Tamara Seredenina

Teresa Ferraro

Georg C Terstappen

Andrea Caricasole

Renza Roncarati
email: rroncarati@sienabiotech.com

Molecular cloning and characterization of a novel human variant of RIC-3, a putative chaperone of nicotinic acetylcholine receptors

Keywords: alpha7-nAChR, alpha-bungarotoxin, chaperone, expression, RIC-3, splice variant

come

3 Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels which are widely distributed in the central and peripheral nervous system. They are involved in a variety of physiological processes including cognition and development and their dysfunction has been associated with a variety of neurological disorders [START_REF] Dajas-Bailador | Nicotinic acetylcholine receptors and the regulation of neuronal signalling[END_REF][START_REF] Gotti | Brain neuronal nicotinic receptors as new targets for drug discovery[END_REF][START_REF] Levin | Nicotinic treatment for cognitive dysfunction[END_REF][START_REF] Rusted | Nicotinic treatment for degenerative neuropsychiatric disorders such as Alzheimer's disease and Parkinson's disease[END_REF][START_REF] Martin | Alpha-7 nicotinic receptor agonists: potential new candidates for the treatment of schizophrenia[END_REF]. Hence, nicotinic receptors represent important and intensively studied targets for therapeutic intervention. The α7-nAChR is of particular relevance for Alzheimer's disease and schizophrenia, but studies focusing on this receptor are challenging since it cannot be easily expressed in heterologous systems [START_REF] Cooper | Host cell-specific folding and assembly of the neuronal nicotinic acetylcholine receptor alpha7 subunit[END_REF][START_REF] Quik | Similarity between rat brain nicotinic alpha-bungarotoxin receptors and stably expressed alpha-bungarotoxin binding sites[END_REF][START_REF] Rangwala | Neuronal alpha-bungarotoxin receptors differ structurally from other nicotinic acetylcholine receptors[END_REF][START_REF] Sweileh | Multistep expression and assembly of neuronal nicotinic receptors is both host-cell-and receptor-subtype-dependent[END_REF]. In fact, the complex process leading to functional expression of α7-nAChR is likely to require the activity of multiple cell-specific assembly and trafficking proteins [10;11]. Recently it was shown that the protein RIC-3 (resistant to inhibitors of cholinesterase) is necessary for functional expression of α7-nAChRs [12;13]. Human RIC-3 belongs to a conserved gene family [START_REF] Halevi | Conservation within the RIC-3 gene family. Effectors of mammalian nicotinic acetylcholine receptor expression[END_REF] and is predicted to contain a cleavable signal peptide in vertebrate species, followed by a proline-rich domain, one transmembrane domain and a cytoplasmic component that consists of a conserved coiled-coil domain and a non conserved C-terminus [START_REF] Cheng | Differential subcellular localization of RIC-3 isoforms and their role in determining 5-HT3 receptor composition[END_REF]. Another experimental evidence suggests the absence of a signal peptide indicating that RIC-3 has two transmembrane domains [START_REF] Castelan | Molecular characterization and localization of the RIC-3 protein, an effector of nicotinic acetylcholine receptor expression[END_REF]. The function of this protein is not yet clear; the prevailing hypothesis states that RIC-3 acts in the endoplasmic reticulum (ER) as a chaperone which regulates the expression of nicotinic and 5-HT 3 receptors by either promoting their maturation and functional expression or by retaining the receptors in the ER, depending on the composition and type of receptor [17;18]. The precise mechanism of action of this protein is not known, however experimental evidence supports an interaction of RIC-3 with unassembled nAChR subunits [START_REF] Lansdell | RIC-3 enhances functional expression of multiple nicotinic acetylcholine receptor subtypes in mammalian cells[END_REF]. To date, the existence of four distinct transcripts of human RIC-3 has been demonstrated [START_REF] Halevi | Conservation within the RIC-3 gene family. Effectors of mammalian nicotinic acetylcholine receptor expression[END_REF] but little is known about the function of these variants. Here, we report the identification, cloning and detailed pattern of expression of a new variant of human RIC-3, and demonstrate that this variant is able to promote the maturation and surface expression of α7-nAChRs in cells that are otherwise not permissive for the expression of this receptor.

Materials and methods

Molecular cloning.

PCR amplification was carried out using a proofreading enzyme (Phusion High-Fidelity DNA Polymerase, Finzymes) following the manufacturer's instructions. 3% DMSO was added to the reactions. Primer sequences were as follows: 5'-CACCATGGCGTACTCCACAGT-3' (forward) and 5'-TCACTCTAAACCCTGGGGGT-3' (reverse). Reaction details were as follows: 98°C/1 min; 10 cycles of (30 sec at 98°C; 30 sec at 65°C, decreasing by 1°C every cycle; 30 sec at 72°C), followed by 30 cycles of (30 sec at 98°C; 30 sec at 55°C, 30 sec at 72°C), followed by 7 min at 72°C. cDNA from adult human hippocampus (see below) was used as template. PCR products were purified using Qiagen gel extraction kit, cloned into pCR-Blunt II-TOPO (Invitrogen) and subcloned into pcDNA3.1 Zeo(+). The inserted DNA was sequenced by standard protocols (MWG-Biotech). The alignment of the amino acid sequences was performed using MUSCLE 3.41 and visualized with GeneDoc 2.6.002. Real-time quantitative PCR. cDNA was synthesized from 2 µg of polyadenylated RNA (Clontech) employing Superscript II reverse transcriptase (Invitrogen) and oligo(dT) primer (Proligo France SAS) according to the manufacturer's instructions. Real-time PCR was performed using an I-Cycler and the iQ SYBR Green Supermix (Bio-Rad, Hercules, CA) according to the instructions of the manufacturer. One µl of cDNA was used as template. Primer concentration was 10 µM. Reactions were carried out in triplicate. Reaction conditions were as follows: 5 min at 95°C; 45 cycles of (30 s at 95°C; 30 s at 55°C). As control, amplification without template was performed. The fluorescence threshold cycle (Ct) was determined using the iCycler iQ real-time PCR detection system software (BioRad). The relative starting quantities of each transcript were determined by interpolating the Ct values of the unknown samples to the standard curve. Real time PCR A c c e p t e d M a n u s c r i p t 4 experiments were repeated twice for each target sequence. Quantification of β-actin expression was carried out in parallel in order to normalize the levels of expression in each sample. Primer sequences were designed to different exons of the human RIC-3, RIC-3e and α7-nAChRs genes. Primer sequences were as follows: 5'-CAGATCATTTGGGTTGGGAAA-3' (forward) and 5'-CACACGAGGTAACAGAATTATCTTCCT-3' (reverse) to amplify a product located within exon 6 of RIC-3; 5'-GGGAAAACAACTGCAGAGGAT-3' (forward) and 5'-TCTTCAGGGTAACTAATTTTCCTG-3' (reverse) to amplify specifically the new variant RIC-3e; 5'-GCAAGAGGAGTGAAAGGTTCTATGA-3' (forward) and 5'-CATGGTCACTGTGAAGGTGACA-3' (reverse) was designed to amplify a product within human α7-nAChRs;

5'-CCTGGCACCCAGCACAAT-3' (forward) and 5'-GCCGATCCACACGGAGTACT-3' (reverse) to amplify a fragment within human β-actin. Cell culture and transfection. CHO-K1 cells were cultured in Ham's F12 medium with 10% FBS, HEK 293 cells were cultured in DMEM with 10% FBS. All media were supplemented with 2 mM Glutamax, 100 U/ml penicillin and 100 µg/ml streptomycin. All media and supplements for cell culture were from Gibco, if not specified otherwise. For transient transfections 7 x 10 4 cells/cm 2 were plated one day before transfection on 13 mm coverslips treated with 10 µg/ml poly-D-lysine. For FLIPR-Ca 2+ measurements HEK 293 cells were plated in 96-well clear-bottom, black-well plates (Costar) at a density of 4 x 10 4 cells/well and cultured for 24 hours prior to transfection. All transfections were carried out using Lipofectamine-2000 (Invitrogen) following the instructions of manufacturer. 0.1 µg of DNA per cm 2 was used for each transfection. The ratio DNA/Lipofectamine was 1/3 w/v. Transfection mix was prepared in OptiMEM. Incubation time with transfection mix was 4h. After the transfection, fresh medium was added and cells were incubated at 37°C. Assays were performed 24-48 h after transfection. All manipulations with cells were carried out in sterile conditions. Cells were maintained at 37°C in a humidified atmosphere of 5% (v/v) CO 2 /air. α-Bgt fluorescent staining. For fluorescence microscopy, HEK 293 and CHO-K1 cells were cultured on PDL-coated glass coverslips at a density of 7x10 4 cells per cm 2 . Labeling was performed with 0.5 µg/ml of α-Bgt conjugated with Alexa Fluor TM -555 (Molecular Probes); for 45 min at 4°C. After three washes with PBS cells were fixed with 3% paraformaldehyde in PBS for 15 min at room temperature. Coverslips were subsequently mounted with Prolong ® gold antifade reagent (Molecular Probes) on glass slides and analyzed with a Zeiss LSM510 confocal microscope equipped with Argon and He/Ne lasers. Ca 2+ flux measurements using a FLIPR TM system FLIPR-Ca 2+ experiments were performed as described previously [START_REF] Roncarati | Functional properties of alpha7 nicotinic acetylcholine receptors co-expressed with RIC-3 in a stable recombinant CHO-K1 cell line[END_REF]. In brief, 24 hours after transfection the medium was replaced with 100 µl of Hank's Balanced Salt Solution-Hepes 20 mM, pH 7.4 (assay buffer) containing 4 µM Fluo-4-AM, 0.02% pluronic acid and 5 mM probenecid. After 30 minutes of incubation at 37˚C the labeling solution was removed and 200 µl of assay buffer containing 2.5 mM probenecid and 10 µM MLA where appropriate were added. Plates were then transferred to a Fluorometric Imaging Plate Reader system (FLIPR, Molecular Devices) with excitation and emission wavelengths set to 488 nm and 540 nm, respectively. Basal fluorescence was recorded for 30 seconds, followed by addition of 50 µl of a 5x concentrated solution of nicotine or PNU-120596 and measurement of fluorescence for 10 minutes. Subsequently, 50 µl of a 6x solution of nicotine were added and fluorescence was measured for 3 minutes. Measurements were made at 1 second intervals for 1 minute after compound addition and at 30 second intervals for the remaining time. The results were exported from the FLIPR raw data as MAX-MIN of fluorescence signal intensity in two intervals corresponding to the first and the second addition of compounds.

Results and discussion

Identification of a novel variant of human RIC-3

In order to clone RIC-3 transcripts, proofreading PCR was carried out using cDNA from adult human hippocampus as template. Primers employed in the reactions were designed based on the known human RIC-3 sequence (RefSeq NM_024557) and comprised initiation and stop codons. Four distinct products were obtained in the RT-PCR (Fig. 1). The specificity of the primers employed was confirmed by performing the reactions with single primer controls; also, a negative control PCR lacking template cDNA did not produce any amplification (data not shown). The band of ca. 1100 bp (Fig. 1) corresponds to the major form of RIC-3 (AY326435 [START_REF] Halevi | Conservation within the RIC-3 gene family. Effectors of mammalian nicotinic acetylcholine receptor expression[END_REF]), as confirmed by DNA sequencing. Four human RIC-3 variants (a, b, c and d) have been previously described [START_REF] Halevi | Conservation within the RIC-3 gene family. Effectors of mammalian nicotinic acetylcholine receptor expression[END_REF]. RIC-3a is the major sequence of RIC-3; for simplicity we will refer to this splice variant as "RIC-3". Based on the apparent length, the two faster migrating products most likely correspond to the shorter variants of RIC-3 (GenBank accession numbers AK021670 and AL832601) [START_REF] Halevi | Conservation within the RIC-3 gene family. Effectors of mammalian nicotinic acetylcholine receptor expression[END_REF]. The minor product of ca. 900 bp does not correspond to any of the previously identified RIC-3 variants. This putative human RIC-3 variant was cloned into the pCR-Blunt II-TOPO vector and sequenced. The sequence (accession number AM422214), demonstrates that the cloned fragment corresponds to human RIC-3. We named the novel variant "RIC-3e", following the nomenclature proposed by Halevi et al. [START_REF] Halevi | Conservation within the RIC-3 gene family. Effectors of mammalian nicotinic acetylcholine receptor expression[END_REF]. The initiation and stop codons are the same as those reported for RIC-3 [START_REF] Halevi | Conservation within the RIC-3 gene family. Effectors of mammalian nicotinic acetylcholine receptor expression[END_REF]. A comparison with the gene structure of the human RIC-3 locus shows that RIC-3e contains the exons 1, 2, 3 and 6. The deletion of 240 bp corresponds to the lack of exons 4 and 5, probably due to alternative splicing. The prediction of the amino acidic sequence and alignment with the known human RIC-3 variants confirmed that the novel cDNA carries a deletion of 240 bp and encodes for a RIC-3 protein of 288 amino acids which lacks the coiled-coil domain and a part of the C-terminal sequence (Fig. 2).

Expression profile of RIC-3 variants and α7-nAChR in human tissues

Little is known about tissue specific distribution of RIC-3. Studies in C. elegans [START_REF] Halevi | The C. elegans ric-3 gene is required for maturation of nicotinic acetylcholine receptors[END_REF] show that RIC-3 is expressed in almost all neurons and the body wall muscles. In situ hybridization of mouse brain with a RIC-3 probe produces a low signal in most regions of the brain with higher levels in cerebellum and some hippocampal areas. Northern blot analysis demonstrates RIC-3 expression in human brain and in some peripheral tissues [START_REF] Halevi | Conservation within the RIC-3 gene family. Effectors of mammalian nicotinic acetylcholine receptor expression[END_REF]. The detailed analysis of the distribution of RIC-3 expression is an important step towards the understanding of its biological function. Therefore, a series of quantitative PCR (qPCR) studies was carried out to determine the expression profile of RIC-3 and RIC-3e transcripts in human tissues. A panel of cDNAs synthesized from mRNA samples derived from different human tissues, including several distinct brain regions, was used. The first set of primers (see Materials and methods) was designed to amplify a product located within exon 6 of RIC-3. This set of primers recognizes all known transcripts except for the truncated form AY326436/AY358475 (see Fig. 2B). A second set of primers was designed to specifically amplify the new variant RIC-3e. Since exons 4 and 5 are missing in this variant, the reverse primer was designed across the junction of exon 3 with exon 6. As expected, a single product corresponding to RIC-3e was amplified from cDNAs from different tissues using this set of primers (data not shown). The results presented in Fig. 3 (A,B) indicate a tight correlation of the distribution and level of expression of the novel RIC-3e variant and the major RIC-3 isoform. The human RIC-3 gene is expressed predominantly in the central nervous system with the highest levels in cerebellum and pituitary gland. These data on mRNA expression levels in different areas of human brain are in general in accordance with recently published work which describes RIC-3 protein localisation in the rat brain [START_REF] Castelan | Molecular characterization and localization of the RIC-3 protein, an effector of nicotinic acetylcholine receptor expression[END_REF]. In peripheral tissues high levels of RIC-3 are observed in pancreas and testis with much lower levels of expression in other tissues. Since RIC-3e has a similar expression profile as compared to RIC-3, the functional significance of this new variant remains elusive. However despite the similarity of tissue distribution of the two variants cell-type or cell compartment-specific expression can not be excluded. A detailed comparison of RIC-3 and RIC-3e proteins in tissues, at the single-cell level resolution will be required to verify this possibility. RIC-3 was shown to be necessary for functional expression of α7-nAChRs [13;14]. However, a detailed comparative expression analysis of both genes is not available. Therefore, qPCR of α7-nAChR was performed on the same panel of human tissues in order to determine if RIC-3 and α7-nAChR transcripts are coherently expressed (Fig. 3C). Figure 3C shows that α7-nAChRs are widely expressed within the central nervous system. The highest levels of α7-nAChR transcripts were found in the cerebral cortex, hippocampus and amygdala. This quantitative analysis correlates well with the distribution pattern of α7-nAChR described by in situ hybridization and [I 125 ]-Bgt-binding studies [20;21]. Analysis of the abundance of α7-nAChR transcripts in the peripheral tissues shows high level of expression in human testis. Interestingly, some recent publications suggest a function for α7-nAChR in the reproductive system [22;23]. Comparison of the distribution pattern of RIC-3 and α7-nAChR shows an overlapping profile of the two genes, which are both expressed at higher levels in the CNS than in the periphery. In general, all the tissues that express α7-nAChR also express significant levels of RIC-3, consistent with the proposed function of RIC-3 as a chaperone of nicotinic receptors [START_REF] Halevi | The C. elegans ric-3 gene is required for maturation of nicotinic acetylcholine receptors[END_REF]. Nevertheless, high levels of expression of RIC-3 are also seen in some brain regions (corpus callosum, pituitary gland, cerebellum) which express relatively low levels of α7-nAChR. This suggests that RIC-3 might possess other, as yet unknown functions not confined to the process of maturation of α7-nAChR.

Analysis of functional activity of RIC-3e

RIC-3 was shown to promote the folding, assembly and membrane targeting of recombinant α7-nAChR in different mammalian cell lines [13;18]. In particular, co-expression of RIC-3 and α7-nAChR resulted in the formation of surface α-Bgt-binding receptors in host cells that are otherwise unable to form functional α7-nAChRs, such as HEK 293, COS and tsA201 [13;17;18]. As the novel variant RIC-3e lacks the evolutionarily conserved coiled-coil domain and a part of the C-terminal region present in RIC-3 proteins, we determined the capacity of RIC-3e to mediate proper membrane expression of α7-nAChRs in a cellular background which is non-permissive to functional α7-nAChRs expression. To this end, HEK 293 cells where transfected with α7-nAChR, either alone or in combination with RIC-3 or RIC-3e. In order to detect α7-nAChR on the cell surface, live cells were incubated with fluorescently labeled α-Bgt. As expected, expression of human α7-nAChR alone did not result in the formation of α-Bgt-binding receptors (Fig. 4A,B), On the contrary, co-expression of α7-nAChR with either RIC-3 (Fig. 4C,D) or RIC-3e (Fig. 4E,F) resulted in α-Bgt binding to cell membranes, indicating that both isoforms are able to promote surface expression of mature α7-nAChRs. Similar results were also observed using CHO-K1 cells (data not shown). In order to demonstrate that co-expression of RIC-3e does not only lead to expression of α7-nAChRs on the plasma membrane but also promotes formation of functionally active channels, we performed measurements of Ca 2+ influx as this channel is known to possess a high conductivity for Ca 2+ ions in the activated state. To this end HEK 293 cells were transiently transfected with constructs encoding human α7-nAChR together with RIC-3 or RIC-3e and with each plasmid alone as control. Subsequently, cells were stimulated with nicotine in the absence or presence of the positive allosteric modulator PNU-120596 [24]. The latter was necessary in order to increase the open-time of the α7-nAChR (which is known to desensitize very rapidly) such that the Ca 2+ influx is sufficient to be measured with a FLIPR system. In fact, we had previously shown thatdepending on the cell line used -potentiation of the activity of α7-nAChR with PNU-120596 was necessary in order to enable functional measurements of Ca 2+ flux with a FLIPR system [START_REF] Roncarati | Functional properties of alpha7 nicotinic acetylcholine receptors co-expressed with RIC-3 in a stable recombinant CHO-K1 cell line[END_REF]. As expected, treatment with nicotine alone did not elicit any measurable changes of the intensity of fluorescence (Fig. 5). Conversely, the addition of nicotine after treatment with PNU-120596 resulted in a significant increase of intracellular Ca 2+ concentrations. Importantly, this effect was only observed in the cells co-transfected with α7-nAChR and RIC-3 or α7-nAChR and RIC-3e. Moreover, the increase of fluorescence intensity was completely abolished when the cells were pretreated with methyllycaconitine (MLA), a selective antagonist of α7-nAChR, thus confirming specificity of the obtained results. In all experiments, the magnitude of the functional response of co-transfected α7-nAChR and RIC-3e was comparable to α7-nAChR in combination with RIC-3. More detailed studies of the two splice variants cannot be made in this system as subtle differences may be masked by the strong potentiation induced by PNU-120596. Taken together, we can conclude that RIC-3e is a functional splice variant leading to expression of functional α7-nAChRs on the plasma membrane. As compared to RIC-3, the variant RIC-3e carries a deletion of the coiled-coil domain and part of the C-terminal domain. Previously published deletion analyses of RIC-3 suggested that ablation of the coiled-coil domain does not influence the capability of RIC-3 to modulate the expression of nicotinic and 5-HT 3 receptors [17;25]. No changes were found in the chaperone activity of naturally occurring RIC-3 variants lacking the coiled-coil domain in Drosophila melanogaster [26]. Our results demonstrate that a variant, which does not contain the coiled-coil domain region (plus an adjacent C-terminal part) naturally exists, promotes α7-nAChR surface expression and functional receptor activity. Coiled-coil domains are known to be important for protein-protein interactions in the organization of molecular scaffolds, in addition to other functions [27]. At least two other regions of RIC-3 have been predicted to provide protein-protein interactions [START_REF] Halevi | Conservation within the RIC-3 gene family. Effectors of mammalian nicotinic acetylcholine receptor expression[END_REF] that may be sufficient to form the scaffold recruiting the complex necessary for maturation of α7-nAChRs. The role of the missing segment may be to gather additional regulatory partners, to specify differential subunit composition of mature receptors, or be related to as yet undiscovered functions of RIC-3.

Conclusions

In conclusion, here we report the existence of a new human variant of RIC-3 which lacks the coiledcoil domain but is still capable to promote functional expression of α7-nAChRs in mammalian cells. The detailed analysis of the expression patterns of RIC-3, RIC-3e and α7-nAChRs has shown that both variants are very similarly distributed in human tissues, with a major overlap to α7-nAChRs in the nervous system. Further studies regarding the biological function of RIC-3 and its variants are necessary to clarify the specific roles of the various isoforms and to identify new targets of this important regulator of neurotransmitter receptor expression.

Acknowledgments. We would like to thank Dr. Giuseppe Pollio for excellent support provided for cloning and molecular biology. We would also like to thank Dr. Hendrick Bothmann for helpful discussions regarding FLIPR experiments.

Bioscience

  Reports Immediate Publication. Published on 11 Aug 2008 as manuscript BSR20080055 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BSR20080055 A c c e p t e d M a n u s c r i p t

Bioscience

  Reports Immediate Publication. Published on 11 Aug 2008 as manuscript BSR20080055 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BSR20080055 A c c e p t e d M a n u s c r i p t

Bioscience

  Reports Immediate Publication. Published on 11 Aug 2008 as manuscript BSR20080055 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BSR20080055 A c c e p t e d M a n u s c r i p t

BioscienceFigure legends Fig. 1 .

 legends1 Figure legends

Fig. 2 .

 2 Fig.2. Amino acid sequence of RIC-3e and comparison to known variants. A. Alignment of the deduced amino acid sequences of RIC-3 (AY326435) and RIC-3e (AM 422214) was performed using MUSCLE 3.41 and visualized with GeneDoc 2.6.002. Black color indicates 100% sequence identity. RIC-3e carries a deletion of 240 bp and encodes a RIC-3 protein of 288 amino acids. B. Schematic representation of known human RIC-3 transcripts. The empty boxes E1-E6 correspond to RIC-3 exons. The predicted protein domains are represented with different colors: SP: signal peptide; Nterm: amino-terminal domain; TM: transmembrane domain; Cterm: carboxy-terminal domain; CCD: coiled-coil domain.

Fig. 3 .

 3 Fig.3. Quantitative real-time PCR expression analysis of RIC-3 variants and α7-nAChR in a panel of human tissues. Results shown are the means and standard errors of three measurements per tissue. Absolute levels of expression were normalized to the expression levels of β-actin. A. Expression profile of RIC-3. B. Expression profile of RIC-3e. C. Expression profile of α7-nAChR.

Fig. 4 .

 4 Fig.4. Bungarotoxin labeling of α7-nAChR in HEK293 cells. HEK293 cells transfected with human α7-nAChR (A, B), co-transfected with human α7-nAChR and RIC-3 (C, D), or co-transfected with human α7-nAChR and RIC-3e (E, F). Scale bar: 5 µm.

Fig. 5 .

 5 Fig.5. Determination of functional α7-nAChR activity using a FLIPR system. HEK 293 cells were transfected with α7-nAChR, RIC-3, RIC-3e or combinations of α7-nAChR and RIC-3 variants as indicated under the corresponding bars (NT, non transfected). Applied compounds are as indicated. MLA was added in the wash buffer and cells were pretreated for 10 min prior to addition of agonist and modulator. All compounds were used at the concentration of 10 µM. Data obtained from one representative experiment (four replicates for each treatment) are presented as mean ± SEM.

Figure 1 Bioscience

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Figure 2 Bioscience

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Figure 3 Bioscience

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Figure 4 Bioscience

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  Figure 5

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

© 2008 The Authors Journal compilation © 2008 Biochemical Society


Alessandro Magini

Simona Mencarelli

Brunella Tancini

Virginia V Ciccarone

Lorena Urbanelli

Andrej Hasilik

Carla Emiliani
email: emiliani@unipg.it

Identification and characterization of mature β-hexosaminidases associated to human placenta lysosomal membrane

Keywords: β-hexosaminidase, lysosomal membrane, membrane associated glycohydrolases CPK: creatine phosphokinase, GAPDH: glyceraldehyde 3phosphate dehydrogenase, β-Gluc: β-glucosidase, Hex: β-hexosaminidase, LAMP-2: lysosomal associated membrane protein 2, LM: lysosomal membrane, α-Man: αmannosidase

INTRODUCTION

In the past glycohydrolases were believed to be mainly concentrated inside endosomal/lysosomal compartments where take part to the degradation of macromolecules into monomeric subunits [START_REF] Gravel | The Metabolic Basis of Inherited Disease[END_REF][START_REF] De Duve | Lysosomes revisited[END_REF]. Nevertheless, new studies show the presence of glycohydrolases at the plasma membrane (PM) and point to their potential role in modifying glycosphingolipids during signaling and cellular communication [START_REF] Hakomori | Tumor malignancy defined by aberrant glycosylation and sphingo(glyco)lipid metabolism[END_REF][START_REF] Hakomori | Structure, organization, and function of glycosphingolipids in membrane[END_REF]. For instance, evidence for PM sialidase Neu3 activity towards gangliosides exposed to the surface of adjacent cells has been recently demonstrated by co-culture experiments [START_REF] Papini | The plasma membrane-associated sialidase MmNEU3 modifies the ganglioside pattern of adjacent cells supporting its involvement in cell-to-cell interactions[END_REF][START_REF] Valaperta | Plasma membrane production of ceramide from ganglioside GM3 in human fibroblasts[END_REF]. Moreover, the presence of the lysosomal sialidase Neu1 anchored to both lysosomal and plasma membranes has been established and an important immunoregulatory role for this enzyme has been suggested [START_REF] Lukong | Intracellular distribution of lysosomal sialidase is controlled by the internalization signal in its cytoplasmic tail[END_REF][START_REF] Wang | Induction of lysosomal and plasma membrane-bound sialidases in human T-cells via T-cell receptor[END_REF]. In particular the translocation of sialidase Neu1 from lysosomes to PM has been observed during T-cell activation, where its ability to desialylate GM3 is required for IL-4 production in activated T-cells [START_REF] Chen | The control of IL-4 gene expression in activated murine T lymphocytes: a novel role for neu-1 sialidase[END_REF][START_REF] Chen | Ganglioside control over IL-4 priming and cytokine production in activated T cells[END_REF][START_REF] Liang | Monocyte differentiation up-regulates the expression of the lysosomal sialidase, Neu1, and triggers its targeting to the plasma membrane via major histocompatibility complex class II-positive compartments[END_REF].

β-Hexosaminidase (Hex, EC 3.2.1.52) is an acidic glycohydrolase that catalyses the cleavage of terminal β-N-acetylglucosamine or β-N-acetylgalactosamine from a broad range of glycoconjugated substrates into lysosomes [START_REF] Lemieux | Crystallographic structure of human β-hexosaminidase A: interpretation of Tay-Sachs mutations and loss of GM2 ganglioside hydrolysis[END_REF][START_REF] Mark | Crystal structure of human β-hexosaminidase B: understanding the molecular basis of Sandhoff and Tay-sachs disease[END_REF]. However, the presence of Hex has also been described in cytosol [START_REF] Gao | Dynamic Oglycosylation of nuclear and cytosolic proteins: cloning and characterization of Bioscience Reports Immediate Publication[END_REF] and associated to PM of different cell populations, such as ascidia and Drosophila melanogaster sperm [START_REF] Lambert | Ascidian eggs release glycosidase activity which aids in the block against polyspermy[END_REF][START_REF] Pasini | Plasma membrane association and preliminary characterization of Drosophila sperm surface glycosidases[END_REF][START_REF] Cattaneo | Purification and characterization of the plasma membrane glycosidases of Drosophila melanogaster spermatozoa[END_REF], human erythrocytes [START_REF] Massaccesi | Isoenzyme pattern and partial characterization of hexosaminidases in the membrane and cytosol of human erythrocytes[END_REF], laeukaemic cells [START_REF] Martino | β-Nacetylhexosaminidase A and S have similar sub-cellular distributions in HL-60 cells[END_REF], lymphocytes and monocytes purified from peripheral blood of multiple sclerosis patients [START_REF] Orlacchio | Beta-Nacetylhexosaminidase in peripheral blood lymphocytes and monocytes in the different forms and stages of multiple sclerosis[END_REF]. In normal human tissues there are two major Hex isoenzymes: the heterodimer Hex A (pI= 4,8) and the homodimer Hex B (pI= 6,9) [START_REF] Mahuran | Biochemical consequences of mutations causing the GM2 gangliosidoses[END_REF]. Minor forms of Hex, characterized by intermediate pI, have also been described in both normal and pathological cells [START_REF] Mahuran | Characterization of human placental β-Hexosaminidase I 2[END_REF]. Hex isoenzymes originate from the association of two different subunits, α and β, which are encoded by two evolutionarily related genes mapped on chromosome 15 and 5, respectively [START_REF] Proia | Organization of the gene encoding the human beta-hexosaminidase alpha-chain[END_REF][START_REF] Proia | Gene encoding the human beta-hexosaminidase beta chain: extensive homology of intron placement in the alpha-and beta-chain genes[END_REF]. Subunit dimerisation occurs into the endoplasmic reticulum, where neo-synthesized precursors form of Hex A (αβ)    and Hex B (ββ  ) undergo to extensive post-translational modifications such as glycosylation and culminate in the final proteolytic processing into lysosomes. The pro-α  subunit is cleaved into α m and α p polypeptides while the mature β-subunit is composed of three polypeptides, β a , β b and β p [START_REF] Mahuran | Biochemical consequences of mutations causing the GM2 gangliosidoses[END_REF][START_REF] Hasilik | Biosynthesis of lysosomal enzymes in fibroblasts. Synthesis as precursors of higher molecular weight[END_REF].

In a recent study we demonstrated the existence of a mature form of Hex A associated with the PM of human fibroblasts and proved its ability to hydrolyse the natural substrate GM2 [START_REF] Mencarelli | Identification of plasma membrane associated β-Hexosaminidase A, active towards GM2 ganglioside, in human fibroblasts[END_REF]. This observation suggests that PM-associated Hex should have a lysosomal origin, in fact only into lysosomes this enzyme undergoes to the final maturative process [START_REF] Mahuran | Biochemical consequences of mutations causing the GM2 gangliosidoses[END_REF]. It is evident that the presence of Hex, as that of other acidic (lysosomal origin) glycohydrolases open new perspectives on the role of these enzymes.

In this study we demonstrate an association of mature Hex with the lysosomal membrane (LM). To this end, highly purified lysosomes were further processed in order to remove the soluble content and to recover purified lysosomal membraneenriched fraction. Moreover the nature of the association of the Hex to lysosomal membrane was further elucidated. Our results show that a fraction of fully processed

Lysosomal membrane-enriched fraction preparation

Purified human placenta lysosomes (PL), 2 ml, obtained from fresh human placenta by a Percoll gradient as described [START_REF] Diettrich | Purification of lysosomal membrane proteins from human placenta[END_REF], were thawed and mixed with 40 µl of protease inhibitor cocktail. The mixture was sonicated (3 times for 15 s each) and diluted up to 10 ml with 10 mM sodium phosphate buffer (Na/P), pH 6.0. This lysosomal homogenate was subjected to ultracentrifugation at 4 °C for 2 h at 183000 g in a Beckman Optima Max MLS-50 rotor, and the supernatant (S1) and the pellet that contained the enriched lysosomal membranes (P1) were separated. P1 was resuspended in 0.5 ml of 10 mM Na/P pH 6.0 in presence of protease inhibitors, sonicated as above and diluted up to 10 ml with the same buffer. Subsequently it was centrifuged at 4 °C for 2 h at 129000 g using the same rotor as above. This yielded S2 supernatant and P2 pellet fractions. The latter was extracted once more and supernatant S3 and pellet P3 fractions were obtained.

Determination of enzyme activity and protein concentration

Total Hex and Hex A activity was measured using 3 mM MUG and 3 mM MUGS, respectively, in 0.1 M citric acid/0.2 M disodium phosphate buffer, pH 4.5 [START_REF] Emiliani | An enzyme with properties similar to those of beta Nacetylhexosaminidase S is expressed in the promyelocytic cell line HL-60[END_REF].

Optimum pH of Hex in lysosomal membrane-enriched fraction and Hex isoenzymes, recovered after DEAE-chromatography, was determined using the substrate MUG in 0.1 M citric acid/0.2 M disodium phosphate buffers at different pH values ranging from pH 3.5 to 7.5.

Thermal stability of Hex isoenzymes, recovered after DEAE-chromatography, was determined by incubating samples at 52 °C for 1 h. Samples were cooled on ice for 1 h and then assayed for Hex activity by using the MUG substrate. The activities of acid α-mannosidase (α-Man, EC 3.2.1.24) and β-glucosidase (β-Gluc, EC 3.2.1.45) were determined using 3 mM solutions of corresponding 4methylumbelliferyl-glycoside substrates as previously described [START_REF] Emiliani | Partial sequence of the purified protein confirms the identity of cDNA coding for human β-mannosidase B[END_REF][START_REF] Leonova | Fate and sorting of acid β-glucosidase in transgenic mammalian cells[END_REF].

Fluorescence of the liberated 4-methylumbelliferone was measured using a Perkin Elmer LS B50 fluorimeter (excitation, 360 nm; emission, 446 nm). One enzymatic unit (U) is the amount of enzyme that hydrolyses 1 µmole of substrate/min at 37 °C.

Protein concentration was determined by the method of Bradford [31] using bovine serum albumin as standard.

Specific activity was expressed as mU/mg of protein.

DEAE-chromatography

Soluble lysosomal content S1 and lysosomal membrane-enriched fraction P3 were resuspended with 0.1% (v/v) NP-40 in 10 mM Na/P pH 6.0, sonicated and centrifuged at 14000 g for 15 min. The recovered supernatants were analyzed by ion-exchange chromatography on DE-52 DEAE-cellulose as previously described [START_REF] Emiliani | An enzyme with properties similar to those of beta Nacetylhexosaminidase S is expressed in the promyelocytic cell line HL-60[END_REF]. Briefly, a 1 ml column was equilibrated with 10 mM Na/P buffer, pH 6.0; protein retained by the column was eluted using a linear 0.0-0.5 M gradient of NaCl in 50 ml of the above buffer. Finally, the proteins were eluted with 1.0 M NaCl in the same buffer. The flow rate was 1 ml/min. Fractions (1 ml each) were collected and assayed for the Hex activity with the MUG and MUGS substrates.

SDS-PAGE and Western blotting analysis

PL and fractions resulting from lysosomal membrane enrichment procedure (P1 and S1, P2 and S2, P3 and S3) were subjected to 12% SDS-PAGE under reducing conditions according to Laemmli [START_REF] Laemmli | Cleavage of structural protein during the assembly of the head of bacteriophage T4[END_REF]. Proteins were transferred to nitrocellulose, and the α and β peptides of  Hex detected using respectively an antiserum specific for αsubunit, raised to a mixture of synthetic peptides belonging to the sequence of mature human enzyme, and a goat anti-β-subunit raised to Hex B purified from human placenta [START_REF] Hasilik | Biosynthesis of lysosomal enzymes in fibroblasts. Synthesis as precursors of higher molecular weight[END_REF]. The filter was also developed with a monoclonal antibody raised to LAMP-2. Blots were analyzed by ECL detection system and densitometric analysis performed using the ImageMaster2D software.

Bioinformatics analysis

Analysis of amino acid sequences of Hex α-subunit (primary accession number P06865) and β-subunit (primary accession number P07686 ) was performed using free internet software. Prediction of transmembrane domains was performed using TMpred-Prediction of Transmembrane Regions and Orientation software (http://www.ch.embnet.org/software/TMPRED_form.html).

Prediction of glycosylphosphatidylinositol (GPI)-anchor motifs was performed using big-PI Predictor GPI Modification Site Prediction software (http://mendel.imp.ac.at/gpi/gpi_server.html). For the prediction of fatty acyl-or prenyl-anchor motifs the following software were using: Myristoylator predicts N- (http://www.expasy.ch/tools/myristoylator/PrePS), and PrePS Prenylation Prediction Suite (http://mendel.imp.ac.at/sat/PrePS/index.html).

NaCl and carbonate extractions

Lysosomal membrane-enriched fraction P3 was resuspended in 0.1 M Na 2 CO 3 pH 11.5 [START_REF] Fujiki | Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum[END_REF] or in 10 mM Na/P containing 1M NaCl pH 6.0. As control, fraction P3 was resuspended in 10 mM Na/P pH 6.0. After 30 min incubation on ice, samples were ultracentrifuge at 4 °C for 2 h at 129000 g. Both supernatant and pellet were adjusted to the same final volume and analyzed by immunoblotting.

Triton X-114 phase separation

Triton X-114 phase separation was performed as described by Bordier [START_REF] Bordier | Phase separation of Integral Membrane Proteins in Triton X-114 Solution[END_REF]. Briefly, fraction P3 was resuspended in 0.2 ml of 10 mM Tris-HCl pH 7.4 containing 150 mM NaCl and 0.6% (v/v) of the detergent Triton X-114 and incubated at 0 °C for 1 h. Detergent-treated sample was then layered onto 6% (w/v) sucrose cushion (0.3 ml), incubated at 30 °C for 3 min and finally centrifuge d at 300 g for 3 min. After centrifugation, the upper aqueous phase was recovered, re-extracted with 0.5% (v/v) Triton X-114 and subjected to a second separation through the same sucrose cushion. The detergent and aqueous phases were adjusted to the same final volume and analyzed by immunoblotting.

2-D Electrophoresis and Western blotting analysis of lysosomal membrane associated Hex

Soluble lysosomal content S1 and lysosomal membrane-enriched fraction P3 were resuspended in 40 mM Tris base, containing 2% (w/v) SDS and 60 mM DTT.

Samples were heated at 95 °C for 5 min and insolubl e debris were removed by centrifugation. 2-D Clean-Up Kit was used for preparing concentrated protein samples, that were then resuspended in rehydration solution (7 M urea, 2 M thiourea, 2% (w/v) ASB-14, 0.5% (v/v) IPG-buffer pH 3-10, 40 mM DTT) with the addition of carbamylated protein standards for 2-DE. Precast linear IPG-strips 11 cm, pH 3-10 were swollen for 14 h 30 V at 20 °C. Isoelectric fo cusing was conducted at 20 °C, with a current limit of 50 µA/IPG-strip in a IPGphor IEF System from Amersham, according to the following steps: 1 h 200 V, 1 h 500 V, 1 h 1000 V, 0.5 h 4000 V (gradient), 6 h 4000 V. After IEF the IPG-strips were transferred to equilibration buffer (6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, 50 mM Tris-HCl pH 8.8) in presence of 1% (w/v) DTT for 15 min, then incubated in equilibration buffer with 4% (w/v) iodoacetamide for further 15 min. The second dimension was performed on ExcelGel 2-D Homogeneous 12.5 (Amersham Bioscience). The resolved proteins were transferred on nitrocellulose membrane using Multiphor II NovaBlot Unit (Amersham Bioscience). After blotting proteins were visualised by Ponceau red staining. The filter was then incubated with an anti-serum raised to Hex B [START_REF] Hasilik | Biosynthesis of lysosomal enzymes in fibroblasts. Synthesis as precursors of higher molecular weight[END_REF] and developed by ECL.

RESULTS

Hex activity and isoenzymes in lysosomal membrane-enriched fractions

Fractions enriched in lysosomal membranes were obtained from PL through three successive steps of sonication and centrifugation as described in the experimental section. For each of the three steps of the experimental procedure we recovered a pellet further enriched in lysosomal membrane (P1, P2, P3) and supernatant containing the soluble lysosomal content (S1, S2, S3). All P and S fractions were assayed for the Hex activity using two substrates, MUG and MUGS, the former being hydrolysed by both αand β-subunits and is the latter specifically by the α-subunit.

We also assayed the activity of another soluble lysosomal glycohydrolase, α-Man [START_REF] Emiliani | Partial sequence of the purified protein confirms the identity of cDNA coding for human β-mannosidase B[END_REF], and of the lysosomal membrane marker β-Gluc, [START_REF] Emiliani | An enzyme with properties similar to those of beta Nacetylhexosaminidase S is expressed in the promyelocytic cell line HL-60[END_REF][START_REF] Leonova | Fate and sorting of acid β-glucosidase in transgenic mammalian cells[END_REF]. All fractions displayed Hex activity toward both MUG and MUGS subtrates, with an acidic optimum pH 4.5.

Results reported in Figure 1 show that the increasing purification level of lysosomal membrane achieved by three steps of sonication and ultracentrifugation give rise to a gradual increase of the specific activity of β-Gluc. On the contrary, the specific activity of α-Man decreased progressively. In P3 it was only barely detectable. The specific activity of Hex, assayed toward the MUG substrate, engaged a peculiar behaviour: we observed a decrease from PL to P2 that can be attributed, as for α-Man, to the removal of soluble Hex, and then, in P3 fraction we detected a reproducible enrichment of total Hex specific activity with respect to P2. A further step of sonication and ultracentrifugation of P3 fraction did not increase the specific activity of Hex (data not shown). A similar trend was detected also when Hex activity was assayed with the MUGS substrate, but the increase in specific activity in P3 fraction with respect to P2 was less pronounced than for MUG activity (Figure 1). The Hex isoenzyme pattern in lysosomal membrane preparation P3 and in soluble lysosomal content (S1) was investigated by DEAE-cellulose chromatography. The subunit composition in peaks of activity that were eluted from the column was calculated from the relative rates of hydrolysis of the MUG and MUGS substrates. With both fractions similar elution profiles were obtained. Hex B (ββ dimer, as demonstrated by its inability to hydrolyse MUGS substrate) was not retained by the column and Hex A, αβ dimer, was eluted with a maximum activity at approximately 0.12 mM NaCl. Nevertheless, the elution profiles disclosed different proportions of Hex B and Hex A between membrane associated and soluble form (Table 1). The same amounts of Hex activity (detected using MUG substrate) were loaded on the columns and the loss of the activity was attributed to an inactivation of α-subunit during the purification procedure. To better characterize the Hex isoenzymes we determined the pH optimum and thermal stability of Hex B and Hex A eluted by ion-exchange chromatography of S1 and P3 fractions. Concerning the thermal stability and, thus, the relative isoenzyme contents, there were no differences between the soluble and the membrane-associated Hex forms (Table 1).

SDS-PAGE and Western blotting analysis of Hex in lysosomal membraneenriched fractions

The pellets containing the enriched lysosomal membranes (P1, P2 and P3) and the supernatants containing the lysosomal matrix proteins (S1, S2 and S3) were In order to verify the effectiveness of our lysosomal membrane enrichment procedure we also monitored LAMP-2, a typical lysosomal integral membrane protein [START_REF] Eskelinen | At the acicic edge: emerging function for lysosomal membrane proteins[END_REF] using the specific antibody (Figure 2, panel C). The results highlighted a gradual increase in the intensity of the LAMP-2 band up to 24-fold in P3 as compared to PL. In a separate experiment we repeated the sonication and ultracentrifugation steps for the fourth time but this did not produce any further enrichment of LAMP-2 in the final pellet (data not shown).

Peripheral membrane protein extraction and Triton X-114 phase separation

In order to understand the nature of the anchoring of the predominantly soluble matrix enzyme to the lysosomal membrane, we first analysed the amino-acid sequence of Hex subunits. Bioinformatic analysis (see materials and methods) of the Hex αand β-subunit amino acid sequences did not reveal the presence of any motif compatible with the addition of GPI, fatty acyl or prenyl lipid bilayer anchors. In fact, no consensus sequences for these post-translational modifications have been found. Instead TMpred-Prediction of Transmembrane Regions and Orientation software highlighted the presence of two putative membrane-spanning domains (Met 13 -Thr 31 and Val 436 -Gln 458 ) in the Hex β-subunit and one hydrophobic amino acid stretch (Leu 6 -Trp 24 ) in the Hex α-subunit, but it has been demonstrated that the amino acid sequences between Met 13 -Thr 31 and Leu 6 -Trp 24 in αand β-subunits, respectively, are removed in endoplasmic reticulum by signal peptidase during N-terminal signal peptide cleavage [START_REF] Mark | Crystal structure of human β-hexosaminidase B: understanding the molecular basis of Sandhoff and Tay-sachs disease[END_REF]. Crystallographic analysis of the Hex B structure showed that the Val436-Gln458 stretch comprises amino acid residues involved in subunit dimerization, a crucial event in acquiring the enzymatic activity [START_REF] Mark | Crystal structure of human β-hexosaminidase B: understanding the molecular basis of Sandhoff and Tay-sachs disease[END_REF].

Based on these considerations we examined the possibility that Hex may be associated with the lipid bilayer as a peripheral membrane protein. Using the procedure of Fujiki [START_REF] Fujiki | Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum[END_REF], aliquots of the lysosomal membrane-enriched fraction P3 were incubated with 0.1 M Na 2 CO 3 pH 11.5, with 1 M NaCl in 10 mM Na/P pH 6.0, and with 10 mM Na/P pH 6.0, for 30 min. After the incubation, the mixtures were subjected to ultracentrifugation and the supernatant (S) and pellet (P) fractions obtained were analyzed by immunoblotting. Hex αand β-subunits and LAMP-2 polypeptides were as before. The results (Figure 3) demonstrated an almost complete solubilisation of both Hex αand β-subunits in the presence of sodium carbonate. Both αand β-subunit bands appeared in the S fraction whereas LAMP-2 was completely recovered in the insoluble P fraction. In contrast, the treatments of the P3 fraction with the buffered 1 M NaCl and the buffer alone caused no appreciable solubilisation of the Hex αand β-subunits or of LAMP-2.

To obtain more information about the nature of the interaction between Hex enzyme and lipid bilayer we also performed Triton X-114 extraction of P3 fraction followed by aqueous/detergent phase separation. The detergent (Det) and aqueous (Aq) phases obtained after centrifugation were analyzed by immunoblotting. As shown in Figure 4, Hex αand β-subunits partitioned in the aqueous phase, whereas LAMP-2 was present exclusively in the detergent phase as expected.

Comparative analysis of soluble and membrane-associated Hex by 2-D electrophoresis

To investigate possible differences between soluble and membrane-associated Hex, we performed 2-D electrophoresis and Western blotting analysis of both S1 and P3 fractions. Creatine phosphokinase (CPK) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) carbamylated standards provided a series of spots across a pI range of 4.9-7.1 and 4.7-8.3 respectively, which allowed the identification of pI of the protein by ImageMaster2D analysis. Spots identified on the blot by the specific antibody raised to Hex B, are pointed out in Figure 5. The results obtained after Western blotting analysis of the lysosomal matrix protein (Figure 5A) demonstrated the presence of two spots of same apparent molecular weight with pIs of 6.00 and 6.66, corresponding to the mature Hex β a and β b -subunits, respectively [START_REF] Mahuran | Biochemical consequences of mutations causing the GM2 gangliosidoses[END_REF].

These polypeptides were generated by the final proteolytic processing of Hex B precursor that takes place in lysosomes [START_REF] Ahn | An alternate targeting pathway for procathepsin L in mouse fibroblasts[END_REF]. The 2-D pattern of P3 (Fig. 5B) showed two spots of slightly different molecular weight and characterized by pIs of 5.80 and 6.63. Analogous experiments performed using specific antibody raised to Hex A did not reveal any differences between soluble and membrane associated Hex α-subunit (data not shown).

DISCUSSION

Glycosphingolipids are ubiquitous membrane components of the eukaryotic cells that are abundant in the plasma membrane where they function as modulators of signal transduction regulating the proliferation, survival, and differentiation of the cells [START_REF] Hakamori | Bifunctional role of glycosphingolipids. Modulators for transmembrane signaling and mediators for cellular interactions[END_REF]. These lipids play key role as signaling molecules in both physiological and pathological processes regulating cell-to-cell and/or cell-environment interactions [START_REF] Hakomori | Tumor malignancy defined by aberrant glycosylation and sphingo(glyco)lipid metabolism[END_REF][START_REF] Hakamori | Glycosilation defining cancer malignancy: new wine in an old bottle[END_REF]. Several recent studies revealed the presence of glycohydrolases not only in lysosomes but also at cell surface [START_REF] Papini | The plasma membrane-associated sialidase MmNEU3 modifies the ganglioside pattern of adjacent cells supporting its involvement in cell-to-cell interactions[END_REF][START_REF] Massaccesi | Isoenzyme pattern and partial characterization of hexosaminidases in the membrane and cytosol of human erythrocytes[END_REF][START_REF] Cordero | Cell surface human alpha-L-fucosidase[END_REF][START_REF] Goi | Membrane anchoring and surface distribution of glycohydrolases of human erythrocyte membranes[END_REF]. They stress the potential role of a remodelling of glycosphingolipids during cellular processes [START_REF] Valaperta | Plasma membrane production of ceramide from ganglioside GM3 in human fibroblasts[END_REF][START_REF] Lukong | Intracellular distribution of lysosomal sialidase is controlled by the internalization signal in its cytoplasmic tail[END_REF]. In a recent study we demonstrated that a mature form of hexosaminidase (Hex A isoenzyme) is associated to the human fibroblast PM and is catalytically active toward its natural substrate GM2 [START_REF] Mencarelli | Identification of plasma membrane associated β-Hexosaminidase A, active towards GM2 ganglioside, in human fibroblasts[END_REF].

Here we demonstrate that lysosomal membranes that were purified from human placenta lysosomes [START_REF] Diettrich | Purification of lysosomal membrane proteins from human placenta[END_REF] by repeated cycles of sonication and sedimentation in the ultracentrifuge also contain mature Hex isoenzymes in non covalently attached forms.

It is conceivable that the procedure used here for the purification of the lysosomal membrane may result in a contamination of the final preparation with lipofuscin-like lysosomal inclusions which tend to sediment with the membranes. However, Hex enzymes are not present in the lipofuscin-like lysosomal inclusion body [START_REF] Schröder | Characterisation of lipofuscin-like lysosomal inclusion bodies from human placenta[END_REF], therefore this kind of contamination is rather unlikely.

We assayed Hex activity with MUG and MUGS substrates and examined the presence of the two polypeptides, α and β, that constitute the Hex isoenzymes using specific antibodies in all fractions obtained during the purification. The results revealed that in the first two steps a major portion of Hex behaved as a soluble lysosomal enzyme similar to α-Man, which was used as control. In the third step Hex, unlike α-Man, behaved as a lysosomal membrane-associated protein and displayed an enrichment comparable to that of  β-Gluc. The latter is known to bind to the lysosomal integral membrane protein LIMP-2 [START_REF] Reczek | LIMP-2 is a receptor for lysosomal mannose-6-phosphate-independent targeting of betaglucocerebrosidase[END_REF]. The conclusion is that a significant amount of Hex is associated with the lysosomal membrane. A further step of sonication and centrifugation was not useful to detach this fraction of the enzyme from the membrane since it resulted in a selective loss of the enzymatic activity of the α-subunit. This was demonstrated by a rapid decrease in MUGS activity, but not in the amount of α-polypeptide that was detected by Western blotting.

Lysosomal membrane-associated Hex activity displayed an acidic optimum pH and thermal stability, as the soluble counterparts. Western blotting analysis clearly demonstrated that both αand β-subunits associated to lysosomal membrane fraction in fully processed forms. In an attempt to elucidate the isoenzyme composition of lysosomal membrane-associated Hex we run a DEAE cellulose chromatography of P3 fraction. The subunit composition of peaks eluted from the column was determined by assay of the eluates with MUG and MUGS substrates. This analysis clearly revealed the presence of both Hex A and Hex B isoenzymes, eluting at the same position as the soluble lysosomal counterpart. The relative amount of Hex A and B is different between the soluble and the lysosomal membrane-enriched fraction Bioinformatic analysis of the Hex αand β-subunit amino acid sequences excluded the presence of sequence compatible with GPI-anchor protein, fatty acylated or prenylated proteins because no post-translational modification consensus sequences have been found. In order to verify if Hex behave as a peripheral membrane protein associated with the lipid bilayer by ionic and/or hydrogen interaction involving transmembrane proteins or phospholipids resident in LM, lysosomal membraneenriched fraction P3 was treated with 1 M NaCl or with sodium carbonate at the extreme pH value of 11.5 [START_REF] Fujiki | Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum[END_REF]. In the first condition Hex remained associated to LM as well as the lysosomal transmembrane protein LAMP-2, while the extreme pH conditions produced the complete solubilisation of both Hex subunits but not of the integral lysosomal membrane LAMP-2 (Figure 4). Triton X-114 phase separation of lysosomal membrane-enriched fraction P3 allowed the completed segregation of Hex αand β-subunits in aqueous phase (Figure 5), thus demonstrating the hydrophilic nature of Hex subunits [START_REF] Bordier | Phase separation of Integral Membrane Proteins in Triton X-114 Solution[END_REF]. In contrast, as expected LAMP-2 protein partitioned completely into the detergent phase. All together these findings support the hypothesis that a portion of the lysosomal Hex behaves as a peripheral membraneassociated protein.

Comparative analysis by 2-D electrophoresis and Western blotting revealed that mature β a and β b polypeptides that were recovered in the lysosomal membraneassociated form displayed a slightly different apparent molecular weight and pI as compared to their soluble lysosomal counterparts (Figure 3B). In contrast, no differences were observed between the soluble and the membrane-associated forms of the α-subunit (data not shown). We speculate that the observed differences between the apparent molecular weights and isoelectric points of the matrix and the membrane-associated forms of Hex β-polypeptides could be due to an as yet unknown post-translational modification. This may result in binding to a component of the membrane.

This study demonstrated the existence of membrane-associated forms of both Hex A and Hex B isoenzymes in lysosomal membranes from human placenta. The lysosomal membrane-associated forms displayed the same catalytic properties as the soluble forms and also the enzyme that has been found at the plasma membrane in an earlier study [START_REF] Lambert | Ascidian eggs release glycosidase activity which aids in the block against polyspermy[END_REF]. Therefore, we consider the existence of two pools of mature Hex: a membrane-associated and a soluble. The membrane-associated Hex is present in both lysosomal and plasma membranes. It should be of interest to examine the pathway delivering the membrane-associated lysosomal Hex to the cell surface and the possible functions of the cell surface-associated enzyme in modulating the glycolipid pattern at the cell boundary.

LEGEND TO THE FIGURES Figure 1 Specific activity of lysosomal glycohydrolases during enrichment procedure

The histograms represent specific activities of total Hex, Hex A, α-Man and β-Gluc as detected in PL and in pellets P1, P2 and P3 that were obtained at three stages of the purification of lysosomal membranes from PL. The results represent means of three independent experiments ± S.D.

Figure 2 Western blots and densitometric analysis of the fractions obtained from lysosomal membrane-enriched procedure

The same amounts of protein (10 µg) of PL and of three stages of lysosomal membrane purification (pellets P1, P2 and P3) and constant aliquots (30 µl) of supernatants S1, S2 and S3 from this purification were separated using SDS-PAGE. After blotting, polypeptides of Hex were detected with antisera against Hex B (A), Hex α-subunit (B). For comparison, LAMP-2 was detected with the appropriate antibody (C). The detection was performed by ECL (the stripes on the left hand side) and densitometry using the ImageMaster2D software (the diagrams on the right hand side). Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Bioscience

  Reports Immediate Publication. Published on 30 Jun 2008 as manuscript BSR20080075 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BSR20080075 S t a g e 2 ( a ) P O S T -P R I N T forms of Hex A and Hex B isoenzymes are non-covalently attached to the inner aspect of the lysosomal membrane. Bioscience Reports Immediate Publication. Published on 30 Jun 2008 as manuscript BSR20080075 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BSR20080075 S t a g e 2 ( a ) P O S T -P R I N T EXPERIMENTAL Materials 4-Methylumbelliferyl-β-N-acetylglucosaminide-6-sulphate (MUGS) was from Toronto Research Chemicals Inc. 4-Methylumbelliferyl-β-N-acetylglucosaminide (MUG), 4methylumbelliferyl-α-mannopyranoside, 4-methylumbelliferyl-β-glucoside, 4methylumbelliferone, Protease Inhibitor Cocktail for Mammalian Cell Extracts, the detergents Triton X-114, NP-40, ASB-14, monoclonal anti-rabbit IgG HRP conjugate, polyclonal goat anti-mouse IgG HRP conjugate and monoclonal anti-goat/sheep IgG HRP conjugate, were from Sigma-Aldrich Fine Chemicals Co. Monoclonal mouse anti-LAMP-2 was from Santa Cruz Biotechnology, Inc. Diethylaminoethyl cellulose (DEAE) was from Whatman Biochemicals, Maidstone, Kent, UK. ECL, IPG strips, ExcelGel 2-D Homogeneous 12.5, 2-D Clean-Up Kit, Carbamylate Calibration Kit for 2-D were from Amersham Bioscencies, Aylesbury, Buckinghamshire, UK. Trans-Blot Nitrocellulose Membrane, bovine serum albumin and Bio-Rad protein assay reagent were from Bio-Rad Laboratories, Segrate-Milano. Centricon YM-10 were from Amicon, Millipore. All other reagents were of analytical grade.

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  Reports Immediate Publication. Published on 30 Jun 2008 as manuscript BSR20080075 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BSR20080075 S t a g e 2 ( a ) P O S T -P R I N T

  Publication. Published on 30 Jun 2008 as manuscript BSR20080075 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BSR20080075 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.

  Bioscience Reports Immediate Publication. Published on 30 Jun 2008 as manuscript BSR20080075 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BSR20080075 S t a g e 2 ( a ) P O S T -P R I N T

Bioscience

  Reports Immediate Publication. Published on 30 Jun 2008 as manuscript BSR20080075 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BSR20080075 S t a g e 2 ( a ) P O S T -P R I N T analysed by Western blotting and immunodetection of Hex using a specific antiserum raised to placental Hex B and able to detect specifically the β-subunit (Figure 2, panel A). A constant amount of protein was loaded in each well. As shown in the left hand column in this Figure, in PL, P1, P2, P3 and S1 fractions, different amounts of mature Hex β-subunit as indicated by a single band of approx. 28 kDa [21,25] were detected. Densitometric analysis (shown in the right hand column in the same panel) indicated a trend in Hex β-subunit polypeptide abundance in the above fractions that was observed for the specific activity, with the MUG substrate. A strong enrichment was observed in fraction P3. Similar results were obtained when Hex α-subunit was immunodetected (Figure 2, panel B): mature α-subunit (54 kDa) was found in all samples except for S3. The clear enrichment of this subunit in P3 as compared to P2 highlighted the presence of both mature Hex αand β-subunits in the membraneassociated enzyme and suggested that the low enrichment of MUGS activity in P3 as compared to P2 may have been caused by an inactivation of Hex A during the fractionation.

Bioscience

  Reports Immediate Publication. Published on 30 Jun 2008 as manuscript BSR20080075 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BSR20080075 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.

Bioscience

  Reports Immediate Publication. Published on 30 Jun 2008 as manuscript BSR20080075 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BSR20080075 S t a g e 2 ( a ) P O S T -P R I N T

Bioscience

  Reports Immediate Publication. Published on 30 Jun 2008 as manuscript BSR20080075 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BSR20080075 S t a g e 2 ( a ) P O S T -P R I N T P3 being Hex B the predominant form, but this last can be attributed to the instability of α-subunit activity.

Figure 3

 3 Figure 3 Extraction of Hex polypeptides from purified lysosomal membranes in the presence of NaCl and carbonate solutionsAliquots of fraction P3 (10 µg protein) were incubated in the presence of 10 mM Na/P pH 6.0 (Control), 10 mM Na/P, 1 M NaCl pH 6.0 (NaCl) or 0.1 M sodium carbonate pH 11.5 (Na 2 CO 3 ) and separated into soluble (S) and membrane subfractions (P) using ultracentrifugation. Hex polypeptides were analysed in an untreated P3 aliquot (T) and the subfractions using SDS-PAGE and Western blotting and anti-Hex B, anti-Hex α-subunit antibodies. For comparison, LAMP-2 was detected with the appropriate antibody.

Figure 4

 4 Figure 4 Partitioning of Hex isoenzymes and LAMP-2 using Triton X-114 phase separationFraction P3 (10 µg protein) were subjected to a Triton X-114 phase separation. After ultracentrifugation the aqueous (Aq) and detergent (Det) phases, and a non-treated aliquot of P3 (T) were analyzed by immunoblotting using anti-Hex B, anti-Hex αsubunit and anti-LAMP-2 antibodies.

Figure 5 Figure 1 Bioscience

 51 Figure 5 Characterisation of Hex polypeptides in the soluble lysosomal content S1 and purified lysosomal membranes P3 using 2-D electrophoresis and Western blottingAliquots of S1 (A) and P3 fraction (B) containing the same amounts of Hex (10 mU as determined with the MUG substrate) were loaded on 11 cm IPG-strips pH 3-10 and separated by isoelectric focusing, and 12.5% SDS-PAGE. After blotting, major βsubunit polypeptides of Hex were detected using an anti-Hex B antiserum as the primary antibody. The isoelectric points (pI) of the detected spots were determined by a comparison with those of the CPK and GAPDH carbamylated standards. Image analysis was performed by using ImageMaster 2D.

Figure 2 Bioscience

 2 Figure 2

Figure 3 Bioscience

 3 Figure 3

Figure 4 Bioscience

 4 Figure 4

Figure 5 Bioscience

 5 Figure 5

Table 1 Separation of A and B isoenzymes of Hex in soluble lysosomal content S1 and purified lysosomal membrane fraction P3 using DEAE-DE52 cellulose chromatography a

 1 ) Values represent the % of Hex activity towards MUG substrate recovered as Hex B and Hex A, with respect to the total MUG activity loaded. Same amounts of MUG activity was loaded on the column in each experiment. b) Salt concentration required for Hex A elution within the gradient.

	c) Optimum pH for Hex isoenzyme activity determined dissolving MUG substrate in
	buffers at different pH values ranging from pH 3.5 to 7.5.
	d) Values represent the % of residual MUG activity of Hex B and Hex A, after 1 h
	incubation at 52 °C, respected to non-heated contro ls.
	The results represent the means of three independent experiments ± S.D.

Bioscience Reports Immediate Publication. Published on 30 Jun 2008 as manuscript BSR20080075 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BSR20080075 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

ACKNOWLEDGEMENTS

Work supported by COFIN-PRIN 2004050497_002 and FIRB RBAUO1MSFR grants to C.E. Bioscience Reports Immediate Publication. Published on 30 Jun 2008 as manuscript BSR20080075 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BSR20080075 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.