Joaquín Fernández-Irigoyen

Mónica Santamaría

Virginia Sánchez-Quiles

Maria Ujue Latasa

Enrique Santamaría

Javier Muñoz

Manuel Mateo Sánchez

Maria Luz Pino

Jesús Valero

Matías Antonio Prieto

Ávila

Manuel M Sánchez

Del Pino

María L Valero

Jesús Prieto

Matías A Ávila

Fernando J Corrales
email: fjcorrales@unav.es

Redox regulation of methylthioadenosine phosphorylase in liver cells. Molecular mechanism and functional implications

Keywords: Methylthioadenosine phosphorylase, methylthioadenosine, oxidative stress, inflammation, cyteine oxidation, sulfenic acid MTAP, 5'-methylthioadenosine phosphorylase, MTA, 5'-deoxy-5'- methylthioadenosine, ROS, reactive oxygen species, SAH, adenosine triphosphate, HCC, hepatocellular carcinoma, DTT,

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Introduction 5'-deoxy-5'-methylthioadenosine (MTA) is a sulphur containing nucleoside that is present in all cell types including prokaryotes, yeast, plants and higher eukaryotes. In mammalian tissues MTA is generated from its precursor, decarboxylated Sadenosylmethionine (SAM), during the synthesis of polyamines spermine and spermidine [START_REF] Pegg | Polyamine metabolism and its importance in neoplastic growth and a target for chemotherapy[END_REF] or, alternatively, from the spontaneous splitting of SAM under physiological conditions [START_REF] Wu | Chiral instability at sulfur of S-adenosylmethionine[END_REF]. Although MTA is known for more than a century, its biological importance was firstly proposed in 1952 [START_REF] Williams-Ashman | Trends in the biochemical pharmacology of 5' -deoxy-5' -methylthioadenosine[END_REF]. The enzyme catalyzed conversion of MTA to adenine (Ade) and 5-methylthioribose-1-phosphate (MTR1P) provides the first and rate limiting step in purine and methionine salvage pathways.

MTR1P is recycled into methionine and Ade is used to replenish the AMP and ATP pools [START_REF] Williams-Ashman | Trends in the biochemical pharmacology of 5' -deoxy-5' -methylthioadenosine[END_REF]. Additionally to its central role in cellular metabolism, MTA also participates in the regulation of a wide variety of cellular functions. Intracellular fluctuations in MTA levels could participate in the regulation of proliferative responses [START_REF] Mato | S-Adenosylmethionine: a control switch that regulates liver function[END_REF], as well as in the modulation of inflammatory responses [START_REF] Hevia | 5' -methylthioadenosine modulates the inflammatory response to endotoxin in mice and in rat hepatocytes[END_REF]. These effects might be mediated by its inhibitory effect on polyamine biosynthesis [START_REF] Pascale | Chemoprevention of hepatocarcinogenesis: S-adenosyl-L-methionine[END_REF], by its capacity to interfere with key signalling pathways through the inhibition of growth-factor induced protein tyrosine phosphorylation and cAMP phosphodiesterase [START_REF] Maher | Inhibition of the tyrosine kinase activity of the fibroblast growth factor receptor by the methyltransferase inhibitor 5'methylthioadenosine[END_REF], and by the inhibition of protein methylation [START_REF] Williams-Ashman | Trends in the biochemical pharmacology of 5' -deoxy-5' -methylthioadenosine[END_REF], a well known posttranslational modification involved in the modulation of cellular signalling and gene expression. Therefore, it appears clear that regulation of MTA levels is crucial to preserve cellular homeostasis. This concept is further reinforced by studies suggesting the therapeutic potential of this natural compound in different diseased conditions [8,[START_REF] Moreno | Methylthioadenosine reverses brain autoimmune disease[END_REF]. 5'-methylthioadenosine phosphorylase (5'-deoxy-5'methylthioadenosine:orthophosphate methylthioribosyltransferase), (EC 2.4.2.28) (MTAP) catalyzes the reversible phosphorolytic cleavage of MTA leading to the production of MTR1P and Ade [START_REF] Pegg | Phosphate-stimulated breakdown of 5' -methylthioadenosine by rat ventral prostate[END_REF]. After the pioneer description of this enzymatic activity, MTAP has been purified and characterized from different organisms and tissues [11,[START_REF] Della Ragione | Purification and characterization of recombinant human 5'methylthioadenosine phosphorylase: definite identification of coding cDNA[END_REF]. Biochemical and structural evidence indicate that mammalian MTAP is a trimer constituted by three identical subunits of 32 kDa [START_REF] Appleby | The structure of human 5'deoxy-5' -methylthioadenosine phosphorylase at 1.7 A resolution provides insights into substrate binding and catalysis[END_REF] that in humans contain 283 amino acid residues. MTAP is abundantly expressed in normal cells and tissues [14] where it accounts for the production of most of the free adenine generated in human cells [START_REF] Kamatani | Dependence of adenine production upon polyamine synthesis in cultured human lymphoblasts[END_REF]. In contrast, lack of MTAP activity is frequently observed in tumour cells [START_REF] Christopher | Methylthioadenosine phosphorylase, a gene frequently codeleted with p16(cdkN2a/ARF), acts as a tumor suppressor in a breast cancer cell line[END_REF]. The MTAP gene has been mapped to human chromosome 9p21 [14] in a region that is often deleted in tumour cells. In most cases, it appears that the loss of MTAP activity or mRNA is the result of homozygous deletions of MTAP gene [START_REF] Illei | Homozygous deletion of CDKN2A and codeletion of the methylthioadenosine phosphorylase gene in the majority of pleural mesotheliomas[END_REF].

However alternative mechanisms must also exist since MTAP impairment was detected in lymphoma and melanoma-derived cell lines with intact MTAP gene [18,[START_REF] Berasain | Methylthioadenosine phosphorylase gene expression is impaired in human liver cirrhosis and hepatocarcinoma[END_REF]. It has been recently reported the presence of a CpG island proximal to the transcription start of the MTAP promoter [18], suggesting a transcriptional regulation of the gene through a methylation-demethylation mechanism. This notion was confirmed in HCC cell lines

showing down-regulation of MTAP gene expression that could not be attributed to genomic losses or mutations but to promoter hypermethylation [20]. Additionally, it has been found that the transcriptional activation of the human MTAP gene is mediated by the binding of CCAAT binding factor to a distal CCAAT motif in the promoter [21].

Besides the regulation at the transcriptional level, there are also evidences suggesting modulation of MTAP activity. Phosphorylation of serine 183 and threonine 188 residues have been recently reported, although the biological transcendence of these modifications must be demonstrated [START_REF] Beausoleil | Large-scale characterization of HeLa cell nuclear phosphoproteins[END_REF]. Oxidation arises also as a mechanism to modulate MTAP activity as suggested by the requirement of the enzyme for thiol reducing agents and its specific and rapid inactivation by thiol-blocking groups [START_REF] Della Ragione | Purification and characterization of 5' -deoxy-5'methylthioadenosine phosphorylase from human placenta[END_REF].

However, although it has been established the implication of cysteine residues in the stabilization of the MTAP protein from hyperthermophilic organisms through the formation of disulfide bridges [START_REF] Zhang | The crystal structure of 5' -deoxy-5' -methylthioadenosine phosphorylase II from Sulfolobus solfataricus, a thermophilic enzyme stabilized by intramolecular disulfide bonds[END_REF], the role of specific thiols in the modulation of MTAP activity is less evident.

In this study we provide data supporting MTAP inactivation in the liver of mice after induction of an inflammatory reaction by administration of LPS. Reduction of MTAP activity was also demonstrated in MTAP expressing HepG2 cells after exposure to tert-butyl hydroperoxide. Reversible inactivation of MTAP by reactive oxygen species (ROS) occurs by specific oxidation of C136 and C223 to sulfenic acid as illustrated by the loss of sensitivity of mutants in which these residues were substituted by serine. MTAP inactivation by ROS arises then as a specific regulatory mechanism that might be pivotal in cellular adaptation to physiological or pathological conditions, such as inflammation, that involve oxidative stress.

Materials and methods:

Animal experiments: Male C57Bl6 mice (19-21 g of weight) were from Harlan.

Studies were approved by the University of Navarra Committee on Animal Care and for 15 min. SAM, SAH and MTA levels were measured in the supernatant by HPLC as described previously [START_REF] Perez-Mato | Methionine adenosyltransferase S-nitrosylation is regulated by the basic and acidic amino acids surrounding the target thiol[END_REF]. 100 µl aliquots were loaded onto a Bio-Sil® ODS-5Scolumn equilibrated in 0.01 M ammonium formate, 4 mM heptanesulphonic acid pH 4.0. MTA was eluted with a linear gradient (25-100%) acetonitrile in the same buffer.

Chromatograms were analyzed with the software 32 Karat 5.0 (Beckman Coulter).

Cell transfection. SK-Hep1 cells grown in 60-mm dishes until 70% confluence were transfected with a pcDNA3.1 HisA plasmid harboring the complete human MTAP cDNA, or the empty pcDNA3.1 HisA vector. Transfections were carried out using Tfx-1 (Promega) according to the manufacturer instructions, and transfectants were selected in complete medium containing 0.6 mg/mL of G418 sulfate (Geneticin; Invitrogen) as described [START_REF] Velasco | The product of the cph oncogene is a truncated, nucleotide-binding protein that enhances cellular survival to stress[END_REF]. After 2 weeks, individual colonies were harvested, and clones transfected with the empty vector pcDNA3.1 HisA (SK-EV) or the MTAP expressing construct (SK-WT) were expanded.

MTAP activity in liver cell lines. [START_REF] Laemmli | Cleavage of structural proteins during the assembly of the head of bacteriophage T4[END_REF]. MTAP containing fractions were pooled, desalted with 5 ml HiPrep desalting columns (GE Healthcare) and the protein concentration was determined using the Bio-Rad protein assay kit based on Bradford assay [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]. The purified protein was then stored at -80º C in the presence of 5 mM DTT.

Enterokinase processing was performed according to the manufacturer recommendations. Then 1 µl of the resulting solutions were mixed with an equal volume of -cyano-4hydroxy-trans-cynnamic acid (CHCA) in 50% acetonitrile with 0.1% TFA and spotted onto a MALDI target plate. The system was calibrated daily with a tryptic digest of ADH, and after analysis near point calibration was performed using ACTH as lockmass standard. Data processing was performed with Masslynx release 4.0 (Waters) to subtract background noise using polynomial order 10 with 10% of the data points below this polynomial. Data were also smoothed by one smooth operation (Savitzky Golay) with a window of two channels. MS spectra were processed automatically with MASCOT and then interpreted by manual inspection using Masslynx 4.0 (Waters). The presence of disulfide bridges was analyzed using the MS-Bridge 4.0.8 from Protein prospector webpage (http://prospector.ucsf.edu/).

MTAP activity

Western blot analysis. Liver tissues and cultured cells were lysed and proteins were extracted as previously described [START_REF] Maher | Inhibition of the tyrosine kinase activity of the fibroblast growth factor receptor by the methyltransferase inhibitor 5'methylthioadenosine[END_REF]. Equal amounts of protein (20 µg) were resolved in 12.5% SDS-polyacrylamide gels. Proteins were electrophoretically transferred to nitrocellulose membranes for 45 min at 120 V. Membranes were probed with a 1:5,000

Results:

Impaired MTA degradation in the liver of LPS challenged mice. To evaluate MTA metabolism under oxidative stress, SAM, SAH and MTA levels were measured in the liver of LPS treated mice, a well known condition inducing a redox imbalance in hepatic cells. As previously shown [START_REF] Payabvash | Nitric oxide modulates glutathione synthesis during endotoxemia[END_REF], the inflammatory reaction associated with LPS administration induced 50% reduction of hepatic SAM and SAH. However, MTA concentration was 2.5 fold higher than that measured in control livers (Fig. 1A). No change in MTAP protein levels was observed upon LPS administration as assessed by

Western blot analysis (Fig. 1B). In agreement with previous studies [START_REF] Schock | Enhanced inflammatory responses in alpha-tocopherol transfer protein null mice[END_REF], heme oxygenase-1 (HO-1) expression was increased in response to the oxidative stress associated with the inflammatory reaction (Fig. 1B). Since MTAP catalyzes the only known MTA catabolic reaction, the increase in hepatic MTA levels in LPS-treated mice, even when the concentration of the precursor SAM was diminished, suggests impairment in MTAP activity. In order to further support this idea, MTAP activity was measured (Fig. 1C) in liver extracts from control and treated mice and a 65% inactivation was observed upon LPS administration (1.52±0.02 vs 0.66±0.08 nmol MTA/min/mg protein in controls and challenged livers respectively).

ROS-mediated MTAP inactivation in hepatic cells.

To investigate the effect of ROS on MTAP activity, HepG2 cells were incubated with tert-butyl hydroperoxide and the rate of exogenous MTA consumption was determined. As a negative control the experiment was also performed with SK-Hep1 cells, which we have previously shown to lack MTAP expression [START_REF] Berasain | Methylthioadenosine phosphorylase gene expression is impaired in human liver cirrhosis and hepatocarcinoma[END_REF] (Fig. 2B). In all cases, the culture medium was deprived of methionine to prevent endogenous MTA synthesis and was supplemented with horse serum to avoid extracellular MTA degradation by the MTAP present in the commonly used bovine serum [START_REF] Riscoe | 5-Methylthioribose. Its effects and function in mammalian cells[END_REF]. stress with tert-butyl hydroperoxide significantly reduced the capacity of HepG2 cells to metabolize the supplemented MTA (only 30% of the initial MTA was degraded under these conditions), likely resulting from the inactivation of the constitutive MTAP (Fig. 2A). The steady state levels of MTAP protein remained unchanged upon tert-butyl hydroperoxide exposure (Fig. 2B). These evidences suggest that cellular MTAP is inactivated by reactive oxygen species. Independently on whether the protein was processed with enterokinase or not, a 60% decrease of the enzymatic activity was measured after oxidation and the inactivation was reversed when the activity assay was performed in the presence of reducing agents (Fig. proteins in which a single cysteine residue was replaced by serine were obtained and their susceptibility to inactivation by H 2 O 2 was analyzed. Mutants were expressed and purified from E. coli extracts and the purity of the corresponding proteins was always more than 95% in all preparations used. Replacement of cysteine by serine had no effect on the enzymatic activity of MTAP mutants. Similarly to the WT enzyme, the activity measured at saturating concentration of MTA was close to 396 nmol MTA consumed min -1 mg -1 in all cases (Fig. 3A). Incubation with 500 µM H 2 O 2 reduced the activity of WT and mutant proteins to about 132 nmol MTA consumed min -1 mg -1 with the exception of C136S and C223S MTAP species that remained fully active after oxidation (Fig. 3A). These results suggest that the oxidation of both, C136 and C223, is required to mediate MTAP inactivation. The loss of sensitivity to inactivation by ROS of C136S and C223S mutants was also investigated in hepatic cells. With this purpose stably transfected SK-Hep1 cells expressing these two MTAP mutants were challenged with 10 µM tert-butyl hydroperoxide and the consumption of extracellular MTA was measured after 30 h. MTAP mutant protein levels (as determined by Western blotting)

and the capacity of MTA degradation by transfected SK-Hep1 cells were similar to those observed in HepG2 cells that express the endogenous enzyme (800-1000 pmol MTA consumed mg -1 min -1 ). SK-Hep1 cells expressing either of the two mutants were not sensitive to tert-butyl hydroperoxide and their MTA metabolizing activity remained unchanged after exposure to the oxidant (Fig. 3B), further supporting the central role of C136 and C223 in mediating MTAP inactivation by ROS. In contrast, SK-Hep1 cells transfected with a vector expressing WT MTAP showed the expected decrease (about 60%) of MTA consumption upon exposure to tert-butyl hydroperoxide.

Identification of Cys

Discussion

The participation of MTA in essential metabolic and signaling pathways suggests that the regulation of MTAP activity might be important in facilitating cellular adaptation to physiological or pathological conditions. The liver parenchyma is one of the normal human tissue types where MTAP gene expression is higher [START_REF] Nobori | Genomic cloning of methylthioadenosine phosphorylase: a purine metabolic enzyme deficient in multiple different cancers[END_REF]. It has been demonstrated that methionine metabolism must be precisely tuned to maintain normal catabolism and salvage pathways to their normal steady state. However, the maintenance of the diseased condition derives in a persistent impairment of liver MAT [START_REF] Avila | Reduced mRNA abundance of the main enzymes involved in methionine metabolism in human liver cirrhosis and hepatocellular carcinoma[END_REF] and MTAP [START_REF] Berasain | Methylthioadenosine phosphorylase gene expression is impaired in human liver cirrhosis and hepatocarcinoma[END_REF] gene expression through epigenetic mechanisms. In light of these observations it might by hypothesized that impairment of SAM synthesis might be compensated, at least partially, by inactivation of MTAP leading to maintenance of MTA levels that may be critical for cellular stability. The idea of a coordinated regulation of MTA metabolism by the redox state of the cell might be further reinforced by evidences indicating the inactivation of SAM decarboxylase by reactive species [START_REF] Hillary | Decarboxylases involved in polyamine biosynthesis and their inactivation by nitric oxide[END_REF].

Interestingly, it has been reported that the hepatic levels of putrescine are increased in murine liver upon treatment with LPS resulting from up-regulation of ornitine decarboxylase. However, the increment of putrescine did not promote a parallel increase in spermidine and spermine (Fig. 6) [START_REF] Endo | Simultaneous induction of histidine and ornithine decarboxylases and changes in their product amines following the injection of Escherichia coli lipopolysaccharide into mice[END_REF]. Our results might explain these observations since MTA, which accumulates in the liver of LPS treated mice as a result of MTAP inactivation, inhibits spermine and spermidine syntases [START_REF] Pegg | Polyamine metabolism and its importance in neoplastic growth and a target for chemotherapy[END_REF][START_REF] Pascale | Chemoprevention of hepatocarcinogenesis: S-adenosyl-L-methionine[END_REF].

Protein oxidation is a common regulatory mechanism of metabolic and signaling pathways [START_REF] Droge | Free radicals in the physiological control of cell function[END_REF]. Native MTAP is a homo-trimer with active sites composed entirely of residues of the same subunit with the exception of His137 and Leu279 that are provided by the neighbouring monomer [START_REF] Appleby | The structure of human 5'deoxy-5' -methylthioadenosine phosphorylase at 1.7 A resolution provides insights into substrate binding and catalysis[END_REF]. The loss of MTAP activity upon exposure to H 2 O 2 is a reversible process that results from the specific oxidation of cysteine residues 136 and 223 as was demonstrated by using the purified enzyme as well as cellular systems whose MTA consumption capacity was severely impaired after an oxidative challenge. or in the catalytic mechanism of MTAP, consistently with our finding that the activity of the enzyme remains as in the WT protein when Cys residues were replaced by serine.

However, specific oxidation of Cys136 and Cys223 resulted in a reduced V max while the K m of the enzyme remained unchanged, suggesting that the binding of MTA to the active site of MTAP is not modified in the oxidized form. Interestingly, Cys223 is located on a flexible loop over the active site cleft of MTAP connecting ß-strand 11 with -helix 5 and is close to Asp220 and Asp222 which participate in the catalytic process and Cys136 is adjacent to His137 that integrates the MTA binding pocket of the neighbouring subunit [START_REF] Appleby | The structure of human 5'deoxy-5' -methylthioadenosine phosphorylase at 1.7 A resolution provides insights into substrate binding and catalysis[END_REF]. Moreover, non-conservative substitution of Cys223 by Tyr resulted in an inactive MTAP mutant [47]. Therefore, it is tempting to speculate that the oxidation of Cys136 and Cys223 might alter the microenvironment of these catalytic residues modifying their chemical reactivity and consequently the activity of the enzyme.

Oxidation of the two cysteines is required to promote MTAP inactivation as indicated by the loss of sensitivity of Cys136S and Cys233S mutants to be inactivated by H 2 O 2 . The idea of a disulfide interaction between these residues arises as a likely explanation to the coordinated effect of the two oxidation events on MTAP inactivation.

However, the distances separating the corresponding sulphur atoms of Cys136 and Cys233 from adjacent subunits are 15.77 Å and 35.54 Å in the same MTAP monomer [START_REF] Appleby | The structure of human 5'deoxy-5' -methylthioadenosine phosphorylase at 1.7 A resolution provides insights into substrate binding and catalysis[END_REF], too far away to allow prediction of their interaction without assuming conformational rearrangements that to our knowledge have not yet been demonstrated. Cys145-S-S-Cys211 were detected, our observations may be explained alternatively assuming the stabilization of Cys136 and/or Cys223 sulfenic derivatives through reaction with residues located in the adjacent monomer leading to the formation of reversible intermediates such as sulfenyl amides [START_REF] Salmeen | Redox regulation of protein tyrosine phosphatase 1B involves a sulphenyl-amide intermediate[END_REF][START_REF] Van Montfort | Oxidation state of the active-site cysteine in protein tyrosine phosphatase 1B[END_REF]. This notion might find further support on the time dependent reduction of sulfenic content per protein subunit observed after oxidation periods longer than 30 min, suggesting their stabilization to derivatives that were recycled back to reduced thiols with reducing agents (Fig. 2 Supp Info). Detailed structural studies are required to demonstrate these hypotheses and to fully elucidate the ROS-induced oxidation intermediates of MTAP.

In summary, we have presented evidences in this study supporting the loss of MTAP activity in the liver of LPS challenged mice as well as in HepG2 cells exposed to tert-butyl hydroperoxide. MTAP activity is regulated by mechanisms involving the specific and reversible oxidation of cysteine residues Cys136 and Cys233

to sulfenic acid that may be further stabilized to a sulfenyl amide intermediate.

Additional oxidation events result in disulfide bonding of Cys145 and Cys211 thiols, although this modification has no impact on the enzymatic activity of MTAP. In the liver this regulatory mechanism might provide means to maintain intracellular MTA levels under stress conditions such as those imposed by inflammatory reactions ensuring hepatocyte viability.

Ciencia, Spain. This laboratory is member of the National Institute of Proteomics Facilities, ProteoRed. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 31 Jan 2008 as manuscript BJ20071569 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071569 S t a g e 2 ( a ) P O S T -P R I N T

Biochemical

  Journal Immediate Publication. Published on 31 Jan 2008 as manuscript BJ20071569 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071569 S t a g e 2 ( a ) P O S T -P R I N T satisfied National Institutes of Health guidelines for humane treatment of animals. Mice were injected intraperitoneally with 15 mg/kg endotoxin (Salmonella typhimurium lipopolysaccharide, Sigma) dissolved in sterile, pyrogen free saline. Liver tissue was removed after 22 h, snap-frozen in liquid nitrogen and stored at -80º C until analysis. SAM, SAH and MTA measurements. Liver samples (50 mg) were homogenized in 250 µl of 0.4 M perchloric acid. Homogenates were centrifuged at 10,000 x g and 4º C

  assays. DTT was removed from the preparation with HiPrep Desalting columns equilibrated in 10 mM Tris/HCl pH 7.4, 10 mM KCl immediately before use. Two independent procedures were used to measure the activity of purified WT and MTAP mutant proteins. Firstly the reaction mixture always contained 1 µM MTAP protein or liver homogenates (400 µg total protein) in 50 mM phosphate buffer pH 7.4 and the appropriate concentration of MTA in a final volume of 400 µl. Preincubation Biochemical Journal Immediate Publication. Published on 31 Jan 2008 as manuscript BJ20071569 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071569 S t a g e 2 ( a ) P O S T -P R I N T with 5 mM DTT or different H 2 O 2 concentrations was performed as indicated. The mixture was then incubated at 37º C for the indicated period of time and the reaction was stopped with perchloric acid to a final concentration of 0.4 M. MTA measurementswere performed by HPLC as indicated previously. Alternatively, MTAP activity was determined according to the procedure described by Singh et al[START_REF] Singh | Picomolar transition state analogue inhibitors of human 5' -methylthioadenosine phosphorylase and X-ray structure with MT-immucillin-A[END_REF]. Conditions were as described above. The reaction was started by the addition of MTA and consumption was followed by the reduction of the absorbance at 275 nm. H 2 O 2 was added when required to the enzyme and the mixture was incubated for 10 min at 37º C before adding MTA. Time course assays were performed in the presence of different concentrations of MTA and initial velocity values were obtained in each case from the linear fitting of MTA variation in the first 3 minutes of the reaction. V i vs MTA concentration plots were fitted to Michaelis Menten equation using Kaleidagraph 3.5b5.Quantification of free thiol groups and sulfenic derivatives. Purified MTAP was preincubated during 5 or 30 min with DTT or the appropriate concentration of H 2 O 2 that were subsequently eliminated with a HiPrep Desalting column equilibrated in 50 mM phosphate buffer pH 7.4. Free thiol groups were quantitated by incubation with DTNB following the absorbance at 412 nm resulting from the liberation of TNB after interaction with SH groups. DTNB reaction was performed in a final volume of 400 µl containing 50 mM phosphate buffer pH 7.4, 1 mM EDTA, 6 M guanidinium chloride, 25 µM protein and 20 mM DTNB. The amount of accessible sulfhydryl groups was calculated by measuring the TNB released using a molar extinction coefficient of 13.600 M -1 cm -1[32,[START_REF] Ellman | Tissue sulfhydryl groups[END_REF]. Determination of sulfenic acid from cysteine SH oxidation was performed using two different approaches. In the first method, sulfenic acid was titrated with TNB according to the procedure described by Poole et al[34]. Protein samples prepared as indicated above were incubated with 20 mM TNB at 25º C for 30 min. The excess of TNB was then removed with HiPrep Desalting columns under the same conditions described above. DTT was added (50 mM) to release the TNB associated to SOH groups that was measured by absorbance at 412 nm. Alternatively, NBD-Cl (20 mM) was used following the procedure described elsewhere[START_REF] Ellis | Novel application of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole to identify cysteine sulfenic acid in the AhpC component of alkyl hydroperoxide reductase[END_REF]. NBD-Cl was added to protein preparations and the mixture was incubated at 25º C for 30 min.After elimination of the NBD-Cl excess with HiPrep Desalting columns, SH derived adducts and sulfenic resulting derivatives were measured at 420 and 347 nm respectively. Biochemical Journal Immediate Publication. Published on 31 Jan 2008 as manuscript BJ20071569 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071569 S t a g e 2 ( a ) P O S T -P R I N T Size exclusion chromatography. WT and MTAP mutants were chromatographed in a Superdex-200 HR 10/30 column (GE Healthcare) equilibrated in 50 mM Tris/HCl pH 7.5. Isocratic elution with the same buffer (0.8 ml/min) was monitored at 280 nm. The column was calibrated in the same conditions using as standard proteins catalase (232 kDa), BSA (67 kDa), trypsin (21 kDa) and ribonuclease A (13.7 kDa). Alternatively MTAP was denatured by 2 h incubation with 6 M guanidinium chloride after the indicated treatments. Unfolded proteins were then analyzed with the same column equilibrated in 100 mM Tris/HCl pH 3.0, 6 M guanidinium chloride. Mass spectrometry analysis. Samples of MTAP protein (50-100 µg) were incubated with 500 µM H 2 O 2 or 5 mM DTT and subsequently prepared for matrix-assisted laser desorption mass spectrometry analysis (MALDI TOF GL-REF mass spectrometer, Waters). After desalting to eliminate the excess of H 2 O 2 or DTT, free cysteines were alkylated by reaction with 100 mM iodoacetamide for 30 min and the protein was precipitated in chloroform-methanol (4:1). Protein pellets were resuspended in 25 mM ammonium bicarbonate and incubated with 2 µl of 50 µg/µl trypsin (Promega, wisconsin, US) at 37ºC for 12h. Alternatively, the protein was dissolved in 10mM Tris /HCl pH 7.8, 10mM calcium chloride and incubated with 2 µl of 25 µg/µl chymotrypsin (Roche) at 25ºC for 12h. Half of the sample was then reduced with 10 mM DTT at 55ºC 30 min. The digestion mix was desalted with reversed phase C18-ZipTip (Millipore).

  Biochemical Journal Immediate Publication. Published on 31 Jan 2008 as manuscript BJ20071569 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071569 S t a g e 2 ( a ) P O S T -P R I N T or 1:1,000 dilution of anti-MTAP (the generous gift of Dr. D.A. Carson, University of California San Diego, USA) or anti-hemoxygenase 1 (HO-1) (Stressgene) antibodies respectively. Anti-chicken and anti-rabbit IgG peroxidase-conjugated secondary antibodies were used for MTAP and HO-1 assays. Blots were developed by enhanced chemoluminiscence (Perkin Elmer).

  MTA was efficiently degraded by HepG2 MTAP activity within the first 30 h of incubation. As expected, the levels of MTA in the culture medium remained unchanged in the presence of SK-Hep1 cells. Induction of oxidative Biochemical Journal Immediate Publication. Published on 31 Jan 2008 as manuscript BJ20071569 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071569 S t a g e 2 ( a ) P O S T -P R I N T

H 2 O 2 -

 22 induced MTAP inactivation. The activity of purified MTAP was measured before and after digestion with enterokinase to determine the effect of the N-terminal tag. Only preparations with more than 95% purity were used for subsequent experiments. Digestion with enterokinase removed the N-terminal tag and resulted in the expected 32 kDa subunits as assessed by SDS-PAGE. Both, tagged and digested enzymes had similar MTA degrading rates of 57.6 and 398 nmol min -1 mg -1 when the assay was performed with 15 and 200 µM MTA respectively (not shown). Since no apparent change of activity was associated with the N-terminal tag, all the experiments were carried out using the His-tagged recombinant proteins. The putative effect of ROS on MTAP activity was studied by preincubation of the enzyme with H 2 O 2 .

Biochemical

  Journal Immediate Publication. Published on 31 Jan 2008 as manuscript BJ20071569 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071569 S t a g e 2 ( a ) P O S T -P R I N T Identification of target cysteine residues involved in the impairment of MTAP activity by ROS. In order to elucidate the participation of cysteine residues on MTAP inactivation by oxidation and to identify the targeted cysteine(s), nine mutant MTAP

  -SH oxidation derivatives. To characterize the thiol oxidation derivatives leading to MTAP inactivation we firstly measured the number of accessible cysteines remaining upon incubation with H 2 O 2 . The protein was oxidized and then denatured in 6 M guanidinium chloride to ensure titration of all free Cys residues with DTNB. This procedure allowed detection of 10 Cys in the reduced protein, which is compatible with the presence of 9 Cys in the MTAP sequence and one additional residue provided by the N-terminal tag. Incubation with H 2 O 2 restricted the access of the thiol reacting reagent to 4 Cys independently of the time of exposure to the oxidant, Biochemical Journal Immediate Publication. Published on 31 Jan 2008 as manuscript BJ20071569 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071569 S t a g e 2 ( a ) P O S T -P R I N T suggesting the oxidation of 6 Cys residues as was further supported by the recovery of reactivity of all 10 Cys with DTT. This evidence indicates oxidation of Cys residues additional to those responsible of MTAP inactivation by ROS. Mass spectrometry analyses were then performed on tryptic and chimotryptic digests from reduced and H 2 O 2 -treated MTAP aiming to identify Cys residues involved in a putative disulfidebonding. Ion assignment from MALDI TOF spectra allowed more than 60% coverage of MTAP sequence in all cases. Interestingly, a 1797.13 Da ion was specifically detected on peptide fingerprints from H 2 O 2 oxidized MTAP and the presence of this peptide species was prevented when oxidation was reversed with DTT (Fig.4). The differential ion might result from interaction between Cys145 and Cys211 leading to the association of the corresponding chymotryptic peptides as suggested by the experimental molecular mass which is 2 Da less than the theoretically predicted from the sequence, in agreement with a disulfide bridged dipeptide. Since no additional cysteine derivatives were identified, the oxidation state of Cys136 and 223, responsible for MTAP inactivation, remained unclear. Oxidation to sulfenic acid was then explored using TNB or, alternatively, NBD-Cl. Control MTAP yielded negative results when incubated with either sulfenic-specific reagents. However, after exposure to H 2 O 2 , sulfenic acids were observed independently of the detection method used and were not detectable if the oxidized and modified enzyme was treated with DTT. TNB reagent allowed titration of 2 sulfenics per MTAP subunit (Fig.5). Substitution of Cys136 or Cys223 by Ser resulted in MTAP variants that contained only one sulfenic acid after oxidation (Fig 5). These results indicate that H 2 O 2 -induced MTAP inactivation results from oxidation of Cys136 and Cys223 to sulfenic acid. A reduction of the 412 nm signal was observed if oxidation was prolonged over 30 min, suggesting that the sulfenic groups might evolve to more stable derivatives (Fig 2 supp. info.).

Biochemical

  Journal Immediate Publication. Published on 31 Jan 2008 as manuscript BJ20071569 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071569 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. ' 2008 The Authors Journal compilation ' 2008 Biochemical Society liver function and that impairment of this pathway correlates with hepatocyte dedifferentiation and proliferation leading to the progression of liver diseases [4, 40]. Consistent with the central role of MTA in the cell, a conservative mechanism based on a coordinated regulation of methionine adenosyltransferase (MAT), the enzyme catalyzing the synthesis of the precursor SAM, and MTAP might prevent deleterious MTA decrease in hepatocytes (Fig 6). Firstly, the oxidative stress resulting from mild and transient liver damage inactivates MAT perhaps providing an adaptive response that allows hepatocyte survival [41]. Down-regulation of MAT might be correlated with reversible inactivation of MTAP, as has been shown in the present study, to compensate the impaired SAM synthesis leading to maintenance of cellular MTA. Cellular antioxidant defences might reactivate MAT [42] and MTAP returning methionine

  These two cysteines are conserved in mammalian MTAPs and in the D. melanogaster ortholog but are not present in other MTAP sequences, explaining the lack of cys-Biochemical Journal Immediate Publication. Published on 31 Jan 2008 as manuscript BJ20071569 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071569 S t a g e 2 ( a ) P O S T -P R I N T mediated redox regulation reported in different Archaea species [11]. No structural or functional evidences support a direct participation of cysteine residues in MTA binding

2 involved

 2 Consistently, the only disulfide bond clearly identified upon oxidation with H 2 O Cys145 and Cys211 as expected from the 3.63 Å separating their thiol groups. Additionally DTNB titration data indicate that six cysteines from the MTAP monomer become oxidized upon incubation with H 2 O 2 and therefore two cysteines supplementary to Cys 136, 145, 211, and 223 may undergo oxidation. Incubation of MTAP with H 2 O 2 led to oxidation of Cys136 and Cys223 to reversible sulfenic acid. Longer exposures of MTAP to H 2 O 2 to those reported in this study resulted in oxidized forms that displayed hydrodynamic volumes larger than those expected for the monomer when analyzed by size exclusion chromatography under denaturing conditions (Fig 2 Supp. Info.). This observation may suggest a reversible inter-subunit interaction that should involve Cys136 and Cys223 since the effect was Biochemical Journal Immediate Publication. Published on 31 Jan 2008 as manuscript BJ20071569 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071569 S t a g e 2 ( a ) P O S T -P R I N T reversed by reducing agents, and mutants lacking these residues failed to accumulate the putatively cross-linked intermediate. Since no disulfide interactions additional to

Figure 1 .

 1 Figure 1. MTAP inactivation in the liver of LPS challenged mice. Hepatic levels of SAM, SAH and MTA in LPS treated mice are represented as the percentage of control values (100%) measured in saline injected animals (n=6 per condition) (A). 100% values were 38+6.12, 12+1.5 and 4.2+1.1 pmol/mg protein for SAM, SAH and MTA respectively. Levels of MTAP and HO-1 proteins were determined in control and LPS challenged mice by Western blotting (representative blots are shown) (B).

Figure 2 .

 2 Figure 2. Impairment of MTAP activity in HepG2 cells upon an oxidative challenge. HepG2 cells were incubated in the absence ( ) or presence of 1 µM tert-butyl hydroperoxide ( ), and their capacity to metabolize MTA was measured. As a negative control the MTA consuming capacity of SK-Hep1 cells ( ) lacking MTAP was measured in the same conditions (A). The initial MTA concentration was 16 µM. The remaining MTA after the different incubation times was measured in the culture

Figure 3 .

 3 Figure 3. Identification of cysteine residues mediating MTAP inactivation by reactive oxygen species. (A) The activity of WT and mutant MTAP proteins was measured in the absence (solid bars) or presence of 500 µM H 2 O 2 (empty bars). MTAP concentration was 1 µM in all cases and the initial and final amount of MTA was measured by HPLC. (B) The MTA metabolizing rate was measured in SK-Hep1 cells stably transfected with an empty vector (SK-EV) or expressing human WT (SK-WT), C136S or C223S MTAP mutants in the absence (solid bars) or presence (empty bars) of 10 µM tert-butyl hydroperoxide. MTA was added to the culture medium at initial concentration of 16 µM. The MTA content in the culture medium was measured by HPLC. The Western blot shows MTAP levels in HepG2 (1) and SK-Hep1 cells expressing WT MTAP (2), C136S(3), C223S (4) or transfected with an empty vector[START_REF] Hevia | 5' -methylthioadenosine modulates the inflammatory response to endotoxin in mice and in rat hepatocytes[END_REF]. All data are the mean±stdev of three independent experiments.

Figure 4 .

 4 Figure 4. Mass spectrometric detection of Cys145-S-S-Cys211 disulfide interaction. MALDI TOF MS spectra of tryptic digests from DTT (A), H 2 O 2 (B) or H 2 O 2 and then DTT treated (C) WT MTAP were obtained. H 2 O 2 was removed before chymotrypsinolysis to avoid peptide crosslinking by disulfide interactions during digestion. The insert on panel B indicates the sequence of the two MTAP peptides containing Cys145 and Cys211 which interaction is consistent with the H 2 O 2 -associated detection of a 1797.13 Da ion.

Figure 5 .

 5 Figure 5. Oxidation of Cys136 and Cys233 to sulfenic acid. Sulfenic acid derivatives were detected by incubation of reduced or oxidized native MTAP with TNB or NBD-CL (A). For TNB experiments, control (dashed line) or H 2 O 2 (solid line), DTT (dotted line) and H 2 O 2 and then DTT (dashed and dotted line) treated MTAP were assayed. For NBD-CL control (solid line) or H 2 O 2 conditions (dashed line) were analyzed. The number of sulfenic derivatives per MTAP subunits was estimated by titration with TNB (B). Results express the mean±stdev of three independent experiments.

Figure 6 .

 6 Figure 6. Schematic representation of MTA metabolism. Checkpoints modulated by redox imbalance are indicated. (1) Methionine adenosyltransferase, (2) methyl transferases, (3) S-adenosylmethionine decarboxylase, (4) ornithine decarboxylase, (5) spermidine synthase, (6) spermine synthase, (7) 5'-methylthioadeosine phosphorylase.

  Figure 1

Figure 3 A

 3 Figure 3

Figure 5 AbsorbanceFigure 6 Methionine

 56 Figure 5

  Since the elution volume of the protein remained unchanged upon incubation with H 2 O 2 (13.22 ml), oxidation associated changes of the trimeric structure of MTAP were ruled out (data not shown). To get insight into the mechanism of H 2 O 2 -mediated inactivation of MTAP, kinetic experiments were performed. V max was reduced from 433±29 to 169±8 nmol MTA consumed min -1 mg -1 in the absence or presence of 500 µM H 2 O 2 , respectively (Table I and Fig 1 Supp Info). The estimated K m for MTA (around 30 µM) remained unchanged upon oxidation (Table I and Fig 1 Supp Info), suggesting that the loss of

1 Supp Info). The hydrodynamic properties of the recombinant MTAP purified from E. coli extracts were analyzed by size exclusion chromatography. The elution volume of recombinant MTAP was 13.28 ml, which is compatible with a molecular mass of 108 kDa according to the elution profile of standard proteins. This result indicates that the purified MTAP is a trimer of tagged subunits. activity might result from impairment of the catalytic mechanism more than from impeded MTA binding to the active site of the enzyme. Therefore, these data suggest that H 2 O 2 is a non-competitive inhibitor with an estimated [I] 50 of about 300 µM.

Table 1 .

 1 V max and K m values for reduced and oxidized MTAP

	Biochemical Journal Immediate Publication. Published on 31 Jan 2008 as manuscript BJ20071569
		V max (nmol MTA consumed min -1 mg -1 )	K m (µM)
	MTAP	433±29,37	32,77±9,42
	MTAP + 100 µM H 2 O 2	271,9±22,45	24,93±9,78
	MTAP + 500 µM H 2 O 2	168,97±8,34	37,18±7,42

S t a g e 2 ( a ) P O S T -P R I N T

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Acknowledgments

The technical assistance of Goretti Azparren, Sonia Beaumont, and María I. Mora is acknowledged. This work was supported by: the agreement between FIMA and the "UTE project CIMA"; grants Plan Nacional I+D+I 2004-03538 and 2004-01855 from Ministerio de Educación y Ciencia to MAA and FJC; grant ROI AA-12677 from the National Institute on Alcohol Abuse and Alcoholism to, MAA; grant R01 AT1576 from the National Center for Complementary and Alternative Medicine to MAA and FJC; grant RO1 AA013847 from the National Center for Complementary and Alternative Medicine to FJC; grant STREP FP6-2004-LIFESCIHEALTH-5 018649 from the 6 th framework programm of the UE to FJC. ISCIII-RETIC RD06/0020 to MAA and FJC. Grant FIS CP04/00123 from Ministerio de Sanidad y Consumo to MS and MAA. MUL is supported by a Juan de la Cierva contract from the Ministerio de Educación y Biochemical Journal Immediate Publication. Published on 31 Jan 2008 as manuscript BJ20071569 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071569 S t a g e 2 ( a ) P O S T -P R I N T


Ivan Kadurin

Andjelko Golubovic

Lilia Leisle

Hermann Schindelin

Stefan Gründer
email: sgruender@ukaachen.de

Differential effects of N-glycans on surface expression suggest structural differences between acid-sensing ion channel (ASIC) 1a and ASIC1b

Keywords: ASIC, acid-sensing ion channel; Endo H, endoglycosidase ASIC, acid-sensing ion channel, N-glycans, trafficking, structure Biochemical

Acid-sensing ion channels (ASICs) are H + -gated Na + channels, with a widespread expression pattern in the central and the peripheral nervous system. ASICs have a simple topology with two transmembrane domains, cytoplasmic termini, and a large ectodomain between the transmembrane domains; this topology has been confirmed by the crystal structure of chicken ASIC. ASIC1a and ASIC1b are two variants encoded by the asic1 gene. The variable part of the protein includes the cytoplasmic N-terminus, the first transmembrane domain and approximately the first third of the ectodomain. Both variants contain two consensus sequences for N-linked glycosylation in the common, distal part of the ectodomain. In contrast to ASIC1a, ASIC1b contains two additional consensus sequences in the variable, proximal part of the ectodomain. Here we show that all extracellular Asn residues within putative consensus sequences for N-glycosylation carry glycans. The two common, distal glycans increase surface expression of the channels but are no absolute requirement for channel activity. In sharp contrast, the presence of at least one of the two proximal glycans, which are specific to ASIC1b, is an absolute requirement for surface expression of ASIC1b. This result suggests substantial differences in the structure of the proximal ectodomain between the two ASIC1 variants.

INTRODUCTION

Acid-sensing ion channels are H + -gated Na + channels [START_REF] Waldmann | H(+)-gated cation channels: neuronal acid sensors in the NaC/DEG family of ion channels[END_REF]. They are abundantly expressed in the central and peripheral nervous system and have a role in synaptic transmission and sensory transduction [START_REF] Wemmie | Acid-sensing ion channels: advances, questions and therapeutic opportunities[END_REF][START_REF] Lingueglia | Acid-sensing ion channels in sensory perception[END_REF]. In mammals, six ASICs are generated from four asic genes. They all share a simple topology with two transmembrane domains (M1 and M2), a large ectodomain (> 350 amino acids) and relatively short (40 -90 amino acids) cytoplasmic termini [START_REF] Saugstad | Analysis of the membrane topology of the acid-sensing ion channel 2a[END_REF]. This topology has been confirmed by the recently determined crystal structure of a chicken ASIC1 deletion mutant [START_REF] Jasti | Structure of acidsensing ion channel 1 and low pH[END_REF]. Moreover, the ASIC1 crystal revealed that three subunits assemble into the functional channel. The extracellular region is composed of five subdomains, which are connected to the membrane-spanning region via an apparently flexible wrist [START_REF] Jasti | Structure of acidsensing ion channel 1 and low pH[END_REF]; as previously predicted [START_REF] Firsov | Mutational analysis of cysteine-rich domains of the epithelium sodium channel (ENaC). Identification of cysteines essential for channel expression at the cell surface[END_REF], this region is stabilized by cysteine-bridges formed by 14 highly conserved cysteines.

ASIC1a and ASIC1b derive from the asic1 gene, probably by using alternative promoters; the N-terminal third of the proteins is different, including the intracellular Nterminus, the first transmembrane domain and the proximal part of the ectodomain [START_REF] Chen | A sensory neuronspecific, proton-gated ion channel[END_REF][START_REF] Bässler | Molecular and functional characterization of acid-sensing ion channel (ASIC) 1b[END_REF]. ASIC1a is broadly expressed in the central and peripheral nervous system [START_REF] Waldmann | A proton-gated cation channel involved in acid-sensing[END_REF], whereas ASIC1b is specifically expressed in the peripheral nervous system [START_REF] Chen | A sensory neuronspecific, proton-gated ion channel[END_REF]. There are also functional differences between the two variants. ASIC1a is the only ASIC that is permeable for Ca 2+ [START_REF] Bässler | Molecular and functional characterization of acid-sensing ion channel (ASIC) 1b[END_REF][START_REF] Waldmann | A proton-gated cation channel involved in acid-sensing[END_REF][10]; the lack of Ca 2+ permeability of ASIC1b can be attributed to differences in a pre-M1 region [START_REF] Bässler | Molecular and functional characterization of acid-sensing ion channel (ASIC) 1b[END_REF]. Moreover, ASIC1a has a higher apparent H + affinity than ASIC1b, which is due to differences in a short stretch of amino acids after M1 [11]. Finally, ASIC1b has a much longer cytoplasmic N-terminus than ASIC1a (88 compared to 43 amino acids) [START_REF] Bässler | Molecular and functional characterization of acid-sensing ion channel (ASIC) 1b[END_REF]; the first 15 amino acids of the ASIC1b N-terminus are conserved in ASIC4 [START_REF] Gründer | A new member of acid-sensing ion channels from pituitary gland[END_REF][START_REF] Akopian | A new member of the acid-sensing ion channel family[END_REF] and may have an unknown functional role [START_REF] Chen | Zebrafish Acid-sensing Ion Channel (ASIC) 4, Characterization of Homo-and Heteromeric Channels, and Identification of Regions Important for Activation by H+[END_REF].

Most membrane proteins are glycoproteins and N-glycans can serve many functions. Among them, they can aid the proper folding of the protein, stabilize the structure of the protein, and promote its intracellular trafficking [START_REF] Helenius | Roles of N-linked glycans in the endoplasmic reticulum[END_REF]16]. Some glycoproteins strictly depend on N-glycans for their function [START_REF] Kundra | Asparagine-linked oligosaccharides protect Lamp-1 and Lamp-2 from intracellular proteolysis[END_REF][START_REF] Turner | N-linked glycosylation sites affect secretion of cryptococcal phospholipase B1, irrespective of glycosylphosphatidylinositol anchoring[END_REF][19] whereas for other glycoproteins N-glycans are completely dispensable.

All ASICs have at least one consensus site for N-glycosylation (Asn-X-Ser/Thr, where X is any amino acid except Pro). Glycosylation has been investigated for ASIC2a [START_REF] Saugstad | Analysis of the membrane topology of the acid-sensing ion channel 2a[END_REF]. Both consensus sites for glycosylation in the ectodomain of ASIC2a (Fig. 1A) are used. Removal of the first N-glycan (at N365) has no effect on current amplitude or apparent H + affinity, whereas removal of the second glycan (at N392) reduces current amplitude 2-fold. Removal of both glycans together reduces current amplitude 3-fold and apparent H + affinity by 0.6 pH units [START_REF] Saugstad | Analysis of the membrane topology of the acid-sensing ion channel 2a[END_REF]. These results show that although glycans are necessary for the full activity and H + affinity of ASIC2a, they are no absolute requirement for channel activity. Apparently, ASIC2a devoid of glycans can assemble into oligomeric channels, traffic to the cells surface and form functional H + -gated channels.

The two glycosylation sites of ASIC2a are conserved in ASIC1a and ASIC1b (Fig. 1A). ASIC1b, but not ASIC1a, has two additional glycosylation consensus sites in the alternative, proximal part of the ectodomain. In the present study we investigated the glycosylation pattern of ASIC1a and ASIC1b. We found that all Asn residues within potential extracellular glycosylation sites of ASIC1a and 1b carry glycans. The two distal N-glycans, which are shared with ASIC2a, are not absolutely required for channel activity. Surprisingly, however, one glycan at either of the two proximal sites of ASIC1b is mandatory for surface expression of ASIC1b. We propose that the proximal glycans are essential for the proper folding of ASIC1b, suggesting substantial structural differences in the proximal part of the ectodomain of ASIC1a and ASIC1b.

MATERIALS AND METHODS

cDNAs and Site-directed Mutagenesis cDNAs for rat ASIC1a and ASIC1b have been described previously [START_REF] Bässler | Molecular and functional characterization of acid-sensing ion channel (ASIC) 1b[END_REF]. The point mutations were constructed by recombinant PCR using Pwo DNA polymerase (Roche Applied Science). In some experiments, ASIC1a was tagged with the hemagglutinin (HA) epitope (YPYDVPDYA); ASIC1b was always tagged with the HA and the vesicular stomatitis virus glycoprotein (VSV-G) epitope (YTDIEMNRLGK). The HA-epitope was inserted into the extracellular loop of the proteins -for ASIC1a between F147 and K148 and for ASIC1b between F180 and S181. Thus, according to the crystal structure of chicken ASIC [START_REF] Jasti | Structure of acidsensing ion channel 1 and low pH[END_REF], the epitopes localize to a loop between α helix 1 and α helix2 of the finger subdomain, where they should be well accessible (Fig 1B). Introduction of the HA-epitope required injection of larger amounts of cRNA for current amplitudes comparable to the wild-type (ASIC1a ~10fold and ASIC1b ~2-fold larger amounts). The VSV-G epitope was added in frame to the C terminus of ASIC1b; introduction of the VSV-G epitope did not alter functional expression of the channel. All PCR-derived fragments were entirely sequenced (MWG-Biotech).

Maintenance of oocytes

Animal care and experiments followed approved institutional guidelines at the University of Würzburg. Ovaries were surgically removed under anaesthesia from adult Xenopus laevis females and oocytes were isolated. Using mMessage mMachine (Ambion, Austin, TX), capped cRNA was synthesized by SP6 RNA polymerase from cDNAs, which had been linearized by Nae I. cRNA was injected into oocytes of Xenopus laevis; after injection, oocytes were kept in OR-2 medium (concentrations in mM: 82.5 NaCl, 2.5 KCl, 1.0 Na 2 HPO 4 , 5.0 HEPES, 1.0 MgCl 2 , 1.0 CaCl 2 , and 0.5 g/liter polyvinylpyrrolidone) at 19 o C.

Immunoblot analysis

0.1-10 ng of cRNA coding for the tagged proteins was injected into oocytes. Microsomal membranes were prepared 2 days after injection as previously described [START_REF] Gründer | A mutation causing pseudohypoaldosteronism type 1 identifies a conserved glycine that is involved in of the epithelial sodium channel[END_REF]. Some samples were treated with Endo H (Roche) in a sodium citrate buffer (150 mM, pH 5.5) or with PNGase F (Roche) in a sodium phosphate buffer (80 mM, pH 8.0) in the presence of 1% Triton X100. Proteins from microsomal membranes of two oocytes were separated on a 8-14 % gradient polyacrylamide-SDS gel and transferred to a polyvinylidene fluoride membrane (PolyScreen; PerkinElmer Life Sciences). Either peroxidase-coupled anti-HA antibody (1:1000; Roche Applied Science) or anti-VSV-G antibody (0,5 µg/ml; Roche Applied Science) in combination with peroxidase-coupled anti-mouse antibody (1:5000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was used for detection. Bound antibodies were revealed by chemiluminescence (ECL kit, GE Healthcare). Electrophysiology 1-10 ng of ASIC1b cRNA and 0.01 ng of ASIC1a cRNA was injected into individual Xenopus oocytes. 24-48 hours after injection, whole-cell currents were measured using twoelectrode voltage-clamp as previously described [21]. During recording, oocytes were bathed in solutions containing (in mM): 140 NaCl, 1.8 CaCl 2 , 1.0 MgCl 2 , 10 HEPES, pH as indicated in figures. For solutions with a pH lower than 6.8, HEPES was replaced by MES. Membrane voltage was held at -70 mV. Data were filtered at 50 Hz and acquired at 0.1-1 kHz. All measurements were performed at room temperature (20-25 °C). Data were analyzed with the software IgorPro (Wave metrics). pH response curves were fit with a Hill function:

I = a + (I max -a)/(1 + (EC 50 /[H]) n ),
where I max is the maximal current, a is the residual current, [H] is the H + -concentration, EC 50 is the half-maximal response occurs, and n is the Hill coefficient. Before fitting, current amplitudes were normalized to the amplitude at the lowest pH used; I max was fixed to 1. The pH of half-maximal activation, pH 50 , was calculated as -log(EC 50 ). Time constants of desensitization were determined by fitting the decay phase of current traces to a monoexponential function. Results are reported as means ± SEM. Statistical analysis was done with the unpaired t-test.

Quantification of surface expression

10 ng of cRNA coding for ASIC1b constructs were injected into Xenopus laevis oocytes. After 2 days of protein expression at 19°C, the surface expression of the HA-tagged ASIC1b constructs was determined as previously described [START_REF] Chen | Permeating protons contribute to tachyphylaxis of 1a[END_REF]. Rat anti-HA antibody (clone 3F10; 1:200; Roche) and HRP-conjugated anti-rat antibody (1:400; Jackson ImmunoResearch Laboratories) were used to label the surface expressed HA-tagged ASIC1b constructs. Single oocyte chemiluminescence was detected with an Orion II Microplate Luminometer (Berthold detection systems; Pforzheim, Germany) in relative light units (RLUs) per sec. RLUs of HAtagged channels were at least 100-fold higher than RLUs of untagged channels. Results from three different batches of oocytes (for each: n = 5-14) are reported as mean ± S.E.M. Statistical analysis was performed with the unpaired t-test.

RESULTS

ASIC1a is a glycoprotein, which carries two glycans

Rat ASIC1a has a predicted molecular mass of ∼60 kD. The inserted HA epitope should increase the molecular weight to ∼61 kD. But immunoblot analysis of membrane protein extracts from Xenopus oocytes injected with HA-tagged ASIC1a revealed an apparent molecular weight of more than 70 kD for this protein (Fig. 2B, lanes 1 and 4). Treatment of protein extracts with endoglycosidase H (Endo H) or N-glycosidase F (PNGase F) reduced the apparent molecular mass of ASIC1a by a few kD (Fig. 2B, lanes 2 and 3), demonstrating that ASIC1a is a glycoprotein. There are two types of N-glycans: high mannose-type, synthesized in the ER, and complex-or mature-type glycans, which result from further processing of high-mannose glycans in the Golgi apparatus. Endo H selectively removes high mannose-type sugars, while PNGase F removes both types of N-glycans. We found no evidence for an Endo H resistant form of ASIC1a, suggesting that high mannose-type ASIC1a traffics to the oocyte plasma membrane without further modification of the sugars in the Golgi. Alternatively, the complex-type form may be too rare to be detected. Closer inspection of the ASIC1a immunoreactivity shows that ASIC1a ran as a double band (Fig. 2B, lanes 1 and 4); after glycosidase treatment, however, only a single band could be observed (Fig. 2B, lanes 2 and 3), suggesting that the ASIC1a population was heterogeneous with respect to N-glycans.

ASIC1a has two consensus sequences for N-glycosylation (NXS/T): one at N366 and one at N393, both in the distal part of the ectodomain (Fig. 2A). Individual substitution of N366 and N393 slightly reduced the apparent molecular mass of ASIC1a and combined substitution of both residues further reduced the molecular mass (Fig. 2C, lanes 2, 3 and 4), showing that both potential glycosylation sites are used. Closer inspection reveals that the N366A (Fig. 2C, lane 2) mutant ran as a single band whereas the N393S mutant ran as a doublet (Fig. 2C, lane 3), like wild-type ASIC1a . Overall these results suggest that one part of the ASIC1a population carries sugars at both glycosylation sites (N366 and N393) whereas the other part of the population carries sugars only at N393. Such inefficient glycosylation of N366 could, for example, be due to rapid folding and sequestering of the consensus site [START_REF] Allen | Intracellular folding of tissue-type plasminogen activator. Effects of disulfide bond formation on N-linked glycosylation and secretion[END_REF]. In summary, N393 seems to be the prime glycosylation site of ASIC1a.

N-glycosylation is dispensable for ASIC1a function

We next studied the implications of N-glycans on the function of ASIC1a by measuring whole cell currents of oocytes expressing ASIC1a with substitutions at the glycosylation consensus sites. Expression of ASIC1a mutants N366A and N393S, as well as of the double mutant N366A/N393S gave rise to pH gated currents (Fig. 3A). Desensitization of mutants N366A and N393S had a tendency to be faster than desensitization of ASIC1a (desensitization time constant τ = 1.7 ± 0.4 sec and 1.7 ± 0.3 sec, respectively, compared to 2.1 ± 0.4 sec; p > 0.1; n = 12); desensitization of double mutant N366A/N393S was significantly faster than desensitization of ASIC1a (τ = 1.1 ± 0.1 sec; p = 0.01; n = 12). Moreover, current amplitudes were reduced compared to the wild type channels (Fig. 3B): the N366A substitution reduced the amplitude about 2-fold, the N393S substitution about 3-fold, and the double-substitution also about 2-fold. All three mutant channels had a slightly but significantly (p ≤ 0.05) lower apparent H + -affinity than wild-type channels (Fig. 3C): the pH 50 was shifted from pH 6.43 ± 0.02 for ASIC1a wild-type to 6.36 ± 0.02 for ASIC1a-N366A, to 6.32 ± 0.03 for ASIC1a-N393S, and to 6.33 ± 0.02 for the double mutant N366A/N393S (n = 12). The reduction in apparent affinity was especially evident at low H + concentrations (Fig. 3C).

In summary, although N-glycans slowed desensitization, increased ASIC1a current amplitude and apparent H + affinity, they were largely dispensable for ASIC1a function, similar to the N-glycans of ASIC2a [START_REF] Saugstad | Analysis of the membrane topology of the acid-sensing ion channel 2a[END_REF].

ASIC1b is a glycoprotein with four N-glycans

Rat ASIC1b has a predicted molecular mass of ∼62 kD [START_REF] Bässler | Molecular and functional characterization of acid-sensing ion channel (ASIC) 1b[END_REF]. Insertion of the hemagglutinin (HA) epitope in the extracellular loop should increase the molecular mass to ∼63 kD. In addition, ASIC1b was tagged at its C-terminus with the vesicular stomatitis virusglycoprotein (VSV-G) epitope, further increasing the predicted molecular mass to ∼65 kD. Immunoblot analysis revealed an apparent molecular mass of ∼70 kD for HA-and VSVtagged ASIC1b (Fig. 4B, lanes 1 and 4). Treatment of protein extracts with Endo H and PNGase F reduced the apparent molecular mass of ASIC1b by several kD (Fig. 4B, lanes 2 and 3), demonstrating that also ASIC1b is a glycoprotein. As for ASIC1a, we found no evidence for an Endo H resistant form of ASIC1b. In contrast to ASIC1a, however, the ASIC1b population seemed to be homogenous with respect to N-glycans (Fig. 4).

ASIC1b has four consensus sequences for N-glycosylation: two of them are in the distal part of the extracellular loop (N399 and N426; Fig. 4A) and are identical to those of ASIC1a. The two others are in the alternatively spliced, proximal part of the protein -one at N192 and one at N216. Individual substitution of the two proximal residues N192 and N216 slightly reduced the apparent molecular mass of ASIC1b and combined substitution of both residues further reduced the molecular mass (Fig. 4C, lanes 2, 3 and 4), suggesting that both sites carry glycans. Individual substitution of residue N399 only slightly reduced the apparent molecular mass of ASIC1b whereas substitution of residue N426 more strongly reduced the apparent molecular mass (Fig. 4C, lanes 6 and 7). Combined substitution of both residues did not visibly further reduce the molecular mass compared to substitution of N426 alone (Fig. 4C, lanes 7 and 8). These results suggest that both residues carried a glycan; this glycan seems to be small for N399, however. Since glycans are added in the ER as a preformed 14- residue oligosaccharide, this interpretation suggests that the glycan at N399 had been further trimmed in the Golgi complex. Combined substitution of all four glycosylation consensus sequences together further reduced the molecular mass of ASIC1b (Fig. 4C, lane 9). In addition, also the total amount of protein was reduced. This reduction of the protein amount suggests reduced protein stability and was consistently observed only for the N1 -N4 construct.

N-glycosylation at one of the proximal sites is indispensable for ASIC1b function

We next studied the implications of N-glycans on the function of ASIC1b. We started with the distal glycans at N399 and N426. Substitution of N399 by Ala did not significantly reduce the current amplitude (Fig. 5B). This finding is consistent with its minor posttranslational modification. In contrast, substitution of N426 reduced the current amplitude about 5-fold (p < 0.01), similar to its effect on ASIC1a. Combined substitution of the two residues similarly reduced the current amplitude 5-fold (p < 0.01). Apparent H + -affinity was slightly reduced for N399A and the double mutant N399A/N426S (Fig. 5C): the pH 50 was shifted from pH 5.78 ± 0.01 to 5.72 ± 0.01 (p < 0.01) and to 5.69 ± 0.02 (p < 0.01), respectively (n = 12); there was no significant shift for the single mutant N426S (pH 50 : 5.76 ± 0.01; n = 13; p = 0.3). These results show that both distal sites are necessary for the proper working of ASIC1b; a glycan at N399 slightly increased apparent H + -affinity whereas a glycan at N426 increased current amplitude. Similar to the glycans of ASIC1a and ASIC2a, however, the two distal glycans of ASIC1b were not absolutely required for channel function.

The two proximal sites (N192 and N216) are not present in ASIC1a. Therefore, we expected no dramatic functional effect of substitutions at these two sites. Individual substitution of N192 and N216 reduced the current amplitude 4-to 8-fold (p < 0.01), however, similar to the N426 substitution (Fig. 5B). The apparent proton affinity was also reduced: the pH 50 was shifted from pH 5.63 ± 0.01 (n = 6) for ASIC1b wild-type to 5.57 ± 0.03 for N192A (n = 6; p < 0.01) and to 5.46 ± 0.02 for N216S (n = 6; p < 0.01; Fig. 5C). Combined substitution of the two residues almost completely silenced the channels (1% of the wild-type amplitude; p < 0.01; Fig. 5B), making it impossible to determine an apparent affinity for H + . This result shows that a glycan at least at one of the two proximal sites is indispensable for ASIC1b activity.

Desensitization was faster for ASIC1b (τ = 0.5 ± 0.02 sec, n = 18; Fig. 5A) than for ASIC1a. Due to the small desensitization time constants it was not possible to determine with precision the effect of N-glycans on desensitization kinetics. The two proximal glycans had no significant effect on desensitization, whereas the N399A substitution as well as the N399S/N426A double substitution significantly speeded desensitization (τ = 0.4 ± 0.01 sec and τ = 0.4 ± 0.02 sec, respectively; p < 0.05; n = 18; Fig. 5A), consistent with the faster desensitization of the glycan mutants of ASIC1a (Fig. 3A).

N-glycosylation at one of the proximal sites is indispensable for ASIC1b surface expression

Since substitution of some of the ASIC1b glycosylation sites dramatically reduced the current amplitude, we asked whether these substitutions compromised trafficking of the channels to the plasma membrane. ASIC1b wild-type and mutant channels used in this study carried an HA epitope within the extracellular loop; we used a monoclonal anti-HA antibody and a luminescence assay to compare the surface expression of these channels. As expected from the unchanged current amplitude, surface expression of ASIC1b-N399A was not significantly reduced compared to the wild-type protein (Fig. 6). Surface expression of ASIC1b-N426, however, was reduced ∼5-fold (Fig. 6). Additional introduction of the N399 substitution (ASIC1b N399A/N426S) did not further reduce surface expression. Removing individually the proximal N-glycans at N192 and N216 caused a strong decrease in surface expression by a factor of ∼5, similar to removal of the distal N-glycan at N426. Simultaneous removal of the two proximal N-glycans abolished surface expression of the protein. Thus, the reduction in surface expression (Fig. 6) almost perfectly matched the reduction in current amplitude (Fig. 5B), suggesting that the reduced current amplitudes are mainly due to a reduced number of functional ion channels at the plasma membrane rather than to a reduced activity of single ion channels.

Together our results show that ASIC1b carries N-glycans at four sites: N192, N216, N399, and N426. The N-glycans on N192, N216 and N426 strongly promoted the surface expression of ASIC1b; one glycan either at position N192 or at position N216 was indispensable for the surface expression of ASIC1b.

ASIC1a and ASIC1b are glycoproteins. This was an expected finding. Moreover, the glycosylation pattern of ASIC1a was very similar to the published glycosylation pattern of ASIC2a [START_REF] Saugstad | Analysis of the membrane topology of the acid-sensing ion channel 2a[END_REF]: both proteins have two conserved consensus sequences for N-glycosylation in the distal part of the ectodomain. Both sites carry glycans, which are dispensable for channel activity, however. Even in the absence of any glycan, ASIC1a and ASIC2a are able to assemble into functional oligomeric channels that traffic to the plasma membrane and are gated by H + . Reduced current amplitude and slightly altered apparent H + affinity suggest, however, that the N-glycans assist in channel assembly and/or trafficking as well as channel function. The two distal sites are conserved in ASIC1b and have a similar role also for this channel.

The two distal glycosylation sites are also conserved in chicken ASIC1, for which the crystal structure is available [START_REF] Jasti | Structure of acidsensing ion channel 1 and low pH[END_REF]. The chicken ASIC1 crystal reveals the position of these two glycans within the three-dimensional structure of an ASIC (Fig. 1B). The first site (corresponding to N366 of rat ASIC1a) is at the beginning of β-sheet 10 within the palm domain whereas the second site (corresponding to N393) is at the end of α-helix 6 of the knuckle domain [START_REF] Jasti | Structure of acidsensing ion channel 1 and low pH[END_REF]. Both glycans, though close in the primary sequence, are thus on opposite parts of the ectodomain pointing away from the protein core (Fig. 1B). Hence both glycans seem easily accessible, rendering unlikely the possibility that modifying enzymes of the Golgi complex cannot access these glycans to trim them to complex-type glycans. The position of the two distal glycans at points where the secondary structure of the protein changes is in agreement with a general trend for the frequent occurrence of glycans at such points [START_REF] Petrescu | Statistical analysis of the protein environment of N-glycosylation sites: implications for occupancy, structure, and folding[END_REF]; glycans may actually favor the reorientation of the peptide chain [START_REF] Petrescu | Statistical analysis of the protein environment of N-glycosylation sites: implications for occupancy, structure, and folding[END_REF]. The first of these two distal sites is highly conserved in ASICs from rat and fish [21], the one exception is ASIC3, which has the second distal site, however (Fig. 1). Thus, it seems that each ASIC has at least one of the two distal glycosylation sites, suggesting some functional role for the distal glycans.

In this context, it is important to remember that subtle functional impacts of glycans may have been missed in the Xenopus oocyte expression system. In a living animal, proteins likely are exposed to other conditions than in Xenopus oocytes. Glycans may, therefore, stabilize the protein structure in a living animal although no functional requirement is observed in oocytes. For example, the temperature of 19 o C at which we kept the oocytes is well below the body temperature of mammals. Glycans were, however, also dispensable for ASIC2a activity in HEK293 cells [START_REF] Saugstad | Analysis of the membrane topology of the acid-sensing ion channel 2a[END_REF] cultured at 37 o C, arguing against strong temperaturedependent effect of the distal glycans.

The surprise came when analyzing the two proximal sites that are specific for ASIC1b. Eliminating both proximal sites together completely abolished ASIC1b activity; this loss of channel function could be clearly attributed to a loss of surface expression (Fig. 6). There are different possibilities how glycans could promote surface expression: they could assist in folding of the protein, stabilize the protein structure, or promote trafficking to the cell surface. We consider the last possibility as less likely since the related ASIC1a apparently does not need glycans for surface targeting. The first two possibilities (folding of the protein, stabilization of its structure) are related and both require that the structure of ASIC1b is significantly different form ASIC1a in the proximal part of the extracellular loop.

N192 and N216 of ASIC1b are predicted to localize to a short α-helix (α-helix at the beginning of a short β-sheet (β-sheet 4) of the finger and β-ball domains, respectively [START_REF] Jasti | Structure of acidsensing ion channel 1 and low pH[END_REF]. The side chains of the corresponding amino acids in chicken ASIC1 (D160 and D184) are both on the surface of the protein core in close proximity (~18 Å) to each other (Fig. 1B). D160 seems to be more readily accessible than D184, which is in a minor groove and partially shielded on one side by K205 (R235 in ASIC1b) and on the other side by the main chain (C180, S181, and P182 in chicken ASIC1 and C212, G213, and P214 in rat ASIC1b). Assuming that the protein structure in this part of the ectodomain is indeed different between ASIC1b and ASIC1a, the exact position of these amino acids may well be different in ASIC1b, however, and glycans may assist in adopting the proper conformation of the proximal ectodomain of ASIC1b. C180 of chicken ASIC1 forms a disulfide bridge with C173; in ASIC1b, glycans may, for example, influence formation of this bridge.

The two proximal glycosylation sites are at the very end of the region that is different between ASIC1a and ASIC1b [START_REF] Chen | A sensory neuronspecific, proton-gated ion channel[END_REF][START_REF] Bässler | Molecular and functional characterization of acid-sensing ion channel (ASIC) 1b[END_REF], immediately upstream of the splice-junction that connects the variable exon to the common exons of ASIC1a and 1b (Fig. 4A). This part of the ectodomain has been proposed to be the binding site of the spider toxin PcTx1 on ASIC1a [25] or, alternatively, to determine the functional consequences of PcTx1 binding to ASIC1a and 1b [START_REF] Chen | /N393S (lane 4) mutants, showing that both N-glycosylation sites carry glycans. ASIC1a was detected with an anti-HA antibody[END_REF]. These proposals are in agreement with our interpretation that the differential glycosylation in the proximal ectodomain reflects structural differences in this region. The additional glycans on ASIC1b do not simply prevent binding of PcTx1, however, since PcTx1 also binds to ASIC1b [START_REF] Chen | /N393S (lane 4) mutants, showing that both N-glycosylation sites carry glycans. ASIC1a was detected with an anti-HA antibody[END_REF].

Proximal glycosylation sites are present also in other ASICs. The second of the two proximal glycosylation sites of ASIC1b (N216) is conserved in ASIC2b, ASIC3, and ASIC4 (Fig. 1A). ASIC1b glycosylation may thus be prototypical for a subgroup of ASICs and proximal glycosylation sites may assist in folding or stabilize the structure of all members within this subgroup. ASIC4 is distinguished from other ASICs by the presence of 8 consensus sequences for N-glycosylation, scattered over its ectodomain (Fig. 1A). Treatment of ASIC4 with PNGase F reduces its molecular weight by more than 10 kD (I.K. & S.G., unpublished), suggesting that most of these sites indeed carry glycans. This result suggests that the structure of the ASIC4 ectodomain may be substantially different from that of other ASICs. Given that ASIC4 is not gated by H + and that its activation mechanism is still unknown [START_REF] Gründer | A new member of acid-sensing ion channels from pituitary gland[END_REF][START_REF] Chen | Zebrafish Acid-sensing Ion Channel (ASIC) 4, Characterization of Homo-and Heteromeric Channels, and Identification of Regions Important for Activation by H+[END_REF], this is a likely hypothesis.

In summary, we propose that N-glycosylation makes an important contribution in shaping the structure of the proximal ectodomain of several ASICs. (B) Top, scheme showing the positions of the two distal consensus sites for N-glycosylation of chicken ASIC1. The positions of the two proximal sites of ASIC1b are shown as dashed lines; they are not glycosylated in chicken, however, because the consensus site is not conserved. The position corresponding to the insertion site of the HA epitope in ASIC1a and 1b is shown by an arrowhead. Bottom, ribbon diagram of the trimeric chicken ASIC1 structure (PDB entry 2QTS) [START_REF] Jasti | Structure of acidsensing ion channel 1 and low pH[END_REF] with subunits coloured magenta, cyan and yellow; only the ectodomains are shown. For one subunit, the N-acetylglucosamines of the two distal glycans together with the connecting asparagine residues are shown in all-bonds representation with carbon atoms in green, oxygen in red and nitrogen in blue and the positions corresponding to the two proximal glycans of ASIC1b are highlighted by green spheres. Similarly, the position where the HA epitope was introduced into the ectodomain of ASIC1a and 1b is marked by two red spheres.

Biochemical

  Journal Immediate Publication. Published on 29 Feb 2008 as manuscript BJ20071614 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071614 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 29 Feb 2008 as manuscript BJ20071614 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071614 S t a g e 2 ( a ) P O S T -P R I N T

Biochemical

  Journal Immediate Publication. Published on 29 Feb 2008 as manuscript BJ20071614 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071614 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Figure 1

 1 Figure 1 Positions of consensus sites for N-glycosylation of ASICs. (A) Scheme showing the positions of putative consensus sites for N-glycosylation of different ASICs. Symbols with an identical color mark glycosylation sites at corresponding positions.(B) Top, scheme showing the positions of the two distal consensus sites for N-glycosylation of chicken ASIC1. The positions of the two proximal sites of ASIC1b are shown as dashed lines; they are not glycosylated in chicken, however, because the consensus site is not conserved. The position corresponding to the insertion site of the HA epitope in ASIC1a and 1b is shown by an arrowhead. Bottom, ribbon diagram of the trimeric chicken ASIC1 structure (PDB entry 2QTS)[START_REF] Jasti | Structure of acidsensing ion channel 1 and low pH[END_REF] with subunits coloured magenta, cyan and yellow; only the ectodomains are shown. For one subunit, the N-acetylglucosamines of the two distal glycans together with the connecting asparagine residues are shown in all-bonds representation with carbon atoms in green, oxygen in red and nitrogen in blue and the positions corresponding to the two proximal glycans of ASIC1b are highlighted by green spheres. Similarly, the position where the HA epitope was introduced into the ectodomain of ASIC1a and 1b is marked by two red spheres.

Figure 2 FIGURE 1 FIGURE 2

 212 Figure 2 Immunoblot analysis of N-linked glycosylation of ASIC1a (A) Linear scheme of ASIC1a showing the approximate positions of N-glycans. M1 and M2, membrane spanning domains; HA, hemagglutinin epitope. (B) Effect of Endo H and PNGase F on apparent mass of ASIC1a. Samples of ASIC1a untreated with enzymes (lanes 1 and 4) were loaded in parallel with Endo H-or PNGase F-treated samples (lanes 2 and 3). (C) Immunoblot analysis of ASIC1a-wt (lane 1), -N366A (lane 2), -N393S (lane 3), and -

FIGURE 3 Biochemical 4 FIGURE 5 FIGURE 6

 3456 FIGURE 3

© 2008 The Authors Journal compilation © 2008 Biochemical Society

S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Licenced copy. Copying is not permitted, except with prior permission as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Acknowledgements

This study was supported by DFG grant GR1771/3-4 to S.G.