Brian Christensen

Torben E Petersen

Esben S Sørensen

Posttranslational modification and proteolytic processing of urinary osteopontin

Keywords: Osteopontin, phosphorylation, sulphation, O-glycosylation, uropontin OPN, osteopontin, PTM, posttranslational modification, RGD, Arg-Gly-Asp, COM, calcium oxalate monohydrate, COD, calcium oxalate dihydrate, ALP, bovine alkaline phosphatase, μRPC, μ reverse-phase chromatography

Osteopontin (OPN) is a highly phosphorylated glycoprotein present in many tissues and body fluids. In urine OPN is a potent inhibitor of nucleation, growth and aggregation of calcium oxalate crystals suggesting a role in the prevention of renal stone formation.

The role of OPN in nephrolithiasis is however somewhat unclear as it may also be involved in urinary stone formation, and it has been identified among the major protein components of renal calculi. Most likely the function of OPN in urine is dependent upon the highly anionic character of the protein. Besides a very high content of aspartic-and glutamic acid residues, OPN is subjected to significant posttranslational modification (PTM), such as phosphorylation, sulphation and glycosylation, which may function as regulatory switches in promotion or inhibition of mineralization. In the present study we have characterized the PTMs of intact human urinary OPN and N-terminal fragments thereof. Mass spectrometric (MS) analysis showed a mass of 37.7 kDa for the intact protein. Enzymatic dephosphorylation and peptide mass analyses demonstrated that the protein contains approximately eight phosphate groups distributed over 30 potential phosphorylation sites. In addition one sulphated tyrosine and five O-linked glycosylations were identified in OPN, whereas no N-linked glycans were detected. Peptide mapping and immunoblotting using different monoclonal antibodies showed that the N-terminal fragments present in urine are generated by proteolytic cleavage at Arg 228 -Leu 229 and Tyr 230 -Lys 231 .

Introduction

Osteopontin (OPN) is a phosphorylated glycoprotein containing an integrin binding RGD (Arg-Glu-Asp) sequence. The protein was originally purified from the mineralized matrix of bovine bone [START_REF] Franzén | Isolation and characterization of two sialoproteins present only in bone calcified matrix[END_REF], however, the expression of OPN is not limited to mineralized tissues but extends to most tissues and body fluids including milk, blood and urine [START_REF] Sodek | Osteopontin[END_REF]. OPN consists of approximately 300 amino acid residues with a very high content of aspartic-and glutamic acid. The protein is involved in a broad range of functions in both physiological and pathological processes like cytokine production, tumor growth, inflammation and mineralization [START_REF] Ashkar | Eta-1 (osteopontin): an early component of type-1 (cell-mediated) immunity[END_REF][START_REF] Rittling | Role of osteopontin in tumour progression[END_REF][START_REF] Giachelli | Osteopontin: a versatile regulator of inflammation and biomineralization[END_REF]. Furthermore, OPN is a key molecule in bone remodelling [START_REF] Giachelli | Osteopontin: a versatile regulator of inflammation and biomineralization[END_REF][START_REF] Chellaiah | Rho-dependent Rho kinase activation increases CD44 surface expression and bone resorption in osteoclasts[END_REF] and it hinders ectopic calcification by inhibiting the formation of hydroxyapatite [START_REF] Hunter | Modulation of crystal formation by bone phosphoproteins: structural specificity of the osteopontinmediated inhibition of hydroxyapatite formation[END_REF][START_REF] Boskey | Osteopontin-hydroxyapatite interactions in vitro: inhibition of hydroxyapatite formation and growth in a gelatin-gel[END_REF], which is the main mineral phase of bone and teeth, as well as calcium oxalate [START_REF] Shiraga | Inhibition of calcium oxalate crystal growth in vitro by uropontin: another member of the aspartic acid-rich protein superfamily[END_REF], the main mineral in renal calculi.

OPN is found at high levels in urine with an average of approximately four mg secreted into human urine daily [START_REF] Min | Quantitative studies of human urinary excretion of uropontin[END_REF]. In contrast, OPN is undetectable in normal kidney tissues [START_REF] Rittling | Detection of mouse osteopontin by western blotting[END_REF], indicating that the OPN produced in the renal tubules is immediately secreted in the urine. OPN can inhibit nucleation, growth and aggregation of calcium oxalate crystals in vitro [START_REF] Shiraga | Inhibition of calcium oxalate crystal growth in vitro by uropontin: another member of the aspartic acid-rich protein superfamily[END_REF][START_REF] Wesson | Control of calcium oxalate crystal structure and cell adherence by urinary macromolecules[END_REF][START_REF] Asplin | Contribution of human uropontin to inhibition of calcium oxalate crystallization[END_REF], and it blocks binding of the crystals to renal epithelial cells [START_REF] Lieske | Adhesion of calcium oxalate monohydrate crystals to renal epithelial cells is inhibited by specific anions[END_REF]. In addition, calcium oxalate monohydrate (COM) crystal growth and aggregation are inhibited by 50% at an OPN concentration between 16 to 28 nM, a concentration that is an order of magnitude lower than the OPN concentration of 131 nM found in normal urine [START_REF] Asplin | Contribution of human uropontin to inhibition of calcium oxalate crystallization[END_REF]. OPN also favours the formation of calcium oxalate dihydrate (COD) over COM crystals [START_REF] Wesson | Control of calcium oxalate crystal structure and cell adherence by urinary macromolecules[END_REF]. The induction of COD by OPN may be protective against calcium oxalate nephrolithiasis since COD has demonstrated less affinity for attachment to renal epithelial cells than COM [START_REF] Wesson | Control of calcium oxalate crystal structure and cell adherence by urinary macromolecules[END_REF]. Besides reducing crystal affinity for renal cells, OPN may act as an intracrystalline protein assisting stone prevention by rendering crystals more susceptible to intracellular degradation [START_REF] Ryall | Intracrystalline proteins and urolithiasis: a comparison of the protein content and ultrastructure of urinary calcium oxalate monohydrate and dihydrate crystals[END_REF]. Furthermore, OPN knockout mice fed an crystal-inducing diet developed renal calcium oxalate stones while wild type mice were completely unaffected, perhaps due to an upregulated OPN expression in response to the induced hyperoxaluria suggesting a protective role of OPN [START_REF] Wesson | Osteopontin is a critical inhibitor of calcium oxalate crystal formation and retention in renal tubules[END_REF]. Similarly, a recent study showed that 10% of mice lacking OPN spontaneously formed renal stones and 65% developed renal crystals under experimentally induced hyperoxaluria, whereas these events were not observed in wild type mice [START_REF] Mo | Renal calcinosis and stone formation in mice lacking osteopontin, Tamm-Horsfall protein, or both[END_REF].

There are many indications that OPN is an important inhibitor of renal stone formation, however, the protein has also been implicated as a promoter of stone formation. One study showed increased calcium oxalate crystal adherence to kidney cells when they were incubated with OPN [START_REF] Yamate | The effect of osteopontin on the adhesion of calcium oxalate crystals to Madin-Darby canine kidney cells[END_REF]. Furthermore, inhibition of OPN synthesis has been shown to suppress the deposition and adhesion of calcium oxalate crystals to rat renal cells [START_REF] Yasui | Osteopontin regulates adhesion of calcium oxalate crystals to renal epithelial cells[END_REF], and addition of polyclonal OPN antibodies reduced the deposition of crystals on the surface of kidney cells [START_REF] Yamate | Interaction between osteopontin on madin darby canine kidney cell membrane and calcium oxalate crystal[END_REF]. It has also been suggested that OPN is involved in urinary stone formation [START_REF] Kohri | Molecular cloning and sequencing of cDNA encoding urinary stone protein, which is identical to osteopontin[END_REF], and the protein has been identified as a prominent constituent of the major matrix of renal calculi [START_REF] Mckee | Ultrastructural immunodetection of osteopontin and osteocalcin as major matrix components of renal calculi[END_REF]. Reduced OPN levels in stone formers compared to non-stone formers have been reported in one study [START_REF] Yasui | Quantification of osteopontin in the urine of healthy and stoneforming men[END_REF], while no difference in urinary OPN levels has been reported in other studies [START_REF] Bautista | Lowmolecular-weight variants of osteopontin generated by serine proteinases in urine of patients with kidney stones[END_REF][START_REF] Kleinman | Osteopontin and calcium stone formation[END_REF].

OPN is extensively altered through posttranslational modification (PTM), such as phosphorylation, sulphation, glycosylation and proteolytic processing, which have significant implications on the structure and the biological function of the protein [START_REF] Kazanecki | Control of osteopontin signaling and function by post-translational phosphorylation and protein folding[END_REF].

The PTMs of OPN from bovine and human milk as well as rat bone have been thoroughly characterized [START_REF] Sørensen | Posttranslational modifications of bovine osteopontin: identification of twenty-eight phosphorylation and three O-glycosylation sites[END_REF][START_REF] Christensen | Post-translationally modified residues of native human osteopontin are located in clusters: identification of 36 phosphorylation and five Oglycosylation sites and their biological implications[END_REF][START_REF] Keykhosravani | Comprehensive identification of posttranslational modifications of rat bone osteopontin by mass spectrometry[END_REF]. Recently, a comparative study of OPN produced by murine ras-transformed fibroblasts and differentiating osteoblasts demonstrated remarkably different phosphorylation patterns between OPNs originating from the same species, which translated into significant functional differences in cellular adhesion [START_REF] Christensen | Cell Type-specific Post-translational Modifications of Mouse Osteopontin Are Associated with Different Adhesive Properties[END_REF].

The PTMs of OPN provide a basis for its regulatory functions in mineralization processes, as dephosphorylated OPN has been shown to loose the ability to inhibit hydroxyapatite formation [START_REF] Hunter | Modulation of crystal formation by bone phosphoproteins: structural specificity of the osteopontinmediated inhibition of hydroxyapatite formation[END_REF][START_REF] Boskey | Osteopontin-hydroxyapatite interactions in vitro: inhibition of hydroxyapatite formation and growth in a gelatin-gel[END_REF]. Emphasizing the importance of the phosphorylations, it was recently shown that highly phosphorylated milk OPN promoted hydroxyapatite formation and growth, whereas the less phosphorylated bone OPN inhibited these processes [START_REF] Gericke | Importance of phosphorylation for osteopontin regulation of biomineralization[END_REF]. Similarly, recombinant (non-phosphorylated) OPN increases mineralization of vascular smooth muscle cells, whereas enzymatically phosphorylated OPN is a potent inhibitor of this process [START_REF] Jono | Phosphorylation of osteopontin is required for inhibition of vascular smooth muscle cell calcification[END_REF]. Recently, it was shown that only phosphorylated OPN dose-dependently inhibited mineralization of MC3T3-E1 osteoblast cell cultures [START_REF] Addison | Pyrophosphate inhibits mineralization of osteoblast cultures by binding to mineral, up-regulating osteopontin, and inhibiting alkaline phosphatase activity[END_REF]. Phosphorylation of OPN peptides also markedly enhanced the inhibition of hydroxyapatite and COM crystal growth [START_REF] Pampena | Inhibition of hydroxyapatite formation by osteopontin phosphopeptides[END_REF][START_REF] Hoyer | Phosphorylated osteopontin peptides suppress crystallization by inhibiting the growth of calcium oxalate crystals[END_REF] and C-terminal fragments of urinary OPN can inhibit COM growth as efficient as the intact protein, suggesting that different parts of the protein can regulate crystal formation [START_REF] Worcester | Osteopontin production by cultured kidney cells[END_REF].

Western blotting of human urinary OPN has shown the presence of different forms in the 40-70 kDa range [START_REF] Ryall | Intracrystalline proteins and urolithiasis: a comparison of the protein content and ultrastructure of urinary calcium oxalate monohydrate and dihydrate crystals[END_REF][START_REF] Bautista | Lowmolecular-weight variants of osteopontin generated by serine proteinases in urine of patients with kidney stones[END_REF][START_REF] Kleinman | Osteopontin and calcium stone formation[END_REF]. The structural variation underlying these differences in migration is unknown. Normal rat kidney cells have been shown to produce both phosphorylated and non-phosphorylated OPN which also diverge in glycosylation and physiological properties [START_REF] Singh | Physiological properties and differential glycosylation of phosphorylated and nonphosphorylated forms of osteopontin secreted by normal rat kidney cells[END_REF]. Furthermore, low molecular weight OPN variants probably resulting from cleavage by serine proteases are present at a higher frequency in urine from individuals with kidney stone disease than in urine from healthy individuals [START_REF] Bautista | Lowmolecular-weight variants of osteopontin generated by serine proteinases in urine of patients with kidney stones[END_REF]. Recently, a series of C-terminal OPN fragments were identified by two-dimensional electrophoresis and mass spectrometry in patients with ovarian cancer, but not in healthy controls [START_REF] Ye | Proteomic-based discovery and characterization of glycosylated eosinophilderived neurotoxin and COOH-terminal osteopontin fragments for ovarian cancer in urine[END_REF]. In the present study we have separated and characterized different forms of human urinary OPN with regard to PTMs and

Experimental

Materials

The monoclonal mouse antibodies 1H3 and 3D9 [START_REF] Kazanecki | Characterization of anti-osteopontin monoclonal antibodies: Binding sensitivity to post-translational modifications[END_REF] were a kind gift from Dr. David T.

Denhardt, Rutgers University, NJ. Swine anti-rabbit and rabbit anti-mouse immunoglobulins conjugated to alkaline phosphatase were from DAKO (Glostrup, Denmark). Bovine pancreas trypsin and Staphylococcus aureus V8 proteinase were obtained from Worthington Biochemical (Freehold, NJ). Thermolysin, bovine alkaline phosphatase (ALP), human plasma thrombin and arylsulphatase were from Sigma (St.

Louis, MO). The μRPC (μ reverse-phase chromatography) C 2 /C 18 PC 2.1/10 column, Protein A sepharose CL-4B resin, DEAE sepharose material and PD-10 desalting columns were from GE Healthcare (Uppsala, Sweden). Vydac C 4 and C 18 RP (reversephase) resin were obtained from The Separations Group (Hesperia, CA). Reagents used for sequencing were purchased from Applied Biosystems (Warrington, UK). 2,5dihydroxybenzoic acid was from LaserBio Labs (Sophia-Antipolis Cedex, France). The Peptide-N-Glycosidase F (PNGase F) kit was obtained from New England Biolabs (Beverly, MA). All other chemicals used were of analytical grade.

Polyclonal OPN Antibodies

Polyclonal antiserum against human milk OPN was raised in rabbits at DAKO (Glostrup, Denmark). OPN specific polyclonal antibodies were isolated from the rabbit serum on a Protein A sepharose column. The specificity of the antibodies was checked and verified by Western blot analyses of milk samples and purified OPN.

Western Blotting

OPN (50 ng/lane) and urine (10 uL) were loaded onto 16% tris-tricine gels, fractionated by SDS-PAGE and electrophoretically transferred to Hybond-P polyvinylidene difluoride membranes (GE Healthcare) for immunodetection. The membranes were blocked in 2% tween in tris-buffered-saline before addition of either polyclonal rabbit OPN antibodies (5 µg/ml) or monoclonal mouse OPN antibodies 1H3 and 3D9 (1.5 μg/ml). OPN was detected with alkaline phosphatase conjugated secondary

Purification of OPN

Urine samples were collected from seven healthy donors with normal renal function and no history of urinary disease and frozen at -80 °C. Thawed urine samples were centrifuged at 4500 g for 30 minutes and the resulting supernatant was incubated with DEAE sepharose material at pH 8.1 for 2 h at room temperature. Adsorbed proteins were batch eluted with stepwise increasing concentrations of ammonium bicarbonate (pH 8.1). The batch containing OPN was lyophilized, followed by passage through a PD10 column in 20% formic acid. The salt free eluate was lyophilized and subjected to further purification by RP-HPLC on a Vydac C 4 column connected to a GE Healthcare LKB system. Separation was carried out at 40 °C in 0.1% trifluoroacetic acid (TFA) (buffer A) and eluted with a gradient of 80% acetonitrile in 0.1% TFA (buffer B) developed over 50 min (0-5 min, 0% B; 5-40 min, 0-50% B; 40-50 min, 50-95% B) at a flow rate of 0.85 ml/min. The peptides were detected in the effluent by measuring the absorbance at 226 nm. The fraction containing OPN was identified by immunodetection and N-terminal sequencing. The full-length protein (OPN60) and N-terminal fragments (OPN45 and OPN50) were separated by RP-HPLC on a Vydac C 18 column and eluted with stepwise increasing concentrations between 10% and 30% of 75% 2-propanol in 0.1% TFA. The fractions were analyzed by N-terminal sequencing and SDS-PAGE, and proteins were visualized by Coomassie staining or by Western blotting. The amount of purified OPN was determined by amino acid analysis.

Phosphate content of OPN

Native and dephosphorylated OPN isoforms were analyzed by MS using a Voyager DE-PRO MALDI-TOF (matrix-assisted laser desorption ionization time-of-flight) mass spectrometer (Applied Biosystems). Samples for MS analyses were prepared by mixing the sample with a saturated solution of 2,5-dihydroxybenzoic acid in a 1:1 ratio directly on the MS-target probe. All spectra were obtained in positive linear-ion mode using a nitrogen laser at 337 nm and an acceleration voltage of 20 kV. Typically, 50-100 laser shots were added per spectrum and calibrated with external standards. The masses were assigned using the half-height width method. For dephosphorylation, 2.5 µg OPN was incubated with 0.07 U ALP in 10 mM ammonium bicarbonate (pH 8.5) overnight at 37 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

°C. The average phosphate content estimated by MS was confirmed with a phosphoprotein phosphate estimation assay kit (Pierce, Rockford, IL).

Carbohydrate analyses

Full-length OPN (OPN60) was quantified by amino acid analysis and hydrolyzed in 2 M TFA for 4 h at 100 °C under oxygen-free conditions. α-L-rhamnose was added as internal standard. For quantification of the more stable amino sugars, Nacetylgalactosamine (GalNAc) and N-acetylglucosamine (GlcNAc), an additional OPN sample was hydrolyzed in 4 M HCl for 6 h at 110 °C. For sialic acid (Sia) analysis, the protein was hydrolyzed in 0.1 M HCl for 60 min at 80 °C. Monosaccharides were separated by high-pH anion-exchange chromatography using a CarboPac PA1 column Removal of potential N-linked glycosylations was performed as described by the manufacturer using a PNGase F deglycosylation kit. OPN was incubated with 500 U PNGase F at 37 °C for 20 h. The reaction products from the enzymatic deglycosylation were analyzed by SDS-PAGE.

Generation and separation of peptides

OPN was digested with trypsin using an enzyme to substrate ratio of 1:30 (w/w) in 0.1 M ammonium bicarbonate at 37 °C for 6 h. Tryptic peptides were separated by RP-HPLC on a μRPC C 2 /C 18 PC 2.1/10 column connected to a GE Healthcare SMART system. Separation was carried out in 0.1% TFA (buffer A) and eluted with a gradient of 60% acetonitrile in 0.1% TFA (buffer B) developed over 54 min (0-9 min, 0% B; 9-49 min, 0-50% B; 49-54 min, 50-100% B) at a flow rate of 0. (residues Gln 36 /Ser 62 -Arg 143 ) from the tryptic digest was further digested with thermolysin in 0.1 M pyridine-acetate (pH 6.5), 5 mM CaCl 2 at 56 °C for 6 h. The resulting peptides were separated as described for the tryptic digest. A glycopeptide (residues Leu 116 -Thr 139 ) from the thermolysin digest was further digested with S. aureus V8 proteinase in 0.1 M ammonium bicarbonate (pH 8.1) at 37 °C for 3.5 h. OPN45/OPN50 and OPN60 were digested with thrombin (0.05 U/µg OPN) in 0.1 M ammonium bicarbonate at 37 °C for 1 h. The generated fragments were separated by RP-HPLC on a Vydac C 18 column connected to a GE Healthcare LKB system.

Separation was carried out in 0.1% TFA (buffer A) and eluted with a gradient of 75% 2propanol in 0.1% TFA (buffer B) developed over 80 min (0-5 min, 0% B; 5-70 min, 0-50% B; 70-80 min, 50-95% B) at a flow rate of 0.85 ml/min.

Characterization of peptides

Peptides and fragments were characterized by mass spectrometric and amino acid sequence analyses. All MS spectra were obtained in both positive reflector-ion and positive linear-ion mode as described above. The theoretical peptide masses and sequence coverage were calculated using the GPMAW program (Lighthouse Data, Odense, Denmark). Amino acid sequence analyses were performed on an Applied Biosystems PROCISE HT protein sequencer with on-line identification of phenylthiohydantoin (PTH)-derivatives. Glycosylated threonine residues were identified by the lack of a PTH-derivate in the cycles where these amino acids are modified.

Sulphate analysis

Modified peptides containing both potential phosphorylation (serines and threonines) and sulphation (tyrosines) sites were dephosphorylated with ALP as described above and analysed by MALDI-TOF-MS. The peptides Phe 158 -Lys 187 and Arg 160 -Lys 187 were further treated with 0.5 U arylsulphatase in 0.2 M sodium acetate (pH 5.0) at 37 °C for 2 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

RESULTS

Purification of OPN from urine -Urine samples from seven healthy donors, with normal renal function and no history of urinary disease, were analysed by Western blotting to examine the molecular forms of OPN in urine (Fig. 1). Urinary OPN was detected with variable intensity in all individuals by a polyclonal antibody. The observed OPN patterns show that all analyzed urine samples contain OPN forms of relative homogenous molecular weight in the range of 45-60 kDa. No, low molecular weight OPN forms were observed in any of the tested individuals.

OPN was purified from human urine by anion-exchange followed by RP-HPLC separation on a Vydac C 4 column (Fig. 2). Sequencing of the OPN containing fraction showed two sequences: a major, I 1 PVKQA 6 , and a minor, V 3 KQADS 8 , both corresponding to the N-terminal part of human OPN. SDS-PAGE revealed the presence of three bands migrating at approximately 45 kDa, 50 kDa and 60 kDa, respectively and Western blotting confirmed their OPN nature (Fig. 3A, Lane 1 and 2). In the following these components will be referred to as OPN45, OPN50 and OPN60.

To separate the different forms, the OPNs were subjected to RP-HPLC on a Vydac C 18 column and eluted with stepwise increasing concentrations of 2-propanol.

The elution profile revealed two fractions (insert in Fig. 2). As judged by SDS-PAGE the first fraction contained OPN45 and OPN50 and the second fraction contained OPN60 (Fig. 3A, lane 3 and 4). The monoclonal antibody 1H3 (epitope: S 218 AETHSH 224 [START_REF] Kazanecki | Characterization of anti-osteopontin monoclonal antibodies: Binding sensitivity to post-translational modifications[END_REF]) recognized all three OPN forms (Fig. 3B, lane 1 and 2), whereas Western blotting with the monoclonal 3D9 (epitope: K 283 FRISHELDSASSEVN 298 [START_REF] Kazanecki | Characterization of anti-osteopontin monoclonal antibodies: Binding sensitivity to post-translational modifications[END_REF]) showed that only OPN60 contains the extreme C-terminal part of OPN (Fig. 3B, lane 3 and 4). This indicates that OPN60 represents the full-length OPN molecule from human urine, since it contains both the N-and C-terminal, whereas OPN45 and OPN50 are truncated or cleaved forms lacking a C-terminal part of the protein.

Identification of cleavage sites in OPN -To identify the C-terminus and thereby

the exact cleavage site that generates OPN45 and OPN50, these proteins were subjected to thrombin cleavage. Reverse-phase separation of the products from the thrombin digest followed by sequence and MS analysis showed the presence of two N-terminal fragments, Ile 1 -Arg 152 and Val 3 -Arg 152 , and two C-terminal fragments, Ser 153 -Arg 228 and Ser 153 -Tyr 230 (Table 1). This indicates that OPN45 and OPN50 are generated by Molecular masses -The masses of the OPNs were determined by linear MALDI-TOF-MS. MS analysis showed a mass of approximately 37.7 kDa for OPN60 (Fig. 4A), whereas the sample containing OPN45 and OPN50 showed one broad mass peak with an average value of approximately 29.3 kDa (Fig. 4B). To estimate the total number of phosphate groups present, OPN60 and the fragments OPN45/OPN50 were treated with ALP. The molecular weight of dephosphorylated OPN60 was 37.1 kDa corresponding to a loss of approximately eight phosphate groups (Fig. 4A). Phosphatase treatment of the N-terminal OPN fragments reduced the mass to 28.8 kDa corresponding to a loss of approximately six phosphorylations (Fig. 4B). This was confirmed by use of a phosphoprotein phosphate estimation kit revealing a total of 7.8 phosphate groups in OPN60 and 6.9 phosphate groups in the sample containing OPN45/OPN50. Subtraction of the theoretical mass of the human OPN polypeptide (33714 Da) from the observed average mass of dephosphorylated OPN60 (37.1 kDa) leaves approximately 3.4 kDa, which must then be accounted for by other PTMs.

Phosphorylations -To identify potential phosphorylation sites in urinary OPN, the OPN fraction (Fig. 2) was digested with trypsin and the resulting peptides were separated by RP-HPLC (data not shown). A large acidic fragment of OPN (Gln 36 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Supplementary Table I and II). In total, the approximately eight phosphate groups in

OPN60 were distributed over 30 potential phosphorylation sites. Some of the phosphopeptides contained more serines/threonines than the observed number of phosphorylations. In these cases the phosphate groups were assigned to residues fitting the target sequence of the Golgi kinase/mammary gland casein kinase (S/T-X-E/S(P)/D) based on the localization of phosphorylated residues in other OPN isoforms [START_REF] Sørensen | Posttranslational modifications of bovine osteopontin: identification of twenty-eight phosphorylation and three O-glycosylation sites[END_REF][START_REF] Christensen | Post-translationally modified residues of native human osteopontin are located in clusters: identification of 36 phosphorylation and five Oglycosylation sites and their biological implications[END_REF][START_REF] Keykhosravani | Comprehensive identification of posttranslational modifications of rat bone osteopontin by mass spectrometry[END_REF].

Data from the peptide phosphorylation analysis are summarized in Table 2 and the resulting map of PTMs is shown in Figure 5, which also contains a comparison with the PTMs of human milk OPN.

Sulphation of OPN -The linear MALDI-TOF-MS spectrum of a peptide covering R 160 PDIQYPDATDEDITSHMESEELNGAYK 187 showed m/z values of 3307.18, 3387.53, and 3466.42 (Fig. 6A). These masses correspond to Arg 160 -Lys 187 with excess masses of ~80 Da, ~160 Da, and ~240 Da, respectively. These masses could correspond to different phosphorylation variants, or they could represent a combination of phosphate and sulphate groups on the peptide. After treatment with ALP an excess mass of ~80 Da remained associated with the peptide (Fig. 6B) strongly indicating that Arg 160 -Lys 187 is also sulphated. The presence of a sulphate group was verified by treatment of the dephosphorylated peptide with arylsulphatase which removed the excess mass of 80 Da (Fig. 6C). The peptide Arg 160 -Lys 187 contains two potential sites for sulphation (Tyr 165 and Tyr 186 ). The residue corresponding to Tyr 165 has been shown to be sulphated in OPN from rat bone [START_REF] Keykhosravani | Comprehensive identification of posttranslational modifications of rat bone osteopontin by mass spectrometry[END_REF], and it is therefore most likely that this residue is sulphated in urinary OPN. Two other tyrosine containing peptides, Gln 5 -Lys 35 and Gly 205 -Lys 225 , were observed with excess masses that could correspond to both phosphorylations and/or sulphate groups (Supplementary Table I). Treatment of these peptides with ALP showed that they only contained phosphate groups. Peptides containing Tyr 165 in human milk OPN was also analysed for sulphation. However, no indication of this modification was observed in the milk isoform (data not shown).

Glycosylations -All O-glycosylated residues in OPN from urine were located in a single thermolytic peptide, Leu 116 -Thr 139 . This peptide was further digested with S. aureus V8 proteinase resulting in the two peptides, L 116 VTDFPTDLPATE 128 and V 129 FTPVVPTVDT 139 , which were separated by RP-HPLC. The peptide L 116 VTDFPTDLPATE 128 was observed in linear MS with m/z values at 3679.83, 3970.97 and 4261.24 (Table 3). These masses correspond to Leu 116 -Glu 128 with three Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

GalNAc-galactose units and four to six Sia units attached. The lack of a PTH-Thr at the positions of Thr 118 , Thr 122 and Thr 127 , when subjected to Edman sequencing, indicates that these threonines are glycosylated in the peptide. Similarly, MS analysis of the peptide V 129 FTPVVPTVDT 139 showed that this peptide contained glycosylations composed of up to two GalNAc, one GlcNAc, two galactose and five sialic acid (Sia) units (Table 3). Edman sequencing showed that Thr 131 and Thr 136 are modified.

Collectively these data show that Thr 118 , Thr 122 , Thr 127 , Thr 131 and Thr 136 are glycosylated in urinary OPN. The absence of any trace of PTH-amino acids in these positions indicates that they are fully glycosylated. The masses of the glycopeptides show that each glycosylated threonine can be modified by different glycan structures, but based on the major mass peaks for both Leu 116 -Glu 128 and Val 129 -Thr 139 they seem mainly to consist of the disialylated core-1 O-glycan Sia-galactose-[Sia]-GalNAc structure.

OPN60 was subjected to mild acid hydrolysis to determine the monosaccharide composition of the glycans on the protein and thereby evaluate the structures observed in MS. The obtained monosaccharides were separated by high-pH anion exchange chromatography and subsequently monitored by pulsed electrochemical detection. The hydrolysates of OPN60 showed the presence of four types of monosaccharides GalNAc, GlcNAc, galactose and Sia (Table 3). The detected amounts of monosaccharides give approximately one mol of GalNAc/galactose and two moles of Sia per glycosylated threonine on OPN corresponding well with the glycans observed by MS.

The human OPN sequence contains two asparagines (Asn 63 and Asn 90 ) in putative N-glycosylation motifs. MS analysis of peptides containing these residues showed that none of these asparagines are glycosylated in urinary OPN. In addition, incubation of urinary OPN with PNGase F did not result in altered migration when subjected to SDS-PAGE (data not shown). Furthermore, no trace of mannose was detected in the analysis of OPN monosaccharides (Table 3), substantiating the absence of N-linked glycosylations in human urinary OPN.

Discussion

The present work describes the purification and characterization of urinary OPN.

Western blotting of urine obtained from seven healthy donors shows that urinary OPN exists in different forms migrating between 45 and 60 kDa (Fig. 1). In the OPN peak Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

eluting from the Vydac C 4 column three dominating forms were observed by Coomassie staining and immunoblotting (Fig. 3A). The strongly staining top band observed by Western blotting may consists of more than one isoform. This assumption is supported by Western blot detection of two bands in the samples containing OPN60 using the monoclonal antibodies 1H3 (faint) and 3D9 (Fig. 3B). OPN is present in human urine in different fragmented forms and it is therefore relevant to speculate on how these forms are generated. The protein has previously been shown to be a substrate for metalloproteinase (MMP)-3 and -7 [START_REF] Agnihotri | Osteopontin, a novel substrate for matrix metalloproteinase-3 (stromelysin-1) and matrix metalloproteinase-7 (matrilysin)[END_REF] and thrombin [START_REF] Senger | Purification of a human milk protein closely similar to tumor-secreted phosphoproteins and osteopontin[END_REF].

In Figure 5 the cleavage sites of these proteolytic enzymes are compared to the observed fragmentation patterns of the characterized urinary OPN, showing that neither MMP-3, MMP-7 nor thrombin have been reported to cleave OPN at the sites reported in this study. It is not clear whether OPN45/OPN50 is the result of cleavage by specific proteases in the urinary secretory system. The cleavage sites identified in this study points in two directions. The cleavage of Arg 228 -Leu 229 could be catalysed by a thrombin-type of protease, whereas the cleavage site Tyr 230 -Lys 231 fits the specificity of a chymotrypsin-type of protease. However, it remains to be shown which proteases that are actually responsible for the cleavage of OPN in urine. Kidney cells have been reported to produce an OPN peptide that are thought to represent tissue-specific intracellular processing of the intact protein [START_REF] Ullrich | Galactose 3 , Sia 4 ) 2387.61 (GalNAc 2 , GlcNAc, Galactose 2 , Sia 5 ) 1894.14 (GalNAc 2 , Galactose 2 , Sia 4 ) 1602.86 (GalNAc 2 , Galactose 2 , Sia 3 ) Carbohydrate composition (mol monosaccharide/mol OPN) GalNAc: 5.15 GlcNAc: 1.34 Galactose: 4.95[END_REF], which supports the hypothesis that OPN45 and OPN50 are proteolytically derived fragments of the intact protein.

Alternative mRNA splicing could also be speculated to be responsible for the presence of the truncated OPN forms. However, the region containing the cleavage sites is encoded by exon 7 of the OPN gene and no intron-exon junction is located in the vicinity. It is therefore a much more plausible explanation that the N-terminal fragments are products of proteolytic activity rather than alternative mRNA splicing. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Interestingly, no C-terminal fragments corresponding to Leu 229 /Lys 231 -Asn 298 were detected by the polyclonal antibody in the Western blot of urine (Fig. 1). This indicates that the C-terminal of urinary OPN is completely degraded when OPN45 and OPN50 are generated from OPN60. In support of this, no low molecular weight Cterminal OPN fragments were detected in Western blots of human urine using different monoclonal OPN antibodies with epitopes mapped to the C-terminal part of the protein [START_REF] Kazanecki | Characterization of anti-osteopontin monoclonal antibodies: Binding sensitivity to post-translational modifications[END_REF]. OPN forms below the 40 kDa electrophoretic mobility range have been observed in urine from patients with kidney stones [START_REF] Bautista | Lowmolecular-weight variants of osteopontin generated by serine proteinases in urine of patients with kidney stones[END_REF], whereas normal individuals had OPN forms migrating between 55-to 66-kDa as observed in this study. These low molecular weight forms of OPN were generated by serine proteases, and could reflect that augmented cleavage of urinary OPN could be a critical step in the formation of kidney stones.

OPN60 was shown to contain approximately eight phosphorylations and six phosphate groups were estimated to decorate OPN45/OPN50. This indicates that the region Leu 229 /Lys 231 -Asn 298 missing in these cleaved forms contains about two phosphate groups. It is not clear whether some serines or threonines in OPN are phosphorylated to a higher degree than others. However, the fragment Leu 229 /Lys 231 -Asn 298 that is missing in OPN45/OPN50 constitutes approximately one fourth of the amino acids in OPN. So, the observation that this fragment contains two out of eight phosphorylations suggests that the phosphate groups decorating the intact molecule are evenly distributed. The degree of phosphorylation on urinary OPN is significantly lower than the levels observed in milk OPN [START_REF] Sørensen | Posttranslational modifications of bovine osteopontin: identification of twenty-eight phosphorylation and three O-glycosylation sites[END_REF][START_REF] Christensen | Post-translationally modified residues of native human osteopontin are located in clusters: identification of 36 phosphorylation and five Oglycosylation sites and their biological implications[END_REF], showing tissue-specific phosphorylation.

A comparison of OPN from human milk and urine with regard to PTMs is shown in Figure 5. It is interesting to observe that of the 36 phosphorylation sites previously located in human milk OPN, 30 sites were also identified in human urinary OPN. However, the average phosphate content of the urinary isoform is only about one third of the milk isoform. In both urine and milk the highly anionic regions of OPN composed of multiple acidic amino acids and phosphorylations could constitute potential binding sites for minerals and calcium salts. These anionic regions could therefore enable OPN to form soluble complexes with calcium ions and thereby inhibit unintentional calcium precipitation and crystallization. Whether the different degrees of phosphorylation of human milk and urinary OPN are due to tissue-specific phosphorylation or dephosphorylation is not known. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Urinary OPN was shown to be sulphated at Tyr 165 . This residue has recently been shown to be sulphated in rat bone OPN [START_REF] Keykhosravani | Comprehensive identification of posttranslational modifications of rat bone osteopontin by mass spectrometry[END_REF], and OPN produced from canine kidney cells has also been shown to be sulphated by metabolic labelling [START_REF] Ullrich | Galactose 3 , Sia 4 ) 2387.61 (GalNAc 2 , GlcNAc, Galactose 2 , Sia 5 ) 1894.14 (GalNAc 2 , Galactose 2 , Sia 4 ) 1602.86 (GalNAc 2 , Galactose 2 , Sia 3 ) Carbohydrate composition (mol monosaccharide/mol OPN) GalNAc: 5.15 GlcNAc: 1.34 Galactose: 4.95[END_REF]. Tyr 165 is conserved in mouse, rat and rabbit but not present in cow, sheep, pig and chicken indicating that sulphation of the protein is specie-dependent. No indication of a sulphate group on Tyr 165 was observed in human milk OPN showing that this modification is also tissue-specific.

In this study, urinary OPN has been shown to contain five O-glycosylated threonines (Thr 118 , Thr 122 , Thr 127 , Thr 131 , and Thr 136 ). As shown in Figure 5, all O-linked glycans identified in human urine OPN are also present in human milk OPN. It appears that the glycans in urinary OPN predominantly consist of a disialylated GalNAcgalactose core. This glycan structure has also been described in OPN from rat bone and mouse fibroblasts and osteoblasts [START_REF] Keykhosravani | Comprehensive identification of posttranslational modifications of rat bone osteopontin by mass spectrometry[END_REF][START_REF] Christensen | Cell Type-specific Post-translational Modifications of Mouse Osteopontin Are Associated with Different Adhesive Properties[END_REF]. No N-linked glycosylations were identified on urinary OPN at the two putative motifs (Asn 63 and Asn 90 ). This is consistent with data on milk, bone and fibroblast OPN [START_REF] Sørensen | Posttranslational modifications of bovine osteopontin: identification of twenty-eight phosphorylation and three O-glycosylation sites[END_REF][START_REF] Christensen | Post-translationally modified residues of native human osteopontin are located in clusters: identification of 36 phosphorylation and five Oglycosylation sites and their biological implications[END_REF][START_REF] Keykhosravani | Comprehensive identification of posttranslational modifications of rat bone osteopontin by mass spectrometry[END_REF][START_REF] Christensen | Cell Type-specific Post-translational Modifications of Mouse Osteopontin Are Associated with Different Adhesive Properties[END_REF]. Normal rat kidney cells have been shown to produce both phosphorylated and non-phosphorylated forms of OPN in vitro, and the non-phosphorylated isoform contained N-linked carbohydrates [START_REF] Singh | Physiological properties and differential glycosylation of phosphorylated and nonphosphorylated forms of osteopontin secreted by normal rat kidney cells[END_REF], whether this is also the case in vivo remains to be shown. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. Coomassie staining of the peak containing OPN45 and OPN50 (from insert Fig. 2). Lane 4: Coomassie staining of the peak containing OPN60 (from insert Fig. 2). (B) Western blot detection with the monoclonal antibody 1H3 of peaks containing OPN45/OPN50 (lane 1) and OPN60 (lane 2) (from insert in Fig. 2). Western blot detection with the monoclonal antibody 3D9 of peaks containing OPN45/OPN50 (lane 3) and OPN60 (lane 4) (from insert in Fig. 2). Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Figure legends

Table 1: Thrombin cleavage of OPN45/OPN50

The fragments were identified by N-terminal sequence and MALDI-TOF-MS analyses. The observed molecular masses were determined by MALDI-TOF-MS in linear mode. Mass difference is that between the observed and calculated average masses. The type of modification corresponding to the mass difference is given in parentheses: phosphorylation (P), sulphation (S) and oxidation of methionine (Mox). The peptides were characterized by Edman sequencing and MALDI-TOF-MS. Glycosylated residues are indicated with bold letters.The observed molecular masses were determined by MALDI-TOF-MS in linear mode. Mass difference is that between the observed and calculated average masses. Based on the MS data, the composition of the individual glycans is suggested in parentheses: GalNAc, Nacetylgalactosamine; GlcNAc, N-acetylglucosamine; Sia, sialic acid. The carbohydrate composition of purified OPN was determined as described in Experimental. n.d.: not detected. Licenced copy. Copying is not permitted, except with permission and as allowed by law.

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 12 Dec 2007 as manuscript BJ20071021 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071021 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society proteolytic processing. These modifications are likely to be key factors in the regulatory roles of OPN in renal stone formation.

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 12 Dec 2007 as manuscript BJ20071021 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071021Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  immunoglobulins.

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 12 Dec 2007 as manuscript BJ20071021 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071021

( 4 x

 4 250 mm) (Dionex, Sunnyvale, CA) and monitored by pulsed electrochemical detection. The neutral monosaccharides were eluted isocratically with 16 mM NaOH, whereas Sia was eluted with 100 mM NaOH, 150 mM sodium acetate. Standard monosaccharide mixtures of α-L-fucose, D-galactosamine, D-glucosamine, D-galactose, D-glucose (all 0.1 mM), D-mannose (0.5 mM), α-L-rhamnose (0.2 mM) and Nacetylneuraminic acid (0.02 mM) (all from Sigma) were used. Linearity was observed between the injected amounts of monosaccharide standards and the peak areas, and hence the amounts of the individual monosaccharides in the samples were deduced from standard curves.

  15 ml/min. The peptides were detected in the effluent by measuring the absorbance at 214 nm. A fragment S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 12 Dec 2007 as manuscript BJ20071021 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071021Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  h and then reanalysed by MS. S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 12 Dec 2007 as manuscript BJ20071021 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071021

  cleavage at Arg 228 -Leu 229 and/or Tyr 230 -Lys 231 in human urine. In an additional S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 12 Dec 2007 as manuscript BJ20071021 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071021 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. experiment digests of OPN45/OPN50 and OPN60 were compared by peptide mapping analysis. Peptides from the N-terminal part (Ile 1 -Leu 229 /Arg 232 ) were found in all OPN isoforms, whereas peptides from the C-terminal part (Leu 229 -Asn 298 ) only were identified in OPN60, further supporting that OPN45 and OPN50 are generated by cleavage in the region around Leu 229 -Arg 232 . To test whether OPN45/OPN50 could be generated from OPN60 by thrombin cleavage, the full-length protein was incubated with this protease. This resulted in two major fractions in RP-HPLC containing the N-terminal part (Ile 1 -Arg 152 and Val 3 -Arg 152 ) and the expected C-terminal part of OPN (Ser 153 -Asn 298 ). Furthermore, we observed a minor fraction representing a fragment corresponding to Ser 153 -Arg 228 /Tyr 230 . Judged by RP-HPLC and MS analyses this fraction only represents a very minor portion of the cleavage products and hence we cannot on this basis conclude that thrombin would cleave OPN at this site in vivo.

  /Ser 62 -Arg 143 ) not susceptible to tryptic cleavage was further digested with thermolysin and the resulting peptides were separated by RP-HPLC. All fractions from the RP-HPLC separations of peptides were analyzed by MALDI-TOF-MS (Table 2 and S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 12 Dec 2007 as manuscript BJ20071021 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071021

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 12 Dec 2007 as manuscript BJ20071021 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071021

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 12 Dec 2007 as manuscript BJ20071021 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071021

  This study indicates that the N-terminal fragments OPN45 and OPN50 are generated from the full-length molecule by proteolytic cleavage at Arg 228 -Leu 229 and/or Tyr 230 -Lys 231 in urine. Though two bands are seen in SDS-PAGE, representing OPN45 and OPN50, only one broad peak is observed by MS suggesting that the mass difference between the fragmented OPNs is not large enough to give separation in linear MALDI-TOF-MS. No heterogeneity at the identified O-glycosylation sites was observed. However, differences in the glycans at the individual sites might account for the observed migration of OPN45 and OPN50 during SDS-PAGE.

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 12 Dec 2007 as manuscript BJ20071021 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071021

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 12 Dec 2007 as manuscript BJ20071021 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071021

  In summary, human urine contains different forms of OPN representing the fulllength protein and truncated forms lacking the C-terminal fragment Leu 229 /Lys 231 -Asn 298 . The intact protein contains approximately eight phosphate groups distributed over 30 phosphorylation sites, whereas the fragments contain approximately six phosphate groups. In addition, one sulphate group and five O-glycosylated threonines were identified in urinary OPN. The PTMs of urinary OPN reported here are likely to play key roles in the protein's regulation of mineral crystallization in urine. Further studies are necessary to investigate if different PTMs could account for the pleiotropic role of OPN as both an inhibitor and promoter of renal stone formation. S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 12 Dec 2007 as manuscript BJ20071021 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071021

Fig. 1 .

 1 Fig. 1. Western blot analysis of human urinary OPN. Urine obtained from seven healthy donors analysed on a 16% tris-tricine gel by SDS-PAGE and detected by a polyclonal OPN antibody. Lane 1. Molecular weight standards. Lane 2-8: Urine samples from seven individuals.

Fig. 2 .

 2 Fig. 2. RP-HPLC chromatography of human urine proteins eluted from DEAE material.Separation was performed on a Vydac C 4 column in 0.1% TFA and proteins were eluted with a gradient of 80% acetonitrile (dashed line) at a flow rate of 0.85 ml/min. Proteins were monitored at 226 nm (solid line). Peaks containing OPN and trypsin inhibitor (HI30) are indicated. The insert shows the separation of proteins in the OPN containing peak on a Vydac C 18 column by stepwise increasing concentrations of 75% 2-propanol.

Fig. 3 .

 3 Fig. 3. Analysis of urinary OPN. Samples were separated on 16% tris-tricine gels by SDS-PAGE, and proteins visualized by Coomassie staining or Western blotting with a polyclonal antibody or the monoclonal antibodies 1H3 or 3D9. (A) Lane 1: Coomassie staining of the OPN containing peak (from Fig.2). Lane 2: Western blot detection with a polyclonal OPN antibody of the OPN containing peak (from Fig.2). Lane 3:

Fig. 4 .

 4 Fig. 4. MALDI-TOF mass spectrometric analysis of native and dephosphorylated urinary OPN. (A) The average mass peaks at ~37.7 kDa (black trace) and 37.1 kDa (grey trace) represent OPN60 before and after treatment with ALP. (B) The average mass peaks at ~29.3 kDa (black trace) and 28.8 kDa (grey trace) represent the Nterminal OPN fragments before and after treatment with ALP.

Fig. 5 .

 5 Fig. 5. Posttranslational modifications in human urinary and milk OPN [28]. Phosphorylations (black), a sulphation (black) and glycosylations (grey) are highlighted. The glycan structures observed in urinary OPN are indicated; GalNAc (black squares), galactose (grey diamonds) and Sia (white circles). The cleavage sites observed in OPN45, OPN50 and OPN60 from urine are indicated by black arrows. Known proteolytic cleavage sites for MMP-3 and MMP-7 [40] and thrombin [41] are indicated by grey arrows.

Fig. 6 .

 6 Fig. 6. MALDI-TOF MS in linear mode of a sulphated peptide. (A) MS spectrum of a fraction containing the peptides Gln 36 -Lys 61 and Arg 160 -Lys 187 . The mass at m/z 3466.42 corresponds to the peptide Arg 160 -Lys 187 modified by three phosphate/sulphate groups. (B) MS after treatment with alkaline phosphatase (ALP). The mass at m/z 3306.43 corresponds to Arg 160 -Lys 187 with an excess mass of ~80 Da indicating the presence of a sulphate group. (C) MS after treatment with alkaline phosphatase and subsequently arylsulphatase. The mass loss of 80 Da from Arg 160 -Lys 187 upon treatment with the sulphatase shows that this peptide contained two phosphorylations and one sulphate group. S t a g e 2 ( a ) P O S T -P R I N T

Figure 1 SFigure 2 S

 12 Figure1

Figure 3 SFigure 4 SFigure 5 S t a g e 2 (Figure 6 S t a g e 2 (

 345262 Figure3

Table 1 S

 1

	Fragment	Observed mass	Calculated mass	Mass difference
		(Da)	(Da)	(Da)
	Ile 1 -Arg 152	20600	16890.5	3709.5 (P and O-glycans)
	Val 3 -Arg 152	20600	16680.2	3919.8 (P and O-glycans)
	Ser 153 -Arg 228	8820.9/8740.5/8658.9	8642.2	178.7/98.3/16.7 (P and/or S, Mox)
	Ser 153 -Tyr 230	9173.6/9093.6/9013.5	8918.5	255.1/175.1/95.0 (P and/or S, Mox)

t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 12 Dec 2007 as manuscript BJ20071021 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071021 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

Table 3 : Characterization of glycosylations on urinary OPN

 3

© 2007 The Authors Journal compilation © 2007 Biochemical Society

Table 2: Characterization of phosphorylated peptides

The peptides from the tryptic and thermolytic (Gln 36


Andreas Pföstl

Sonja Zayni

Andreas Hofinger

Paul Kosma

Christina Schäffer
email: christina.schaeffer@boku.ac.at

Paul Messner
email: paul.messner@boku.ac.at

Biosynthesis of dTDP-3-acetamido-3,6-dideoxy-α-D-glucose (dTDP-α-D-Quip3NAc)

Keywords: dTDP-α-D-Quip3NAc, Thermoanaerobacterium thermosaccharolyticum, S-layer glycoprotein, nucleotide-activated sugar precursors, 3,4-ketoisomerase, COSY, correlated spectroscopy, DTT, 1,4-dithio-D,L-threitol, His 6 , hexahistidine, HMQC, heteronuclear multiple quantum correlation spectroscopy, GST, glutathione-S-transferase, PCR, polymerase chain reaction, IPTG, isopropyl-1-thio-β-galactopyranoside, NMR, nuclear magnetic resonance spectroscopy, RP-HPLC, reversed phase high performance liquid chromatography, SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis

Derivatives of 3-amino-3,6-dideoxyhexoses are widely spread in nature. They are part of the repeating units of lipopolysaccharide O-antigens, of the glycan moiety of surface (S-) layer glycoproteins, and also of many antibiotics. In this study, we focused on the elucidation of the biosynthesis pathway of dTDP-3-acetamido-3,6-dideoxy-α-D-glucose (dTDP-α-D-Quip3NAc) from the Gram-positive, anaerobic, thermophilic organism Thermoanaerobacterium thermosaccharolyticum E207-71, which carries Quip3NAc in its S-layer glycan. The biosynthesis of dTDP-α-D-Quip3NAc involves five enzymes -a transferase, a dehydratase, an isomerase, a transaminase, and a transacetylase -and follows a pathway similar to that of dTDP-3-acetamido-3,6-dideoxy-α-D-galactose (dTDP-α-D-Fucp3NAc) biosynthesis in Aneurinibacillus thermoaerophilus L420-91 T . The open reading frames of interest were cloned, overexpressed in E. coli, and purified. To elucidate the enzymatic cascade, the different products were purified by HPLC and characterized by nuclear magnetic resonance spectroscopy. The initiating reactions catalyzed by the glucose-1-phosphate thymidylyltransferase RmlA and the dTDP-D-glucose-4,6-dehydratase RmlB are well established. The subsequent isomerase was shown to be capable of forming a dTDP-3-keto-6-deoxy-D-glucose intermediate from the RmlB product dTDP-4-keto-6-deoxy-D-glucose, whereas the isomerase involved in the dTDP-α-D-Fucp3NAc pathway synthesizes dTDP-3-keto-6-deoxy-D-galactose.

The subsequent reaction steps of either pathway involve a transaminase and a transacetylase, leading to the specific production of nucleotide-activated 3-acetamido-3,6-dideoxy-α-Dglucose and 3-acetamido-3,6-dideoxy-α-D-galactose, respectively. Sequence comparison of the open reading frames responsible for the biosynthesis of dTDP-α-D-Quip3NAc revealed homologs in Gram-negative as well as in antibiotic-producing Gram-positive bacteria. There is strong evidence that the elucidated biosynthesis pathway may also be valid for LPS O-antigen structures and antibiotic precursors.

INTRODUCTION

S-layer proteins frequently constitute the outermost cell envelope of archaea and bacteria, and many of them are glycosylated. Typically, bacterial S-layer glycoproteins consist of a long heteropolymeric glycan chain composed of individual repeating units and of a short oligosaccharide core, making them structurally similar to the O-antigens of LPS (for review see [START_REF] Messner | Prokaryotic glycoproteins[END_REF]). The assembly of the sugar chains of LPS is quite well characterized [START_REF] Keenleyside | Genetics and biosynthesis of lipopolysaccharides[END_REF], whereas the biosynthesis of S-layer glycoproteins remains still to be explored in detail. Several reports have shown that the biosynthesis pathways of nucleotide-activated sugar precursors follow identical routes, both in Gram-positive and Gram-negative organisms [START_REF] Graninger | Homologs of the Rml enzymes from Salmonella enterica are responsible for dTDP-β-L-rhamnose biosynthesis in the gram-positive thermophile Aneurinibacillus thermoaerophilus DSM 10155[END_REF][START_REF] Kneidinger | Biosynthesis pathway of ADP-L-glycero-β-Dmanno-heptose in Escherichia coli[END_REF][START_REF] Zayni | The dTDP-4-dehydro-6-deoxyglucose reductase encoding fcd gene is part of the surface layer glycoprotein glycosylation gene cluster of Geobacillus tepidamans GS5-97 T[END_REF]. The genetic information of S-layer glycan biosynthesis is organized in S-layer glycosylation (slg) gene clusters [START_REF] Novotny | Genetic organization of chromosomal S-layer glycan biosynthesis loci of Bacillaceae[END_REF].

Thermoanaerobacterium thermosaccharolyticum E207-71 is a Gram-positive, anaerobic, thermophilic organism. It possesses a glycosylated S-layer protein with hexasaccharide repeating units consisting of D-glucose, D-galactopyranose, D-galactofuranose, D-mannose, L-rhamnose, and 3-acetamido-3,6-dideoxy-α-D-glucose (α-D-Quip3NAc) [START_REF] Altman | Characterization of the glycan structure of a major glycopeptide from the surface layer glycoprotein of Clostridium thermosaccharolyticum E207-71[END_REF]. Among the biosynthesis pathways of nucleotide sugar precursors required for the biosynthesis of the T. thermosaccharolyticum E207-71 S-layer glycan, the reaction sequences leading to TDP-L-Rha [START_REF] Giraud | The rhamnose pathway[END_REF], UDP-D-Galp [9] and UDP-D-Galf [START_REF] Köplin | UDP-galactofuranose precursor required for formation of the lipopolysaccharide O antigen of Klebsiella pneumoniae serotype O1 is synthesized by the product of the rfbD KPO1 gene[END_REF] are well described in literature. The biosynthetic route for dTDP-α-D-Quip3NAc, in contrast, is still unknown. D-Quip3NAc and other 3-amino substituted sugars, such as D-Fucp3NAc, have already been identified as part of LPS O-antigens of several Gram-negative organisms [START_REF] Aspinall | Lipopolysaccharide of Campylobacter coli serotype O:30. Fractionation and structure of liberated core oligosaccharide[END_REF][START_REF] Perry | Structural characterization of the antigenic O-chain of the lipopolysaccharide of Escherichia coli serotype O65[END_REF][START_REF] Kocharova | Structure and cross-reactivity of the O-antigen of Providencia stuartii O18 containing 3-acetamido-3,6-dideoxy-Dglucose[END_REF][START_REF] Zych | Structures of the O-polysaccharides and classification of Proteus genomospecies 4, 5 and 6 into respective Proteus serogroups[END_REF]. Only for D-Fucp3NAc occurring in the Aneurinibacillus thermoaerophilus L420-91 T S-layer glycan, the biosynthetic route has been elucidated so far [START_REF] Pfoestl | Biosynthesis of dTDP-3acetamido-3,6-dideoxy-α-D-galactose in Aneurinibacillus thermoaerophilus L420-91 T[END_REF].

Perelle and coworkers [START_REF] Perelle | Identification of the O-antigen biosynthesis genes of Escherichia coli O91 and development of a O91 PCR serotyping test[END_REF] identified the gene cluster responsible for the biosynthesis of the O-antigen in E. coli O91, which contains a modified D-Quip3N among other sugar residues.

These authors identified a gene that may possibly code for an isomerase catalyzing the conversion of dTDP-4-keto-6-deoxy-D-glucose into dTDP-3-keto-6-deoxy-D-glucose. However, neither biochemical experiments nor complementation experiments with knock-out mutants have been performed to demonstrate the proposed protein function. Derivatives of D-Quip3N have not only been described as components of S-layer glycoproteins and LPS O-antigens, but also as important biosynthetic precursors of D-desosamine and D-mycaminose [START_REF] He | Formation of unusual sugars: mechanistic studies and biosynthetic applications[END_REF], both being sugar residues that are frequently found in antibiotics. Among these 3-amino-6-deoxy-sugar containing antibiotics are erythromycin [START_REF] Summers | Sequencing and mutagenesis of genes from the erythromycin biosynthetic gene cluster of Saccharopolyspora erythraea that are involved in L-mycarose and D-desosamine production[END_REF], oleandomycin [START_REF] Aguirrezabalaga | Identification and expression of genes involved in biosynthesis of S t a g e 2 ( a ) P O S T -P R I N T L-oleandrose and its intermediate L-olivose in the oleandomycin producer Streptomyces antibioticus[END_REF][START_REF] Doumith | Interspecies complementation in Saccharopolyspora erythraea : elucidation of the function of oleP1, oleG1 and oleG2 from the oleandomycin biosynthetic gene cluster of Streptomyces antibioticus and generation of new erythromycin derivatives[END_REF], and tylosin [START_REF] Cundliffe | The tylosin-biosynthetic genes of Streptomyces fradiae[END_REF]. There are several reports on the biosynthesis of the respective sugar precursors. For instance, in Streptomyces fradiae, which produces the antibiotic tylosin, a 3,4-keto isomerase was found [START_REF] Melançon | TDP-mycaminose biosynthetic pathway revised and conversion of desosamine pathway to mycaminose pathway with one gene[END_REF]. The biosynthesis of nucleotide-activated D-mycaminose follows a similar pathway as described for D-Fucp3NAc and as postulated for D-Quip3NAc, starting from glucose-1-phosphate. This compound is converted by RmlA-and RmlB-homologous enzymes into dTDP-4-keto-6-deoxy-D-glucose, which is the substrate for the 3,4-isomerase. The produced 3-keto-6-deoxy sugar is subsequently aminated and methylated. Recently, the 3,4-isomerase of S. fradiae was characterized as a dTDP-4-keto-6-deoxy-D-glucose-3,4ketoisomerase (designated Tyl1a) [START_REF] Melançon | Characterization of TDP-4-keto-6-deoxy-D-glucose-3,4-ketoisomerase from the D-mycaminose biosynthetic pathway of Streptomyces fradiae: in vitro activity and substrate specificity studies[END_REF]. A comparable pathway is also proposed for the biosynthesis of dTDP-D-desosamine. The putative isomerase involved is designated OleP1 and belongs to the cytochrome P450 monooxygenase family [START_REF] Aguirrezabalaga | Identification and expression of genes involved in biosynthesis of S t a g e 2 ( a ) P O S T -P R I N T L-oleandrose and its intermediate L-olivose in the oleandomycin producer Streptomyces antibioticus[END_REF]. The occurrence of D-Quip3N derivatives as part of macrolide antibiotics and the presence of similar isomerases in organisms, which produce these antibiotics illustrate the importance of the elucidation of the biosynthesis pathway of dTDP-α-D-Quip3N. The implication of these studies for the development of novel antibiotics was demonstrated recently. Replacement of the TDP-4-keto-6-deoxy-D-glucose-3,4ketoisomerase (Tyl1a) activity with FdtA from Aneurinibacillus thermoaerophilus L420-91 T , synthesizing non-naturally occurring TDP-4-epi-D-mycaminose, resulted in the formation of a derivative of the macrolide antibiotic tylosin [START_REF] Melançon | Engineered biosynthesis of macrolide derivatives bearing the non-natural deoxysugars 4-epi-D-mycaminose and 3-Nmonomethylamino-3-deoxy-D-fucose[END_REF].

In this report, we show that the biosynthesis of dTDP-α-D-Quip3NAc follows a similar pathway as described for dTDP-α-D-Fucp3NAc [START_REF] Pfoestl | Biosynthesis of dTDP-3acetamido-3,6-dideoxy-α-D-galactose in Aneurinibacillus thermoaerophilus L420-91 T[END_REF]. This pathway starts from glucose 1-phosphate, representing a compound available from the general sugar metabolism. Glucose 1-phosphate is activated by the glucose 1-phosphate thymidylyltransferase RmlA to form dTDP-D-glucose and dehydrated by the dTDP-D-glucose-4,6-dehydratase RmlB to form dTDP-4-keto-6-deoxy-D-glucose. Both proteins are key enzymes in the biosynthetic pathway of dTDP-β-L-rhamnose [START_REF] Giraud | The rhamnose pathway[END_REF]. We could demonstrate that the isomerases of A. thermoaerophilus L420-91 T and T. thermosaccharolyticum E207-71 use the same precursor -dTDP-4-keto-6deoxy-D-glucose -but the synthesised reaction products are dTDP-3-keto-6-deoxy-D-hexose epimers that are further processed by subsequent amination and acetylation reactions. G-10 were purchased from Amersham-Biosciences (Uppsala, Sweden). For RP-HPLC analysis a Nucleosil 120-3C18 column (Macherey-Nagel, Düren, Germany) was obtained by ARC (Seibersdorf, Austria). All primers used in this study were synthesized by Invitrogen (Lofer, Austria). Glutathione (reduced form) and imidazole were obtained from ICN Chemicals (Eschwege, Germany).

EXPERIMENTAL

Bacterial strains and culture conditions

Thermoanaerobacterium thermosaccharolyticum E207-71 was grown under anaerobic conditions in S-medium at 60 °C [START_REF] Altman | Characterization of the glycan structure of a major glycopeptide from the surface layer glycoprotein of Clostridium thermosaccharolyticum E207-71[END_REF]. E. coli DH5α (Invitrogen) was used for cloning purposes.

Enzyme overexpression was performed in E. coli BL21(DE3). For selective growth, ampicillin (Sigma) and kanamycin (Invitrogen) were used at a concentration of 100 µg/ml and 50 µg/ml, respectively.

DNA manipulations, DNA sequencing, polymerase chain reaction and gene identification

Standard DNA manipulation procedures were performed according to the methods described by Sambrook et al. [START_REF] Sambrook | Molecular cloning: a laboratory manual[END_REF]. To identify the genes involved in dTDP-D-Quip3NAc biosynthesis on the chromosome of T. thermosaccharolyticum E207-71, PCR with degenerate primers was performed (Table I). Primer design was based on sequence comparison of known transaminases of organisms containing isomerase-like enzymes. For further sequencing, chromosome walking [START_REF] Pfoestl | Biosynthesis of dTDP-3acetamido-3,6-dideoxy-α-D-galactose in Aneurinibacillus thermoaerophilus L420-91 T[END_REF] and inverse PCR [START_REF] Sambrook | Molecular cloning: a laboratory manual[END_REF] was used. The obtained PCR products were purified by agarose gel elution (Qiagen, Hilden, Germany) and sequenced (AGOWA, Berlin, Germany). PCR was performed using a PCR Sprint thermocylcer from Hybaid (Ashford, UK).

The nucleotide sequences were analyzed with the sequence analysis program OMIGA TM (Accelrys, Unterhaching-München, Germany). Homology searches and sequence alignments were performed with the BLAST tool at NCBI [26] and Multalign [START_REF] Corpet | Multiple sequence alignment with hierarchical clustering[END_REF], respectively.

Cloning, overexpression and purification of the open reading frames of interest

To characterize the newly identified enzymes QdtA, QdtB and QdtC, they were cloned and overexpressed in E. coli. To clone the ORFs the Gateway™ system from Invitrogen was used.

Briefly, the genes of interest were amplified by PCR with primers containing defined nucleotide sequences at their ends (Table 1) in order to allow insertion into the vectors. For qdtA and qdtB, pDest17 vectors containing a His 6 -tag, for qdtC, pDest15 vector containing a GST-tag were used. E. coli BL21(DE3) was used as expression host. Cells carrying the plasmids were grown in 0.7 L culture volumes of Luria Bertani medium containing 50 µg/ml ampicillin at 37 °C. At an optical density of 0.6 at 600 nm, the cells were induced for expression with 1 mM isopropyl-1-thio-β-D-galactopyranoside (IPTG) for three hours. The cells were harvested, washed and resuspended in 10 ml of 20 mM phosphate buffer, pH 7.4. To lyse the cells, ultrasonication on ice was performed and cell debris was removed by ultracentrifugation at 331000 x g. Protein purification was performed as recommended by the manufacturer, using HiTrap chelating columns with bound Ni 2+ ions for His 6 -tagged proteins, or GSTrap columns for GST-tagged proteins. Protein containing fractions were pooled and concentrated with Ultrafree-MC centrifugal devices (cutoff 10 kDa; Millipore, Vienna, Austria). The protein concentrations were adjusted to 1 mg/ml and 1,4-dithio-D,L-threitol (DTT) and glycerol were added as stabilizers to a final concentration of 1 mM and 20%, respectively. The purity of the enzymes was checked by SDS-PAGE [START_REF] Laemmli | Cleavage of structural proteins during the assembly of the head bacteriophage T4[END_REF] and the protein content was determined according to the method of Bradford [START_REF] Bradford | A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF], using the BioRad protein assay (BioRad, Vienna, Austria).

Enzyme assays and substrate synthesis

dTDP-4-keto-6-deoxy-D-glucose III, which is the substrate of the isomerase QdtA, and dTDP-D-Fucp3N VIII, for the incubation with the transacetylase QdtC, were obtained in mg-amounts as described previously [START_REF] Pfoestl | Biosynthesis of dTDP-3acetamido-3,6-dideoxy-α-D-galactose in Aneurinibacillus thermoaerophilus L420-91 T[END_REF]. Standard assays contained 50 nmol of the nucleotide-activated sugar and 5 nmol of enzyme and were performed in 50 mM K 2 HPO 4 buffer (pH 7.4) with 5 mM MgCl 2 . Mixtures for the transamination and transacetylation reactions additionally contained 50 nmol of L-glutamate, 5 nmol of pyridoxal phosphate and 50 nmol of acetyl-CoA, respectively. The assays were performed at 37 °C for 30 min. The reaction products were analyzed by RP-HPLC with 0.5 M NaH 2 PO 4 (pH 6.0) as mobile phase at a flow rate of 0.6 ml/min and monitored by UV detection at 254 nm [START_REF] Tonetti | Synthesis of GDP-L-fucose by the human FX protein[END_REF]. Large scale purification as required for NMR analysis was performed by RP-HPLC using 0.2 M TEAA (pH 6.0) as eluent.

To investigate the substrate specificity of the isomerase QdtA and the transaminase QdtB, and due to the impossibility to isolate the 3-keto substrate IV, cross incubations of these enzymes with the corresponding enzymes from the dTDP-D-Fucp3NAc pathway were performed. In detail, the isomerase FdtA from A. thermoaerophilus L420-91T was incubated

NMR analysis

Spectra were recorded at 300 K at 300.13 MHz for 1 H and at 75.47 MHz for 13 C with a Bruker AVANCE 300 spectrometer equipped with a 5 mm quadrupole nuclear probehead (QNP) with z-gradients. Data acquisition and processing were performed with the standard XWINNMR software (Bruker). 1 H spectra were referenced to internal 2,2-dimethyl-2-silapentane-5-sulfonic acid (δ = 0), 13 C spectra were referenced externally to 1,4-dioxane (δ = 67.40). 1 H/ 13 C HMQCspectra were recorded in the phase-sensitive mode using time-proportional phase increments (TPPI) and pulsed field gradients for coherence selection. Spectra resulted from a 256 x 4096 data matrix, with 1800 or 800 scans per t1 value, respectively. For in-situ experiments of the isomerase reaction, samples containing dTDP-4-keto-6-deoxy-D-glucose III (dTDP-6-deoxy-D-xylo-hex-4-ulose; 9 mg) in 20 mM K 2 HPO 4 buffer (in 0.6 ml D 2 O, pD 7.4) were prepared and kept at 318 K. After adding QdtA (0.113 mg) or FdtA (0.1 mg), respectively, and mixing in the NMR vial, NMR measurements were immediately started and recorded at distinct time intervals.

RESULTS AND DISCUSSION

Identification of the dTDP-α-D-Quip3NAc gene locus

Based on protein homology of the isomerase FdtA involved in the biosynthesis of dTDPα-D-Fucp3NAc in A. thermoaerophilus L420-91 T [START_REF] Pfoestl | Biosynthesis of dTDP-3acetamido-3,6-dideoxy-α-D-galactose in Aneurinibacillus thermoaerophilus L420-91 T[END_REF] and the proposed isomerase involved in dTDP-D-Quip3NAc biosynthesis in E. coli O91 [START_REF] Perelle | Identification of the O-antigen biosynthesis genes of Escherichia coli O91 and development of a O91 PCR serotyping test[END_REF], it is conceivable that both nucleotidesugar pathways follow similar routes. The crucial step of either pathway is the transition of the Based on the amino acid sequence next to the N-terminus (EGDVI) and the C-terminus (DELQAA) of the compared enzymes two degenerate primers (wQdtB_for1 and wQdtB_rev1) were designed. A PCR fragment of 450 bp was amplified using DNA from T. thermosaccharolyticum E207-71 as template, whose sequence revealed an incomplete ORF, similar to that encoding FdtB of the dTDP-α-D-Fucp3NAc pathway. Further sequencing starting from this fragment by chromosome walking and inverse PCR revealed the complete ORF for QdtB and, in addition, the presence of the genes encoding an isomerase and a transacetylase upstream of qdtB (Figure 1). The rml genes, which are responsible for the biosynthesis of dTDP-β-L-rhamnose as well as for the 4-keto precursor III of dTDP-α-D-Quip3NAc, were identified downstream of qdtB. Between the clustered genes encoding the enzymes of either nucleotide sugar biosynthesis pathway, an ORF coding a Wzx homologous protein, possibly involved in polysaccharide export was found. In summary, sequencing experiments revealed a genetic locus which contains eight ORFs (Figure 1). It can be assumed that this 7000-bp comprising sequence represents a partial slg gene cluster of T. thermosaccharolyticum E207-71 (Genbank accession number AY422724). Following the nomenclature for dTDP-α-D-Fucp3NAc biosynthesis [START_REF] Pfoestl | Biosynthesis of dTDP-3acetamido-3,6-dideoxy-α-D-galactose in Aneurinibacillus thermoaerophilus L420-91 T[END_REF] the identified genes, whose gene products are responsible for the biosynthesis of dTDP-α-D-Quip3NAc, were designated QdtA (dTDP-4-keto-6-deoxy-D-glucose-3,4-ketoisomerase), QdtB (transaminase), and QdtC (transacetylase). In this nomenclature, "Q" codes for quinovosamine, "d" for the D-configuration and "t" for the C-3 atom of the sugar ring. O-antigen, was identified by sequence comparison to be an isomerase [START_REF] Zych | Structures of the O-polysaccharides and classification of Proteus genomospecies 4, 5 and 6 into respective Proteus serogroups[END_REF]. Interestingly, S. fradiae is known to be a producer of tylosin, which contains the 3-amino-6-deoxy sugar residue D-mycaminose in its structure. While the QdtA-like enzyme Tyl1a was not annotated in the original Genbank submission [START_REF] Melançon | TDP-mycaminose biosynthetic pathway revised and conversion of desosamine pathway to mycaminose pathway with one gene[END_REF], the in vitro activity of this enzyme was demonstrated recently [START_REF] Melançon | Characterization of TDP-4-keto-6-deoxy-D-glucose-3,4-ketoisomerase from the D-mycaminose biosynthetic pathway of Streptomyces fradiae: in vitro activity and substrate specificity studies[END_REF].

Sequence comparison of

QdtB homologs are present in organisms which contain isomerase-like proteins. QdtB shares highest homology with the transaminase FdtB from A. thermoaerophilus L420-91 T (60/74, AAO06353). Among other enzymes presumably involved in the biosynthesis of nucleotide-activated D-Quip3N and D-Fucp3N, the transaminases from the D-desosamine (OleN2 47/67, AAD55458, and DesV 45/63, AAC68680) and D-mycaminose (TylB 46/64) biosynthesis pathways share high similarities with QdtB. All these enzymes are members of the DegT/DnrJ/EryC1/StrS family of amino-transferases.

Alignment of QdtC with enzymes from the database revealed that this enzyme shows to some extent homology to other known acetyltransferases, but almost no homology to acetyltransferases catalyzing the acetylation of 3-amino-6-deoxy sugar residues. Nevertheless, like all other aligned acetyltransferases, QdtC possesses a hexapeptide repeat motif [START_REF] Vaara | Eight bacterial proteins, including UDP-N-acetylglucosamine acyltransferase (LpxA) and three other transferases of Escherichia coli, consist of a sixresidue periodicity theme[END_REF], which unambiguously identifies it as a transferase.

The rml genes of T. thermosaccharolyticum E207-71 responsible for the biosynthesis of dTDP-β-L-rhamnose are highly homologous to the corresponding enzymes identified in Clostridium acetobutylicum [START_REF] Nölling | Genome sequence and comparative analysis of the solvent-producing bacterium Clostridium acetobutylicum[END_REF] and A. thermoaerophilus DSM 10155 [START_REF] Graninger | Homologs of the Rml enzymes from Salmonella enterica are responsible for dTDP-β-L-rhamnose biosynthesis in the gram-positive thermophile Aneurinibacillus thermoaerophilus DSM 10155[END_REF], and occur in the order rmlB, rmlA, rmlD and rmlC.

Based on the genetic data obtained in the present study we propose the biosynthetic pathway for dTDP-α-D-Quip3NAc VI as depicted in Figure 2.

Cloning of qdtA, qdtB and qdtC and overexpression of the encoded proteins of

T. thermosaccharolyticum E207-71 in E. coli

All enzymes involved in the biosynthesis of dTDP-α-D-Quip3NAc were cloned with the Gateway TM system from Invitrogen. Prior to heterologous expression, the plasmids were verified by nucleotide sequencing. In order to allow rapid and efficient purification, QdtA and QdtB were expressed as His 6 -fusions and QdtC as GST-tagged protein. Since affinity chromatography resulted in enzymes of high purity, no further purification steps were applied.

According to the SDS-PAGE evidence the molecular mass of the enzymes QdtA, QdtB, and QdtC of 18.6 kDa, 42.1 kDa, and 56.9 kDa, respectively, corresponded to the masses predicted form the amino acid sequences (data not shown).

The isomerase QdtA forms dTDP-3-keto-6-deoxy-D-glucose

BLAST search revealed the high similarity of QdtA with FdtA, which is the isomerase involved in the biosynthesis of dTDP-α-D-Fucp3NAc IX. Thus, it was concluded that QdtA may also act as an isomerase. The dTDP-4-keto-6-deoxy-D-glucose III educt of this enzyme is obtained by the sequential action of RmlA and RmlB. Initial experiments performed with QdtA, QdtB and QdtC, acting on the required substrates and co-substrates, revealed a rather fast conversion of dTDP-4-keto-6-deoxy-D-glucose III into dTDP-α-D-Quip3NAc VI. In contrast, experiments without addition of the isomerase to the reaction mixture showed only poor yields of the final product dTDP-α-D-Quip3NAc VI. This observation is in agreement with previously published data [START_REF] Pfoestl | Biosynthesis of dTDP-3acetamido-3,6-dideoxy-α-D-galactose in Aneurinibacillus thermoaerophilus L420-91 T[END_REF] and clearly showed the necessity of QdtA in the pathway.

Conversion of the 4-keto substrate III into the 3-keto product IV was initially monitored by RP-HPLC. The elution profile showed a similar chromatographic pattern as described for the FdtA-catalyzed reaction, implicating that this method was not suitable for the separation of educt and product due to chemical similarities of the substances (Figure 3).

To verify the function of the enzyme, online-NMR monitoring of the enzymatic reaction was applied. Although highest enzyme activities were obtained between 60 °C and 65 °C (data not shown), the direct conversion of dTDP-4-keto-6-deoxy-D-glucose III into the 3-keto product IV by the action of QdtA was studied by NMR measurements recorded at 45 °C in order to get detectable quantities of the isomerization product and to prevent its degradation.

Formation and increase of a new anomeric signal at 5.87 ppm ( 3 J H-1,H-2 4.3, 3 J H-1,P 7.1 Hz) was observed within 10 min and reached a maximum signal intensity of ~25% after ~3 h. The signal of H-1 was correlated to a solvent-hidden signal of H-2 at 4.65 ppm by a COSY experiment lending support to the formation of a keto group at C-3, thus causing the observed downfield shift of H-2. Due to concomitant degradation of the material, a full spectral assignment of the 3-keto intermediate IV was not possible. The spectral characteristics, however, are in agreement with those of the reaction product reported for a Tyl1a-catalyzed conversion of III [START_REF] Melançon | Characterization of TDP-4-keto-6-deoxy-D-glucose-3,4-ketoisomerase from the D-mycaminose biosynthetic pathway of Streptomyces fradiae: in vitro activity and substrate specificity studies[END_REF] and with the NMR data published for dTDP-3-keto-6-deoxy-α-Dgalactose VII (dTDP-6-deoxy-D-ribo-hex-3-ulose), obtained from a reversed transamination reaction [START_REF] Chen | Expression, purification, and characterization of TylB, an aminotransferase involved in the biosynthesis of mycaminose[END_REF] and prepared via a non-enzymatic conversion of the substrate [START_REF] Naundorf | Substrate specificity of native dTDP-D-glucose-4,6dehydratase: chemo-enzymatic syntheses of artificial and naturally occurring deoxy sugars[END_REF]. For comparison, a control experiment without the addition of enzyme was performed, which revealed the formation and increase of the new anomeric 1 H signal within 30 min to a maximum of ~ 5 % of the mixture, followed by a subsequent decrease in its signal intensity and appearance of signals arising from degradation of the sample (Figure 4). Due to inherent instability of the 3-keto intermediate direct conversion was not attempted.

QdtB aminates the 3-keto-6-deoxy-gluco-epimer forming dTDP-α-D-Quip3N

The formation of dTDP-α-D-Quip3N V was observed in a combined reaction of QdtB with the isomerase QdtA and, in another reaction, without the isomerase. In an assay containing the isomerase, quantitative turnover of the dTDP-4-keto-6-deoxy-D-glucose III substrate was obtained over a time period of 30 min at 37 °C. However, also without addition of QdtA a turnover of about 25 % was observed after 12 hours of incubation. These results are in agreement with previous data from the biosynthesis of dTDP-D-Fucp3NAc IX, where the transaminase FdtB was able to directly convert dTDP-4-keto-6-deoxy-D-glucose III into dTDP-D-Fucp3N VIII (over 36 hours) [START_REF] Pfoestl | Biosynthesis of dTDP-3acetamido-3,6-dideoxy-α-D-galactose in Aneurinibacillus thermoaerophilus L420-91 T[END_REF]. Obviously, over a certain time span, both transaminases, QdtB and FdtB, are capable of synthesizing the respective 3-aminated 6-deoxy sugar product V and VIII without the involvement of an isomerase.

To investigate the specificity of QdtB, enzyme assays were also performed with the substrate VII for the transaminase FdtB, formed by the isomerase FdtA from the dTDPα-D-Fucp3NAc biosynthesis pathway. Incubations were done at 37 °C for three hours and the reaction products were monitored by RP-HPLC. Inspection of the HPLC chromatograms showed that QdtB has a clear preference for the substrate provided by the isomerase QdtA from T. thermosaccharolyticum E207-71. The amination of the 3-keto-gluco-epimer IV is approximately three times more efficient than that of the 3-keto-galacto-epimer VII.

The introduction of the 3-amino-group into the product of the enzymatic reaction was clearly visible in the 300 MHz 1 H NMR spectrum of V. The spectrum revealed two upfieldshifted triplets for H-3 and H-4, respectively, with large values of the vicinal coupling constants and with H-3 being correlated to a nitrogen-bearing carbon occurring at 56.04 ppm (Table II).

Acetylation is catalyzed by QdtC

QdtC turned out to be responsible for the transfer of the acetyl group from acetyl-CoA to dTDP-α-D-Quip3N V. dTDP-α-D-Quip3NAc VI obtained in this reaction was purified by II). In addition, small downfield shifts were observed for the neighbouring carbons. Signals of the pyranose unit were in good agreement with the data of a terminal α-D-Quip3NAc unit observed in an O-antigenic bacterial polysaccharide [START_REF] Toukach | Structure of a new acidic O-antigen of Proteus vulgaris O22 containing O-acetylated 3-acetamido-3,6-dideoxy-D-glucose[END_REF].

The substrate specificity of the transacetylase QdtC was determined by incubating the enzyme also with dTDP-α-D-Fucp3N VIII. According to the chromatographic profile the transacetylation reaction catalyzed by QdtC turned out to be bifunctional, acetylating both epimers, dTDP-α-D-Quip3N V and dTDP-α-D-Fucp3N VIII. Interestingly, alignment of QdtC from T. thermosaccharolyticum E207-71 and FdtC from A. thermoaerophilus L420-91 T revealed that their amino acid sequences are not highly homologous.

Conclusions

Biosynthesis of dTDP-α-D-Quip3NAc and dTDP-α-D-Fucp3NAc follows similar pathways.

The crucial reaction of either pathway is the conversion of dTDP-4-keto-6-deoxy-D-glucose into the respective dTDP-3-keto-6-deoxy-D-hexose epimer by the 3,4-ketoisomerases QdtA and FdtA, respectively. In a previous report, it was shown that FdtA is able to provide a 3-keto substrate in the galacto-configuration at C-4 [START_REF] Pfoestl | Biosynthesis of dTDP-3acetamido-3,6-dideoxy-α-D-galactose in Aneurinibacillus thermoaerophilus L420-91 T[END_REF]. In this report, we could show that QdtA forms a similar sugar residue, but retains the gluco-configuration at C-4. The transition of the 4-keto group into the 3-keto group is the crucial event for a component to be recognized as a substrate for the transaminases (QdtB and FdtB), followed by the last step of the cascade, the acetylation by the transacetylases (QdtC and FdtC).

Only recently, Davis and coworkers have elucidated the X-ray structure of FdtA [START_REF] Davis | The X-ray structure of dTDP-4keto-6-deoxy-D-glucose-3,4-ketoisomerase[END_REF].

The authors have determined the active site of the enzyme to be built by conserved His 49 and His 51 residues. The third His 95 of the active site is supposed to be specific for 3,4-ketoisomerases, which convert the configuration at C-4. Furthermore, they speculate that His 95 is replaced by an arginine residue in 3,4-ketoisomerases with retaining function. All of these relevant amino acids can be found in the QdtA sequence (Figure 5).

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 18 Oct 2007 as manuscript BJ20071044 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071044 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

MaterialsL

  -glutamate and acetyl-CoA were obtained from Sigma (Vienna, Austria), dTDP-D-glucose from Fluka (Buchs, Switzerland). GSTrap, HiTrap Chelating, MonoQ HR5/5, and Sephadex S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 18 Oct 2007 as manuscript BJ20071044 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071044 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 18 Oct 2007 as manuscript BJ20071044 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071044 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 18 Oct 2007 as manuscript BJ20071044 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071044 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society with QdtB, the transaminase from the dTDP-D-Quip3NAc pathway of T. thermosaccharolyticum E207-71. The reaction mixtures contained the same co-factors and co-substrates as described above for the single incubations.

4 -

 4 keto educt, dTDP-4-keto-6-deoxy-D-glucose III, into a 3-keto product, dTDP-3-keto-6deoxy-D-hexose IV, which is catalyzed by a 3,4-ketoisomerase. Thus, an isomerase similar to that involved in the biosynthesis of dTDP-α-D-Fucp3NAc was very likely to be present in the dTDP-α-D-Quip3NAc biosynthesis pathway of T. thermosaccharolyticum E207-71. To obtain entry into the chromosomal locus encoding the isomerization event several aminotransferases of different organisms, which are located in close vicinity to the respective 3,4-ketoisomerase S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 18 Oct 2007 as manuscript BJ20071044 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071044 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society on the chromosome, were aligned. Especially the amino acid composition of the hypothetical pyridoxal-phosphate binding site seemed to be a typical feature of the compared transaminases.

  QdtA with proteins from the database at NCBI revealed high homologies with more than 80 ORFs exhibiting pfam05523 domain (pfam05523: FdtA, WxcM-like, C-terminal). This family includes also FdtA from Aneurinibacillus thermoaerophilus, which has been characterized as a dTDP-6-deoxy-3,4-keto-hexulose isomerase, and WxcM from Xanthomonas campestris pv. campestris [31]. Some of these homologous proteins originate from organisms which are known to synthesize derivatives of D-Quip3N and D-Fucp3N, such as E. coli, X. campestris and A. thermoaerophilus. High similarities were observed with FdtA from A. thermoaerophilus L420-91 T (identity/similarity (%) 55/69, Genbank accession number AAO06351) and WbsB from E. coli O91 (48/64, AAK60451). Biochemical data are available for FdtA, which demonstrate that this enzyme catalyzes the formation of dTDP-3-keto-6-deoxy-D-galactose VII from the substrate dTDP-4keto-6-deoxy-D-glucose III. WbsB from E. coli O91, harbouring D-Quip3NAc residues in its S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 18 Oct 2007 as manuscript BJ20071044 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071044 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 18 Oct 2007 as manuscript BJ20071044 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071044 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 18 Oct 2007 as manuscript BJ20071044 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071044 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 18 Oct 2007 as manuscript BJ20071044 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071044 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society RP-HPLC (Figure 3 D) and further analyzed by NMR spectroscopy . The product VI of the N-acetylation reaction (Figure 4) displayed signals of an N-acetyl group at 2.05 ppm/22.45 ppm for the 1 H-and 13 C-NMR signals of the methyl group and a substantial downfield shift of the proton signal of H-3 (Table

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 18 Oct 2007 as manuscript BJ20071044 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071044 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

Figure legends: Figure 1 .

 1 Figure legends:

Figure 2 .

 2 Figure 2. Comparison of the biosynthesis pathways for dTDP-α-D-Quip3NAc and dTDP-α-D-Fucp3NAc. I, D-glucose-1-phosphate; II, dTDP-D-glucose; III, dTDP-4-keto-6-deoxy-Dglucose; IV, dTDP-3-keto-6-deoxy-D-glucose; V, dTDP-D-Quip3N; VI, dTDP-D-Quip3NAc; VII, dTDP-3-keto-6-deoxy-D-galactose; VIII, dTDP-D-Fucp3N, IX, dTDP-D-Fucp3NAc. RmlA, glucose-1-phosphate thymidylyltransferase; RmlB, dTDP-D-glucose 4,6-dehydratase; QdtA, dTDP-4-keto-6-deoxy-D-glucose-3,4-ketoisomerase; QdtB, dTDP-3-keto-6-deoxy-Dglucose aminase; QdtC, dTDP-D-Quip3N acetylase; FdtA, dTDP-4-keto-6-deoxy-D-glucose-3,4-ketoisomerase; FdtB, dTDP-3-keto-6-deoxy-D-galactose aminase; FdtC, dTDP-D-Fucp3N acetylase.

Figure 3 .

 3 Figure 3. HPLC analysis of the products of the enzymatic reactions catalyzed by QdtA, QdtB and QdtC. A, dTDP-glucose II; B, dTDP-4-keto-6-deoxy-D-glucose III, synthesized from II by RmlB; C, dTDP-D-Quip3N V, synthesized from III by sequential action of QdtA and QdtB; D, dTDP-D-Quip3NAc VI, final reaction product of the concerted action of RmlB, QdtA, QdtB and QdtC using II as substrate. The numbers used for designation of enzymatic products are identical to those used in Figure 2.

Figure 4 .

 4 Figure 4. 300 MHz 1 H-NMR spectrum of dTDP-Quip(3)NAc VI recorded at 300 K in D 2 O. The signal at 3.7 ppm (indicated by an arrow) corresponds to residual Tris-buffer; the signal at ~7.4 ppm (indicated by an arrow) is due to residual imidazole. S t a g e 2 ( a ) P O S T -P R I N T

Figure 5 .

 5 Figure 5. Amino acid sequence alignment of T. thermosaccharolyticum E207-71 QdtA, Aneurinibacillus thermoaerophilus L420-91 T FdtA, and Streptomyces fradiae Tyl1a. The amino acids of the active site are in bold letters, the characteristic signature sequence is highlighted in grey. The positions of the conserved histidines (His 49 and His 51 in FdtA) are indicated by black dots. Note that His 95 (indicated by a grey dot) in FdtA is an arginine in QdtA and in Tyl1a.S t a g e 2 ( a ) P O S T -P R I N T

S t a g e 2 Figure 1 of

 21 Figure 1 of Pföstl et al

Figure 2 ofFigure 3 ofFigure 4 of

 234 Figure 2 of Pföstl et al. S t a g e 2 ( a ) P O S T -P R I N T

Table I .

 I Strains, primers and vectors used in the study

	Strains	Description	Source
	T. thermosaccharolyticum E207-71	Wild type strain
	A. thermoaerophilus L420-91T	Wild type strain
	E. coli DH5α	K-12 F-Φ80d lacZΔM15 endA1 recA1	Invitrogen
		hsdR17 (rK-mK-) supE44 thi-1 gyrA96
		relA1 Δ(lacZYA-argF) U169
	E. coli BL21(DE3)	(F-ompT hsdSB (rB-mB-) gal dcm (DE3))	Invitrogen
	Primers
	wQdtB_for1	GARGGNGAYGARGTNAT
	wQdtB_rev1	GCNGCYTGNARYTCRTC
	gQdtA_for1	GCATTTTGTATAACGTTGCGTTAATA
	gQdtA_rev1	TTCATTTTTGCACTCTCTCC
	gQdtB_for1	GCATGAAAATATCATTTGCAAGCT
	gQdtB_rev1	GCTAATAACACTTCCTAATCCG
	gQdtC_for1	GCATGCCAAATAATATTTCTAAAAGTG
	gQdtC_rev1	CGTTATACAAAATTCTTCCCCC
	Vectors
	pBCKS II	Chromosome walking	Stratagene
	pDonr201	Cloning vector	Invitrogen
	pDest15	Expression vector (GST-tag)	Invitrogen
	pDest17	Expression vector (His-tag)	Invitrogen
	gFdtA15	FdtA in pDest15	[15]
	gFdtB17	FdtB in pDest17	[15]
	gFdtC15	FdtC in pDest15	[15]
	gQdtA	QdtA in pDonr201	This study
	gQdtB	QdtB in pDonr201	This study
	gQdtC	QdtC in pDonr201	This study
	gQdtA17	QdtA in pDest17	This study
	gQdtB17	QdtB in pDest17	This study
	gQdtC15	QdtC in pDest15	This study

S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 18 Oct 2007 as manuscript BJ20071044 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071044 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

Table II .

 II NMR spectroscopic data a of dTDP-α-D-Quip3N V, and of dTDP-α-D-Quip3NAc VI Spectra were recorded at 300 K for solutions in D 2 O at pD 7.4.

				V		VI
	Atom	1 H	J, [J het ]	13 C	1 H	J, [J het ]	13 C
		(ppm)	(Hz)	(ppm)	(ppm)	(Hz)	(ppm)
	1"	5.58	3.5 / [7.4] 95.00	5.56	3.4 / [7.0] 95.32
	2"	3.78	[3.4]	69.57	3.60	[3.0]	70.85
	3"	3.42	10.0	56.04	4.04	10.5	54.62
	4"	3.35	9.2	72.01	3.17	9.8	74.28
	5"	4.01	6.4	69.73	4.02	6.2	70.39
	6"	1.28	-	17.16	1.26	-	17.12
	NH(Ac)				2.05		22.45
	1'	6.34	7.0	85.83	6.33	7.2	85.65
	2a'/2'b	2.39/2.35 7.0	39.38	2.39/2.34 7.2	38.92
	3'	4.62	n.d. b	71.79	4.63	n.d.	71.94
	4'	4.17	n.d.	86.03	4.18	n.d.	86.06
	5a'/ 5'b 4.17/4.15 n.d.	66.27	4.17	n.d.	66.09
	2			152.55			n.d.
	4			167.38			n.d.
	5			112.53			n.d.
	6	7.72	n.d.	138.15	7.72	1.2	138.37
	Me	1.92	-	12.44	1.92	-	12.01

a b n.d. not determined.

ACKNOWLEDGEMENTS

This work was supported by the Austrian Science Fund, projects P18013-B10 (to P. M.), and P19047-B12 (to C. S.).