David A Zeevi

Ayala Frumkin

Gideon Bach
email: bach@hadassah.org.il

TRPML and lysosomal function

Keywords: Lysosomal storage disorder, mucolipin protein family, endocytosis and lysosomal function, mucolipidosis

Mucolipin 1 (MLN1), also known as TRPML1, is a member of the mucolipin family. The mucolipins are the only lysosomal proteins within the TRP superfamily. Mutations in the gene coding for TRPML1 result in a lysosomal storage disorder (LSD). This review summarizes the current knowledge related to this protein and the rest of the mucolipin family.

(MIM#252650), an autosomal recessive, neurodegenerative disease characterized by severe psychomotor retardation and vision impairment due to retinal degeneration and cornea opacity ( Reviewed by [START_REF] Altarescu | The neurogenetics of Mucolipidosis type IV[END_REF][START_REF] Bach | Mucolipidosis type IV. A review[END_REF][START_REF] Bach | Mucolipin 1: Endocytosis and cation channel (A review)[END_REF] ). Achlorhydria and iron deficiency are also part of the clinical symptoms [START_REF] Schiffmann | Constitutive achlorhydria in mucolipidosis type IV[END_REF][START_REF] Lubensky | Lysosomal inclusions in gastric parietal cells in mucolipidosis type IV: A novel cause of achlorhydria and hypergastremia[END_REF]. Unlike most other lysosomal disorders MLIV progresses extremely slowly, thus patient ages range from 1 year to the late thirties and forties and life expectancy is not clear yet. Milder patients, particularly in the psychomotor impairment, were also reported [START_REF] Chitayat | Mucolipidosis type IV: Clinical manifestations and natural history[END_REF][START_REF] Casteels | Mucolipidosis type IV: presentation of a mild variant[END_REF][START_REF] Reiss | Mucolipidosis type IV: A late onset and mild form[END_REF].

MLIV is found in increased frequency among Ashkenazi Jews (AJ) with a heterozygote frequency of 1:100 [START_REF] Bargal | Mucolipidosis type IV: Novel mutations in Jewish and non-Jewish patients and the frequency of the disease in the Ashkenazi Jewish population[END_REF][START_REF] Bach | The frequency of Mucolipidosis type IV in the Ashkenazi Jewish population and the identification of 3 novel MCOLN1 mutations[END_REF]. Over 15 mutations were identified in MLIV patients, Jews and non-Jews, including two MCOLN1 founder mutations in the AJ population comprising 95% of the mutated alleles among AJ MLIV patients [START_REF] Bach | Mucolipin 1: Endocytosis and cation channel (A review)[END_REF]. These founder mutations date approximately 40 generation ago [START_REF] Risch | Geographic distribution of disease mutations in the Ashkenazi Jewish population supports genetic drift over selection[END_REF].

The identification of MCOLN1 mutations opened the door for accurate patient diagnosis as well as prenatal diagnosis and heterozygote identification. Lately MLIV is included in the population screening program operated in various countries with high concentrations of AJ in an attempt to ascertain high risk couples, in which both parents are heterozygotes and offer them options for family planning (prenatal diagnosis, marriage decisions), as a preventative program to reduce the number of newly born MLIV patients in the high risk population [START_REF] Bach | Prevention of lysosomal storage disorders in Israel[END_REF].

Lysosomal storage: MLIV is classified as a mucolipidosis due to the simultaneous lysosomal accumulation of lipids together with water soluble substances [START_REF] Leroy | Mutant enzymatic and cytological phenotypes in cultured human fibroblasts[END_REF]. This characteristic heterogeneous storage is observed in cells of every tissue and organ of MLIV patients and principally best demonstrated by electron microscopy [START_REF] Berman | Congenital Corneal clouding with abnormal systemic storage bodies: A new variant of Mucolipidosis[END_REF][START_REF] Merin | Mucolipidosis IV: Ocular, systemic and ultrastructural findings[END_REF][START_REF] Newell | A new Mucolipidosis with psychomotor retardation, corneal clouding and retinal degeneration[END_REF][START_REF] Tellez-Nagel | Mucolipidosis IV: Clinical, ultrastructural, histochemical and chemical studies of a case, including a brain biopsy[END_REF][START_REF] Goebel | Morphological and chemical biopsy findings in Mucolipidosis IV[END_REF]. The storage

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material in MLIV is autofluorescent [START_REF] Goldin | Cultured skin fibroblasts derived from patients with Mucolipidosis 4 are auto-fluorescent[END_REF]. This typical finding is also present in cultured amniotic fluid cells obtained from MLIV affected fetuses and thus this technique was used for prenatal diagnosis purposes in the era preceding the gene discovery [START_REF] Kohn | Prenatal diagnosis of Mucolipidosis IV by electron microscopy[END_REF][START_REF] Arnon | Cultured amniotic fluid cells for prenatal diagnosis of lysosomal storage disorders: A methodological study[END_REF]. Chemical analysis led to the identification of the stored substances as a variety of lipids namely, gangliosides, phospholipids and neutral lipids while the water soluble materials included primarily mucopolysaccharides and glycoproteins [START_REF] Bach | Mucolipidosis type IV. A review[END_REF][START_REF] Bach | Mucolipin 1: Endocytosis and cation channel (A review)[END_REF]. Histochemical analysis indicated that gangliosides were predominately stored in neural tissues while phospholipids were mostly accumulating in visceral organs [START_REF] Folkerth | Mucolipidosis IV: Morphology and histochemistry of an autopsy case[END_REF]. It should be noted that despite the early onset of the disease in early infancy and the clear evidence of the characteristic lysosomal storage in these early ages, the fact that the disease remains in an apparent steady state or very slow deterioration for at least 2-3 decades may suggest that the storage is not as massive and progressive as is found in most other lysosomal storage disorders.

What leads to the storage in MLIV? Earlier studies indicated that the heterogeneous storage does not stem from the impairment or deficiency of a lysosomal hydrolase that could account for this phenomenon [START_REF] Bargal | Mucolipidosis type IV: Abnormal transport of lipids to the Lysosomes[END_REF]. Studies in cultured fibroblasts with radiolabeled compounds such as phosphatidylcholine or gangliosides, which constitute the bulk of the storage in MLIV, or with fluorescent lipid analogs, indicated that the abnormal accumulation results from a defect in the late stages of the endocytosis process of membrane compounds leading to the impairment of lysosomal biogenesis or, alternatively, a defect in the normal maintenance of the mature lysosomes [START_REF] Bargal | Mucolipidosis type IV: Abnormal transport of lipids to the Lysosomes[END_REF][START_REF] Chen | Abnormal transport along the lysosomal pathway in Mucolipidosis type IV disease[END_REF][START_REF] Jansen | Delayed lysosomal metabolism of lipids in mucolipidosis type IV fibroblasts after LDLreceptor-mediated endocytosis[END_REF][START_REF] Pryor | Mucolipin-1 is a lysosomal membrane protein required for intracellular lactosylceramide traffic[END_REF]. It should be pointed out that another LSD, namely, Niemann Pick disease type C (NPC) (MIM#257220) [START_REF] Patterson | Niemann-Pick type C: a lipid trafficking disorder[END_REF] is also suggested to be involved in defective vesicular trafficking of late endosomes, although with different cellular and biochemical characteristics [START_REF] Kobayashi | Late endosomal membranes rich in lysobisphosphatidic acid regulate cholesterol transport[END_REF][START_REF] Vruchte | Accumulation of glycosphingolipids in Niemann-Pick C disease disrupts endosomal transport[END_REF][START_REF] Vance | Lipid imbalance in the neurologiacal disorder, Niemann-Pick C disease[END_REF], thus, these two disorders (MLIV and NPC) constitute a distinct group within the LSD of endosomal/lysosomal trafficking impairment leading to defective lysosomal biogenesis or function.

Catabolism of the accumulated materials in MLIV still does occur in the storage vacuoles (early lysosomes? Multivesicular bodies?) of MLIV patients, though in an altered rate and pattern (for instance; degradation products are not exported normally from these organelles to the Golgi apparatus) [START_REF] Chen | Abnormal transport along the lysosomal pathway in Mucolipidosis type IV disease[END_REF][START_REF] Pryor | Mucolipin-1 is a lysosomal membrane protein required for intracellular lactosylceramide traffic[END_REF]. This helps to explain the clinical manifestations of this disease, as outlined above; since despite the early onset of the disease and severe neurological involvement, MLIV patients remain in an apparent steady state for decades. This may indicate that indeed the slower catabolism rate prevents a massive storage as is the case in most other LSD, and hence, the extremely slow progression of the clinical picture in these patients.

Genetics:

The gene MCOLN1 was identified by linkage studies [START_REF] Bargal | Identification of the gene causing Mucolipidosis type IV[END_REF][START_REF] Bassi | Cloning of the gene encoding a novel integral membrane protein, Mucolipin -and identification of the two major founder mutations causing Mucolipidosis type IV[END_REF][START_REF] Sun | Mucolipidosis type IV is caused by mutations in a gene encoding a novel transient receptor potential channel[END_REF] following its mapping to human chromosome 19p13.2-13.3 [START_REF] Slaugenhaupt | Mapping of the Mucolipidosis type IV gene to chromosome 19p and definition of founder haplotypes[END_REF]. The identification of this gene triggered the identification of two other homologous genes, namely, MCOLN2 and MCOLN3, which mapped to chromosome 1p22. shown to be associated with early deafness, vestibula defects, pigmentation abnormality and perinatal lethality [START_REF] Dipalma | Mutations in MCOLN3 associated with deafness and pigmentation defects in varitintwadder (Va) mice[END_REF]. At present no human counterpart was reported. Mutations in a mucolipin-like gene in C. elegans -CUP5 was reported to be associated with general lysosomal storage phenomena of a similar nature as is present in cells of MLIV patients, as well as increased cell death leading to embryonic lethality [START_REF] Fares | Regulation of endocytosis by CUP-5, the Caenorhabditis elegans mucolipin 1 homolog[END_REF][START_REF] Treusch | Caenorhabditis elegans functional orthrologue of human protein h-mucolipin-1 is required for lysosomal biogenesis[END_REF]. Recent reports have linked the embryonic lethality in CUP5-null C. elegans to starvation of embryonic cells and general developmental defects [START_REF] Schaheen | Basis of lethality in C. elegans lacking CUP-5, the Mucolipidosis type IV orthologue[END_REF]. These observations were suggested to stem from defective endocytosis of nutrients and the general accumulation of a genetic suppressor, the ATP-binding cassette (ABC) transporter MRP-4, in CUP5-null cells [START_REF] Schaheen | Basis of lethality in C. elegans lacking CUP-5, the Mucolipidosis type IV orthologue[END_REF][START_REF] Schaheen | Suppression of the CUP-5 Mucolipidosis type IV related lysosomal dysfunction by the inactivation of an ABC transporter in C. elegans[END_REF].

Abnormal mitochondrial function due to increased mitochondrial fragmentation is observed in MLIV cultured fibroblasts, as well as in cell lines from other lysosomal storage disorders, as a result of inefficient autophagolysosomal recycling of mitochondria [START_REF] Jennings | Mitochondrial aberrations in Mucolipidosis type IV[END_REF]. This leads to decreased Ca 2+ buffering capacity in MLIV cells which increases susceptibility to a caspase-8-dependent apoptotic pathway. This phenomenon might also explain the increased sensitivity to apoptosis in C. elegans as described above. The functional link between mutations in the MCOLN1 gene, mitochondrial aberrations and the clinical manifestations in MLIV still remains to be seen.

Mucolipins and subcellular localization:

Three proteins are encoded by the MCOLN genes, namely, TRPML1, TRPML2, and TRPML3 also referred as MLN1-3 or Mucolipin 1-3, respectively. Following the identification of the relevant genes the putative structure of the protein products was deduced by computerized analysis [START_REF] Bargal | Identification of the gene causing Mucolipidosis type IV[END_REF]. This indicated that TRPML1 is 580 amino acids long consisting of 6 transmembrane domains which attribute this protein to the TRP superfamily [START_REF] Nilius | TRP channels: a TR(I)P through a world of multifunctional cation channels[END_REF][START_REF] Montell | The TRP superfamily of cation channels[END_REF], although it does not contain ankirin domains. The two other TRPML proteins have similar structure.

The physiological function of the mucolipins has not yet been fully elucidated, but electrophysiological analyses indicate that they function as cation channels. This was particularly demonstrated in TRPML1 channels. Based on previous findings (see above) it is expected that TRPML1 function would be related to the biogenesis of lysosomes or their normal maintenance. Indeed, studies with expression vectors and immunohistochemistry demonstrated that TRPML1 is located to lysosomal vacuoles [START_REF] Pryor | Mucolipin-1 is a lysosomal membrane protein required for intracellular lactosylceramide traffic[END_REF][START_REF] Manzoni | Overexpression of wild-type and mutant mucolipin proteins in mammalian cells: effects on the late endocytic compartment organization[END_REF][START_REF] Vergarajauregui | Two di-leucine motifs regulate trafficking of mucolipin-1 to lysosomes[END_REF][START_REF] Miedel | Two di-leucine motifs regulate trafficking of mucolipin-1 to lysosomes[END_REF][START_REF] Kiselyov | TRP-ML1 is a lysosomal monovalent cation channel that undergoes proteolytic cleavage[END_REF][START_REF] Venkatachalam | Lysosomal localization of TRPML3 depends on TRPML2 and the mucolipidosis-associated protein TRPML1[END_REF].

Site directed mutagenesis indicated the role of two dileucine motifs, one at the N-terminal and the second at the C-terminal tail of TRPML1, as subcellular targeting motives. Experiments with a C-terminal construct of TRPML1 (consisting of amino acids 518-580 fused to the Cterminus of Tac) identified a C-terminal dileucine motif (amino acids 573-578: EEHSLL) that represents an AP2-dependent internalization signal responsible for targeting TRPML1 to early endosomes from the plasma membrane [START_REF] Vergarajauregui | Two di-leucine motifs regulate trafficking of mucolipin-1 to lysosomes[END_REF]. In addition, it was found that palmitoylation of cysteines in close proximity to the C-terminal dileucine motif (on amino acids 565-567) regulates this endocytotic signaling. However, the full length TRPML1 protein proved to be independent of this targeting signal since removal of the C-terminal dileucine motif was insufficient to mistarget the full length protein from its proper lysosomal subcellular localization [START_REF] Pryor | Mucolipin-1 is a lysosomal membrane protein required for intracellular lactosylceramide traffic[END_REF][START_REF] Vergarajauregui | Two di-leucine motifs regulate trafficking of mucolipin-1 to lysosomes[END_REF][START_REF] Miedel | Two di-leucine motifs regulate trafficking of mucolipin-1 to lysosomes[END_REF][START_REF] Kiselyov | TRP-ML1 is a lysosomal monovalent cation channel that undergoes proteolytic cleavage[END_REF][START_REF] Venkatachalam | Lysosomal localization of TRPML3 depends on TRPML2 and the mucolipidosis-associated protein TRPML1[END_REF]. As such, another dileucine motif in the N-terminus of TRPML1

(consisting of amino acids 11-16: ETERLL) was identified, containing sufficient lysosomal targeting information [START_REF] Vergarajauregui | Two di-leucine motifs regulate trafficking of mucolipin-1 to lysosomes[END_REF][START_REF] Venkatachalam | Lysosomal localization of TRPML3 depends on TRPML2 and the mucolipidosis-associated protein TRPML1[END_REF]. Miedel et al. [START_REF] Miedel | Two di-leucine motifs regulate trafficking of mucolipin-1 to lysosomes[END_REF] characterized the trafficking of TRPML1 by reporting a strict AP1 dependence for the lysosomal localization of the full length protein

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. Thus, taken together, it appears that the N-terminal dileucine motif is the primary targeting signal of TRPML1 and it is apparently also AP1 dependent. A simple AP1 knockdown experiment with a Tac-TRPML1 N-terminus construct should clarify this point.

Furthermore, it has recently been reported in human fibroblasts that TRPML1 may play a role in lysosomal exocytosis [START_REF] Laplante | Lysosomal exocytosis is impaired in Mucolipidosis type IV[END_REF]. This process is characterized by fusion of lysosomes with the plasma membrane whereby lysosomal contents are exocytosed. In such a case, the normally lysosomal TRPML1 may find itself in the "foreign territory" of the plasma membrane. Hence, the AP2-dependent C-terminal dileucine internalization motif may be of critical importance to retrieve TRPML1 to lysosomes from the plasma membrane following such an exocytotic event.

The cellular localization of TRPML2 and TRPML3 is less clear, but a recent study which is confirmed in the present report, indicates that these proteins together with TRPML1 are capable of interacting with each other (Figure 3 and [START_REF] Venkatachalam | Lysosomal localization of TRPML3 depends on TRPML2 and the mucolipidosis-associated protein TRPML1[END_REF]. While TRPML1 and TRPML2

can each transport independently from the Golgi to lysosomes, TRPML3 requires one of the other two for its lysosomal localization. This data suggests that in fact all the members of the mucolipin family are lysosomal proteins but this should await further confirmation and characterization. Nevertheless, these observations imply that transfection studies leading to overexpression of TRPML3, unlike the other two TRPML proteins, will not reflect the natural localization of this protein as it will preclude stoichiometric association of the excess TRPML3 with the other TRPML members thereby diverting it from targeting to lysosomes.

The fate of TRPML1 in MLIV patients depends upon the nature of the mutations in the MCOLN1 gene. Naturally, gross genetic alterations, such as partial gene deletions or splicing mutations (both of which are the AJ founder mutations), as well as insertions or nonsense mutations, will basically lead to the absence of any functional protein. On the other hand, several missense mutations were shown to cause the misfolded protein to be retained in the endoplasmic reticulum, apparently due to the chaperon "proof reading" system, thereby preventing these mutant proteins from reaching the lysosome [START_REF] Manzoni | Overexpression of wild-type and mutant mucolipin proteins in mammalian cells: effects on the late endocytic compartment organization[END_REF][START_REF] Kiselyov | TRP-ML1 is a lysosomal monovalent cation channel that undergoes proteolytic cleavage[END_REF]. Other missense mutations do reach the lysosomes, but have been shown to affect TRPML1 channel activity.

Table 1 summarizes the published data regarding the fate of some of the TRPML1 missense mutations. It should be noted, however, that with the exception of the mild F408 in-frame deletion, all MLIV mutations to date cause the same, or similar, severe clinical manifestations.

Mucolipin structure and protein interactions:

The putative structure of TRPML1 is pictured in Figure 1. TRPML1 is palmitoylated on its C terminus at residues 565-567 [START_REF] Vergarajauregui | Two di-leucine motifs regulate trafficking of mucolipin-1 to lysosomes[END_REF] (and unpublished results). The suggested function of this palmitoylation has been described above with regard to trafficking of the protein.

In addition, TRPML1 is also a glycoprotein with four putative N-linked glycosylation sites (at amino acid positions 159, 179, 220, and 230) in its long luminal loop between transmembrane domains 1 and 2. TRPML1's N-linked sugars are sialylated, at least in part, in the Golgi prior to the protein's transport to lysosomes [START_REF] Miedel | Two di-leucine motifs regulate trafficking of mucolipin-1 to lysosomes[END_REF][START_REF] Kiselyov | TRP-ML1 is a lysosomal monovalent cation channel that undergoes proteolytic cleavage[END_REF]. Interestingly, the N- prelysosomal compartment. In addition, both studies also reported that each cleavage product co-immunoprecipitates the other, suggesting that the cleavage products may remain associated with each other after cleavage. However, one very important discrepancy was observed between these two studies as Kiselyov et al. [START_REF] Kiselyov | TRP-ML1 is a lysosomal monovalent cation channel that undergoes proteolytic cleavage[END_REF] were able to inhibit cleavage of TRPML1 with cathepsin B protease inhibitors while Meidel et al. [START_REF] Miedel | Two di-leucine motifs regulate trafficking of mucolipin-1 to lysosomes[END_REF] resorted to the broad lysosomal serine, plasmin, and cysteine protease inhibitor, leupeptin, to prevent cleavage. Nonetheless, both studies did qualify their results by not ruling out the possibility of a number of different proteases being responsible for the cleavage of the protein.

The most significant finding, with regard to TRPML1 cleavage, is that the endogenous protein has also been found to undergo cleavage. In addition, it has also been found that the N Nonetheless, all such electrophysiological recordings must be qualified in light of the fact that the lysosomal membrane is different, topologically and structurally, from the plasma membrane. Aside from both membranes featuring different populations of glycolipids, sphingolipids, and phospholipids; the lysosomal membrane is also a specialized single lipid bilayer membrane as opposed to the double lipid bilayer plasma membrane [START_REF] Holzman | Lysosomes[END_REF][START_REF] Zimmerberg | The physical chemistry of biological membranes[END_REF][START_REF] Winchester | Lysosomal membrane proteins[END_REF]. These and other differences between the lysosomal membrane and the plasma membrane likely reflect important influences upon electrophysiological recordings.

The second observation that predominates in TRPML1 but also TRPML2, and TRPML3 overexpression, is the formation of indissociable aggregates. These aggregates have appeared in pictured immunoblots of TRPML1 in a number of different publications [START_REF] Manzoni | Overexpression of wild-type and mutant mucolipin proteins in mammalian cells: effects on the late endocytic compartment organization[END_REF][START_REF] Vergarajauregui | Two di-leucine motifs regulate trafficking of mucolipin-1 to lysosomes[END_REF][START_REF] Miedel | Two di-leucine motifs regulate trafficking of mucolipin-1 to lysosomes[END_REF][START_REF] Song | TRPML cation channels regulate the specialized lysosomal compartment of vertebrate B-lymphocytes[END_REF]. We also observed a similar observation with regard to TRPML2 and TRPML3 (Figure 3). While endogenous expression of TRPML2 and TRPML3 cannot be verified due to the lack of antibodies that recognize these endogenous proteins, characterization of the endogenous TRPML1 has revealed no indissociable aggregates in immunoblots carried out on control HEK 293 cells (Figure 2). It is assumed that due to the highly hydrophobic nature of the TRPML integral membrane proteins, aggregation in overexpression would be unavoidable.

Nevertheless, overexpressed protein aggregation does not preclude the possibility of protein-protein interactions existing between the different TRPMLs and each other. One common feature of many TRP proteins is the ability to homo-and hetero-multimerize with closely related members of the same subfamily [START_REF] Hellwig | Homo-and heteromeric assembly of TRPV channel subunits[END_REF][START_REF] Strübing | Formation of novel TRPC channels by complex subunit interactions in embryonic brain[END_REF]. Indeed, Venkatachalam et al. [START_REF] Venkatachalam | Lysosomal localization of TRPML3 depends on TRPML2 and the mucolipidosis-associated protein TRPML1[END_REF] reported interactions between TRPML1 and both TRPML2 and TRPML3, respectively, using a FRET-based approach. This same approach demonstrated an interaction between TRPML2 and TRPML3 as well. Here we further report that TRPML1 also co-immunoprecipitates

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TRPML2 and TRPML3 and vice-versa (Figure 3). These protein interactions and others await verification with controlled experiments on the endogenous TRPML proteins in the future.

Nonetheless, the concept of TRPML1 being part of a complex is important when considering optimization of future experiments to characterize the structure and function of the protein.

The concept of a complex structure involving the different mucolipins and the implications for MLIV awaits further elucidation.

TRPML1 channel activity:

The mechanism which accounts for the pathogenesis of MLIV is not clear. The initial assessment of the function of TRPML1 was suggested by its predicted protein structure [START_REF] Bargal | Identification of the gene causing Mucolipidosis type IV[END_REF],

which indicated one transmembrane (TM) helix in the amino-terminal region and at least five transmembrane domains in the carboxy-terminal half of the protein (Figure 1). The putative structure of six transmembrane domains along with a TRP cation channel motif spanning the third to the sixth TM domains suggested that the protein belongs to the TRP superfamily [START_REF] Minke | The roles of trp and calcium in regulating photoreceptor function in Drosophila[END_REF] with a high resemblance to the polycystin-2/TRPP2 protein [START_REF] Delmas | Polycystins: from mecanosensation to gene regulation[END_REF]. Thus it was expected that TRPML1 function as a cation channel.

The ability to perform electrophysiological analyses of channel activity, in order to prove the above assumption, suffers from serious limitation since the protein is located in the lysosomal and/or late endosomal membrane. Therefore electrophysiological recordings have been made possible only after overexpressing the protein in different cell types in ectopic locations.

Overexpression of the protein to the Xenopus oocyte plasma membrane observed TRPML1 as a novel channel with a large conductance and permeability to Na + , K + and Ca 2+ [START_REF] Laplante | Identification and characterization of the single channel function of human mucolipin-1 implicated in mucolipidosis type IV, a disorder affecting the lysosomal pathway[END_REF]. Since an overexpressed protein which is located in an ectopic location does not necessarily reflect the endogenous activity of the channel, especially in an environment where other channel proteins might interact with the overexpressed protein, a different approach to record the channel activity of the overexpressed protein in a relatively isolated environment was taken [START_REF] Cantiello | Cation channel activity of mucolipin-1: the effect of calcium[END_REF]. The channel activity was assessed in endosomal vesicles of MLIV patient and in TRPML1 over-expressing cells, as well as in proteoliposomes which contained the in vitro translated protein [START_REF] Raychowdhury | Molecular pathophysiology of mucolipidosis type IV: pH dysregulation of the mucolipin-1 cation channel[END_REF]. Electrical recordings of the channel from both preparations indicated that wild type TRPML1 is a multiple subconductance non-selective cation channel whose function is inhibited by Ca 2+ transport and a reduction of pH [START_REF] Cantiello | Cation channel activity of mucolipin-1: the effect of calcium[END_REF][START_REF] Raychowdhury | Molecular pathophysiology of mucolipidosis type IV: pH dysregulation of the mucolipin-1 cation channel[END_REF]. Since it was shown in

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this system that mutated channels lost their ability to be regulated by Ca 2+ and by lowering pH, it was suggested that this might hint to the mechanism which underlies the pathophysiology of the disease. Intravesicular pH is critical in endosomal/lysosomal fusion, suggesting that its regulation (possibly by Ca 2+ ) is essential for the ability of vesicular organelles to fuse [START_REF] Overly | Quantitative measurement of intraorganelle pH in the endosomal-lysosomal pathway in neurons by using ratiometric imaging with pyranine[END_REF][START_REF] Luzio | Membrane dynamics and the biogenesis of lysosomes[END_REF].

In a different approach, the channel conductance was measured by whole cell current recording after mistargeting the protein to the plasma membrane in HEK 293 cells. In this system TRPML1 showed characteristic strong outwardly rectifying current [START_REF] Kiselyov | TRP-ML1 is a lysosomal monovalent cation channel that undergoes proteolytic cleavage[END_REF]. Following these recordings a new mechanism was suggested for the pathogenesis of MLIV. Soyombo et al. [START_REF] Soyombo | TRP-ML1 regulates lysosomal pH and acidic lysosomal lipid hydrolytic activity[END_REF] indicated that TRPML1 in fact functions as a proton channel and demonstrated a decrease in the lysosomal pH in cultured cells of MCOLN1 mutants when compared to normal. This suggests that TRPML1 might function as a pH regulator of lysosomes, whose function is to pump H + ions out of these organelles when this milieu becomes acidified during the catabolism process. Thus, TRPML1 functions as a pH regulator in maintaining the required acidity for the normal activity of lysosomal hydrolases. It was also demonstrated that supplying lysosomes with weak bases lead to the correction of lysosomal storage as judged by electron microscopy observations. This data remains to be confirmed and further characterized.

The most recent study of TRPML1 channel activity claims that none of the above electrophysiological recordings could be reproduced [START_REF] Pryor | Mucolipin-1 is a lysosomal membrane protein required for intracellular lactosylceramide traffic[END_REF]. This study proves that correct targeting of TRPML1 to lysosomes and the integrity of its predicted ion pore are essential for its physiological function. The authors mention the following reasons to explain the difficulty of obtaining electrophysiological data from TRPML1 in transfected cells: 1) the channel may be inactivated by proteolytic cleavage; 2) subunits which might be needed for channel oligomerization may be missing; and 3) possible TRPML1-interacting proteins, which could be needed for gating the channel, may also be missing.

MLIV research has presented with conflicting results well before the cloning of the MCOLN1 gene. Nowadays, the conflicting electrophysiological recordings of the overexpressed protein are confounded by confusing data with regard to lysosomal pH of MLIV patient fibroblasts. While we found elevated pH [START_REF] Bach | Elevated lysosomal pH in Mucolipidosis type IV cells[END_REF], others reported normal [START_REF] Pryor | Mucolipin-1 is a lysosomal membrane protein required for intracellular lactosylceramide traffic[END_REF][START_REF] Kopitz | C 14 -Methylamine accumulation in cultured human skin fibroblasts--a biochemical test for lysosomal storage and lysosomal diseases[END_REF] or reduced pH [START_REF] Soyombo | TRP-ML1 regulates lysosomal pH and acidic lysosomal lipid hydrolytic activity[END_REF]. While the pH discrepancies could be explained by the autoflouresence of the MLIV fibroblasts, the other conflicting results could be explained by the artificial conditions of the mistargted and overexpressed proteins. In their review which deals with "Cellular and molecular function of mucolipins and polycystin 2," Qian and Noben-Trauth [START_REF] Qian | Cellular and molecular function of mucolipins (TRPML) and polycystin 2 (TRPP2)[END_REF] mention a variety of experiments with polycystin 2 that describe its localization at various intracellular locations. They suggest that these discrepancies could be explained by differing methodologies, however, they also predict that differing results may stem from dynamic intracellular trafficking of polycystin-2 and that this trafficking may display variable regulation in various tissues. These principles might hold for TRPML1, the closest relative of polycystin 2, but only further investigation into the dynamic trafficking of this protein would give a firm support to this idea. arrows. This data confirms that the endogenous TRPML1 protein is also cleaved in vivo as described elsewhere [START_REF] Kiselyov | TRP-ML1 is a lysosomal monovalent cation channel that undergoes proteolytic cleavage[END_REF].

  TRPMLs are encoded by the MCOLN genes. TRPML1 is encoded by MCOLN1 localized in humans at chromosome 19p13.2-13.3 while the 2 other genes -MCOLN2 and MCOLN3 are localized at chromosome 1p22.3 encoding TRPML2 and TRPML3 respectively. MCOLN1 is mutated in humans in a lysosomal storage disorder -Mucolipidosis type IV (MLIV)

  glycosylation sites have been used to map a physical and temporal pattern of TRPML1 cleavage in its long luminal loop. TRPML1 is cleaved into two distinct cleavage products resulting from a directed cleavage of TRPML1 between two glycosylation sites at amino acid positions 179 and 220, respectively[START_REF] Miedel | Two di-leucine motifs regulate trafficking of mucolipin-1 to lysosomes[END_REF]. Kiselyov et al.[START_REF] Kiselyov | TRP-ML1 is a lysosomal monovalent cation channel that undergoes proteolytic cleavage[END_REF] further characterized the cleavage site by performing N-terminal sequencing on the C-terminal cleavage product yielding a putative cleavage site just upstream of the proline at amino acid position 201. Both studies observed that the N-linked sugars on both the N and C-terminal cleavage products are sialylated, suggesting that the temporal cleavage of TRPML1 occurs post-ER, in a

8 TRPML1

 8 and C-terminal cleavage products are in excess with relation to the full length protein in HEK 293, human skin fibroblast, and bovine brain cells [46 and Figure 2]. This suggests that A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT cleavage may play an important role in regulating the function of the protein. It has already been proposed that cleavage inactivates the channel function of TRPML1 [46]. Overexpression of TRPML1 has led to the discovery of two predominant observations. The first is the distinct possibility of plasma membrane localization predominating over the widely agreed upon lysosomal localization of the protein. This phenomenon was used in HEK 293 cells where plasma membrane localization facilitates simple electrophysiological recordings via standard patch-clamp methods [46, 49].

FiguresFigure 1 .Figure 2 .

 12 Figures Figure 1. The putative structure of TRPML1

Figure 3 .

 3 Figure 3. TRPML1 co-immunoprecipitates TRPML2 and TRPML3. C311UP was used to

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Acknowledgment: This work was supported by the Rita Altura Foundation

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Appendix

Materials and Methods

TRP-ML Expression Constructs


Ke Lei

Haruaki Ninomiya
email: ninomiya@grape.med.tottori-u.ac.jp

Michitaka Suzuki

Takehiko Inoue

Miwa Sawa

Masami Iida

Hiroyuki Ida

Yoshikatsu Eto

Seiichiro Ogawa

Kousaku Ohno

Yoshiyuki Suzuki

Enzyme enhancement activity of N-octyl-β-valienamine on β-glucosidase mutants associated with Gaucher disease

Keywords: Gaucher disease, β-glucosidase, valienamine, chaperone

est destinée au dépôt

Introduction

Gaucher disease (GD) is an inherited lipid storage disorder characterized by lysosomal accumulation of glucocerebroside (glucosylceramide) in monocyte-macrophage cells [START_REF] Beutler | Gaucher disease[END_REF]. It is caused by mutations in a gene that encodes acid β-glucosidase (β-Glu; glucocerebrosidase EC3.2.1.45). Patients with GD exhibit visceral symptoms such as hepatosplenomegaly, anemia, bone lesions and respiratory failure, with or without progressive neurological symptoms. Patients without neurological symptoms are classified as type 1, whereas those with neurological symptoms are classified into type 2 (acute infantile form) and type 3 (juvenile form).

At present, there are two established therapeutic strategies for GD: enzyme replacement therapy and substrate reduction therapy. Enzyme replacement has been achieved by intravenous administration of macrophage-targeted recombinant β-Glu [START_REF] Barton | Replacement therapy for inherited enzyme deficiency -macrophage-targeted glucocerebrosidase for Gaucher's disease[END_REF] whereas substrate reduction has been achieved by oral administration of N-butyl-deoxynojirimycin (OGT918), which inhibits glucosyltransferase and decreases substrate biosynthesis [START_REF] Cox | Novel oral treatment of Gaucher's disease with Nbutyldeoxynojirimycin (OGT918) to decrease substrate biosynthesis[END_REF]. Both therapies have been proven to be effective for visceral, hematologic and skeletal abnormalities [START_REF] Grabowski | Enzyme therapy for Gaucher disease: the first 5 years[END_REF][START_REF] Erikson | Ten years' experience of enzyme infusion therapy of Norrbottnian (type 3) Gaucher disease[END_REF][START_REF] Platt | Inhibition of substrate synthesis as a strategy for glycolipids lysosomal storage disease therapy[END_REF]. Unfortunately, the efficacy of these therapies to neurological manifestations is, if any, limited [START_REF] Schiffmann | Prospective study of neurological responses to treatment with macrophage-targeted glucocerebrosidase in patients with type 3 Gaucher's disease[END_REF][START_REF] Prows | Gaucher disease: enzyme therapy in the acute neuronopathic variant[END_REF][START_REF] Aoki | Improvement of neurological symptoms by enzyme replacement therapy for Gaucher disease type IIIb[END_REF][START_REF] Aerts | Substrate reduction therapy of glycosphingolipid storage disorders[END_REF].

It has been known that some of the disease-causing mutations of β-Glu do not interfere with its catalytic activity but disrupt either its proper folding in the endoplasmic reticulum (ER) or intracellular trafficking out of this compartment. Recent evidence suggested that the mutant proteins, retained in the ER because of improper folding or trafficking, were degraded by the proteasome in a series of processes summarized as ERAD (ER-associated protein degradation) [START_REF] Ron | ER retention and degradation as the molecular basis underlying Gaucher disease heterogeneity[END_REF][START_REF] Schmitz | Impaired trafficking of mutants of lysosomal glucocerebrosidase in Gaucher's disease[END_REF]. This bases the idea of "enzyme enhancement therapy", a novel therapeutic strategy for GD, which aims at stabilization and rescue of the mutant enzyme by using cell-permeable small molecules [START_REF] Sawkar | Therapeutic strategies to ameliorate lysosomal storage disorders -a focus on Gaucher disease[END_REF][START_REF] Desnick | Enzyme replacement and enhancement therapies: lessons from lysosomal disorders[END_REF]. In pursuit of this idea, we have shown that a carbohydrate mimic N-octyl-βvalienamine (NOV), an inhibitor of β-Glu, could increase the protein level and enzyme activity of F213I mutant β-Glu in cultured GD fibroblasts [START_REF] Lin | N-Octyl-β-valienamine up-regulates activity of F213I mutant β-glucosidase in cultured cells: a potential chemical chaperone therapy for Gaucher disease[END_REF], whereas Sawkar and colleagues reported that N-nonyl-deoxynojirimaycin (NN-DNJ), another inhibitor of β-Glu, could increase the enzyme activity of N370S and G202R mutants [START_REF] Sawkar | Chemical chaperones increase the cellular activity of N370S β-glucosidase: A therapeutic strategy for Gaucher disease[END_REF][START_REF] Sawkar | Gaucher disease-associated glucocerebrosidases show mutationdependent chemical chaperoning profiles[END_REF]. In a recent review by Bernier et al. [START_REF] Bernier | Pharmacological chaperones: potential treatment for conformational diseases[END_REF], the term "chemical chaperone" was used to describe molecules that help folding of proteins in a nonspecific manner, whereas those with specific effects on the target protein were termed "pharmacological chaperone". In addition, the term "enzyme enhancement activity (EEA)" is defined as an activity of a molecule to increase the cellular enzyme activity when the molecule is applied to live cells [START_REF] Desnick | Enzyme replacement and enhancement therapies: lessons from lysosomal disorders[END_REF].

The purpose of the current study was to further explore the potential of NOV as a pharmacological chaperone for β-Glu mutant proteins. For this purpose, we examined EEA of NOV in cultured human GD fibroblasts as well as in COS cells transiently expressing recombinant mutant proteins. Since we found that NOV was effective on both N370S and G202R mutants, we compared EEA of NOV and that of NN-DNJ. We also tested activities of Nalkyl-β-valienamines with various lengths of the acyl chain on the F213I mutant.

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M A N U S C R I P T Endoglycosidase-H was from New England Biolabs.

Construction of β-Glu expression plasmids -Human β-Glu cDNA (a kind gift from Dr. S. Tsuji, Tokyo University) was subcloned into a mammalian expression vector pCAGGS. A Flagepitope was introduced to the C-terminus of the cDNA by PCR. The following mutations were introduced by using the Quick Change site-directed mutagenesis kit: N188S, G193W, G202R, F213I, N370S and L444P. All the mutations were confirmed by direct sequencing.

Cell culture -Human skin fibroblasts and COS cells were cultured in DMEM/10% BCS at 37 o C in 5% CO 2 . We used one control cell line (H8) and 9 lines of GD cells. 6 lines of GD cells were from Japanese patients. 4 cell lines carried β-Glu mutations of F213I/F213I, G202R/L444P, N188S/G193W and nt1447del20insTG/L444P, whereas in the other 2 cell lines, only one β-Glu mutation, nt1447del20insTG or D409H, was identified and the mutation on the other allele was left unknown [START_REF] Eto | Clinical and molecular characteristics of Japanese Gaucher disease[END_REF]. The other 3 lines of GD cells that carried the N370S mutation were from Caucasian patients: two cell lines (DMN00.41 and DMN87.30) carried N370S homozygous mutations and one cell line carried N370S/84GG. Both 84GG and nt1447del20insTG are predicted to cause premature termination of the encoded protein. Culture medium was replaced every 2 days with fresh media supplemented with or without N-alkyl-β-valienamines at the indicated concentrations. COS cells in 35-mm dishes were transfected with β-Glu cDNA by using LipofactAMINE according to manufacture's instructions. 24 h post-transfection, cells were treated with or without the compounds for 24 h.

Immunoprecipitation and immunoblotting -All procedures were carried out at 4 o C. COS cells were lysed by sonication in PBS supplemented with 1% Triton X-100 and a protease inhibitor cocktail (Boehringer). After a brief centrifugation to remove insoluble material, the supernatant was precleared with an aliquot of agarose beads. For immunoprecipitation of Flag-β-Glu, the lysates (500 μl from a 35-mm dish) were incubated for 16 h with anti-Flag M2 agarose beads (20 μl of 50% slurry). The beads were washed with PBS/1% Triton X-100, rinsed with H 2 O and the final volume of the precipitates was adjusted to 40 μl with H 2 O. For the enzyme assay, 4 μl of the precipitates was used as described below. For immunoblotting, bound proteins were eluted by incubation of 20 μl of precipitates with the same volume of 2×SDS-PAGE sample buffer at 100 o C for 3 min. SDS-PAGE and Western transfer were carried out as previously described [START_REF] Lin | N-Octyl-β-valienamine up-regulates activity of F213I mutant β-glucosidase in cultured cells: a potential chemical chaperone therapy for Gaucher disease[END_REF]. The blots were probed with rabbit polyclonal anti-Flag antibody and developed using an ECL kit (Amersham Pharmacia).

In vitro enzyme assay -β-Glu activities in cell lysates or immunoprecipitates were determined by using 4-methylumbelliferone-conjugated β-D-glucopyranoside as a substrate [START_REF] Vaccaro | Effect of a heatstable factor in human placenta on glucosylceramidase, glucosylsphingosine glucosyl hydrolase, and acid β-glucosidase activities[END_REF]. For preparation of cell lysates, cells in 35-mm dishes were scraped into 100 μl of ice-cold H 2 O and lysed by sonication. Insoluble materials were removed by centrifugation and protein concentrations were determined with a BCA microprotein assay kit. Anti-Flag immunoprecipitates were prepared as described above. 4 μl of the lysates or immunoprecipitates was incubated at 37 o C with 8 μl of the substrate solution in 0.1 M citrate buffer, pH 5.2, supplemented with sodium taurocholate (0.8% w/v). The reaction was terminated by adding 0.4 ml of 0.2 M glycine sodium hydroxide buffer (pH 10.7). Liberated 4-methylumbelliferone was measured with Perkin Elmer Luminescence Spectrometer (excitation wave length: 340 nm; emission: 460 nm). One unit of enzyme activity was defined as nmol of 4-methylumbelliferone released per hour and normalized for the amount of protein contained in the lysates.

Intact cell enzyme assay -β-Glu activities in live cells were estimated by the methods described by Sawkar et al. [START_REF] Sawkar | Chemical chaperones increase the cellular activity of N370S β-glucosidase: A therapeutic strategy for Gaucher disease[END_REF]. Briefly, cells in 24-well plates were treated with NOV or NN-DNJ for 4 days. After washing with PBS, the cells were incubated in 80 µl of PBS and 80 µl of 0.2 M acetate buffer (pH 4.0). The reaction was started by addition of 100 µl of 4-methylumbelliferyl-β-D-glucoside (5 mM), followed by incubation at 37°C for 1 h. The reaction was stopped by lysing the cells by the addition of 2 ml of 0.2 M glycine buffer (pH 10.7) and liberated 4methylumbelliferone was quantified. Every experiment was performed in parallel with cells that had been preincubated with or without conduritol B epoxide (CBE: Toronto Research Chemicals) at 0.5 mM for 1 h. The CBE-sensitive component was ascribed to lysosomal β-Glu, whereas the CBE-insensitive component was ascribed to non-lysosomal β-Glu.

Endoglysosidase-H treatment -Cell lysates (in H 2 O containing ~40 μg protein) from transfected COS cells were incubated at 100 o C for 10 min with 0.5% SDS and 40 mM DTT to denature proteins. They were then incubated at 37 o C for 1 h with 0.5 unit of endoglysosidase-H (Endo-H) in 50 mM citrate buffer (pH 5.5). The reaction mixture was subjected to SDS-PAGE, Western transfer and anti-Flag immunoblotting as described above.

Immunofluorescence -We used staining procedures described previously [START_REF] Lin | N-Octyl-β-valienamine up-regulates activity of F213I mutant β-glucosidase in cultured cells: a potential chemical chaperone therapy for Gaucher disease[END_REF]. Briefly, COS cells grown on cover glasses were transfected with Flag-β-Glu cDNA and 24 h post-transfection, exposed to Lysotracker Red (0.5 μM; Molecular Probe) for 1 h. Cells were fixed with 4% paraformaldehyde and permeabilized with 1% TritonX-100. They were incubated with rabbit polyclonal anti-Flag antibody (1:500), followed by Alexa488-conjugated anti-rabbit IgG. Fluorescent images were collected by using a Leica TSC SP2 confocal laser microscope.

Results

EEA of NOV in human GD fibroblasts assessed by ex vivo enzyme assay

In our previous study [START_REF] Lin | N-Octyl-β-valienamine up-regulates activity of F213I mutant β-glucosidase in cultured cells: a potential chemical chaperone therapy for Gaucher disease[END_REF], EEA of NOV was evaluated by ex vivo experiments in which GD cells were treated with NOV for 4 days followed by determination of the enzyme activity in cell lysates. By this method, we found significant EEA of NOV in F213I homozygous and heterozygous (F213I/L444P) cells. The maximum response was obtained at an NOV concentration of 30 μM in F213I homozygous cells. NOV was ineffective in cells with mutations of N370S/84GG, L444P/L444P and L444P/RecNcil. N-alkyl-β-valienamine preparations, including NOV, used in our previous study were poorly soluble in water, and hence in culture medium. Although NOV had the best solubility among the N-alkyl-β-valienamines, it was barely soluble in water above 3 mM. To test whether the water-solubility affected EEA, we prepared hydrochloride forms of N-alkyl-β-valienamines (see Fig. 4a), which were easily soluble in water above 30 mM. By using the hydrochloride form of NOV, we could reproduce EEA of NOV in F213I homozygous cells but with a different doseresponse profile: the maximum response was obtained at 3 μM (Fig. 1a). Because of this improved efficacy, hydrochloride preparations were used in all the subsequent experiments.

In the 7 lines of GD cells newly included in the screening, we found statistically significant EEA of NOV in four lines of cells with mutations of N188S/G193W, G202R/L444P and N370S/N370S. NOV effects on the two cell lines with N370 homozygous mutations were remarkably similar to each other, causing an ~2-fold increase at concentration of 3 and 30 μM. In accordance with the results in our previous study [START_REF] Lin | N-Octyl-β-valienamine up-regulates activity of F213I mutant β-glucosidase in cultured cells: a potential chemical chaperone therapy for Gaucher disease[END_REF], NOV elicited weak EEA in N370S/84GG cells, although the increase was not statistically significant. In contrast, NOV elicited no effects in cells with mutations of nt1447del20insTG/L444P. In two cell lines, only one mutation of β-Glu, D409H or nt1447del20insTG, was identified, and the mutation on the other allele was left unknown. NOV effects were also negative in these two lines of cells (Fig. 1a).

EEA of NOV and NN-DNJ assessed by intact cell enzyme assay

The above ex vivo enzyme assay did not indicate whether the lysosomal enzyme activity was enhanced by NOV. To compensate for this, we employed "intact cell enzyme assay" described by Sawkar et al. [START_REF] Sawkar | Chemical chaperones increase the cellular activity of N370S β-glucosidase: A therapeutic strategy for Gaucher disease[END_REF], in which the cellular enzyme activity was estimated by application of the substrate (4-methylumbelliferone-conjugated β-D-glucopyranoside) to intact cells followed by quantification of liberated 4-methylumbelliferone. In this assay, cells were pretreated with or without a high concentration (0.5 mM) of conduritol B epoxide (CBE), an irreversible inhibitor of lysosomal β-Glu, before exposure to the substrate. In wild-type cells, more than 95% of the total cellular enzyme activity was sensitive to CBE, suggesting that the degradation was mostly due to lysosomal enzyme activity. There were no significant differences in the absolute values of the CBE-insensitive activity (i.e., non-lysosomal enzyme activity) between control and GD cells (data not shown).

In accordance with the results of ex vivo enzyme assay, NOV was effective in cells with mutations of F213I/F213I, N188S/G193W, G202R/L444P and N370S/N370S, but not in cells with mutations of L444P/RecNcil, L444P/L444P and D409H/unknown (Fig. 1b). In contrast to the results of ex vivo enzyme assay, however, NOV was clearly effective on N370S/84GG cells. We also evaluated EEA of NN-DNJ and found significant effects of this compound in cells with mutations of N370S/N370S and N370S/84GG. This result was in accordance with the report by Sawkar et al. [START_REF] Sawkar | Gaucher disease-associated glucocerebrosidases show mutationdependent chemical chaperoning profiles[END_REF] who found EEA of NN-DNJ in cells with homozygous mutations of N370S and G202R. NN-DNJ was also effective in cells with mutations of F213I/F213I. Although the increases were not statistically significant, NN-DNJ elicited weak EEA in cells with mutations of N188S/G193W and G202R/L444P, whereas it was not at all effective in cells with mutations of L444P/RecNcil, L444P/L444P and D409H/unknown. Thus, it appeared that the two compounds, NOV and NN-DNJ, shared the same selectivity on β-Glu mutations. In addition, neither NOV nor NN-DNJ affected the levels of CBE-insensitive, non-lysosomal enzyme activity (data not shown).

Given the similar effects of NOV and NN-DNJ, we examined whether these two compounds acted synergistically or not. When F213I/F213I cells were exposed to NOV and NN-DNJ (both at 3 μM) simultaneously, the effects were not additive, but EEA of NOV was rather diminished (Fig. 1b). Similar findings were reproduced in N188S/G193W and G202R/L444P cells. In two lines of N370 homozygous cells and N370S/84GG cells, NN-DNJ did not reduce EEA of NOV, but again the effects of these two compounds were not additive. To further confirm this lack of co-operation, we evaluated in vitro inhibitory activity of NN-DNJ in the presence or absence of NOV and found that NOV caused a dose-dependent decrease in the efficacy of NN-DNJ (Fig. 1c). The IC 50 values for NN-DNJ were 0.31 + 0.03 and 1.6 + 0.31 μM (means + SEM, n=3), in the absence and presence of NOV (3 μM), respectively. This NOV effect was not specific for NN-DNJ but a similar decrease in the inhibitory activity was also observed for CBE. As a negative control, N-butyl-β-valienamine, which has a very weak in vitro inhibitory activity (see Fig. 4), failed to affect the activities of NN-DNJ and CBE.

EEA of NOV on recombinant mutant β-Glu expressed in COS cells

Primary-cultured human cells have several disadvantages for evaluation of EEA, including their genetic heterogeneity and heterozygousity of the mutations. For example, we could not tell from the positive effect of NOV in N188S/G193W cells which of the two mutants (or both of them) responded to NOV. As an alternative method to evaluate EEA, we used heterologous expression of recombinant β-Glu in COS cells. A problem with this system was the endogenous β-Glu activity of COS cells. To circumvent this problem, we placed a Flag-epitope at the C-terminus of recombinant β-Glu and determined the enzyme activity recovered in anti-Flag immunoprecipitates. Practically, 24 h after transfection, the cells were exposed to NOV for another 24 h, and cell lysates were subjected to anti-Flag M2 immunoprecipitation. As shown in Fig. 2a, the immunoprecipitates from mock-transfected cells contained virtually no activity whereas those from cells transfected with mutant Flag-β-Glu contained various levels of activities. The relative levels of the activities in immunoprecipitates (Fig. 2a, right panel) faithfully reflected the relative levels in lysates (left panel), suggesting similar efficacies of immunoprecipitation between the different constructs.

By using this immunoprecipitation/enzyme assay, we evaluated EEA of NOV on the wild-type and 6 kinds of β-Glu mutants and found dose-dependent, positive effects on N188S, G202R, F213I and N370S mutants, and negative effects on G193W and L444P mutants (Fig. 2b). The profiles of dose-dependence were different from those in human cells: with the exception of N188S, the maximum effects of NOV were obtained at 10 μM, the highest concentration applied. The effect on the wild-type β-Glu was negative. EEA of NOV on the 4 kinds of mutants were statistically significant as analyzed using the data at an NOV concentration of 10 μM (Fig. 2c). Anti-Flag Western blotting of immunoprecipitation products showed that NOV caused dosedependent increases in the protein levels of N188S, G202R and F213I mutants. The protein levels

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ACCEPTED MANUSCRIPT 8 of N370S mutant was marginally increased by NOV, whereas the levels of G193W and L444P mutants were not at all affected (Fig. 2b).

Intracellular localization and processing of recombinant β-Glu expressed in COS cells

In our previous study using F213I homozygous human cells [START_REF] Lin | N-Octyl-β-valienamine up-regulates activity of F213I mutant β-glucosidase in cultured cells: a potential chemical chaperone therapy for Gaucher disease[END_REF], we could show by immunofluorescence and cell fractionation experiments that NOV restored lysosomal localization of the mutant β-Glu. Having shown EEA of NOV on the recombinant proteins expressed in COS cells, we examined whether there were any differences in the intracellular localization and processing of the proteins between the wild-type and mutants, and whether NOV caused any alterations.

Anti-Flag immunofluorescence of the wild-type Flag-β-Glu showed reticular distribution throughout the cytosol, indicating its predominant localization in the ER. The anti-Flag signals showed little co-localization with Lysotracker red (Fig. 3a, upper panels). This distribution was in contrast to lysosomal localization of the endogenous, wild-type β-Glu in human fibroblasts [START_REF] Lin | N-Octyl-β-valienamine up-regulates activity of F213I mutant β-glucosidase in cultured cells: a potential chemical chaperone therapy for Gaucher disease[END_REF], but was not specific to COS cells. Similar intracellular distribution was reproduced in CHO, Hela and HEK293 cells (data not shown). This distribution was neither specific for the Flag-tagged protein because similar distribution was observed for recombinant proteins tagged with a myc-epitope or GFP (data not shown).

Like the wild-type protein, F213I mutant Flag-β-Glu (Fig. 3a, middle panels) as well as other mutant proteins (data not shown) were mainly localized in the ER and did not co-localize with Lysotracker red. To confirm this localization, we tested sensitivity of expressed proteins to Endo-H digestion (Fig. 3b). Anti-Flag Western blotting of the wild-type Flag-β-Glu gave two bands: the major band at 66 kDa and the minor band at 62 kDa, which represented the immature ER form and the mature post-Golgi form, respectively [START_REF] Ron | ER retention and degradation as the molecular basis underlying Gaucher disease heterogeneity[END_REF]. As expected, Endo-H digestion abolished the 66 kDa band and yielded a band at 58 kDa, which corresponds to the nonglycosylated protein, whereas it barely affected the 62 kDa band. The same experiments on mutant proteins revealed similar results, confirming predominant localization in the ER of both wild-type and mutant proteins in COS cells.

NOV treatment of transfected cells failed to alter the intracellular distribution and processing of expressed proteins, as assessed by anti-Flag immunofluorescence (Fig. 3a, lower panels) and Endo-H digestion (Fig. 3b). These findings suggested that the site of action of NOV was the ER, consistent with our assumption that it acted as a pharmacological chaperone to protect mutant proteins from ERAD. However, these results also indicated that the heterologous expression was inappropriate as an experimental system to assess lysosomal transport of rescued proteins.

EEA of N-alkyl-valienamines related to NOV

All of the pharmacological chaperones for mutant lysosomal enzymes described so far are inhibitors of the target enzyme [START_REF] Cohen | Therapeutic approaches to protein-misfolding diseases[END_REF]. The hypothesis "the best chaperone is the best inhibitor" postulates that the activity of a molecule as a chaperone depends on its binding affinity to the target enzyme, and hence on its inhibitory activity [START_REF] Fan | Accelerated transport and maturation of lysosomeal α-galactosidase A in Fabry lymphoblasts by an enzyme inhibitor[END_REF]. We have shown previously that the in vitro inhibitory activity of N-alkyl-β-valienamines against β-Glu depended on the number and the length of the acyl chain [START_REF] Ogawa | Synthesis of potent β-D-glucocerebrosidase inhibitors: N-alkyl-β-valienamines[END_REF][START_REF] Ogawa | Chemical modification of β-glucocerebrosidase inhibitor N-octyl-β-valienamine: synthesis and biological evaluation of N-alkanoyl and N-alkyl derivatives[END_REF]. To examine whether the above hypothesis holds true for N-alkylβ-valienamines, we prepared compounds with various lengths of the acyl chain and compared their in vitro inhibitory activities and EEA. Compounds a~e listed in Fig. 4a contained acyl chain with various lengths, whereas compound f contained double acyl chains. Compound g carried a glucopyranosyl head group instead of a valienamine head group, and was included as a negative control.

In vitro inhibition experiments using cell lysates from control human fibroblasts revealed that compound f with double acyl chains was the best β-Glu inhibitor among these compounds, with the following rank order of potency: f > b, e > c, d > a, g (Fig. 4b, and the IC 50 values were given in Fig. 4a). Immunoprecipitation/enzyme assays using COS cells transfected with F213I mutant Flag-β-Glu showed that compound c (N-dodecacyl-β-valienamine) exhibited EEA, which was as potent as that of compound b (NOV). Compound d exhibited week EEA, whereas compounds e and f rather suppressed the enzyme activity. Compounds a and g, both of which had a weak in vitro inhibitory activity, failed to alter the enzyme activity of the F213I mutant (Fig. 4c, and the relative efficacies were summarized in Fig. 4a). These findings indicated that the simple "the best chaperone is the best inhibitor" hypothesis did not hold true for N-alkylβ-valienamines, and suggested that the presence of a single acyl chain with an appropriate length (i.e., carbon numbers between 8 and 14) was necessary for them to act as a chaperone for mutant β-Glu.

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Discussion

In the current study, we evaluated EEA of NOV in human GD cells using two assays: ex vivo enzyme assay (Fig. 1a) and intact cell enzyme assay (Fig. 1b). With the exception of N370S/84GG cells, these two assays gave the same results: NOV exhibited EEA in 5 GD cell lines with mutations of F213I/F213I, N188S/G193W, G202R/L444P and N370S/N370S, whereas it was ineffective in cells with mutations of L444P/L444P, L444P/RecNciI and D409H/unknown. It was also ineffective in cells with mutations of nt1447del20insTG/L444P and nt1447del20insTG/unknown, in ex vivo enzyme assay. NOV was only marginally effective in N370S/84GG cells as assessed by ex vivo enzyme assay whereas it caused clear EEA in the same cells as assessed by intact cell enzyme assay. The precise reason for this discrepancy was left unknown, but it may suggest that intact cell enzyme assay was more sensitive than ex vivo enzyme assay to detect EEA. Using heterologous expression of recombinant Flag-tagged β-Glu in COS cells, we could further confirm EEA of NOV (Fig. 2). It was effective on N188S, G202R, F213I and N370S mutants but was ineffective on G193W and L444P mutants. These results confirmed the selective effects of NOV on individual mutants as expected from its effects in human cells. Besides, this analysis suggested that EEA of NOV on N188S/G193W human cells reflected its selective effect on N188S mutant. Anti-Flag Western blotting showed that NOV-induced increase in the enzyme activity was accompanied by increased protein levels of the mutant proteins, consistent with an action of NOV as a pharmacological chaperone. The identical genetic background of this heterologous expression system is a clear advantage over human cells to assess EEA. However, it also had a disadvantage that, unlike the endogenous protein, the expressed protein was retained in the ER (Fig. 3) and hence the lysosomal transport of rescued protein could not be assessed. Nonetheless, the heterologous expression system will be a useful alternative to evaluate EEA when human cells with specific mutations are not available.

Studies on genotype-phenotype relationships in human patients have shown that except for the N370S mutation, which is exclusively associated with type I, non-neuronopathic form, the other three mutations with positive responses to NOV (N188S, G202R, F213I), can be associated with type 2 or 3, neuronopathic forms [START_REF] Eto | Clinical and molecular characteristics of Japanese Gaucher disease[END_REF][START_REF] Zimmer | Intracellular transport of acid β-glucosidase and lysosome-associated membrane proteins is affected in Gaucher's disease (G202R mutation)[END_REF].

Recently, X-ray crystallography of human β-Glu revealed that it consisted of three structural domains [START_REF] Dvir | Xray structure of human acid-beta-glucosidase, the defective enzyme in Gaucher disease[END_REF]. Domain III contains the catalytic site, whereas the functional significance of domain I and II is left unknown. All the mutations with positive responses to NOV (N188S, G202R, F213I and N370S) were located in domain III, whereas those with negative responses, L444P and D409H were located in domain II and I, respectively. Since NOV is a structural mimic of the substrate, it is expected to bind to domain III. Therefore, localization of the mutations in domain III might be a prerequisite to pharmacological rescue of the mutant proteins by NOV. However, an alternative possibility remains that the selective effects of NOV simply reflected the degree of instability of individual mutant proteins.

By using intact cell enzyme assay in human GD cells, we compared effects of NOV and NN-DNJ and found similar responses of individual cells to the two compounds (Fig. 1b). This was not surprising, given that NN-DNJ was also a structural mimic of the substrate and was expected to act in a manner similar to NOV. An important finding was that these two compounds did not work synergistically, but simultaneous addition of the two rather suppressed the action of each compound. Although the molecular mechanism for this lack of co-operation was left unknown, suppression of in vitro inhibitory activity of NN-DNJ by NOV (Fig. 1c) suggested that 11 the presence of NOV hindered the binding of NN-DNJ to the enzyme, and vice versa.

We have shown that the optimal concentration of the hydrochloride form of NOV to elicit EEA on F213I homozygous cells was 10 times lower than that of the non-hydrochloride form, most likely because of its better solubility in the medium (Fig. 1a). By profiling the activities of N-alkyl-β-valienamines, we also found that N-dodecacyl-β-valienamine elicited EEA as potent as that of NOV (Fig. 4). In addition, NOV appeared to work better than NN-DNJ at the same concentration, at least in intact cell enzyme assay using human cells (Fig. 1b). These findings in cultured cells, however, do not allow us to predict which compound works best as a pharmacological chaperone in whole animals, and hence has the best therapeutic value. To prove the activity of N-octyl-β-epi-valienamine, an isomer of NOV, as a pharmacological chaperone for mutant β-galactosidase, we developed transgenic mice that lacked the endogenous wild-type enzyme and instead expressed a mutant human enzyme [START_REF] Matsuda | Chemical chaperone therapy for brain pathology in G(M1)-gangliosidosis[END_REF]. The same strategy will be used to confirm EEA of NOV and related compounds in whole animals, and to determine which compound works best as a pharmacological chaperone for mutant β-Glu.

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LegendsFig. 1

 1 D M A N U S C R I P T EEA of NOV and NN-DNJ on mutant β-Glu in GD fibroblasts. a. Ex vivo enzyme assay. 9 lines of GD cells were cultured for 4 days in the absence or presence of increasing concentrations of NOV and β-Glu activity in cell lysates was determined. The data from cells with positive responses to NOV were shown in the upper panel whereas those with negative responses were shown in the lower panel. Each point represents the mean + SEM of 3 determinations each done in triplicate. Red marks indicate that the values are statistically different from the values in the absence of the drug (* p<0.05, t test). b. Intact cell enzyme assay. GD cells were cultured for 4 days in the absence or presence of NOV and/or NN-DNJ (both at 3 μM). Lysosomal β-Glu activity was estimated in intact cells as described in Material and Methods. Each bar represents the mean + SEM of 3 determinations each done in triplicate. * p<0.05, statistically different from the values in the absence of the drug (t test). c. Effects of NOV on in vitro inhibitory activities of NN-DNJ and CBE. Inhibitory activities of NN-DNJ and CBE against β-Glu in lysates from a control cells were determined in the absence or presence of NOV (upper panels). As a control, the same experiments were repeated with N-butyl-β-valienamine (lower panels). Each point represents the mean of duplicates obtained in a single experiment. Similar results were reproduced twice.

Fig. 2

 2 Fig.2Effects of NOV on recombinant β-Glu expressed in COS cells. COS cells were transfected with Flag-tagged β-Glu constructs. 24 h post-transfection, cells were treated with or without NOV for 24 h. a. Basal activities of β-Glu in cell lysates (left panel) and anti-Flag immunoprecipitation (IP) products (right panel). The activities were expressed as relative to that of the wild-type protein (100%). The values for the wild-type protein in lysates and IP products were 132 + 16 and 310 + 12 nmol/mg protein/h (means + SEM, n=3), respectively. b, c. Effects of NOV on protein levels and activities of recombinant β-Glu in anti-Flag IP products. In b, cells were treated with or without increasing concentrations of NOV and the IP products were subjected to in vitro enzyme assay or anti-Flag immunoblotting as described in Materials and Methods. Molecular weights are given on the left (kDa). The data with NOV concentrations at 0 and 10 μM were depicted in a bar graph in c for comparison of NOV effects on individual mutants. All in a-c, each bar or point represents mean + SEM of more than 3 determinations. * p<0.05, statistically different from the values in the absence of the drug (t test).

Fig. 3

 3 Fig.[START_REF] Cox | Novel oral treatment of Gaucher's disease with Nbutyldeoxynojirimycin (OGT918) to decrease substrate biosynthesis[END_REF] Intracellular localization and processing of recombinant β-Glu expressed in COS cells. a. Immunofluorescence. Cells were transfected with wild-type Flag-β-Glu (upper panels) or F213I mutant. Cells transfected with F213I mutant were cultured for 24 h in the absence (middle panels) or presence (lower panels) of NOV (10 μM). They were exposed to Lysotracker red before fixation and stained with anti-Flag antibody. Bound antibody was visualized with Alexa488-conjugated secondary antibody. Shown are the images obtained with a confocal microscope. b. Endo-H sensitivity. Lysates from cells transfected with each Flag-β-Glu construct were subjected to Endo-H digestion followed by SDS-PAGE and anti-Flag immunoblotting (upper panel). Cells transfected with wild-type Flag-β-Glu or F213I mutant were treated with or without NOV (lower panel). Molecular weights are given on the left (kDa). All results shown were representative and were reproduced at least twice.

Fig. 4

 4 Fig. 4 Profiling of N-alkyl-β-valienamines. a. Chemical structures of the compounds evaluated in

  Materials and methodsMaterials -Dulbecco's Modified Eagle's Medium (DMEM), bovine calf serum (BCS) and LipofactAMINE reagent were obtained from Life Technologies Inc. N-alkyl-β-valienamine hydrochlorides were synthesized in our laboratory (Central Research Laboratories, Seikagaku Co.). Stock solution of the compounds was prepared in H 2 O at 3 mM and stored at -20 o C. Anti-Flag M2 affinity gel and rabbit polyclonal anti-Flag antibody were from Sigma.

Acknowledgements

We thank Kaneski C. and Brady R.O. for human GD fibroblasts with N370S homozygous mutations (DMN00.41 and DMN87.30) and Tsuji S. for human β-Glu cDNA.

Footnotes

Abbreviations: β-Glu; β-glucosidase, NOV; N-octyl-β-valienamine, ER, endoplasmic reticulum, GD; Gaucher disease, NN-DNJ; N-nonyl-deoxynojirimaycin.

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