison with the X-ray structures of the levansucrases from B. subtilis (74 % identity on amino acid level) and G. diazotrophicus (26 %) indicated an overall similar protein structure, carrying identical essential amino acid motifs (RDP, DXXER) but also important differences [START_REF] Altschul | Gapped BLAST and PSI-BLAST: a new generation of protein database search programs[END_REF][START_REF] Schäffer | Improving the accuracy of PSI-BLAST protein database searches with composition-based statistics and other refinements[END_REF][START_REF] Meng | Structural framework of fructosyl transfer in Bacillus subtilis levansucrase[END_REF][START_REF] Martinez-Fleites | Crystal structure of levansucrase from the Gram-negateive bacterium Gluconacetobacter diazotrophicus[END_REF]. A detailed biochemical investigation of the 16 Ls variants identified common structure-based functional principles and catalytic specifity modifying determinants.

MATERIALS AND METHODS

Identification of the levansucrase, SacB, from Bacillus megaterium B. megaterium was cultivated aerobically at 37 °C in Luria-Bertani (LB) medium [START_REF] Sambrook | Molecular cloning; a laboratory manual[END_REF] supplemented with 0.5 % (w/v) sucrose. Samples collected at indicated time points after cultivation start were separated by centrifugation into cells and cell-free growth medium (2600 x g, 15 min, 4 °C). For SDS-PAGE [START_REF] Sambrook | Molecular cloning; a laboratory manual[END_REF] analysis of the secreted proteins, 2.5 ml of cell-free growth medium were desalted using PD-10 columns (GE Healthcare, Uppsala, Sweden) prior to concentration by lyophilisation. Proteins were separated by 10 % (w/v) SDS-PAGE and stained with Coomassie Brillant Blue.

Proteins were identified by peptide mass fingerprinting, as well as by using postsource decay fragmentation data recorded with a Bruker Ultraflex matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometer (Ultraflex; Bruker Daltonics, Bremen, Germany). The spectra were evaluated with the Biotools software (Bruker Daltonics) along with the MASCOT search engine (version 1.9; Matrix Science, London, United Kingdom). The criteria used to accept protein identifications included the extent of sequence coverage (minimum, 30%), the number of peptides matched (minimum number, 5), and the probability score (minimum Mowse score, 70).

Construction of the levansucrase variants by site-directed mutagenesis

Molecular biology methods were outlined previously by Sambrook et al. [START_REF] Sambrook | Molecular cloning; a laboratory manual[END_REF]. The 1371 bp coding sequence of B. megaterium sacB lacking the signal peptide coding sequence at the 5' end of the gene was amplified by PCR using primers pRBEc1_for (5'-TTATTCCATGGCTAAAGGAAACGACAGCAAAG-3'; Nco I-site italicised) and pRBEc1_rev (5'-TATCAGGATCCGCTATTGCAAAGCGCTCAGTC-3'; BamH I-site itali-cised). The PCR-product was digested with Nco I and BamH I and ligated into the previously Nco I/BamH I cut pET11d (Novagen®, San Diego, CA, USA) yielding pRBEc1.

The sacB gene was mutated using the QuikChange® site-directed mutagenesis kit (Stratagene). The forward primers used to generate the mutations were as follows (the exchanged amino acids are lower case):

Levansucrase production and purification

For cloning and cultivation, E. coli cells carrying the corresponding plasmid were grown in Luria-Bertani (LB) medium [START_REF] Sambrook | Molecular cloning; a laboratory manual[END_REF] supplemented with 0.5 mM ampicillin at 37 °C in shaking flasks. Recombinant gene expression was induced with 0.5 mM IPTG after 3 hours and the temperature was lowered to 26 °C for 20 h. For cell disruption, the cell culture was centrifuged at 4 °C for 20 min at 2600 x g and the supernatant discarded. The cells were washed and subsequently dissolved in 50 mM Sorensen's phosphate buffer (pH 6) and disrupted by sonication (sonoplus, Bandelin electronics with sonotrode MS72) for 4 min at 70 % power and 50 % time. After centrifugation at 4 °C and 5200 x g for 20 min, the supernatant contained the recombinantly produced Ls. The enzyme was purified by FPLC using a 15 ml CM-Sepharose column with a controlled pump mix system (Liquid Chromatography Controller LCC-500 Plus, LKB Pump P-500, Pharmacia) and a UV-detector (Biotech UV-MII, Pharmacia). Loading was done with the crude cell extract in 50 mM Sorensen's phosphate buffer (pH 6.6) with a flow rate of 0.5 ml min -1 . For elution, a linear gradient from 50 mM to 1 M of Sorensen's phosphate buffer (pH 6.6) was used. Fractions of 2 ml were collected (autosampler LKB Frac-100, Pharmacia) and analysed by SDS-PAGE (Figure 1). The protein concentrations of the fractions were measured photometrically at λ = 280 nm (NanoDrop Spectrophotometer ND-100, peqLab Biotechnology).

Characterisation of the wild-type levansucrase

The optimal enzyme concentration was determined in 50 mM Sorensen's phosphate buffer (pH 6.6) at 37 °C and 500 mM sucrose. The tested enzyme concentrations were 73.60, 29.44, 14.72, 7.36, and 4.91 mg l -1 purified Ls, respectively. Reactions were stopped after 1 h by heating (100 °C, 10 min). The release of fructose and glucose from sucrose was analysed by TLC. The optimal substrate concentration for the transfructosylation was determined in 50 mM Sorensen's phosphate buffer (pH 6.6) at 37 °C using 7.36 mg l -1 levansucrase. Sucrose concentrations tested were 1 M, 750, 500, and 250 mM, respectively. Reactions were stopped after 1 h by heating (100 °C, 10 min) and analysed as described above. The pH optimum was determined in 50 mM Sorensen's phosphate buffer at 37 °C, 500 mM sucrose using 7.36 mg l -1 enzyme concentration. The reactions at pH-values of 5, 5.6, 6, 6.6, 7, 7.6, and 8, respectively, were stopped by heating (100 °C, 10 min). Carbohydrate content was analysed by High Performance Anion Exchange Chromatography, (HPAEC, pre column: CarboPac PA1, 4 x 50 mm, main column: CarboPac PA1, 4 x 250 mm Dionex, conductivity detector PAD-2, Dionex at 1 ml min -1 ). The optimal reaction temperature was determined in 50 mM Sorensen`s buffer (pH 6.6) at various temperatures [START_REF] Chambert | Polymerase and hydrolase activities of Bacillus subtilis levansucrase can be separately modulated by site-directed mutagenesis[END_REF][START_REF] Geier | Characterization and influence on virulence of the levansucrase gene from the fireblight pathogen Erwinia amylovora[END_REF][START_REF] Delano | The PyMOL Molecular Graphics System[END_REF]40, and 50 °C). The optimal reaction conditions for the wild-type and the variants were at an enzyme concentration of 7.36 mg l -1 , a substrate concentration of 500 mM sucrose, and a reaction time of 60 min. The release of glucose was measured by HPAEC.

Determination of the kinetic parameters

The kinetic parameters of the wild-type and the variants were determined in 50 mM Sorensen's phosphate buffer (pH 6.6) at 37 °C. Enzyme concentration was 7.36 mg l -1 and the reaction time was 60 min. The substrate concentration varied from 500 to 1 mM sucrose. The release of glucose was measured by HPAEC.

Determination of the product spectrum

The product spectrum was determined for wild-type Ls and each variant using 50 mM Sorenson's phosphate buffer (pH 6.6) and 500 mM sucrose at 37 °C. The enzyme concentration was 100 mg l -1 . Samples were taken at indicated times for a period of 9 d. The reaction products were identified using standards (1-kestose, 6-kestose, nystose, all 10 mg l -1 ) by HPAEC. All unidentified Os were separated by silica gel column chromatography (3.5 x 500 mm, flow rate 500 µl min -1 ). The structures were analysed by NMR and mass spectroscopy.

Characterisation of the polysaccharide

The formation kinetics of Ps were examined in 50 mM Sorenson's phosphate buffer (pH 6.6) at 37 °C, 500 mM sucrose and 7.36 mg l -1 enzyme concentration. Samples were taken after 30 sec, 30 min, 60 min, 24 h, and 72 h, respectively, and analysed by TLC. The production of Ps was performed in Sorensen's phosphate buffer (50 mM, pH 6.6) at 37 °C with 100 mg l -1 Ls. After 10 min the polymer was precipitated with 1 volume of ethanol (99 % (v/v)). After centrifugation (14,000 x g, 5 min, 4 °C) the supernatant was discarded and the precipitate dried by lyophilisation. Size distribution of formed Ps was characterised by Multi Angle Laser Light Diffraction (MALLD, Dawn DSP, Wyatt Technologies) and RI analysis (RI-101, Shodex).

For the identification of the linkage type, the Ps were produced as described above and dialysed against water at 4 °C over night (Slide-A-Lyzer Dialysis cassettes, Pierce, MWCO 30 kDa). The sample was centrifuged for 5 min at 14,000 x g and the supernatant discarded.

The precipitate was washed 4 times with 1 ml of ethanol in water (66 % (v/v)). The Ps were dried by lyophilisation and dissolved in 900 mM NaOD for NMR spectroscopy.

NMR spectroscopy

All samples for NMR analysis were freeze-dried from MilliQ water and dissolved in D 2 O (or 900 mM NaOD). Spectra were recorded on a Bruker DRX600 or Bruker DRX400 operating at 600 and 400 MHz for 1 H, respectively. One-dimensional 1 H and 13 correlation)] were recorded using standard pulse programs at 38.8 °C, and data were analysed using the software packages XWINNMR (Bruker, Germany). Chemical shifts are given in the δ-scale (ppm) and coupling constants J in Hz.

RESULTS AND DISCUSSION

Characterisation of the wild-type levansucrase SacB from Bacillus megaterium

When B. megaterium was cultivated in LB-medium supplemented with and without 0.5 % (w/v) sucrose, SDS-PAGE comparison of the secretome from both cultivations revealed a dominant protein solely produced in the presence of sucrose (Figure 1). This protein with a relative molecular mass of 52,000 was identified as described in materials and methods (Figure 2). The analysed peptide fragments were homologous to fragments predicted for the [START_REF] Sun | A protein database constructed from low-coverage genomic sequence of Bacillus megaterium and its use for accelerated proteomic analysis[END_REF]. The third and last gene in the operon in B. subtilis encodes a 520 amino acid protein which might function as a permease [START_REF] Sun | A protein database constructed from low-coverage genomic sequence of Bacillus megaterium and its use for accelerated proteomic analysis[END_REF]. Interestingly, the protein encoded by a gene sequence following the levanase gene sacB in B. megaterium was annotated as bacterioferritin (Hundertmark, Hiller, Münch, Jahn, 2005, unpublished data). The sacB of B. megaterium was amplified lacking the coding sequence of the N-terminal signal peptide and cloned into an E. coli expression vector.

After production and purification of the recombinant Ls (Figure 3) the optimal reaction conditions of B. megaterium SacB were examined as described in materials and methods. A broad pH optimum between pH 6.0 and 7.0 for the Ls activity was observed. The enzyme activity significantly dropped down at pH values below 5.6 and above 7.6. The temperature optimum of the Ls was found at 45 °C. In standard reactions, long-term temperature treatment revealed a high protein stability at 37 °C for at least 24 h. The optimal reaction conditions for fructosyl transfer were determined with the purified Ls by measuring the initial velocity of the reaction at 37 °C, pH 6.6, 7.36 mg l -1 purified enzyme and 500 mM sucrose.

Kinetic studies and characterisation of the product spectrum of the wild-type levansucrase

The kinetic parameters were determined as described in materials and methods (Table 1). The K m -value 6.6 mM for hydrolysis measured for the wild-type Ls from B. megaterium is low compared to other levansucrases, e.g. SacB from B. subtilis with 13.5 to 40 mM [START_REF] Schomburg | Enzyme data and metabolic information: BRENDA, a resource for research in biology, biochemistry, and medicine[END_REF]. This indicates a high affinity to the substrate sucrose supported by a very high k cat (2272 s -1 ) and a high catalytic efficiency (346 mM -1 s -1 , Table 1). The reaction conditions used for the determination of the product spectrum favour hydrolysis (58.4 %) followed by synthesis of polyfructan (22.1 %). Further analysis showed that polyfructan produced by the Ls from B. megaterium had a molecular weight (M w ) of 2,711 kDa and consists mainly of β(2→6) linkages, while the polyfructan produced by the Ls from B. subtilis had a M w of 6,295 kDa and was described as β(2→6) linked levan with some β(2→1) branches [START_REF] Chambert | Kinetic studies of levansucrase of Bacillus subtilis[END_REF]. Beside the polyfructan formation, the wild-type Ls of B. megaterium also synthesised 5 different detectable Os. Three products were identified by HPAEC as 1-kestose (isokestose), 6-kestose, and nystose which are known acceptors for the transfer of fructosyl units [START_REF] Van Hijum | Structure-function relationships of glucansucrase and fructansucrase enzymes from lactic acid bacteria[END_REF] (Figure 4).

The two unknown by-products were isolated and analysed by 1-and 2D-NMR and mass spectroscopy (Figure 5, Tables 2 and3). The 1-D proton spectrum of the first compound showed two anomeric protons (4.6-5.2 ppm), which correspond to an anomeric mixture of two non reducing sugar residues. The 1-H signal the β-configurated glucoside-residue resonated at higher field (4.59 ppm) with a large coupling constant (d, 7.96Hz), where the 1-H signal (5.16 ppm) of the α-configured glucose appeared as minor signal of a reducing sugar in a ratio of α:β 1:2. Based on the results of the 2-D COSY spectrum, correlations were observed between the H-1s of the glucose residues and the corresponding H-2s (alpha 3.8-4.1 ppm; beta 3.20-3.16 ppm), which was coupled to H-3s (alpha 3.63-3.68 ppm; beta 3.42-3.40 ppm). The assignments for the anomeric carbon signals (alpha 94.94 ppm; beta 98.79 ppm) and C-2s (alpha 74.27 ppm, beta 76.89 ppm) were achieved by 2-D HSQC spectrum analysis.

In addition, 1 H 13 C HMBC long range 2 J and 3 J correlations were observed for the H-6 signal of glucose with the 13

Functional role of the exchanged amino acids and insights into the reaction mechanism of the levansucrase from Bacillus megaterium

Levansucrases belong to glycoside hydrolase family 68 (GH 68) according to CAZy [START_REF] Coutinho | Carbohydrate-active enzymes: an integrated database approach[END_REF] which bind the substrate sucrose in the -1 (fructosyl residue) and +1 site (glucosyl residue). In The residues Asp 95 , Glu 352 and Asp 257 are proposed to form the catalytic triad of the Ls from B. megaterium (Figure 7). The substitution of Asp 95 with alanine inhibited the enzyme activity (Table 4). Sequence alignments with other levansucrases from various bacteria showed that and Tyr of the 2-OH of the glucopyranoside [START_REF] Meng | Structural framework of fructosyl transfer in Bacillus subtilis levansucrase[END_REF]. Due to the low reaction rate hydrolysis was predominantly observed (Tables 6 and7). The substituted amino acid residues Trp 172 and Ser 173 were proposed to be part of interactions with aa of the active site. In the Ls of B.

subtilis, this Trp is situated in the +1 region and functioned to form a pocket for the glucopyranoside of the C-6-OH. Our experiments with Ls variant W172A showed 72-fold increase of the K m and thus confirmed this function. It was very interesting to notice that subsitution of Ser 173 with alanine effected the catalytic efficiency dramatically, while K m was comparable to the wild-type Ls. Thus, it is proposed that Ser 173 does not influence the binding mode of sucrose, but it must somehow participate in subsequent catalysis.

Poly-versus oligosaccharide synthesis

The substitution of Arg 370 with alanine of B. megaterium SacB resulted in fundamental changes in product formation. The corresponding residue in the Ls from B. subtilis in subsite -1 (Arg 360 ), was previously described as a key residue for the formation of 1-kestose [START_REF] Chambert | Polymerase and hydrolase activities of Bacillus subtilis levansucrase can be separately modulated by site-directed mutagenesis[END_REF]. We Ls variant R370A, the β(2→1) linkage is cleaved leading to the hydrolysation product blastose. Blastose was supposed to be no donor substrate of the enzyme indicated by its strong accumulation due to a unfavourable β(2→6) linkage between glucose and fructose.

Residue Asn 252 obviously plays an important role for transfructosylation. A substitution to Ala or Gly completely stopped the Ps production at all, while the K m and k cat -values were not effected (Table 5). Catalysis of this variant was switched from mainly Ps synthesis to hydrolysis (Tables 6 and7). In contrast mutation of Asn 252 to Asp 252 effected just downregulation of Ps synthesis (Figure 8), also without changing K m and k cat values significantly. The X-ray structure of the Ls from B. subtilis (74 % identity on amino acid level) enabled insights into the function of this residue. Due to its position in the +2 site, it might stabilise the third fructosyl unit of the growing Os chain and direct it as an acceptor substrate in the optimal position for further transfructosylation. It is located in a channel where the Os chain is able to enter the active site for the transfructosylation reaction. Indeed, based on the Ls structure of B. subtilis, it seems likely that no other possibilities are existing for the Os chain to access the catalytic site (Figure 9).

The mutation of Asn 252 to Asp 252 obviously reduced the coordination of a fructosyl unit in the +2 site. However, it was still possible as indicated by the formation of Ps (Figure 8). The mutations to alanine, glycine and histidine led to a complete knock-out of Ps formation. Very recently, Ozimek et al. [START_REF] Ozimek | The levansucrase and inulosucrase enzymes of Lactobacillus reuteri 121 catalyse processive and non-processive transglycosylation reactions[END_REF] proposed a model of the sugar-binding subsites in two Lb. reuteri 121 fructosyltransferases. For the fructan polymer synthesis the subsites +2, and +3 need a high affinity for binding the growing fructan polymer chain. No structural determinants have been reported so far.

Residues Arg 370 and Asn 252 which seem to be crucial for the polyfructan synthesis are found to be conserved in Ls from Gram positive bacteria. In contrast, the endophytic Gram negative bacterium G. diazotrophicus SRT4 secretes a constitutively expressed Ls (LsdA, EC 2.4.1.10), which mainly converts sucrose into fructo-oligosaccharides. It contains a His 419 instead of Arg 370 at the equivalent position. This His residue is found strictly conserved in Gram negative levansucrases. Further, Asn 252 is strictly conserved in Gram positive bacteria while in Gram negative this region shows variability. The corresponding part of the threedimensional structure of LsdA was almost perfectly superimposable with the equivalent residues of the B. subtilis SacB in the active site. In contrast the region +2 was not perfectly superimposable (Figure 9).

In conclusion the present study reports the full characterisation of a novel Ls, SacB, from B. megaterium and its product spectra. Site-directed mutagenesis clarified the functional roles of different amino acids which are involved in the formation of different reaction products.

There are only few studies existing which concern the Os vs. Ps formation of levansucrases describing a processive or non-processive mechanism [START_REF] Seibel | Synthesis of sucrose analogues and the mechanism of action of Bacillus subtilis fructosyltransferase (levansucrase)[END_REF][START_REF] Chambert | Polymerase and hydrolase activities of Bacillus subtilis levansucrase can be separately modulated by site-directed mutagenesis[END_REF][START_REF] Ozimek | The levansucrase and inulosucrase enzymes of Lactobacillus reuteri 121 catalyse processive and non-processive transglycosylation reactions[END_REF]. So far, no structural insights or amino acids have been identified to give conclusions or clarify differences regarding Ps and Os synthesis among the levansucrases in detail. Here it was shown for the first time that a single amino acid substitution at the +2 subsite switches the enzymatic reaction from Ps to Os formation conserving a high enzyme activity by changing from a processive to a nonprocessive mechanism. Based on these studies in future more structural details of the +2 and +3 subsites may enable the redesign of this enzyme for the synthesis of taylor-made shortchain fructo-oligosaccharides.

Biochemical Journal Immediate Publication. Published on 4 Jul 2007 as manuscript BJ20070600

Table 1 Optimal reaction conditions

The optimal reaction conditions for the Ls from B. megaterium are shown in comparison to the Ls from B.

subtilis.

Table 2 NMR data for the identification of blastose

Table 3 NMR data for the identification of neokestose Table 4 Kinetic parameters of the wild-type levansucrase and its variants.

The reaction was performed with 7.36 mg l -1 enzyme in Sorensen's phosphate buffer (pH 6.6, 50 mM) with 500 mM sucrose. For the variants D95A, R256A, D257A, E350A, and E352A the determination of kinetic parameters was not possible due to missing or too slow release of glucose under these reaction conditions.

Table 5 Kinetic parameters of the wild-type levansucrase and its variants.

The reaction was performed with 7.36 mg l -1 enzyme in Sorensen's phosphate buffer (pH 6.6, 50 mM) with 500 mM sucrose.

Table 6 Product spectrum after 60 min reaction time

All values are given in mM l -1 . The sample used for the identification of neokestose is indicated.

Table 7 Product spectrum after 19 h reaction time

All values are mM l -1 . The sample used for the identification of blastose is indicated.

Figure 1 Effect of sucrose on the secretome of Bacillus megaterium DSM319

B. megaterium DSM319 was cultivated aerobically in 100 ml of LB medium without (lane 1) or with (lane 2) the addition of 0.5 % (w/v) sucrose at 37°C and 250 rpm. Six h after cultivation start, 1.5 ml of cell-free growth medium were desalted using a PD-10 column (GE Healthcare; Uppsala; Sweden) and subsequently lyophilised.

The precipitated proteins were analysed via 10 % SDS-PAGE and stained with Coomassie Brilliant Blue G250.

Lane M shows Precision Plus Protein Standard (Bio-Rad, Munich, Germany).

Figure 4 Identification of the transfructosylation products

The carbohydrates up to tetrasaccharides were identified by High-Performance Anion Exchange Chromatography (HPAEC). The dotted line is the Os standard (1-kestose, 6-kestose, nystose, all 0.01 g l -1 ), the solid line is the wild-type Ls reaction (stopped after 19 h at 37 °C and 0.5 M sucrose in sorensen phosphate buffer, 50 mM, pH 6.6, enzyme concentration was 0.1 g l -1 ), the dashed line is the Os standard (concentration as above) mixed with the same wild-type Ls sample. Gd is Gluconacetobacter diazotrophicus [START_REF] Martinez-Fleites | Crystal structure of levansucrase from the Gram-negateive bacterium Gluconacetobacter diazotrophicus[END_REF], Ps is Pseudomonas syringae [START_REF] Hettwer | Cloning, nucleotide sequence, and expression in Escherichia coli levansucrase genes from the plant pathogens Pseudomonas syringae pv. glycinea and P. syringae pv. phaseolicola[END_REF], Ra is Rahnella aquatilis [START_REF] Song | Levansucrase of Rahnella aquatilis ATCC33071. Gene cloning, expression, and levan formation[END_REF],

Ea is Erwinia amylovora [START_REF] Geier | Characterization and influence on virulence of the levansucrase gene from the fireblight pathogen Erwinia amylovora[END_REF], Zm is Zymomonas mobilis [START_REF] Song | Nucleotide and derived amino acid sequences of an extracellular sucrase gene (invB) of Zymomonas mobilis ZM1 (ATCC10988)[END_REF], Ss is Streptococcus salivarius [START_REF] Rathsam | The cell-bound fructosyltransferase of Streptococcus salivarius: the carboxyl terminus specifies attachment in a Streptococcus gordonii model system[END_REF], Pp is

Paenibacillus polymyxa [START_REF] Bezzate | Disruption of the Paenibacillus polymyxa levansucrase gene impairs its ability to aggregate soil in the wheat rhizosphere[END_REF], Ba is Bacillus amyloliquefaciens [START_REF] Tang | Isolation and characterization of levansucrase-encoding gene from Bacillus amyloliquefaciens[END_REF], Sm is Streptococcus mutans [35], Bs is

Bacillus subtilis [START_REF] Meng | Structural framework of fructosyl transfer in Bacillus subtilis levansucrase[END_REF], Bm is Bacillus megaterium. The amino acids used for the mutagenesis study are indicated and the resulting variant is labeled above the sequence.

Figure 7 The proposed transfructosylation reaction mechanism

The mechanism is based on SacB from B. megaterium with sucrose as substrate.

- - - - D257A - - - - E350A - - - - E352A - - - - Y421A
51.9 ± 16.3 2.9 ± 0.3 335 6.5

W94A, 5 ' 3 '

 53 All introduced mutations and the integrity of the residual sacB gene were confirmed by DNA sequencing.

  C spectra and phasesensitive two-dimensional spectra [double-quantum-filtered COSY, NOESY (Nuclear overhauser and exchange spectroscopy), TOCSY (Total correlated spectroscopy), 1 H-13 C HSQC (Heteronuclear single quantum coherence) and HMBC (Heteronuclear multiple bond Biochemical Journal Immediate Publication. Published on 4 Jul 2007 as manuscript BJ20070600

  Ls SacB, a 473 amino acid residue exoenzyme of B. subtilis. BLAST searches of the B. subtilis SacB against the translated genome data of B. megaterium led to the identification of the SacB protein sequence and the genomic position of the corresponding sacB gene [21; 22]. B. megaterium SacB consists of 484 amino acids including the 29 amino acids of an Nterminal signal peptide. It shows 74 % identity at the amino acid sequence level to SacB from B. subtilis. Hence, the B. megaterium protein was also named SacB. Nine of the 11 additional amino acids found in B. megaterium SacB compared to SacB of B. subtilis are adjacent to one another at location 82-90 (considering the numbering of B. megaterium SacB). Like sacB of B. subtilis, sacB of B. megaterium is the first gene of an operon. It is followed by a gene consisting of 1,563 bp. The deduced protein sequence shows 66 % identity to the levanase LevB from B. subtilis. B. subtilis levB is also the second gene in an operon

  C signals at δ C 106.52 ppm (alpha, C-2') and 106.55 ppm (beta, C-2') of the fructofuranoside, respectively, which shows correlations for the fructofuranoside H-3' positions (beta 79.78 ppm; alpha 79.68 ppm/4.58 ppm) and H-4' positions (beta 77.45 ppm; alpha 77.31 ppm/4.07 ppm) (Figure 4). All other carbon and proton signals were assigned from a combination of 2-D COSY, TOCSY, HMBC, and HSQC experiments. All experiments provide strong evidence of an 1:2 α:β mixture of β6Fru-α,β-Glc (6-O-β-Dfructofuranosyl-α,β-D-glucopyranoside, blastose). The ESI MS of the second compound showed the molecule peak at m/z = 527 [M + Na + ]. This trisaccharide is a non reducing sugar. In the 1-D proton spectrum the anomeric proton resonated at δ H 5.35 ppm with a small J 12 value of 3.90 Hz. This proton showed correlation in the 2D-HMBC spectrum with quaternary carbon C-2' (106.45 ppm) of a fructofuranoside residue. In the 2-D HMBC spectrum protons corresponding of the H-6 glucose residue showed correlations to the quaternary carbons C-2'' of a second fructofuranoside residue. Taken together, all signals assigned from the one dimensional and correlation spectroscopy (i.e. COSY, TOCSY, HMBC, DEPT and HSQC), the trisaccharide was identified as β2,6Fru-αGlc-β1,2Fru (2,6-O-β-D-fructofuranosyl-α-D-glucopyranosyl-1,2-O-β-D-fructofuranoside, neokestose).

  this family, two X-ray structures of the B. subtilis and G. diazotrophicus enzymes have been elucidated and both show a five-fold β-propeller topology[18; 19]. Amino acid sequence alignments of various Ls enzymes (Figure6) in combination with structural data of family members pointed towards functional relevant residues. Subsequent site directed mutagenesis of Trp 94 , Asp 95 , Val 115 , Leu 118 , Trp 172 , Ser 173 , Asn 252 , Arg 256 , Asp 257 , Glu 350 , Glu 352 , Arg 370 , Tyr 421 was performed in order to explore the structural determinants which are responsible in B. megaterium SacB for the transfructosylation activity.

  this amino acid residue acts as a nucleophile. It usually attacks the glucopyranosyl residue of the sucrose and forms an enzyme-fructosyl-intermediate with inversion of the glycosidic bond in the intermediate state. The corresponding acid/base catalyst was Glu 352 . Consequently, an exchange into alanine led to a high decrease in enzyme activity. The variant D257A showed almost no activity. The residue Asp 257 might have been coordinated by hydrogen bonds to the fructofuranoside in positions 3-OH and 4-OH.Trp 94 , Arg 256 , Glu 350 , and Tyr 421 were selected as their corresponding amino acid residues of the Ls from B. subtilis[18; 24] form hydrogen bonds to sucrose and direct it in a productive orientation. Here, the substitution with alanine of each of these amino acid residues in the Ls from B. megaterium showed different effects. The variants R256A and E350A were nearly inactive. This agrees with the structural information obtained for the Ls from B. subtilis where the corresponding Arg 246 formed hydrogen bonds to the 3-OH of the fructofuranoside and also to 4-OH of the glucopyranoside while Glu 340 coordinated the 3-OH and 4-OH of the glucopyranoside. The Ls variant W94A still carried 9 % of the catalytic efficiency k cat /K m while variant Y421A showed 3 % of the wild-type activity. Considering the X-ray structure of the Ls from B. subtilis, this Trp formed a hydrogen bond with 6-OH of the fructofuranoside

  observed the time dependent accumulation of different Os during Ls variant R370A catalysis. After 60 min reaction time an accumulation of neokestose (2,6-β-Fru-αGlc-1,2-β-Fru, 32.7 mM) was determined while after 19 h blastose (2,6-β-Fru-α,βGlc) was the main reaction product (69.7 mM). A 4-fold K m -value decrease compared to the wild-type enzyme supported the interactions of Arg 370 with the 2-OH and 3-OH of the glucosyl residue in the active site. It is possible that the substitution to Ala favoured an unspecific substrate binding and enabled a fructosylation at C-6 of the glucosyl residue of sucrose yielding neokestose. When the trisaccharide neokestose was accumulating and entering the active site of the B. megaterium

Figure 2 Figure 3

 23 Figure 2 Identification of the levansucrase by MALDI/MSMass spectrum of the peptide fragment VMKPLITSNTVTDEIER. The levansucrase was identified as described in materials and methods).

Figure 5 Figure 6

 56 Figure 5 Correlation spectrum of α,β blastose (HMBC)

Figure 8 TLC

 8 Figure 8 TLC analysis of polymer-vs. oligosaccharide formation activityThe reaction was performed using wild-type levansucrase SacB from B. megaterium and indicated variants carrying amino acid exchanges in position Asn 252 . The reaction conditions were 50 mM Sorensen`s buffer, pH 6.6, and 37 °C. The enzyme concentration was 7.36 mg l -1 each. Std1 and Std2 are carbohydrate standards containing glucose (Glc), fructose (Fru), sucrose (suc) and maltose (not indicated) top down. The sample times are indicated above in[h]. The detectable polymer is encircled.

Figure 9

 9 Figure 9 Local identification of the crucial amino acid residuesThis superimposing shows the levansucrases from Bacillus subtilis (Bs, purple) and Gluconacetobacter diazotrophicus (Gd, cyan) with sucrose in the active site[18; 19; 27; 28]. The analogue amino acid residues of the Bacillus megaterium (Bm) Ls are given in parentheses.

Figure 1 Figure 2 Figure 3 Biochemical

 123 Figure 1

Figure 4 Figure 5 Biochemical

 45 Figure 4

Figure 6 Figure 7

 67 Figure 6

Figure 8 Figure 9 Biochemical

 89 Figure 8

Table 1 Levansucrase

 1

				, SacB, from		Levansucrase, SacB, from
			B. subtilis [23]		B. megaterium
	K m TO number (k cat )	13.5 -40 mM 0.6 -60 s -1		6.6 mM 2272 s -1
	pH optimum	pH 6 for hydrolysis		pH 6.6 for hydrolysis
	T optimum		37 -50 °C		45 °C
	stability		pH 3.5 to 7.5 at 4 °C, no time indicated	> 6 months at pH 6.6 and -20 °C
	Table 2
	C-atom	αblastose δ C	δ H	βblastose δ C	δ H
	C-1	94.94		5.16 (d, 3.70)	98.79	4.59-4.57 (d, 7.96)
	C-2	74.27		3.49-3.46 (m)	76.89	3.20-3.16 (t, 8.70)
	C-3	75.48		3.63-3.68 (m)	78.45	4.42-3.40 (m)
	C-4	72.55		3.34-3.40 (m)	72.49	3.88-3.85 (m)
	C-5	73.49		3.85-3.88 (m)	77.79	3.84-3.80 (m)
	C-6	63.61		3.96-3.89, 3.67-3.62	63.61	3.96-3.89, 3.67-3.62
	C-1'	62.99		3.70-3.62 (m)	62.99	3.70-3.62 (m)
	C-2'	106.52		-	106.55	-
	C-3'	79.68		4.58-4.56 (d, 8.26)	79.78	4.58-4.56 (d, 8.26)
	C-4'	77.31		4.07-4.03 (t, 7.60)	77.45	4.07-4.03 (t, 7.60)
	C-5'	83.97		3.82-3.76 (m)	84.02	3.82-3.76 (m)
	C-6'	65.21		3.77-3.72 (m)	65.32	3.77-3.72 (m)

Biochemical Journal Immediate Publication. Published on 4 Jul 2007 as manuscript BJ20070600 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Table 3 neokestose

 3

	C-atom	δ C	δ H
	C-1	62.89	3.91-3.87, 3.76-3.73
	C-2	106.45	-
	C-3	79.47	4.16-4.13 (d, 8.5)
	C-4	77.03	4.11-4.07 (t, 7.4)
	C-5	83.88	3.84-3.80 (m)
	C-6	65.17	3.83-3.73
	C-1'	94.74	5.35 (d, 3.88)
	C-2'	73.74	3.53-3.49 (dd, 3.90, 10.00)
	C-3'	75.15	3.69-3.66 (m)
	C-4'	74.76	3.92-3.88 (m)
	C-5'	71.89	3.48-3.45 (m)
	C-6'	62.03	3.58 (s)
	C-1''	64.14	3.73-3.69, 3.64-3.61
	C-2''	106.47	-
	C-3''	78.90	4.18-4.16 (d, 8.49)
	C-4''	76.64	4.04-4.00 (t, 8.5)
	C-5''	84.08	3.87-3.83 (m)
	C-6''	65.08	3.80-3.76, 3.68-3.64

Table 4 Variant K m [mM] v max [mM min -1 ] k cat [s -1 ] k cat /K m [mM -1 s -1 ]

 4

	wild-type	6.6 ± 1.1	19.6 ± 0.6	2272	346
	W94A	31.9 ± 11.0	8.6 ± 0.9	1000	31
	D95A	-	-	-	-
	L118A	66.6 ± 22.1	10.6 ± 1.1	1231	19
	W172A	480.4 ± 124.8	20.7 ± 3.2	2396	5
	S173A	2.3 ± 1.2	3.1 ± 0.3	363	159
	N252A	4.1 ± 1.7	12.8 ± 1.1	1480	361
	R256A

Table 5 Table 6

 56

	Variant	K m [mM]		v max [mM min -1 ]		k cat [s -1 ]	k cat /K m [mM -1 s -1 ]
	wild-type	6.6 ± 1.1		19.6 ± 0.6		2272	346
	N252A	4.1 ± 1.7		12.8 ± 1.1		1480	361
	N252D	8.4 ± 2.3		32.3 ± 1.6		3743	445
	N252G	7.5 ± 0.8		19.5 ± 0.4		2256	301
	N252H	35.1 ± 30.7	13.2 ± 3.4		1529	43.5
	R370A	29.2 ± 11.6	1.5 ± 0.2		179	6.1
		wild-type	L118A W172A N252A N252D	N252G N252H R370A Y421A
	glc	428.0	85.1	13.0	447.9	463.4	334.9	118.6	138.7	12.2
	fru	292.2	71.6	11.3	391.4	362.0	279.9	102.3	103.0	9.6
	suc	41.9	407.1	487.0	17.1	0.0	141.8	381.4	321.3	487.5
	1-kestose	15.9	3.4	0.0	5.2	3.5	6.9	0.0	3.4	0.0
	α,β-blastose	6.4	0.0	0.0	7.3	17.3	9.7	0.0	3.9	0.0
	6-kestose	3.5	0.0	0.0	15.0	4.2	6.8	0.0	0.0	0.0
	neokestose	2.8	3.5	0.0	5.8	7.4	6.6	0.0	32.7	0.0
	nystose	1.5	0.5	0.0	1.7	4.1	0.0	0.0	0.0	0.0
	Table 7
		wild-type	L118A W172A N252A N252D N252G N252H R370A Y421A
	glc	449.8	304.1	101.1	459.9	443.8	448.7	431.1	414.4	97.8
	fru	359.8	226.7	89.5	399.4	375.5	360.3	360.3	357.2	83.8
	suc	0.0	172.2	389.7	0.0	0.0	0.0	27.4	0.0	389.5
	1-kestose	7.6	4.5	2.1	4.0	7.7	7.0	6.0	6.0	4.7
	α,β-blastose	24.5	7.4	1.1	14.1	21.5	26.3	16.7	69.7	1.1
	6-kestose	4.9	4.1	0.6	11.9	14.8	6.7	4.5	1.2	0.0
	neokestose	9.5	5.3	5.4	7.0	9.3	15.0	12.3	13.6	6.9
	nystose	3.7	2.5	0.0	3.2	3.5	3.4	2.1	2.1	0.0

Biochemical Journal Immediate Publication. Published on 4 Jul 2007 as manuscript BJ20070600 © 2007 The Authors Journal compilation © 2007 Biochemical Society

© 2007 The Authors Journal compilation © 2007 Biochemical Society

Acknowledgment

This project is supported by DFG (SFB 578) and AiF (Project 14777).


Zrinka Marijanovic

Josiane Ragimbeau

José Van Der Heyden

Gilles Uzé

Sandra Pellegrini
email: pellegri@pasteur.fr

Comparable potency of IFNalpha2 and IFNbeta on immediate JAK/STAT activation but differential down-regulation of IFNAR2

Keywords:

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Type I IFNs are helical cytokines with pleiotropic activities that contribute to immediate defense against pathogens, development of adaptive immunity and protective antitumor responses [START_REF] Vilcek | Fifty years of interferon research: aiming at a moving target[END_REF]. Accordingly, type I IFNs regulate diverse activities of non immune and immune cells and are also regulators of bone homeostasis. A unique feature of this cytokine family is the existence of a multiplicity of ligands. In humans the family comprises 16 IFN subtypes, broadly referred to as IFNα/β, all binding the ubiquitously expressed type I IFN receptor, made of IFNAR1 and IFNAR2. These latter are single membrane-spanning proteins belonging to the class 2 cytokine receptor superfamily [START_REF] Pestka | Interferons, interferon-like cytokines, and their receptors[END_REF]. Upon IFN binding, membraneproximal immediate signaling is initiated through catalytic activation of receptor-associated Tyk2 and Jak1 tyrosine kinases [START_REF] Yeh | The Janus kinase family of protein tyrosine kinases and their role in signaling[END_REF][START_REF] Haan | Jaks and cytokine receptors--an intimate relationship[END_REF]. Stat family members, such as the ubiquitously expressed Stat1/2/3, are then tethered to the activated receptor complex via specific phosphotyrosine recruitment motifs, undergo phosphorylation on tyrosine and translocate to the nucleus to drive gene expression. Non-Stat signaling pathways can also be activated by type I IFNs and are believed to modulate gene expression and to shape IFN-induced bioactivities in defined cellular contexts [START_REF] Rani | Alternative and accessory pathways in the regulation of IFN-beta-mediated gene expression[END_REF][START_REF] Van Boxel-Dezaire | Complex modulation of cell type-specific signaling in response to type I interferons[END_REF]. IFN also activates membrane-proximal signaling events that promote receptor down-modulation and signal termination [START_REF] Basu | The antiviral action of interferon is potentiated by removal of the conserved IRTAM domain of the IFNAR1 chain of the interferon alpha/beta receptor: effects on JAK-STAT activation and receptor downregulation[END_REF][START_REF] Kumar | SCF(HOS) ubiquitin ligase mediates the ligand-induced down-regulation of the interferon-alpha receptor[END_REF].

It is well documented that IFNα and IFNβ engage the receptor components with different efficiencies [START_REF] Cutrone | Contributions of cloned type I interferon receptor subunits to differential ligand binding[END_REF][10][11]. Recent work has extended previous studies and determined the biophysical parameters of binding (association and dissociation rate constants) of type I IFN subtypes to the extracellular domains of the two receptor subunits immobilized on an artificial lipid support [12]. These in vitro analyses showed that affinities and rate constants of IFNα2 and IFNβ interaction with the receptor ectodomains are different. IFNα2 exhibits nanomolar binding affinity and IFNβ exhibits ~100 pM binding affinity towards IFNAR2. Larger differences were observed in the ligand binding affinity to IFNAR1. IFNα2 exhibits micromolar binding affinities to the IFNAR1 ectodomain, while IFNβ exhibits ~50 nM binding affinity towards IFNAR2. It was demonstrated that the kinetics of dissociation of IFNα, but not of IFNβ, from the immobilized complex is strongly affected by the concentration of IFNAR1 [12,13]. Moreover, it was proposed that the differential potency of IFN subtypes to elicit an antiproliferative response lies in their different ability to bind IFNAR1 [14]. Overall, these studies suggested that recruitment of IFNAR1 is a rate limiting step in the assembly of the ternary ligand-receptor complex and that a sufficiently high concentration of IFNAR1 may compensate for its low intrinsic affinity to IFNα2.

Since IFNAR1 is the low affinity component in the formation of the ternary complex, one would predict that immediate receptor-initiated signaling events may be sensitive to IFNAR1 surface concentration. Indeed, the level of cellular IFNAR1 appears to be tightly regulated. We have shown that IFNAR1 is a relatively short lived protein that matures to the cell surface and is rapidly internalized and degraded, unless stabilized through its association with Tyk2 [15]. Upon IFNα binding, IFNAR1 is targeted to degradation through a biochemical cascade that has been in part elucidated and requires Tyk2 catalytic activity [START_REF] Kumar | SCF(HOS) ubiquitin ligase mediates the ligand-induced down-regulation of the interferon-alpha receptor[END_REF]16].

Based on these premises, our primary goal was to investigate the relationship between IFNAR1 and IFNAR2 surface levels and immediate-early signaling events, ie activation of Jak/Stat molecules and receptor down-modulation, induced by IFNα2 or IFNβ. For this, we have measured relative levels of IFNAR1 and IFNAR2 expressed in different human cell lines and monitored herein early kinetics and dose dependence of Jak/Stat activation. We have also studied basal and ligand-induced surface decay of IFNAR1 and IFNAR2. To directly test the effect of variable IFNAR1 levels, we have artificially modulated its concentration and analyzed possible effects on IFN signaling potency.

Despite the heterogeneity in the levels of the two subunits in different cell lines, a unifying concept emerges from this comprehensive analysis that points to comparable level of activation of Jak/Stat molecules in response to the two IFN subtypes and to a differential effect on traffic of the IFNAR2 receptor subunit.

EXPERIMENTAL PROCEDURES

Cell culture and transfection procedures

293T and HeLa cells were cultured in DMEM (Dulbecco's modified Eagle's medium) and Daudi and Jurkat cells in RPMI supplemented with 10% foetal calf serum at 37°C in 6% CO 2 .

Human amnion-derived WISH cells were transfected using the calcium phosphate DNA precipitation technique. To over-express IFNAR1, a plasmid encoding IFNAR1 and pSV2neo were co-transfected at a ratio of 10/1. Clones were selected in 1.2 mg/ml of G418 (Geneticin, Invitrogen).

To silence IFNAR1, a 19 nt siRNA was designed to target the 3'UTR mRNA sequence of IFNAR1. The sequence ATACGGGCAAGCTCTTAAC was inserted into the RNAi vector pSUPERretro (OligoEngine, Seattle) as indicated by the manufacturer. This puro R -siRNA plasmid was co-transfected with pSV2neo at molar ratio of 10/1. Cells were first selected in G418 to favour clones with highest amount of plasmid DNA. G418 R clones were then kept in puromycin to maintain pressure for the siRNA plasmid. Clones were screened for IFNAR1 levels by flow cytometry using mAb AA3. For the experiment shown in Fig. 8, 293T cells were transiently transfected using Lipofectamine (Invitrogen) with an expression plasmid encoding either IFNAR1 or the IFNAR1 specific RNAi described above. All transfections included an EGFP expression plasmid at a ratio of 1/50 of total DNA. Transfection mixtures were normalized for the same concentration of total DNA with the corresponding empty vector. Two days after transfection, cells were treated for 2 hrs with 200 pM IFNα2 or β or left untreated and receptor levels were analyzed by flow cytometry.

Antibodies and other reagents

Recombinant IFNα2b was a gift of D. Gewert (Wellcome, U.K.) or G. Adolf (Ernst Boehringer Institute, Vienna) and IFNβ was from L. Runkel (Biogen Idec, Boston). IFN were purified to specific activities > 10 8 IU/mg of protein. Cycloheximide (CHX) (Sigma) was used at 20 µg/ml on HeLa and 293T cells and at 10 µg/ml on Jurkat and Daudi cells. Monensin (Sigma) was solubilized in absolute ethanol and used at 25 µM. Human transferrin (Sigma) was solubilized in cell culture medium and was used at 50 µg/ml. Phospho-Ser535specific IFNAR1 Abs, described in [17], was a gift of S. Fuchs (University of Pennsylvania, Philadelphia, PA). Tyk2 phospho-specific Abs was from Calbiochem. Tyk2 mAb T10-2 was described in [18]. Phospho-tyrosine Stat1 and Stat2 Abs were from Upstate and phosphotyrosine Stat3 Abs from BioLabs. Stat1, Stat2, Stat3 Abs and 4G10 anti-phosphotyrosine mAB were from Upstate. Ubiquitin FK2 mAb was from Biomol; IFNAR1 mAbs (AA3, EA12) were a gift from L. Runkel and 64G12 from P. Eid (CNRS, Villejuif, France).

IFNAR2 mAb CD118 was purchased from PBL Biochemical Laboratories. IFNAR2 mAbs H10 and D5 were from L. Runkel.

Flow cytometric analysis

Transiently transfected 293T cells (Fig. 8) were stained with mAbs AA3 (IFNAR1) and D5 (IFNAR2) and signals were amplified with biotinylated rat anti-mouse IgG (Jackson Immunochemicals) and APC-conjugated streptavidin (Becton Dickinson) and analyzed with a FACScalibur. IFNAR1 and IFNAR2 expression levels were determined by the Geometric mean APC fluorescence values within subpopulations of transfected cells identified by the intensity of EGFP fluorescence. To assess the effect of monensin in Fig. 9A, down-regulation of the transferrin receptor from the cell surface was studied. Briefly, HeLa cells were starved for 1 hr, preincubated for 15 min with monensin (25 µM) before addition of transferrin (50 µg/ml) for 30 min. In Fig. 9B, cells were preincubated with monensin 15 min before addition of IFNα or IFNβ. Surface IFNAR1 was monitored by incubating cells with 10 µg/ml mAb AA3 and surface IFNAR2 was monitored with 10 µg/ml of mAb CD118 for 30 min on ice, followed by 30 min incubation with 10 µg/ml biotinylated anti-mouse IgG and with streptavidin-phycoerythrin (Jackson ImmunoResearch Laboratories, Inc.). The level of surface transferrin receptor (TrR) was monitored with OKT9 mAb (a gift from A. Alcover, Institut Pasteur, Paris). Cells were analyzed using a FACscan (Becton Dickinson), using CellQuest software.

Protein analysis by immunoblot

Cell lysates were prepared in modified RIPA buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 1% NP40, 0.5% DOC, 0.03% SDS) with 1 mM Na 3 VO 4 and a cocktail of antiproteases from Sigma. For the experiment in Fig. 5, cells were lysed in NP40 buffer (50 mM Tris/HCl pH 8, 150 mM NaCl, 1% NP40, 0.5 mM EDTA, 1 mM Na 3 VO 4 , 10 mM N-ethylmaleimide and antiproteases). IFNAR1 was immunoprecipitated with 1 µg of mAb EA12 and detected with mAb 64G12. IFNAR2 was immunoprecipitated with 1 µg of mAb CD118 and detected with a mixture of H10 and D5 mAbs.

Antiproliferative effect

HeLa cells were plated in 96-well plates at a density of 2500 cells/well. Twenty hours later cells were left untreated or treated with 1-10000 pM of either IFNα or IFNβ for additional 72 h. Cells were then washed with PBS, fixed with paraformaldehyde and stained with 0.5% crystal violet for 5 min. After removing the solution, cells were washed with deionized water and air-dried. Crystal violet was solubilized with 1% Triton-X-100. Optical density was analyzed at 570 nm.

RESULTS

Lack of correlation between relative R2/R1 ratios and Jak/Stat activation

In order to analyze early signaling events in cells expressing different surface levels of IFNAR1, we first measured the relative ratio of IFNAR1 and IFNAR2 present at the surface of four human cell lines. The steady state level of IFNAR1 and IFNAR2 was measured by flow cytometry, using two mAbs (AA3 and CD118) and an identical staining protocol (Experimental procedures). Daudi (Burkitt's lymphoma B lymphocytes), Jurkat (leukemia T lymphocytes), HEK293T (embryonic kidney cells) and HeLa (carcinoma-derived epithelioid cells) were analyzed. As shown in Fig. 1A, the relative expression of the two subunits (referred as the R2/R1 ratio) was remarkably dissimilar, with two extreme profiles represented by Jurkat (R2/R1 around 4) and HeLa cells (R2/R1 around 0.5).

Based on these profiles, we chose Daudi and Jurkat for further studies. These cells express different R2/R1 ratios (around 2 in Daudi and 4 in Jurkat) and, being both of lymphoid origin, are best amenable to direct comparison. We first analyzed early kinetics of Jak/Stat phosphorylation induced by a single dose (500 pM) of either IFN subtype. Within 15 min of stimulation, phosphorylation of examined proteins was close to maximal level (Fig. 1B andC). At around 30 min, phosphorylation started to decline, with the exception of Stat2 which persisted for at least 4 hr. The profiles of IFNα2 and IFNβ stimulated signals in each cell line were remarkably similar. However, phosphorylated Tyk2 and Stat2 were more abundant in Daudi cells, while phosphorylated Stat3 was more abundant in Jurkat cells.

Notably, the extent of phosphorylation of these proteins closely reflected their total content in each cell line (Fig. 1B andC).

Next, we analyzed dose response profiles of Tyk2, Stat1/2/3 phosphorylation after 30 min of treatment with IFNα2 or IFNβ (Fig. 1D). In both cell lines, phosphorylation of Tyk2, Stat1/3 was dose-dependent, reaching saturation at around 100 pM of either IFN subtype. Surprisingly, phosphorylation of Stat2 appeared not to be dose-dependent within the range of cytokine dose tested (Fig. 1D). In conclusion, the magnitude and duration of Jak/Stat activation by IFNα and IFNβ were virtually identical in each of the two cell lines and similar conclusions were reached from analyses of phosphorylation profiles in 293T and HeLa cells (data not shown). Moreover, we could not directly correlate R2/R1 ratios neither with α/β differential signaling nor with sensitivity to IFNα2.

IFNAR1 is limiting for ligand-induced Jak/Stat activation

Comparison of signaling in different cell types presents obvious limitations, one being the variable content of signaling effectors. Thus, we decided to monitor α/β signaling in sister clones expressing different levels of IFNAR1. For this, we chose human WISH cells, well known for their α/β differential antiproliferative response [13]. As shown in Fig. 2 Three stable transfectants were derived from WISH (parental) cells: a control clone (R23) expressing IFNAR1 to the same level as parental cells, a clone that over-expressed IFNAR1 (R16) and a clone with low IFNAR1 (CPO2) which was obtained by RNAi technology (see Experimental procedures). The relative surface level of the two receptor subunits in these clones is shown in Fig. 3A. IFNAR1 was approximately 7 fold higher in R16 cells and 2 fold lower in CPO2 cells with respect to R23 or parental WISH. As expected, IFNAR2 levels were comparable in all clones. In western blot analysis, IFNAR1 level was barely detectable in CPO2 cells and was strongly increased in R16 cells as compared to endogenous IFNAR1 in R23 cells (Fig. 3B, top panels).

To investigate whether surface IFNAR1 concentration affected sensitivity to IFNα2, we compared the Jak/Stat activation profile of these clones. Cells were incubated with 500 pM IFNα2 for 5, 15 and 30 min and protein phosphorylation was monitored (Fig. 3B).

Phosphorylation of all proteins, with the exception of Stat2, was reduced in CPO2 cells as compared to the other clones. Interestingly, Stat2 phosphorylation was comparable in the three clones and did not decay with time. We further compared CPO2 and R23 cells to assess whether the weak response of CPO2 cells could be augmented further by increasing IFNα2 dose. As shown in Fig. 4A, even at the highest dose tested (2500 pM), the response of CPO2 cells remained low, approaching that of control R23 cells stimulated with 10 pM.

Despite the remarkable difference of IFNAR1 levels in R16 and R23 cells, their Jak/Stat phosphorylation profiles by IFNα were quite comparable (Fig. 3B). To explore the possibility of an increased IFNα2 sensitivity of R16 cells, we compared response profiles of the two clones to low ligand doses (Fig. 4B). If anything, R16 cells were slightly less responsive than R23 to low cytokine doses. Altogether, these data showed that a mere 2 fold decrease in surface IFNAR1, as in CPO2 cells, had a considerable impact on the magnitude of signaling and on the IFNα sensitivity threshold. Conversely, a surplus of IFNAR1 over endogenous level did not translate into increased signaling nor in increased sensitivity to IFNα. These results demonstrate that cell surface IFNAR1 concentration is indeed a critical factor for assembly of a functional signaling complex, but its increased concentration does not augment IFNα, nor IFNβ (data not shown), signaling potency. Moreover, these results imply that assembly of the trimeric ligand-receptor complex is the step determining the extent of downstream Jak/Stat activation.

IFNβ, but not IFNα2, induces IFNAR2 down-regulation

Along with Jak/Stat engagement, binding of IFN also triggers membrane-proximal signals which promote internalization and drive endosomal sorting of the engaged receptor.

Numerous studies have implicated ubiquitination of ligand-activated receptors as a signal for endosomal sorting towards degradation [19][20][21]. Recent data showed that, upon IFNα binding, IFNAR1 becomes serine phosphorylated and consequently ubiquitinated prior to lysosomal degradation [START_REF] Kumar | SCF(HOS) ubiquitin ligase mediates the ligand-induced down-regulation of the interferon-alpha receptor[END_REF]. Much less is known about ligand-induced post-translational modification and traffic of IFNAR2. We therefore tested whether IFNα2 and IFNβ led to differential modification of IFNAR1 and/or IFNAR2. As shown in Fig. 5 (left panels), IFNAR1 was inducibly phosphorylated on tyrosine and on Ser535 and heavily ubiquitinated.

IFNAR2 was inducibly phosphorylated on tyrosine but was not ubiquitinated (Fig. 5, right panels). All modifications were induced to a similar extent by IFNα2 and IFNβ. Similar conclusions were reached upon analysis of HeLa cells (data not shown). Jaitin et al. [13] recently showed that a 2 hr stimulation of 293T cells with IFNβ, but not IFNα, causes down-regulation of surface IFNAR2. We thus asked whether this β-specific effect could be generalized to other cell types and whether different surface R2/R1 ratios (as in cell lines shown in Fig. 1A) influenced this α/β differential trait. In order to thoroughly assess the effect of ligand binding on receptor dynamics, we first determined in the four cell lines the surface stability of each receptor subunit in the absence of stimulus. Cells were treated with cycloheximide (CHX) to prevent new protein synthesis and the decay of each subunit was followed by flow cytometry (Fig. 6). Interestingly, IFNAR2 displayed an apparent slow basal turnover, with 90% of the initial content present at 3 h. On the other hand, surface IFNAR1 decayed rapidly in all but Daudi cells, with between 55 to 60% of the initial values remaining at 3 h (Fig. 6). The different stability of the two receptor subunits was also confirmed by western blot analyses (data not shown). Overall, these results showed that the two receptor subunits have different basal turnovers, with IFNAR1 being considerably more dynamic than IFNAR2. Surface IFNAR1 and IFNAR2 levels were then monitored in cells treated with IFNα2 or IFNβ, in the presence of CHX (Fig. 7A). Within the first hour, IFNAR1 decreased more rapidly in cells treated with either ligand than in CHX only treated cells shown in Fig. 6. This ligand-induced acceleration was observed in all cell types (Fig. 7A, left panels). At 90 min, the rate of decay decreased, approaching the basal rate seen in Fig. 6. Importantly, a clear difference in down-regulation of IFNAR2 by the two ligands was observed for 293T and HeLa cells. IFNβ caused a substantial decrease (approaching 50% at 90 min) in surface IFNAR2, whereas IFNα2 had a minimal effect (Fig. 7A, right panels). The differential effect of IFNα2 and IFNβ on IFNAR2 down-regulation was less pronounced in Daudi cells and appeared negligible in Jurkat cells. Since these cells express higher level of IFNAR2 relative to IFNAR1 (high R2/R1 ratio), the percentage of mobilized IFNAR2 may be small and uncomplexed IFNAR2 may remain at the cell surface. Indeed, a differential effect between IFNα2 and IFNβ was evident upon plotting arbitrary IFNAR2 internalization units instead of percentages of initial values (insets in Fig. 7A). Western blot analyses of total receptor content confirmed the ligand-induced decrease of IFNAR1 in the four cell lines. The decrease of IFNAR2 was detectable in IFNβ-treated 293T and Hela cells, but not in Jurkat or Daudi cells (Fig. 7B).

To study the effect of varying IFNAR1 level on IFNAR2 down-regulation, we depleted or over-expressed IFNAR1 in 293T cells (see Experimental procedures) and monitored by FACS receptor levels after a 2 hr-treatment with IFNα2 or IFNβ. As seen in Fig. 8, in response to IFNα2 the surface decay of IFNAR2 was minimal even in cells with a surplus of IFNAR1 as compared to control cells (endogenous IFNAR1 level taken as 1).

Thus, the failure of IFNα2 to promote IFNAR2 down-regulation is not due to limiting amount of IFNAR1. In response to IFNβ, IFNAR2 was down-regulated in control cells as expected.

The amount of down-regulated IFNAR2 diminished in cells depleted of IFNAR1, but did not proportionally increase in cells with a surplus of IFNAR1.

Overall, these results demonstrate a differential effect on post-binding receptor dynamics, with solely IFNβ being able to down-modulate IFNAR2. While this IFNβ specific effect can be generalized to different cell types, the extent by which it is experimentally detectable appears to depend on the relative expression level of the two chains and hence on the proportion of IFNAR2 which is engaged in the trimeric complex.

IFNα2 induces IFNAR2 recycling

We have shown above that IFNα2 failed to down-regulate surface IFNAR2, although it efficiently accelerated IFNAR1 down-regulation. We reasoned that the constant level of IFNAR2 measured at the cell surface could either reflect stability at the plasma membrane or it could result from the dynamic equilibrium between IFNα-induced internalization and recycling. In order to distinguish between these two possibilities, we used monensin, a drug that blocks recycling without affecting the internalization process [22]. The toxic effect of To control the action of the ionophore, we monitored the surface level of the transferrin receptor (TrR), which undergoes continuous recycling in the presence of transferrin [23]. As expected, TrR level decreased approximately 50% in monensin-treated cells. In the same condition, basal IFNAR2 turnover was unaffected (Fig. 9B). HeLa cells were then treated with IFNα2 or IFNβ in the presence or absence of monensin, and surface decay of IFNAR1 and IFNAR2 was measured by flow cytometry. Monensin did not affect IFNAR1 dynamics (Fig. 9C, top panels) nor it affected the disappearance of surface IFNAR2 induced by IFNβ (bottom right panel). However, monensin led to a substantial decrease of surface IFNAR2 in cells treated with IFNα (bottom left panel). Thus, following binding of IFNα2 to the receptor complex, IFNAR2 is indeed internalized but, instead of being routed towards a degradation pathway, it appears to recycle back to the cell surface.

DISCUSSION

The evolutionary conservation of multiple type I IFN subtypes in all eutherian mammals evokes distinct physiological roles despite shared receptor usage. Here, we have investigated whether IFNα2 and IFNβ, well described for their different mode of engagement of the receptor, differ in membrane-proximal signaling potency and in receptor down-regulation processes. In particular, we have studied cell lines that express endogenous IFNAR1 and IFNAR2 at different relative ratios and evaluated potential contribution of this ratio to early Jak/Stat signaling by the two IFN subtypes. Our major observations can be summarized as follows : (i) within each cell line, the phosphorylation profiles of Jak/Stat molecules induced by IFNα2 or IFNβ are virtually identical within the first four hours of stimulation; (ii) the extent of tyrosine phosphorylation of Jak/Stat components appears to be proportional to the amount of protein in the cell, rather than to the R2/R1 ratio; (iii) the activation profile of Stat2 differs from that of Tyk2, Stat1 and Stat3 and this in all cellular contexts analyzed; (iv) most remarkably, IFNα2 and IFNβ lead to differential down-regulation and routing of IFNAR2.

Previous studies have reported some differences in the phosphorylation profile of Jak/Stat proteins by IFNα and IFNβ. In human myocardial fibroblasts selective IFNβ-induced activation of Jak1 and higher Stat1/2 phosphorylation were observed, along with a 120-fold higher sensitivity to the antiviral effect of IFNβ as compared to IFNα [24]. In human vascular endothelial cells, a 2-3 fold higher Stat1 phosphorylation was observed with IFNβ, whereas a 2-3 log difference in α/β potency was measured in functional assays [25]. In monocytederived immature dendritic cells, levels of activation of Stat1/2 by IFNα2 and β were found to be comparable [26], as in the four human cell lines described in the present work. In sum, these results suggest that cell type differences in the potency of the two IFN subtypes to activate membrane-proximal signaling events do exist. However, when present, these differences are relatively modest, do not reflect ligand binding affinities to IFNAR1 and are unlikely to account for the large differences in potencies measured in functional assays.

Indeed, in spite of identical phosphorylation potency, we did measure a 2 log difference in the antiproliferative potencies of IFNα2 and IFNβ on HeLa cells (Fig. 9A).

It has been previously shown that IFNAR1 contributes to enhance the affinity of the cellular receptor complex for the ligand and also modulates ligand selectivity [START_REF] Cutrone | Contributions of cloned type I interferon receptor subunits to differential ligand binding[END_REF]27]. Studies of the assembly of the trimeric complex on artificial surfaces argued against a substantial interaction between the receptor subunits in the absence of ligand. Instead, the local concentration of IFNAR1 was shown to influence the assembly of the IFNα ternary complex [12,13]. Not suprisingly, we found that the relative surface expression level of the two IFN receptor subunits varies in different cell lines. Yet, within each cell line and regardless of the R2/R1 ratio, IFNα 2 and IFNβ-induced phosphorylation profiles were comparable. Furthermore, IFNAR1 does indeed represent a critical factor for both IFNα2 and IFNβ-driven activation of the Jak-Stat signaling complex. In fact, depletion of endogenous IFNAR1 in WISH cells led to a significant reduction of signal activation which could not be compensated by increased ligand dose. On the other hand, a surplus of IFNAR1 in WISH cells did not change the IFN sensitivity profile nor the kinetics of Jak/Stat activation (Fig. 3, data not shown). Thus, in spite of the ability of IFNAR1 to compensate for weak IFNα binding, increased surface concentration of IFNAR1 does not change signaling potency.

Comparison of IFN signaling in Jurkat and Daudi cells indicated that the basal cellular content of Stat1/3, rather than receptor levels, impacts on the extent of their phosphorylation and thus, most likely, on downstream gene induction profiles. While this may contribute to cell type specific differences in IFN action, it also evokes the notion that changes in the content of Stats, as upon cell activation or differentiation, may tune IFN responses and gene induction programs [28][29][30][31]. Interestingly, Stat2 phosphorylation appears quite unique in all cellular contexts tested: it is saturated more readily and at lower IFN doses, is more sustained and barely affected by IFNAR1 depletion. We previously showed that Stat2, as opposed to Stat1/3, is not engaged at all upon induced dimerization of the IFNAR1 cytoplasmic domain [16]. Thus, the unique Stat2 activation profile may, at least in part, be consequent to its constitutive association to the IFNAR2 chain [32].

The lack of correlation between ligand-IFNAR1 affinity and Jak/Stat activation potency suggests that α/β differential bioactivities must be achieved through mechanisms other than immediate Jak/Stat activation. Moreover, these mechanisms are likely to be influenced by ligand binding affinity. Our present analysis of ligand-induced receptor traffic provides some interesting clues to this issue. Despite the heterogeneity in the expression level of the two subunits in different cell lines, consistent differences were observed in the dynamics of the two receptor subunits. With the exception of Daudi cells, where IFNAR1 is highly expressed and stable, in all other cell lines studied IFNAR1 exhibits a relatively short half-life and is down-regulated similarly by IFNα2 and IFNβ. Interestingly, IFNAR2 exhibits a longer half-life which is differentially affected by the two IFN subtypes: following binding of IFNβ, surface IFNAR2 levels decrease, whereas following binding of IFNα2 surface IFNAR2 remains constant as the internalized pool predominantly recycles back to the cell surface. This notion was supported by an experiment that tested the effect of monensin on receptor dynamics. In the presence of this well characterized inhibitor of recycling, IFNα2 led to significant down-regulation of IFNAR2 (Fig. 9). These data evoke previous biochemical demonstrations that IFNAR1 and IFNAR2 can be co-immunoprecipitated after stimulation with IFNβ, but not IFNα [33][34][35][36]. Moreover, in one such study performed on Daudi cells, active IFNβ, but not IFNα, could be recovered in the immunoprecipitated complex [37].

Our results extend to several human cell lines the α/β differential down-regulation of IFNAR2 originally reported in 293T cells [13]. Moreover, our studies in these latter cells demonstrate that the IFNβ-induced down-regulation of IFNAR2 indeed depends on formation of the ternary complex and that increasing IFNAR1 does not modify ligand capacity to downregulate IFNAR2. These observations strongly suggest that it is the strength of ligand binding and/or the lifetime of the ternary complex which may account for the differential control of IFNAR2.

That the strength of ligand binding may be critical for receptor routing is well established. A more stable ligand-receptor interaction at the moderately acidic conditions of early endosomes, may promote receptor degradation and prevent recycling [38,39] [20,21,40]. While ligand-induced ubiquitination of IFNAR1 promotes its efficient sorting towards degradation [START_REF] Kumar | SCF(HOS) ubiquitin ligase mediates the ligand-induced down-regulation of the interferon-alpha receptor[END_REF]16], we showed here that IFNAR2 does not undergo ligand-induced ubiquitination (Fig. 5). Hence, differential ubiquitination is unlikely to underlie the α/β differential effect on IFNAR2 routing. An alternative possibility is that IFNβ, through efficiently bridging IFNAR2 to ubiquitinated IFNAR1, drives the complex towards a degradative pathway. While the molecular steps by which IFNα2 and IFNβ lead to differential intracellular routing of IFNAR2 remain to be elucidated, our results imply that neither ligand properties nor receptor traffic impacts on immediate-early activation of Jak/Stat. Divergent endosomal sorting of activated receptors has emerged as a dynamic process that can tune and diversify intracellular signaling [41]. For example, it was shown that TGF-β receptors, residing in different membrane microdomains, can internalize through distinct endocytic compartments, which in turn mediate either signal activation or receptor degradation [42]. Interestingly, it has been reported that a fraction of IFNAR1 can be found in lipid rafts in HeLa cells and mouse embryo fibroblasts [43,44]. This observation raises the possibility that membrane accessibility of (competent) receptor subunits may control their basal and ligand-induced dynamics. In this scenario, the intrinsic binding property of the ligand combined with cell type differences in availability of competent receptors would contribute to the α/β differential IFNAR2 routing. Whether divergent traffic routes of the engaged receptors may shape IFN subtype-specific signaling and/or contribute to Statindependent signaling [START_REF] Van Boxel-Dezaire | Complex modulation of cell type-specific signaling in response to type I interferons[END_REF] remains an appealing and open question.

  , the comparative analysis performed in these cells did not reveal α/β differential potency in Jak/Stat signaling and confirmed the distinctive profile of Stat2 activation with respect to Stat1/3.

  viability of different cell lines was tested. HeLa cells were chosen as they were minimally affected by the drug. Moreover, in these cells a 2 log difference in the antiproliferative potencies of IFNβ and IFNα2 could be measured(Fig 9A).

  . Recent studies have implicated ubiquitination of activated receptors as a signal for endosomal sorting towards degradation [19] and the absence of ubiquitination was shown to divert internalized Biochemical Journal Immediate Publication. Published on 13 Jul 2007 as manuscript BJ20070605 © 2007 The Authors Journal compilation © 2007 Biochemical Society -14receptors towards the recycling pathway

Figure 1 .

 1 Figure 1. Surface levels of IFNAR1 and IFNAR2 and profiles of IFN-induced Jak/Stat phosphorylation in four cell lines. (A) Steady-state levels of IFNAR1 and IFNAR2 at the cell surface of the indicated cell lines were measured by flow cytometry. (B and C) Kinetics of IFNα and IFNβ-induced Tyk2 and Stat1/2/3 phosphorylation in Daudi and Jurkat cells. Cells were incubated with 500 pM IFNα2 or IFNβ for the indicated time. Total lysates (40 µg) were resolved by SDS/PAGE and the levels of phosphorylated or total Tyk2 and Stat1/2/3 were analyzed by western blot. (D) Dose response profiles of Tyk2 and Stat1/2/3 phosphorylation after IFNα2 or IFNβ treatment of Daudi and Jurkat cells. Cells were treated with the indicated doses (from 2.5 to 500 pM) of IFNα2 or IFNβ for 30 min. Lysates (40 µg) were analyzed as described in (A). Data are representative of at least 3 different experiments.

Figure 2 .

 2 Figure 2. IFNα2 and IFNβ-induced phosphorylation profiles in WISH cells. (A) Kinetics of Tyk2 and Stat1/2/3 phosphorylation in WISH cells treated with 500 pM of IFNα2 or IFNβ, for the indicated time. (B) Dose response profiles of Tyk2 and Stat1/2/3 phosphorylation in WISH cells treated for 15 min with IFNα2 or IFNβ. Lysates (40 µg) were analyzed by western blot. Data are representative of at least 3 different experiments.

Figure 3 .

 3 Figure 3. Levels of IFNAR1 and IFNAR2 and IFNα2-induced phosphorylation profiles in WISH-derived clones. (A) Surface IFNAR1 and IFNAR2 levels in stable WISH clones measured by flow cytometry. Three stable clones were derived from WISH parental cells, one depleted of endogenous IFNAR1 (CPO2), one over-expressing IFNAR1 (R16) and one expressing IFNAR1 as in parental cells (R23). (B) Total IFNAR1 content and IFNα-induced Jak/Stat phosphorylation in CPO2, R23 and R16 clones. Cells were incubated with 500 pM IFNα2 for the indicated time. Lysates (40 µg) were analyzed by western blot. Representatives of at least 3 different experiments are shown. The lower band visible in the R16 samples represents a 95 kDa incompletely processed IFNAR1 species which accumulates when the protein is overexpressed [15].

Figure 4 .

 4 Figure 4. Dose response profiles of IFNα2-induced Jak/Stat phosphorylation in WISHderived clones. (A) CPO2 and R23 cells were treated with increasing doses of IFNα2 for 15 min. Note the maximal dose (2500 pM) added on CPO2 cells. (B) R23 and R16 cells were treated with IFNα2 (from 1 to 100 pM) for 15 min. Total lysates (40 µg) were resolved by SDS/PAGE and levels of phosphorylated Tyk2, Jak1 and Stat1/2/3 were analyzed by western blot. The arrow points to a band, identified as phospho-Tyk2, which cross-reacts with anti-phospho-Jak1 Abs.

Figure 5 .

 5 Figure 5. Ligand-induced post-translational modifications of IFNAR1 and IFNAR2. Daudi cells were treated with 500 pM IFNα2 or IFNβ for the indicated times. Total lysates (5 mg) were used to immunoprecipitate endogenous IFNAR1 and IFNAR2. Immunoprecipitates were resolved by SDS/PAGE and the extent of ubiquitination of the receptors was analyzed using anti-ubiquitin Abs (top panels). Note the shift in migration of IFNAR1 due to induced ubiquitination. The left membrane was stripped and reprobed with anti-phosphotyrosine 4G10 mAb, P-Ser535-IFNAR1-specific Abs and anti-IFNAR1 mAb, as indicated. The right membrane was stripped and reprobed with anti-phosphotyrosine 4G10 mAb and anti-IFNAR2 mAb, as indicated. Migration of the 116 kDa marker is indicated on the left. Data are representative of at least 3 different experiments.

Figure 6 .

 6 Figure 6. Basal decay of surface IFNAR1 and IFNAR2. Surface stability of IFNAR1 and IFNAR2 in unstimulated cells. Decay of IFNAR1 (empty squares) and IFNAR2 (filled squares) from the surface of Daudi, Jurkat, 293T and HeLa cells treated with cycloheximide (CHX) was measured by flow cytometry. The results are plotted as percentages of the mean fluorescence at time 0. Mean values of 4 different experiments are presented.

Figure 7 .

 7

(

  A) IFNAR1 (left panels) and IFNAR2 (right panels) decay from the cell surface were measured by flow cytometry in cells treated with CHX and 500 pM IFNα2 (empty circles) or IFNβ (filled circles) for the indicated time. Data are plotted as the percentage of mean fluorescence at time 0. Mean values of at least 3 different experiments are shown. In the insets the difference between mean fluorescence values measured on untreated and IFN-plotted. (B) Daudi, Jurkat, 293T and HeLa cells were treated with 500 pM IFNα2 or IFNβ for the indicated time. Total lysates (40 µg) were resolved by SDS/PAGE and levels of IFNAR1 and IFNAR2 were analyzed by western blot using corresponding antibodies.

Figure 8 .

 8 Figure 8. Ligand-induced down-regulation of IFNAR2 in 293T cells expressing different levels of IFNAR1. 293T cells were transiently cotransfected with EGFP and either an IFNAR1-silencing vector or an IFNAR1-expression vector. An aliquot of the transfected cells was monitored for EGFP and IFNAR1 levels. In parallel, cells were treated with 200 pM IFNα2 or IFNβ for 2 h, or left untreated, and levels of IFNAR2 were measured in chosen EGFP-positive subpopulations. IFNAR1 relative level (horizontal axis) represents the amount of surface IFNAR1 in transfected cells relative to endogenous IFNAR1 (marked as 1) in untreated cells. The extent of IFNAR2 down-regulation (vertical axis) is expressed as the difference in the Geo Mean of IFNAR2 specific fluorescence between untreated cells and cells treated with IFNα2 (empty circles) or IFNβ (filled circles).

Figure 9 .

 9 Figure 9. IFNα2-induced recycling of IFNAR2 in HeLa cells. (A) IFN-induced antiproliferative effect in HeLa cells. Cells were either left untreated or treated with the indicated concentrations of IFNα2 (open circles) or IFNβ (closed circles) for 72 hr. Cell density was monitored by crystal violet staining as described in Experimental procedures. The mean optical density values of triplicate datasets are presented. (B) HeLa cells were either left untreated or treated with 25 µM monensin for 30 min in the presence of transferrin (50 µg/ml) and the level of TrR at the cell surface was measured by flow cytometry (left panel). Data are plotted as percentages of the mean fluorescence in control condition. The mean value of 4 different experiments is presented. In the right panel, the basal decay of IFNAR2 from the cell surface was monitored in the presence (open squares) or absence (filled squares) of monensin for the indicated times using flow cytometry. The results are plotted as percentages of the mean fluorescence at time 0. The mean values of 3 different experiments are presented. (C) IFNα2-induced IFNAR2 recycling. HeLa cells were treated with 500 pM IFNα2 (left panels) or IFNβ (right panels) in the presence (open circles) or absence (closed circles) of monensin for the indicated times and the surface levels of IFNAR1

Biochemical Journal Immediate Publication. Published on 13 Jul 2007 as manuscript BJ20070605 © 2007 The Authors Journal compilation © 2007 Biochemical Society

ACKNOWLEDGEMENTS

We thank P. Eid, D. Gewert, L. Runkel, S. Fuchs and A. Alcover for providing reagents; the platform of cytometry of the Institut Pasteur; F. Michel and V. Di Bartolo for advice and critical reading of the manuscript. Z. M. was supported by a Marie Curie International Fellowhip (contract MIF1- CT-2004-509400). This work was supported by a collaborative grant of the Association pour la Recherche sur le Cancer (n° 3158 to S.P. and G.U.).