Conserved Amino Acid Residues in the COOH-terminal Tail Are Indispensable for the Correct Folding and Localization and Enzyme Activity of Neutral Ceramidase
2004; Elsevier BV; Volume: 279; Issue: 28 Linguagem: Inglês
10.1074/jbc.m404012200
ISSN1083-351X
AutoresMotohiro Tani, Nozomu Okino, Noriyuki Sueyoshi, Makoto Ito,
Tópico(s)Glycosylation and Glycoproteins Research
ResumoSeveral lines of evidence suggest that neutral ceramidase is involved in the regulation of ceramide-mediated signaling. Recently, the enzymes from mouse and rat were found to be localized at plasma membranes as a type II integral membrane protein, occasionally being detached from the cells after proteolytic processing of the NH2-terminal anchoring region (Tani, M., Iida, H., and Ito, M. (2003) J. Biol. Chem. 278, 10523–10530). We report here that conserved hydrophobic amino acid residues in the COOH-terminal tail are indispensable for the correct folding and localization, and enzyme activity of neutral ceramidase. Truncation of four, but not three, amino acid residues from the COOH terminus of rat neutral ceramidase resulted in a complete loss of enzyme activity as well as cell surface expression in HEK293 cells. Point mutation analysis revealed that Ile758, the 4th amino acid residue from the COOH terminus, and Phe756 are essential for the enzyme to function. The truncated and mutated enzymes were found to be retained in the endoplasmic reticulum (ER) and rapidly degraded without transportation to the Golgi apparatus. Treatment of the cells expressing the aberrant COOH-terminal enzyme with MG-132, a specific inhibitor for the proteasome, increased the accumulation of the enzyme in the ER, indicating that the misfolded enzyme was degraded by the proteasome. It was also found that the COOH-terminal tail was indispensable for the enzyme activity and correct folding of the prokaryote ceramidase from Pseudomonas aeruginosa, indicating that the importance of the COOH-terminal tail of the enzyme has been preserved through evolution. Several lines of evidence suggest that neutral ceramidase is involved in the regulation of ceramide-mediated signaling. Recently, the enzymes from mouse and rat were found to be localized at plasma membranes as a type II integral membrane protein, occasionally being detached from the cells after proteolytic processing of the NH2-terminal anchoring region (Tani, M., Iida, H., and Ito, M. (2003) J. Biol. Chem. 278, 10523–10530). We report here that conserved hydrophobic amino acid residues in the COOH-terminal tail are indispensable for the correct folding and localization, and enzyme activity of neutral ceramidase. Truncation of four, but not three, amino acid residues from the COOH terminus of rat neutral ceramidase resulted in a complete loss of enzyme activity as well as cell surface expression in HEK293 cells. Point mutation analysis revealed that Ile758, the 4th amino acid residue from the COOH terminus, and Phe756 are essential for the enzyme to function. The truncated and mutated enzymes were found to be retained in the endoplasmic reticulum (ER) and rapidly degraded without transportation to the Golgi apparatus. Treatment of the cells expressing the aberrant COOH-terminal enzyme with MG-132, a specific inhibitor for the proteasome, increased the accumulation of the enzyme in the ER, indicating that the misfolded enzyme was degraded by the proteasome. It was also found that the COOH-terminal tail was indispensable for the enzyme activity and correct folding of the prokaryote ceramidase from Pseudomonas aeruginosa, indicating that the importance of the COOH-terminal tail of the enzyme has been preserved through evolution. Ceramide (N-acylsphingosine, Cer), 1The abbreviations used are: Cer, ceramide; CDase, ceramidase; CHOP cells, Chinese hamster ovary cells that express polyoma LT antigen; DMEM, Dulbecco's modified Eagle's medium; ER, endoplasmic reticulum; FBS, fetal bovine serum; GFP, green fluorescent protein; HRP, horseradish peroxidase; NBD, 4-nitrobenzo-2-oxa-1,3-diazole; PBS, phosphate-buffered saline; Sph, sphingosine; S1P, sphingosine 1-phosphate; HEK, human embryonic kidney cells; CHX, cycloheximide.1The abbreviations used are: Cer, ceramide; CDase, ceramidase; CHOP cells, Chinese hamster ovary cells that express polyoma LT antigen; DMEM, Dulbecco's modified Eagle's medium; ER, endoplasmic reticulum; FBS, fetal bovine serum; GFP, green fluorescent protein; HRP, horseradish peroxidase; NBD, 4-nitrobenzo-2-oxa-1,3-diazole; PBS, phosphate-buffered saline; Sph, sphingosine; S1P, sphingosine 1-phosphate; HEK, human embryonic kidney cells; CHX, cycloheximide. sphingosine (Sph), and sphingosine 1-phosphate (S1P) have emerged as a new class of lipid biomodulators for various cell functions (1Pettus B.J. Charlfant C.E. Hannun Y.A. Biochem. Biophys. Acta. 2002; 1585: 114-125Crossref PubMed Scopus (666) Google Scholar, 2Cuvillier O. Biochem. Biophys. Acta. 2002; 1585: 153-162Crossref PubMed Scopus (281) Google Scholar, 3Spiegel S. Kolesnick R. Leukemia. 2002; 16: 1596-1602Crossref PubMed Scopus (114) Google Scholar). Both Cer and Sph have been shown to induce growth arrest and apoptosis (1Pettus B.J. Charlfant C.E. Hannun Y.A. Biochem. Biophys. Acta. 2002; 1585: 114-125Crossref PubMed Scopus (666) Google Scholar, 2Cuvillier O. Biochem. Biophys. Acta. 2002; 1585: 153-162Crossref PubMed Scopus (281) Google Scholar), whereas S1P appears to promote cell growth and proliferation and suppress the apoptosis induced by Cer (3Spiegel S. Kolesnick R. Leukemia. 2002; 16: 1596-1602Crossref PubMed Scopus (114) Google Scholar). Consequently, the balance of the cellular contents of Cer/Sph/S1P is thought to regulate the diversity of cellular responses.Ceramidase (CDase, EC 3.5.1.23) is an enzyme that cleaves the N-acyl linkage of Cer to produce Sph and free fatty acid (4Gatt S. J. Biol. Chem. 1963; 238: 3131-3133Abstract Full Text PDF PubMed Google Scholar). Three categories of CDases: acid, neutral, and alkaline, are clearly distinguished not only by their catalytic pH optima, but also their primary structures (5Ito M. Okino N. Tani M. Mitsutake S. Kita K. Futerman A.H. Ceramide Signaling. Landes Bioscience, Georgetown, TX2003: 41-48Google Scholar). Neutral CDase, which shows an optimum pH of 6.5–8.5, has been cloned from bacteria (6Okino N. Ichinose S. Omori A. Imayama S. Nakamura T. Ito M. J. Biol. Chem. 1999; 274: 36616-36622Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), Drosophila (7Yoshimura Y. Okino N. Tani M. Ito M. J. Biochem. 2002; 132: 229-236Crossref PubMed Scopus (49) Google Scholar), mouse (8Tani M. Okino N. Mori K. Tanigawa T. Izu H. Ito M. J. Biol. Chem. 2000; 275: 11229-11234Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), rat (9Mitsutake S. Tani M. Okino N. Mori K. Ichinose S. Omori A. Iida H. Nakamura T. Ito M. J. Biol. Chem. 2001; 276: 26249-26259Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar), and human (10El Bawab S. Roddy P. Qian T. Bielawska A. Lemasters J.J. Hannun Y.A. J. Biol. Chem. 2000; 275: 21508-21513Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Accumulating evidence suggests that neutral CDase regulates the intracellular content of Cer and thereby Cer-mediated signaling. For example, an increase of Cer caused by the IL-1β-stimulated hydrolysis of SM was gradually normalized by up-regulation of neutral CDase as detected in mRNA and de novo synthesis levels after long term treatment with IL-1β, showing the function of neutral CDase as a cytoprotective enzyme in mesangial cells exposed to inflammatory stress (11Franzen R. Pautz A. Brautigam L. Geisslinger G. Pfeilschifter J. Huwiler A. J. Biol. Chem. 2001; 276: 35382-35389Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). In contrast, nitric oxide (NO) down-regulates the protein expression of neutral CDase via a protein kinase C-dependent mechanism in renal mesangial cells, resulting in an increase of Cer after treatment of the cells with NO (12Franzen R. Fabbro D. Aschrafi A. Pfeilschifter J. Huwiler A. J. Biol. Chem. 2002; 277: 46184-46190Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Very recently, the targeted expression of neutral CDase was found to reverse the retinal degradation by normalizing the Cer level in Drosophila arrestin and phospholipase C mutants (13Acharya U. Patel S. Koundakjian E. Nagashima K. Han X. Acharya J.K. Science. 2003; 299: 1740-1743Crossref PubMed Scopus (117) Google Scholar). These results suggest that neutral CDases may be suitable targets in the therapeutic management of cytokine-induced inflammation and retinal degeneration.Although the primary structure of neutral CDase is highly conserved from bacteria to mammals (8Tani M. Okino N. Mori K. Tanigawa T. Izu H. Ito M. J. Biol. Chem. 2000; 275: 11229-11234Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), the subcellular localization of the enzyme is quite different depending on its origin. Mammalian neutral CDases are present at the plasma membrane as a type II integral membrane protein, occasionally being detached from cells after proteolytic processing of the NH2-terminal anchoring region (14Tani M. Iida H. Ito M. J. Biol. Chem. 2003; 278: 10523-10530Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), whereas the bacterial and invertebrate enzymes are solely secretory proteins (7Yoshimura Y. Okino N. Tani M. Ito M. J. Biochem. 2002; 132: 229-236Crossref PubMed Scopus (49) Google Scholar, 15Okino N. Tani M. Imayama S. Ito M. J. Biol. Chem. 1998; 273: 14368-14373Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). This discrepancy may stem from whether a Ser/Thr-rich domain (mucin box) is present downstream of the NH2-terminal signal/anchor sequence of the enzyme, because all mammalian CDases possess a mucin box, whereas the bacterial and invertebrate enzymes do not, and a mutant mammalian CDase lacking the mucin box or possible O-glycosylation sites in the mucin box was secreted into the medium instead of localizing at the cell surface (14Tani M. Iida H. Ito M. J. Biol. Chem. 2003; 278: 10523-10530Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar).Very recently, a change in the COOH-terminal region of acid CDase was identified to be a novel mutation in Farber disease in which Cer is accumulated in lysosomes due to the inactivation of acid CDase (16Zhang Z. Mandal A.K. Mital A. Popescu N. Zimonjic D. Moser A. Moser H. Mukherjee A.B. Mol. Genet. Metab. 2000; 70: 301-309Crossref PubMed Scopus (26) Google Scholar). In the present study, we found that two conserved amino acid residues, Phe and Ile/Val, in the COOH-terminal domain were completely conserved in neutral CDases from bacteria to humans. Thus, we examine the role of the conserved COOH-terminal tail of neutral CDases using mammalian and bacterial enzymes. In conclusion, we demonstrated that two conserved amino acids in the COOH-terminal tail are indispensable for the correct folding and localization, and enzyme activity of neutral CDase.EXPERIMENTAL PROCEDURESMaterials—Anti-Rab6 antibody was kindly provided by Dr. S. Tanaka (Shizuoka University, Japan). HRP-labeled anti-mouse IgG antibody was purchased from Nacalai Tesque (Japan). Cy3-labeled anti-mouse IgG antibody, anti-FLAG M2 antibody, and benzyl-GalNAc were from Sigma. ECL plus, and HRP- and Cy3-labeled anti-rabbit IgG antibodies were obtained from Amersham Biosciences. MG-132 (benzylcarbonyl-Leu-Leu-Leu-aldehyde) and anti-Myc antibody were purchased from Calbiochem and Invitrogen, respectively. Anti-neutral CDase antibody was raised in a rabbit using the recombinant rat CDase as the antigen (9Mitsutake S. Tani M. Okino N. Mori K. Ichinose S. Omori A. Iida H. Nakamura T. Ito M. J. Biol. Chem. 2001; 276: 26249-26259Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). C12-NBD-Cer was prepared as described in Ref. 17Tani M. Kita K. Komori H. Nakagawa T. Ito M. Anal. Biochem. 1998; 263: 183-188Crossref PubMed Scopus (40) Google Scholar. HEK293 cells (JCRB9068, established by F. L. Graham) were obtained from the Human Science Research Resource Bank. All other reagents were of the highest purity available.CDase Assay—The hydrolysis and reverse hydrolysis activities of neutral CDase were measured using C12-NBD-Cer (for the hydrolysis reaction) and NBD-dodecanoic acid and Sph (for the reverse hydrolysis reaction) as substrates (18Tani M. Okino N. Mitsutake M. Tanigawa T. Izu H. Ito M. J. Biol. Chem. 2000; 275: 3462-3468Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar).Plasmid Construction—The vector pcDNA3.1/Myc-His(+) containing a full-length rat neutral CDase gene tagged with Myc at the COOH terminus (wild-type CDase) was prepared as described previously (9Mitsutake S. Tani M. Okino N. Mori K. Ichinose S. Omori A. Iida H. Nakamura T. Ito M. J. Biol. Chem. 2001; 276: 26249-26259Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Wild-type CDase tagged with FLAG at the NH2 terminus and Myc at the COOH terminus (FLAG-tagged CDase) was constructed as reported (14Tani M. Iida H. Ito M. J. Biol. Chem. 2003; 278: 10523-10530Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The truncation mutants were constructed by PCR as described below. COOH-terminal fragments were amplified with a 5′ primer (5′-TTGATGAAGCAAAGCCTG-3′) and a 3′ primer with a stop codon (double underlined) and a XhoI restriction site (underlined): 5′-AGACTCGAGCTATTCAAATGCTAGTATGACAGCAG-3′ (for Δ11 CDase), 5′-AGACTCGAGCTAAGGGGAAGAAATTCCTTCAAA-3′ (for Δ6 CDase), 5′-AGACTCGAGCTAAAAAGGGGAAGAAATTCCTTCA-3′ (for Δ5 CD-ase), 5′-AGACTCGAGCTATTCAAAAGGGGAAGAAATTCC-3′ (for Δ4 CDase), 5′-AGACTCGAGCTAAATTTCAAAAGGGGAAGAAATT-3′ (for Δ3 CDase). These fragments were digested with EcoRI and XhoI, and subcloned into the vector pcDNA3.1/Myc-His(+) containing FLAG-tagged CDase cDNA. Site-directed mutagenesis of wild-type CDase was carried out by the amplification of COOH-terminal fragments with a 5′ primer (5′-TTGATGAAGCAAAGCCTG-3′) and a 3′ primer with a XhoI restriction site (underlined): 5′-CCGCTCGAGAGTAGTGACACGTTCAAAAGGGGAA-3′ (for I758R CDase), 5′-CCGCTCGAGAGTAGTGACATCTTCAAAAGGGGAA-3′ (for I758D CDase), 5′-ACTCGAGAGTAGTGACAAATTCAAAAGGGGAA-3′ (for I758F CDase), 5′-ACTCGAGAGTAGTGACAACTTCAAAAGGGGAA-3′ (for I758V CDase), 5′-CCGCTCGAGAGTAGTGACAATTTCATCAGGGGAA-3′ (for F756D CDase), 5′-CCGCTCGAGAGTAGTGACAATTTCACGAGGGGAA-3′ (for F756R CDase), 5′-CCGCTCGAGAGTAGTGACAATTTCAATAGGGGAA-3′ (for F756I CDase) and 5′-CCGCTCGAGAGTAGTGACAATTCGAAAAGGGGAA-3′ (for E757R CDase) (double underline shows the location of mutations). These fragments were digested with EcoRI and XhoI, and subcloned into the vector pcDNA3.1/Myc-His(+) containing wild-type CDase cDNA. To obtain a plasmid vector containing Pseudomonas neutral CDase and the mutant DNA, DNA fragments were prepared by PCR using a 5′ primer containing a NheI site (5′-AAAGCTAGCATGTCACGTTCCGCATTCACC-3′) and a 3′ primer with a XhoI restriction site: 5′-TTTCTCGAGGGGAGTGGTGCCGAGCACCTC-3′ (for Pseudomonas wild type), 5′-ACTCGAGGGGAGTGGTGCCGAGATCCTCGA-3′(for Pseudomonas V665D), 5′-TCTCGAGTTACACCTCGAAGGAGCGGGTC-3′ (for Pseudomonas Δ5), and 5′-TCTCGAGTTACTCGAAGGAGCGGGTCGAG-3′ (for Pseudomonas Δ6) (double underline shows a stop codon). These fragments were digested with NheI and XhoI, and subcloned into the vector pET-23a (Novagen). Pseudomonas wild-type and V665D constructs were tagged with poly(His), but Δ5 and Δ6 were not because of the insertion of a stop codon at the COOH terminus. The sequences of these constructs were verified with a DNA sequencer (Applied Biosystems Japan; model 377).Cell Culture and cDNA Transfection—HEK293 cells, human embryonic kidney cells, were grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), and 60 μg/ml of kanamycin in a humidified incubator containing 5% CO2. cDNA transfection was carried out using LipofectAMINE™ Plus (Invitrogen) according to the instructions of the manufacturer.Preparation of Culture Supernatants and Cell Lysates—At 18 h after transfection with the CDase cDNA, the medium was replaced with serum-free Opti-MEM (Invitrogen, 500 μl/well on 24-well plates) and cultured for an additional 24 h. The cell culture medium was collected and subjected to centrifugation at 13,000 × g for 5 min. The supernatant, supplemented with a 1:10 volume of 200 mm Tris-HCl, pH 7.5 containing 1% Triton X-100 and 33 μg/ml of proteinase inhibitors (leupeptin, pepstatin, and chymostatin), was used as cell supernatant. Cells attached to the culture plate were rinsed with PBS and then lysed by adding 200 μl of 10 mm Tris-HCl, pH 7.5, containing 0.5% Triton X-100 and 3.3 μg/ml of the proteinase inhibitors described above. Lysates were collected by pipette and used as cell lysates.Protein Assay, SDS-PAGE, and Western blotting—Protein content was determined by the bicinchoninic acid protein assay (Pierce) with bovine serum albumin as standard. SDS-PAGE was carried out according to the method of Laemmli (19Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206048) Google Scholar). Protein transfer onto a polyvinyldifluoride membrane was performed using TRANS-BLOT S.D. (Bio-Rad) according to the method described in Ref. 20Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44708) Google Scholar. After treatment with 3% skim milk in Tris-buffered saline (TBS) containing 0.1% Tween 20 (T-TBS) for 1 h, the membrane was incubated with primary antibody for 1 day at 4 °C. After a wash with T-TBS, the membrane was incubated with HRP-conjugated secondary antibody for 2 h. After another wash with T-TBS, the ECL reaction was performed for 5 min as recommended by the manufacturer, and chemiluminescent signals were visualized with an ECL™ mini-camera (Amersham Biosciences).Immunocytochemistry and Fluorescence Microscopy—Transfected cells were cultured on cover glass and then fixed with 3% paraformaldehyde in PBS for 15 min. After rinsing with PBS and 50 mm NH4Cl in PBS, cells were permeabilized, if necessary, by 0.1% Triton X-100 in PBS. After treatment with blocking buffer (5% skim milk in PBS) for 15 min, the samples were incubated with primary antibody (diluted 1:1000 with blocking buffer) at 4 °C for 1 day followed by Cy3-labeled secondary antibody at room temperature for 2 h. Immunostained samples were examined with a confocal laser-scanning microscope (Digital Eclipse C1, Nikon, Japan).Generation of Polyclonal Antibodies against Pseudomonas Neutral CDase—The rabbit antiserum against the Pseudomonas neutral CDase was raised against a full-length CDase (residues 1–646, Ref. 6Okino N. Ichinose S. Omori A. Imayama S. Nakamura T. Ito M. J. Biol. Chem. 1999; 274: 36616-36622Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), which had been expressed using a pET23a plasmid vector and Escherichia coli BL21(DE3) cells. To obtain the recombinant CDase, 1 ml of overnight culture was inoculated into 200 ml of LB in the presence of 100 μg/ml of ampicillin and incubated at 25 °C. After a 20-h incubation, isopropylthio-β-d-galactopyranoside was added to a final concentration of 0.1 mm, and incubated at 25 °C for 24 h. Cells were harvested by centrifugation at 5,000 × g for 10 min at 4 °C, and the pellet was sonicated in 10 mm Tris-HCl buffer, pH 7.5, containing 0.5% Triton X-100 and 0.5 mm ABSF. The insoluble fraction (inclusion bodies) was precipitated by centrifugation (15,000 × g for 10 min), lysed, and subjected to SDS-PAGE. After electrophoresis, the gel was stained with Gelcode Blue Stain Reagent (Pierce). The CDase band was cut out and subjected to electroelution. The protein obtained was used for immunization.Expression of Pseudomonas Neutral CDase and the Mutants—E. coli BL21 (DE3)pLysS cells transformed with a plasmid vector containing Pseudomonas neutral CDase and the mutant DNA were grown at 25 °C for 16 h in 10 ml of LB medium containing 100 μg/ml of ampicillin and 35 μg/ml of chloramphenicol with shaking. Then, isopropylthio-β-d-galactopyranoside was added to the culture to a final concentration of 0.1 mm to trigger transcription. After an additional 1 h culture at 25 °C, cells were harvested by centrifugation, suspended in 1 ml of 10 mm Tris-HCl, pH 7.5, containing 0.1% Triton X-100 and 3.3 μg/ml of the proteinase inhibitors (leupeptin, pepstatin, and chymostatin), and sonicated. After sonication, the solution was centrifuged at 13,000 rpm for 5 min, and the supernatant obtained was used as the crude enzyme solution.RESULTSAlignment and Truncation of COOH-terminal Region of Neutral CDases—Although the primary structure of neutral CDase is highly conserved from bacteria to humans, the enzyme is classified into two prototypes; bacteria/invertebrate and mammalian types. The latter possesses a Ser/Thr-rich mucin-like domain (mucin box) downstream of the NH2-terminal signal/anchor sequence while the former does not (Fig. 1A). The mucin box was shown to retain the enzyme on the plasma membranes as a type II integral membrane protein. Thus mucin box-deleted mutant enzymes and bacteria/invertebrate enzymes were found to be secreted into the culture medium (7Yoshimura Y. Okino N. Tani M. Ito M. J. Biochem. 2002; 132: 229-236Crossref PubMed Scopus (49) Google Scholar, 14Tani M. Iida H. Ito M. J. Biol. Chem. 2003; 278: 10523-10530Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 15Okino N. Tani M. Imayama S. Ito M. J. Biol. Chem. 1998; 273: 14368-14373Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). In this study, we found that two amino acid residues, Phe and Ile/Val, in the COOH-terminal tail were completely conserved in both two types of the enzyme (Fig. 1A). To address the role of the conserved COOH-terminal tail, COOH-terminal-truncated mutant enzymes lacking 11, 6, 5, 4, and 3 amino acid residues from the COOH terminus, respectively, were constructed and expressed in HEK293 cells (Fig. 1B, Δ11, Δ6, Δ5, Δ4, Δ3). Wild-type and truncated enzymes were tagged with FLAG at the NH2 terminus. As shown in Fig. 2A, all truncated CDases except Δ3 lost the enzyme activity as measured in terms of the hydrolysis (a) and synthesis (b) of Cer. The enzyme activity of wild-type and Δ3 CDases was found in cell lysates as well as the medium (Fig. 2B). Western blotting analysis of the cell lysate using anti-FLAG antibody revealed that the wild-type and Δ3 CDases showed two protein bands having a molecular mass of 133 kDa and 113 kDa (Fig. 2B). The 133-kDa protein seems to be a mature form in the Golgi and the 113-kDa protein, developing form in the ER (9Mitsutake S. Tani M. Okino N. Mori K. Ichinose S. Omori A. Iida H. Nakamura T. Ito M. J. Biol. Chem. 2001; 276: 26249-26259Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). A 130-kDa mature form was also detected in the culture medium of wild-type and Δ3 CDases when stained with anti-neutral CDase antibody (Fig. 2B). This secretory CDase is likely to be generated by processing of the NH2-terminal signal/anchor sequence from the mature form (14Tani M. Iida H. Ito M. J. Biol. Chem. 2003; 278: 10523-10530Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). In contrast, no mature forms were detected in the cell lysate or medium of Δ11, Δ6, Δ5, and Δ4 enzymes. These results suggest that the truncated CDases except Δ3 were not transported from the ER to the Golgi and lost enzyme activity.Fig. 2Expression of wild-type and COOH-terminal-truncated rat neutral CDases in HEK293 cells. A, time course for hydrolysis (a) and reverse hydrolysis (b) activities. HEK293 cells were transfected with a plasmid vector containing wild-type or truncated enzyme cDNA. The hydrolysis and reverse hydrolysis activities of the cell lysates were determined using C12-NBD-Cer (for the hydrolysis reaction) and Sph and NBD-dodecanoic acid (for the reverse reaction) as substrates. B, enzyme activity and protein expression of wild-type and truncated enzymes. The neutral CDase activity of the cell lysate and medium was measured using C12-NBD-Cer as a substrate, and the protein expression was determined by Western blotting using anti-FLAG (for cell lysates) and anti-neutral CDase antibodies (for culture supernatants).View Large Image Figure ViewerDownload (PPT)Next, we analyzed the subcellular localization of COOH-terminal-truncated enzymes. As shown in Fig. 3A, wild-type and Δ3 CDases were located at plasma membranes while Δ11, Δ6, Δ5, and Δ4 enzymes were likely to be retained in the ER and not be transported to the plasma membrane. Since human neutral CDase was reported to localize in mitochondria (10El Bawab S. Roddy P. Qian T. Bielawska A. Lemasters J.J. Hannun Y.A. J. Biol. Chem. 2000; 275: 21508-21513Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar), we examined whether wild-type and truncated enzymes from rat were expressed in mitochondria. In this experiment, GFP with a mitochondria-targeting signal (mitochondria-GFP) was used as a marker for mitochondria. As shown in Fig. 3B, wild-type, Δ4, and Δ3 CDases were not co-localized with GFP-mitochondria, indicating that the rat enzymes are not transported to mitochondria under the conditions used in this study. In conclusion, the removal of more than three amino acid residues from the COOH terminus of rat neutral CDase resulted in a complete loss of enzyme activity and correct subcellular localization. The truncated enzymes lacking 232, 89, 56, and 23 amino acid residues from the COOH terminus showed no enzyme activity either (data not shown).Fig. 3Subcellular localization of wild-type and COOH-terminal-truncated rat neutral CDases. A, HEK293 cells overexpressing the CDases were fixed, permeabilized with Triton X-100, and then stained with anti-FLAG antibody followed by anti-mouse IgG-Cy3. The arrows indicate the expression of CDase at the plasma membrane. B, HEK293 cells were co-transfected with a plasmid vector containing CDase and that containing pCMV/Myc/mitochondria (Invitrogen) as a mitochondrial marker. Cells were fixed, permeabilized with Triton X-100, and stained with anti-FLAG antibody followed by anti-mouse IgG-Cy3. Left and center panels show the expression of CDase and mitochondria-GFP. Merged images are shown in the right panels.View Large Image Figure ViewerDownload (PPT)Effects of Point Mutation of the Conserved Amino Acids in the COOH-terminal Tail on Enzyme Activity and Intracellular Localization—The truncated enzyme Δ3 retained the activity whereas Δ4 did not. Therefore, Ile758 of the rat enzyme, the 4th amino acid residue from the COOH terminus, is likely to be integral to the enzyme activity and correct subcellular localization (Fig. 1A). In addition to Ile758, Phe756 is also conserved in all enzymes (Fig. 1A). Thus, we examined the effects of point mutations of Ile758 and Phe756 on enzyme activity and subcellular localization. All mutant and wild-type enzymes were tagged with Myc at the COOH terminus and expressed in HEK293 cells. As shown in Fig. 4A, replacement of Ile758 with Asp or Arg almost completely abolished the enzyme activity. On the other hand, no decrease in enzyme activity was observed when Ile758 was replaced with Val, which is conserved in all neutral CDases except the rat enzyme. Replacement of Ile758 with Phe decreased the enzyme activity to 20% in comparison with wild-type CDase. Western blotting analysis using anti-Myc antibody detected the 113-kDa ER form, but not the 133-kDa mature form, in the cell lysates when I758D and I758R mutants were overexpressed in HEK293 cells. No enzyme activity or protein band of I758D and I758R was found in the medium. Similarly, F756D and F756R, but not F756I, showed no enzyme activity in cell lysates and culture medium. Correspondingly, the 133-kDa and 130-kDa mature forms were not found in the cell lysate and culture medium, respectively. The replacement of Phe756 with Ile had little effect. Next, the cell surface distribution of the enzyme was examined with (permeable condition) or without (unpermeable condition) Triton X-100 treatment. Mutants I758D, I758R, F756D, and F756R, all of which lost enzyme activity completely, were not stained with anti-Myc antibody under unpermeable conditions, indicating they were not expressed on the surface of cells (Fig. 4B, lower panels). However, under permeable conditions these mutant CDases were detected in the ER at almost the same level as the wild-type enzyme (Fig. 4B, upper panels). The cell surface expression of the mutants I758F and F756I is somewhat lower than that of the wild-type and I758V enzymes. This result is well consistent with the enzyme activity levels. No reduction in enzyme activity or cell-surface expression was observed when Glu757, a non-conserved amino acid, was replaced with Arg (control experiment). In summary, these results clearly indicate that the conserved amino acid residues in the COOH-terminal tail, Ile758 and Phe756, are integral to the activity and correct subcellular localization of the enzyme. It is noteworthy that Ile758 or Phe756 can be changed with another hydrophobic, but not a charged, amino acid without a marked loss of enzyme activity.Fig. 4The enzyme activity, expression, and intracellular localization of wild-type and point-mutated rat neutral CDases in HEK293 cells. A, enzyme activity and expression of the CDase in HEK293 cells. Cells were transfected with a plasmid vector containing wild-type or point-mutated CDase cDNA. The cell lysates and the culture supernatants were used to measure the neutral CDase activity with C12-NBD-Cer as a substrate, and the protein expression was determined by Western blotting using anti-Myc antibody. B, intracellular localization of the CDase. Cells transfected with a plasmid vector containing wild-type or point-mutated CDase cDNA were fixed, permeabilized with Triton X-100 (+) or not (–), and stained with anti-Myc antibody.View Large Image
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