TRP-ML1 Is a Lysosomal Monovalent Cation Channel That Undergoes Proteolytic Cleavage

Mutations in the gene MCOLN1 coding for the TRP (transient receptor potential) family ion channel TRP-ML1 lead to the lipid storage disorder mucolipidosis type IV (MLIV). The function and role of TRP-ML1 are not well understood. We report here that TRP-ML1 is a lysosomal monovalent cation channel. Both native and recombinant TRP-ML1 are cleaved resulting in two products. Recombinant TRP-ML1 is detected as the full-length form and as short N- and C-terminal forms, whereas in native cells mainly the cleaved N and C termini are detected. The N- and C-terminal fragments of TRP-ML1 were co-immunoprecipitated from cell lysates and co-eluted from a Ni2+ column. TRP-ML1 undergoes proteolytic cleavage that is inhibited by inhibitors of cathepsin B (CatB) and is altered when TRP-ML1 is expressed in CatB-/- cells. N-terminal sequencing of purified C-terminal fragment of TRP-ML1 expressed in Sf9 cells indicates a cleavage site at Arg200 ↓ Pro201. Consequently, the conserved R200H mutation changed the cleavage pattern of TRP-ML1. The cleavage inhibited TRP-ML1 channel activity. This work provides the first example of inactivation by cleavage of a TRP channel. The significance of the cleavage to the function of TRP-ML1 is under investigation. Mutations in the gene MCOLN1 coding for the TRP (transient receptor potential) family ion channel TRP-ML1 lead to the lipid storage disorder mucolipidosis type IV (MLIV). The function and role of TRP-ML1 are not well understood. We report here that TRP-ML1 is a lysosomal monovalent cation channel. Both native and recombinant TRP-ML1 are cleaved resulting in two products. Recombinant TRP-ML1 is detected as the full-length form and as short N- and C-terminal forms, whereas in native cells mainly the cleaved N and C termini are detected. The N- and C-terminal fragments of TRP-ML1 were co-immunoprecipitated from cell lysates and co-eluted from a Ni2+ column. TRP-ML1 undergoes proteolytic cleavage that is inhibited by inhibitors of cathepsin B (CatB) and is altered when TRP-ML1 is expressed in CatB-/- cells. N-terminal sequencing of purified C-terminal fragment of TRP-ML1 expressed in Sf9 cells indicates a cleavage site at Arg200 ↓ Pro201. Consequently, the conserved R200H mutation changed the cleavage pattern of TRP-ML1. The cleavage inhibited TRP-ML1 channel activity. This work provides the first example of inactivation by cleavage of a TRP channel. The significance of the cleavage to the function of TRP-ML1 is under investigation. Mucolipidosis type IV (MLIV) 2The abbreviations used are: MLIVmucolipidosis type IVHAhemagglutininIPimmunoprecipitationNi2+-NTAnickel-nitrilotriacetic acidFLfull-lengthTRPtransient receptor potentialEndo FN-glycosidase FEndo Hβ-endo-N-acetylglucosaminidase HHSFhuman skin fibroblastCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidWTwild typeCatcathepsinPMplasma membrane. is a lipid storage disorder characterized by an abnormal accumulation of membranous lipids in patients' cells (reviewed in Refs. 1Slaugenhaupt S.A. Curr. Mol. Med. 2002; 2: 445-450Crossref PubMed Scopus (65) Google Scholar and 2Bach G. Mol. Genet. Metab. 2001; 73: 197-203Crossref PubMed Scopus (153) Google Scholar). Clinically, the disease manifests as corneal clouding, degeneration of the retina, and severe psychomotor retardation (1Slaugenhaupt S.A. Curr. Mol. Med. 2002; 2: 445-450Crossref PubMed Scopus (65) Google Scholar, 2Bach G. Mol. Genet. Metab. 2001; 73: 197-203Crossref PubMed Scopus (153) Google Scholar, 3Bonavita S. Virta A. Jeffries N. Goldin E. Tedeschi G. Schiffmann R. J. Child Neurol. 2003; 18: 443-449Crossref PubMed Scopus (22) Google Scholar, 4Smith J.A. Chan C.C. Goldin E. Schiffmann R. Ophthalmology. 2002; 109: 588-594Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 5Frei K.P. Patronas N.J. Crutchfield K.E. Altarescu G. Schiffmann R. Neurology. 1998; 51: 565-569Crossref PubMed Scopus (73) Google Scholar, 6Chitayat D. Meunier C.M. Hodgkinson K.A. Silver K. Flanders M. Anderson I.J. Little J.M. Whiteman D.A. Carpenter S. Am. J. Med. Genet. 1991; 41: 313-318Crossref PubMed Scopus (56) Google Scholar). MLIV is associated with mutations in MCOLN1 (TRP-ML1), a member of the TRP (transient receptor potential) family of ion channels (7Sun M. Goldin E. Stahl S. Falardeau J.L. Kennedy J.C. Acierno Jr., J.S. Bove C. Kaneski C.R. Nagle J. Bromley M.C. Colman M. Schiffmann R. Slaugenhaupt S.A. Hum. Mol. Genet. 2000; 9: 2471-2478Crossref PubMed Scopus (342) Google Scholar, 8Bassi M.T. Manzoni M. Monti E. Pizzo M.T. Ballabio A. Borsani G. Am. J. Hum. Genet. 2000; 67: 1110-1120Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 9Bargal R. Avidan N. Ben-Asher E. Olender Z. Zeigler M. Frumkin A. Raas-Rothschild A. Glusman G. Lancet D. Bach G. Nat. Genet. 2000; 26: 118-123Crossref PubMed Scopus (301) Google Scholar). The TRP family includes several members that are implicated in human diseases, such as TRPP2 (10Cai Y. Maeda Y. Cedzich A. Torres V.E. Wu G. Hayashi T. Mochizuki T. Park J.H. Witzgall R. Somlo S. J. Biol. Chem. 1999; 274: 28557-28565Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar), TRPM1 (11Duncan L.M. Deeds J. Hunter J. Shao J. Holmgren L.M. Woolf E.A. Tepper R.I. Shyjan A.W. Cancer Res. 1998; 58: 1515-1520PubMed Google Scholar), and TRPV6 (12Fixemer T. Wissenbach U. Flockerzi V. Bonkhoff H. Oncogene. 2003; 22: 7858-7861Crossref PubMed Scopus (204) Google Scholar). A critical question in MLIV pathogenesis is why do mutations in TRP-ML1 lead to the cellular phenotype of MLIV? mucolipidosis type IV hemagglutinin immunoprecipitation nickel-nitrilotriacetic acid full-length transient receptor potential N-glycosidase F β-endo-N-acetylglucosaminidase H human skin fibroblast 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid wild type cathepsin plasma membrane. Previous work on the ion selectivity and permeation of TRP-ML1 produced conflicting results. Thus, transient expression in Xenopus oocytes and in fibroblasts suggests that TRP-ML1 is targeted to the lysosomes and functions as a Ca2+-permeable channel that may regulate lysosomal Ca2+ release and consequently agonist-evoked Ca2+ signals (13LaPlante J.M. Ye C.P. Quinn S.J. Goldin E. Brown E.M. Slaugenhaupt S.A. Vassilev P.M. Biochem. Biophys. Res. Commun. 2004; 322: 1384-1391Crossref PubMed Scopus (91) Google Scholar, 14LaPlante J.M. Falardeau J. Sun M. Kanazirska M. Brown E.M. Slaugenhaupt S.A. Vassilev P.M. FEBS Lett. 2002; 532: 183-187Crossref PubMed Scopus (132) Google Scholar). On the other hand, TRP-ML1 synthesized in cell-free system and reconstituted into planar lipid bilayers behaves as a monovalent cations permeable, outwardly rectifying channel (15Raychowdhury M.K. Gonzalez-Perrett S. Montalbetti N. Timpanaro G.A. Chasan B. Goldmann W.H. Stahl S. Cooney A. Goldin E. Cantiello H.F. Hum. Mol. Genet. 2004; 13: 617-627Crossref PubMed Scopus (117) Google Scholar). The outward rectification indicates that when present in lysosomes, TRP-ML1 primarily moves ions into the lysosomal lumen. The outward rectification makes it unlikely that in vivo TRP-ML1 would function as a lysosomal Ca2+ release channel, which suggested an alternative role of TRP-ML1 in lysosomal and cellular functions. In the present report we analyzed the expression pattern and channel properties of TRP-ML1 and several disease-associated mutants. We report that TRP-ML1 is an outwardly rectifying monovalent cation-permeable channel that is primarily expressed in the lysosomes. In the lysosomes, TRP-ML1 is inactivated by proteolytic cleavage. These findings suggest a novel mechanism of regulating TRP-ML1 function. Materials—The DNA-modifying enzymes N-glycosidase F (Endo F), and β-endo-N-acetylglucosaminidase H (Endo H) were from New England Biolabs. QuikChange site-directed mutagenesis kit was from Stratagene. Cathepsin B inhibitors were from Calbiochem, and cathepsin B was from Sigma. CatB-/- cells were generously provided by Dr. Terence S. Dermod (Vanderbilt University, Nashville, TN). TRP-ML1-/- Cells—Human skin fibroblasts (HSF), clone WG0909, that is TRP-ML1-/-, and the WG0987 clone, a heterozygous relative, were obtained from the Repository for Mutant Human Cell Strains, Montreal Children's Hospital. Fibroblasts were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, l-glutamine, and non-essential amino acids. TRP-ML1 Expression Constructs—The full-length sequence corresponding to the human TRP-ML1 coding region was amplified by PCR using IMAGE clone BF 529860 as template. The 1.7-kb amplified product was subcloned into the pCMV vectors either with no tag, an N-terminal HA tag, or a C-terminal Myc tag. Insert orientation and polymerase fidelity were verified by restriction enzyme mapping and sequencing. Site-directed Mutagenesis, Cell Transfection, and Immunoblotting—The plasmid pCMV-HA-TRP-ML1 was used as a template to construct mutants using a mutagenesis kit (QuikChange, Stratagene). All mutations were confirmed by sequencing the entire DNA insert to verify the presence of the desired mutation and the absence of extraneous mutations. HEK293 cells were transfected in 60-mm dishes with 5 μg of plasmid DNA and 10 μl of Lipofectamine 2000 (Invitrogen). Cell extracts were prepared by sonication in homogenization buffer containing 50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 2 mm EDTA, 5 mm MgCl2, and Complete protease inhibitor mixture tablet (Roche Applied Science). Microsomal pellets were extracted with 1% CHAPS or 1% Triton X-100 and subjected to SDS-PAGE and immunoblotting with anti-HA, anti-Myc, or anti-TRP-ML1 antibodies raised in rabbits against the N-terminal sequence TAPAGPRGSETERLLTPN (αN1) or against the C-terminal sequence CGRDPSEEHSLLVN (αC1). The specificity of the anti-TRP-ML1 antibodies was verified by recognition of the transfected protein, by blocking the signal with the peptides used to raise the specific antibodies and by the absence of a specific signal in TRP-ML1-/- cells. Electrophysiology—For conventional whole cell recording, cells grown on coverslips were placed in a perfusion chamber that was secured on the stage of an Olympus IX50 inverted microscope equipped with a fluorescent illuminator and filters designed to identify green fluorescent protein-expressing cells. Transmembrane currents were recorded using an Axopatch 200D amplifier, stored in a PC, and analyzed with PClamp6 and Origin software. In the whole cell mode, the pipette solution contained (in mm) 140 cesium aspartate (to cancel endogenous K+ and Cl- conductance), 5 NaCl, 5 Mg-ATP, 10 HEPES, 2 EGTA, or 10 BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid), pH 7.2. The standard bath solution contained (in mm): 140 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, pH 7.5. In some experiments, NaCl was replaced with N-methyl-d-glucamine, and 0 or 10 mm divalent metals were included in the bath solutions. The experiments were performed at room temperature. Confocal Immunocytochemistry—Cells grown on glass coverslips were fixed and permeabilized by a 10-min incubation at -20 °C with 100% methanol or were fixed by a 5-min incubation with 3.7% formaldehyde and permeabilized by incubation with 0.01% Triton X-100 at 4 °C for 5 min. After fixation, nonspecific sites were blocked by incubation in 5% goat serum. Subsequently the cells were incubated with the primary antibodies in blocking solution. Following washout of the primary antibodies with phosphate-buffered saline, the cells were stained with fluorescent secondary antibodies and analyzed using a Bio-Rad 1024 confocal microscope. The images were recorded with a ×40 objective and analyzed off-line using NIH Image™ software. Localization of WT TRP-ML1 and Mutants—Expression in HeLa cells suggested primarily lysosomal localization of TRP-ML1 (16Manzoni M. Monti E. Bresciani R. Bozzato A. Barlati S. Bassi M.T. Borsani G. FEBS Lett. 2004; 567: 219-224Crossref PubMed Scopus (69) Google Scholar). To verify TRP-ML1 localization, HA-tagged human TRP-ML1 (HA-TRP-ML1) was expressed in HEK293 cells, HeLa cells, and HSF. Fig. 1 shows that WT TRP-ML1 is primarily present in intracellular compartments. To identify the compartment in which TRP-ML1 is localized, cells transfected with HA-TRP-ML1 were co-stained with anti-HA antibodies and antibodies against EEA1, the mannose 6-phosphate receptors, or LAMP1 as markers for early endosomes, late endosomes/Golgi, and lysosomes, respectively. Fig. 1, A and B, shows that TRP-ML1 co-localized with mannose 6-phosphate receptors (MPR) only in the Golgi, probably because of TRP-ML1 over-expression. Significant overlap of TRP-ML1 was found only with LAMP1, confirming its lysosomal localization. Several disease-associated TRP-ML1 mutants have been identified (7Sun M. Goldin E. Stahl S. Falardeau J.L. Kennedy J.C. Acierno Jr., J.S. Bove C. Kaneski C.R. Nagle J. Bromley M.C. Colman M. Schiffmann R. Slaugenhaupt S.A. Hum. Mol. Genet. 2000; 9: 2471-2478Crossref PubMed Scopus (342) Google Scholar, 8Bassi M.T. Manzoni M. Monti E. Pizzo M.T. Ballabio A. Borsani G. Am. J. Hum. Genet. 2000; 67: 1110-1120Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 17Bargal R. Avidan N. Olender T. Ben Asher E. Zeigler M. Raas-Rothschild A. Frumkin A. Ben-Yoseph O. Friedlender Y. Lancet D. Bach G. Hum. Mutat. 2001; 17: 397-402Crossref PubMed Scopus (63) Google Scholar, 18Bargal R. Goebel H.H. Latta E. Bach G. Neuropediatrics. 2002; 33: 199-202Crossref PubMed Scopus (25) Google Scholar). The expression pattern of some of these mutants is shown in Fig. 1C. The T232P and D362Y mutations resulted in retention of TRP-ML1 in the endoplasmic reticulum (Fig. 1C). Hence, these mutations cause MLIV probably because they are not targeted to the lysosomes. On the other hand the expression pattern of the F465L mutant was identical to that of WT TRP-ML1 (Fig. 1, D and E). As will shown mutation the channel of which why mutation in TRP-ML1 at a of TRP-ML1 with HA in its N (HA-TRP-ML1) in HEK293 cells resulted in two the full-length TRP-ML1 and a short form of (Fig. expression of TRP-ML1 with Myc in its C resulted in two (Fig. Hence, when a of TRP-ML1 is cleaved to in N- and C-terminal The the first and of TRP-ML1 sites (Fig. To TRP-ML1 is cleaved at or from the endoplasmic lysates from cells HA-TRP-ML1 and were either with which or with which only or that have not been by Fig. shows that the TRP-ML1 into a with only a of the was to with H. with not changed the of the N-terminal fragment of TRP-ML1. the and the N-terminal fragment of TRP-ML1 are to F are or to H. The C-terminal fragment was to The of the entire fragment was only changed by H (Fig. This may because the C-terminal fragment was not modified in the because the cleavage of TRP-ML1 is to the sites (Fig. it is that only of the sites on the C-terminal fragment was modified in the To these we transfected cells with a specific inhibitor of Biophys. 2002; PubMed Scopus Google Scholar). In the presence of the N- and C-terminal fragments to with H. Thus, it is that of the C-terminal fragment is modified in the Golgi, probably only of its sites is confirming that the cleavage the and TRP-ML1 sites and that TRP-ML1 is cleaved at a which is to the TRP-ML1 is in experiments with changed the cleavage of TRP-ML1 resulting in two N-terminal which suggests that may the cleavage of the channel. To the native TRP-ML1 is we raised antibodies against an N-terminal (αN1) and a C-terminal sequence of TRP-ML1. Fig. shows that detected primarily the short product in WT The specificity of is shown by a of specific in TRP-ML1-/- cells clone The in clone is to for expression M.T. Manzoni M. Monti E. Pizzo M.T. Ballabio A. Borsani G. Am. J. Hum. Genet. 2000; 67: 1110-1120Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). WT and TRP-ML1-/- cells with TRP-ML1 the and short not as specific as detected the C-terminal fragment of TRP-ML1 in WT fibroblast and not form of TRP-ML1 in TRP-ML1-/- cells (Fig. the short form of TRP-ML1 was detected in extracts from HEK293 cells and bovine (Fig. The of the short form in native cells indicates that the native is cleaved in the lysosomes. of the N and C termini of TRP-ML1 (Fig. suggests that they associated the proteolytic cleavage. of the C the N and of the N the C-terminal fragments in cell extracts (Fig. The the and N-terminal fragments is because of expressed in Sf9 cells of co-eluted fragments from the with buffer containing 1% CHAPS (Fig. of the TRP-ML1 cleavage expressed in Sf9 cells was purified using column. The purified resulted in of cleaved the is only at the C N-terminal sequencing of the purified C-terminal resulted in the sequence in B, and were transfected in HEK293 cells. the altered cleavage expression pattern of cells TRP-ML1 were with 2 or 10 inhibitor and the were analyzed by with The in and short TRP-ML1 of cell with from experiments is shown in the TRP-ML1 was expressed in WT and CatB-/- the altered cleavage pattern and of TRP-ML1 in CatB-/- of the the cleavage was expressed in Sf9 cells and purified on a column. The C-terminal fragment was subjected to N-terminal sequencing. In two experiments N-terminal sequencing the sequence corresponding to of that the cleavage was Arg200 and Pro201. the were not probably to the of the we verified the cleavage site by the sequence with these mutations not the they the cleavage pattern of TRP-ML1 (Fig. the conserved R200H altered the cleavage pattern (Fig. All mutations the potential cleavage site altered to cleavage. This is because in the lysosomes TRP-ML1 is cleaved by TRP-ML1 a lysosomal at the C In an to of TRP-ML1 to the lysosomes we the which includes the critical J.S. L.M. Biochem. 2003; PubMed Scopus Google Scholar). of sequence not TRP-ML1 localization (Fig. that to the lysosomal of TRP-ML1. the of sequencing and the mutation to Arg200 ↓ as a potential cleavage After the and N-terminal fragments to and in the cells. of in TRP-ML1 localization of TRP-ML1 and the of N- and C-terminal fragments to H raised the that TRP-ML1 is cleaved in the To identify the lysosomal protease that HEK293 cells transfected with TRP-ML1 were with 1 a protease and with a cathepsin B (CatB) inhibitor N. M. J. Biol. Chem. 2002; PubMed Scopus Google Scholar). The two inhibitors cleavage of TRP-ML1 (Fig. As a the cathepsin inhibitor not TRP-ML1 cleavage at as as 10 of cleavage by suggested that is in the cleavage of TRP-ML1. A role for was obtained by human TRP-ML1 in CatB-/- fibroblasts (Fig. We were not to the of the native TRP-ML1 in the CatB-/- fibroblasts because only the human Human TRP-ML1 was cleaved in CatB-/- the cleavage was at a site from that in WT fibroblasts as from the of an N-terminal fragment in TRP-ML1-/- cells. In the findings shown in Fig. 4 that TRP-ML1 is targeted to the lysosomes it is cleaved by or a is that TRP-ML1 is cleaved by lysosomal and the by at the Arg200 ↓ of and expression of TRP-ML1 in CatB-/- cells the cleavage by and the cleavage by other lysosomal resulting in the cleavage in Fig. In either cleavage by a role in the cleavage of TRP-ML1 in the lysosomes. the cleavage the of TRP-ML1 from the endoplasmic reticulum is confirmed by the of the mutants shown in Fig. T232P and D362Y were to by F and confirming that these mutants are in the endoplasmic reticulum or (Fig. Consequently, these mutants were not the F465L mutant was cleaved and the cleaved product was to with and of The that and of the F465L mutant was suggests that the channel function of TRP-ML1 is not for its The of WT TRP-ML1 and the of TRP-ML1 is expressed in intracellular that some of the WT and F465L TRP-ML1 was targeted to the plasma (Fig. no T232P and of the D362Y mutants were found at the plasma membrane. of the by expression of of the and short of TRP-ML1 and only the form of the T232P mutant at the plasma (Fig. was to TRP-ML1 channel properties using the whole cell TRP-ML1 was by whole cell with intracellular solution containing cesium aspartate and solution containing aspartate or these TRP-ML1 outwardly rectifying (Fig. A and The outward rectification indicates of ions from the into the lysosomal at and The of TRP-ML1 expressed in HEK293 cells is to the with cell-free synthesized TRP-ML1 reconstituted into lipid bilayers (15Raychowdhury M.K. Gonzalez-Perrett S. Montalbetti N. Timpanaro G.A. Chasan B. Goldmann W.H. Stahl S. Cooney A. Goldin E. Cantiello H.F. Hum. Mol. Genet. 2004; 13: 617-627Crossref PubMed Scopus (117) Google Scholar). as shown in Fig. is that expression of the F465L mutant not in channel expression and of the F465L mutant was to that of is to in the region of on the of sequence with other members of the TRP channel family G. M. M. R. E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, T. A. G. B. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar), and the F465L mutation channel Hence, the T232P and D362Y are the F465L is a channel to the disease TRP-ML1 by that the two fragments of TRP-ML1 associated (Fig. and that and short TRP-ML1 were present at the (Fig. B and raised the question of which form of TRP-ML1 the and is the role of the channel cleavage. The first that the cleavage TRP-ML1 was obtained by the in cells the T232P the T232P mutant (Fig. A and not as as cells WT TRP-ML1. only the T232P was present at the (Fig. the TRP-ML1 by the that cleavage TRP-ML1 is in Fig. In the first of experiments, TRP-ML1 expressed in HEK293 cells was with the whole cell Fig. A and B, shows that of with or recombinant at pH the by The cells were incubated at pH 5 for several of TRP-ML1 channel was detected TRP channel, was used as a for the specificity of the of were with at pH and the of the by of the receptors with was in cells and cells with with not the of the (Fig. In the of experiments HEK293 cells TRP-ML1 were with the inhibitor that of which the cleavage to the of TRP-ML1 (Fig. the of TRP-ML1 at the plasma (Fig. This with an (Fig. Hence, the with the T232P with and the of the inhibitor suggest that the cleavage to TRP-ML1 channel function. report here that TRP-ML1 is a cation channel that is inactivated by cleavage. The cleavage may by lysosomal of which is which to the critical or cleavage. native TRP-ML1 is the cleaved form is the form of the channel found in native cells. The cleavage to the channel function of TRP-ML1. The question that is why do cells to TRP-ML1 a role in lipid and as from the disease phenotype and as will shown in is that inactivation by cleavage a mechanism to the of TRP-ML1 channel activity. TRP-ML1 may to lipid TRP-ML1 is probably targeted to the lysosomes, its function is and it is inactivated by cleavage to of lysosomal which in the accumulation of the cleaved form of the channel. We Dr. for

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