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J. Biol. Chem., Vol. 277, Issue 19, 17300-17307, May 10, 2002

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Molecular Cloning and Characterization of Sphingolipid Ceramide N-Deacylase from a Marine Bacterium, Shewanella alga G8^*

Masako Furusato, Noriyuki Sueyoshi, Susumu Mitsutake, Keishi Sakaguchi, Katsuhiro Kita, Nozomu Okino, Sachiyo Ichinose^§, Akira Omori^§, and Makoto Ito^¶

From the  Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581 and the ^§ Mitsubishi Kasei Institute of Life Sciences, 11 Minamiooya, Machida 194-8511, Tokyo, Japan

Received for publication, November 7, 2001, and in revised form, January 15, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Recently, lyso-sphingolipids have been identified as ligands for several orphan G protein-coupled receptors, although the molecular mechanism for their generation has yet to be clarified. Here, we report the molecular cloning of the enzyme, which catalyzes the generation of lyso-sphingolipids from various sphingolipids (sphingolipid ceramide N-deacylase). The 75-kDa enzyme was purified from the marine bacterium, Shewanella alga G8, and its gene was cloned from a G8 genomic library using sequences of the purified enzyme. The cloned enzyme was composed of 992 amino acids, including a signal sequence of 35 residues, and its molecular weight was estimated to be 109,843. Significant sequence similarities were found with an unknown protein of Streptomyces fradiae Y59 and a Lumbricus terrestris lectin but not other known functional proteins. The 106-kDa recombinant enzyme expressed in Escherichia coli hydrolyzed various glycosphingolipids and sphingomyelin, although it seems to be much less active than the native 75-kDa enzyme. In vitro translation using wheat germ extract revealed the activity of a 75-kDa deletion mutant lacking a C terminus to be much stronger than that of the full-length enzyme, suggesting that C-terminal processing is necessary for full activity.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Glycosphingolipids (GSLs)¹ and sphingomyelin (SM) are ubiquitous components of the plasma membranes of vertebrates. Recently, they were found to be accumulated in cell-surface microdomains together with cholesterol and glycosylphosphatidylinositol-anchored proteins (1, 2). Lyso-GSLs and lyso-SM, the N-deacylated derivatives of GSLs and SM, respectively, are detected in normal tissues at very low levels (3) but accumulated in several types of inherited sphingolipid storage diseases (4-6). Several lines of evidence suggest a biological role for lyso-GSLs in cell activities. For example, lyso-GM3 strongly inhibited the tyrosine-specific autophosphorylation of the epidermal growth factor receptor of A431 cells, in which lyso-GM3 was actually detected with GM3 (7). Various lyso-sphingolipids inhibit protein kinase C, and this may be responsible for the pathogenesis of sphingolipidoses (8). Psychosine (galactosylsphingosine) inhibited cytokinesis to induce the formation of multinuclear cells that are observed in the brains of patients with globoid cell leukodystrophy (9). Lyso-SM (sphingosylphosphorylcholine, SPC), was found to accumulate in the brains of patients with Niemann-Pick disease type A (10) or in the skin of patients with atopic dermatitis (11) and is considered to be related to their symptoms. SPC as well as lyso-GM1a were found to induce apoptosis in neuronal cells (12).

Recently, the G protein-coupled receptors for lyso-sphingolipids have been cloned and characterized; ovarian cancer G protein-coupled receptor (OGR1) (13) and T cell death-associated gene 8 (TDAG8) (14) were identified as a receptor for SPC and psychosine, respectively, and GPR4, with high sequence similarity to OGR1, was a receptor with high affinity for SPC and low affinity for lyso-phosphatidylcholine (15).

Although the molecular mechanism underlying the generation of lyso-sphingolipids in mammals remains unclear, lyso-GSL-generating enzymes have been found in several microorganisms, including Nocardia sp. (16), Pseudomonas sp. (17), and Streptomyces sp. (18). The enzyme from Pseudomonas sp. TK4 was found to hydrolyze not only GSLs but also SM, generating corresponding lyso-sphingolipids and fatty acids (17). The TK4 enzyme is clearly distinguishable from ceramidase in that it hydrolyzes the same linkage of free ceramide but not GSLs or SM (19, 20) and thus has been tentatively designated sphingolipid ceramide N-deacylase (SCDase) (17).

Herein we report the molecular cloning and characterization of a novel SCDase from a newly isolated strain of Shewanella alga G8. Although both the TK4 and G8 enzymes efficiently hydrolyzed various GSLs and SM, a distinct difference in specificity was found in the hydrolysis of NBD-GalCer, in which the -position of the fatty acid was labeled with NBD, and 2-palmitoyl-5-nitrophenyl--D-glucoside (2P5N-Glc). That is, NBD-GalCer was efficiently hydrolyzed by G8 SCDase but not TK4 SCDase, whereas 2P5N-Glc was hydrolyzed by the TK4 enzyme but completely resistant to the G8 enzyme. This study also suggests that the processing of the C-terminal region of the G8 enzyme is necessary for the full expression of activity.

This is the first report of the gene cloning of SCDase, information that should prove indispensable for the elucidation of the structure and functions of the enzyme and facilitate developments in lyso-sphingolipid-related biology and pathophysiology.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Crude ganglioside mixture was prepared from bovine brain as described previously (21), and GM1a was prepared from the crude ganglioside mixture using sialidase-producing bacteria, Pseudomonas sp. YF-2, as a microbial catalyst by the method described in a previous study (22). GD1b, GM3, asialoGM1, lactosylceramide (LacCer), galactosylceramide (GalCer), glucosylceramide (GlcCer), restriction endonucleases, and Ligation Pack were purchased from Wako Pure Chemical Industries (Osaka, Japan). Other GSLs were obtained from Iatron Laboratories, Inc. (Tokyo, Japan). SM and ceramide from bovine brain were purchased from Matreya. D-erythro-(2S,3R)-SPC was purchased from Biomol Co. [¹⁴C]SM (labeled at the choline residue) was obtained from ARC. Triton X-100 and Lubrol PX were obtained from Sigma Chemical Co. and Nacalai Tesque Inc. (Kyoto, Japan), respectively. A precoated Silica gel 60 TLC plate was obtained from Merck (Darmstadt, Germany). pET23b and modifying enzymes were purchased from Takara Shuzo Co. (Kyoto, Japan) and [-³²P]dCTP was from Amersham Biosciences, Inc. (Buckinghamshire, England). All other reagents were of the highest purity available.

Isolation and Identification of Strain G8-- The SCDase-producing bacterium G8 was isolated from sea sand using medium A (0.05% NH₄Cl, 0.05% K₂HPO₄, 0.1% yeast extract, 2.0% NaCl, and 0.01% bovine brain gangliosides, pH 7.0), and identified according to the 8th edition of Bergey's Manual of Determinative Bacteriology (23). This strain is currently maintained in a medium containing crude gangliosides (0.5% polypeptone, 0.1% yeast extract, 2% NaCl, 0.1% bovine brain gangliosides, and 1.6% agar, pH 7.0) in our laboratory.

Phylogenetic Analysis-- 16 S rDNA was amplified using the universal primers p27f (5'-AGA GTT TGA TCM TGG CTC AG-3'; positions 8-27; Escherichia coli numbering (24)) and p1492r (5'-GGC TAC CTT GTT ACG ACT T-3'). PCR products were purified from 1.0% agarose gel and sequenced directly using nine different sequencing primers (25). The nucleotide sequences were aligned and analyzed using Clustal W (26), with other 16 S rDNA sequences obtained from the Ribosomal Data base Project II (27).

Enzyme Assay-- The activity of SCDase was measured using GM1a as the substrate as described previously (17). In a standard assay, the reaction mixture contained 10 nmol of GM1a and an appropriate amount of the enzyme in 20 µl of 25 mM sodium acetate buffer, pH 6.0, containing 0.1% Triton X-100. Following incubation at 37 °C for 30 min, the reaction was stopped by heating in a boiling water bath for 5 min. The reaction products were freeze-dried in a Speed Vac concentrator, dissolved in 15 µl of chloroform/methanol (1:2, v/v), and analyzed by TLC using solvent I (chloroform/methanol/0.02% CaCl₂, 5/4/1, v/v) as the developing solvent. GSLs and lyso-GSLs were visualized by spraying the plates with orcinol-H₂SO₄ reagent and scanning them with a Shimadzu CS-9300 chromatoscanner with the reflectance mode set at 540 nm. The extent of hydrolysis was calculated as follows: hydrolysis (%) = (peak area for lyso-GM1a generated) × 100/(peak area for remaining GM1a + peak area for lyso-GM1a generated). One enzyme unit was defined as the amount capable of catalyzing the release of 1 µmol of lyso-GM1a/min from GM1a under the conditions indicated above. A value of 10³ and 10⁶ units of enzyme was expressed as 1 milliunit (mU) and 1 microunit (µU), respectively. The activity of SCDase was also measured using C12-NBD-GalCer, [¹⁴C]SM (labeled at the choline residue) or [¹⁴C]GM1a (labeled at the stearic acid) as a substrate as described below. C12-NBD-GalCer was prepared by the method described by Nakagawa et al. (28). The reaction mixture contained 100 pmol of C12-NBD-GalCer and an appropriate amount of the enzyme in 20 µl of 25 mM sodium acetate buffer, pH 6.0, containing 0.1% Triton X-100. Following incubation at 37 °C for 30 min, the reaction was terminated by heating in a boiling water bath for 5 min. The sample was evaporated, dissolved in 15 µl of chloroform/methanol (2/1, v/v), and applied to a TLC plate, which was developed with solvent I. NBD-dodecanoic acid released by the action of the enzyme and the remaining C12-NBD-GalCer were separated on a TLC and then analyzed and quantified with a Shimadzu CS-9300 chromatoscanner (excitation 475 nm, emission 525 nm). The hydrolysis of [¹⁴]SM or [¹⁴]GM1a was performed as follows; the reaction mixture contained 100 pmol of ¹⁴C-substrate, 900 pmol of each cold substrate, and 60 µU of SCDase in 20 µl of 25 mM sodium acetate buffer, pH 6.0, containing 0.1% Triton X-100. After incubation at 37 °C for the periods indicated, the sample was evaporated, dissolved in 20 µl of chloroform/methanol (2/1, v/v), and applied to a TLC plate, which was developed with solvent I. An imaging analyzer (BAS1500, Fuji Film, Japan) was used for analysis and quantification of the ¹⁴C-substrate and ¹⁴C-product. 2-Palmitoyl-5-nitrophenyl--D-glucoside (2P5N-Glc) was used as an alternative substrate for SCDase. 2P5N-Glc was prepared according to the method described in a previous study (29). The reaction mixture contained 50 µg of 2P5N-Glc and an appropriate amount of the enzyme in 200 µl of 25 mM sodium acetate buffer, pH 6.0, containing 0.8% Triton X-100. Following incubation at 37 °C for 30 min, the optical density at 405 nm was measured to estimate the release of 2-amino-5-nitrophenyl--D-glucoside from 2P5N-Glc. For quantification of hydrolysis of 2P5N-Glc, 10 µl of reaction mixture was dried and re-dissolved in 20 µl of chloroform/methanol (2/1, v/v) and applied to a TLC plate, which was developed with solvent I. The released 2-amino-5-nitrophenyl--D-glucoside was determined by the orcinol-H₂SO₄ method. Reverse hydrolysis was measured by the method described previously (30). The mixture contained 100 pmol of [¹⁴C]stearic acid, 900 pmol of stearic acid, 2 nmol of D-erythro-SPC (Biomol Co.), and 60 µU of SCDase in 20 µl of 25 mM sodium acetate buffer, pH 6.0, containing 0.1% (w/v) Triton X-100. Following incubation at 37 °C for the periods indicated, the reaction was terminated by heating in a boiling water bath for 5 min. The sample was evaporated, dissolved in 20 µl of chloroform/methanol (2/1, v/v), and applied to a TLC plate, which was developed with solvent I. An imaging analyzer (BAS1500, Fuji Film, Japan) was used for analysis and quantification of the [¹⁴C]SM generated and [¹⁴C]stearic acid unreacted. Exoglycosidases were assayed using p-nitrophenylglycosides (31) as a substrate.

Purification of SCDase-- Inocula from an agar slant of G8 were introduced into a cotton-plugged 500-ml flask containing 100 ml of sterilized peptone-yeast extract-ganglioside (PYG) medium (0.5% polypeptone, 0.1% yeast extract, 2.0% NaCl, 0.1% bovine brain gangliosides, pH 7.0) and incubated at 25 °C for 22 h with vigorous shaking. The culture fluid was centrifuged at 8300 rpm for 30 min, and the supernatant (4,600 ml) was then concentrated using an Ultrafiltration System (Millipore) with a 30,000 cut membrane. The concentrated fluid was applied to a Butyl-Toyopearl 650M column (80 ml; Tosoh Co., Japan), previously equilibrated with 20 mM Tris-HCl, pH 7.5 (buffer A), containing 2.0 M NaCl. The enzyme was eluted from the column with an increasing Lubrol PX from 0 to 2.0% in buffer A, using a BPLC-600FC HPLC system (Yamazen Co., Japan) at a flow rate of 5 ml/min. The active fractions were pooled (130 ml) and then applied to a DEAE-Sepharose FF column (30 ml, Amersham Biosciences, Inc.), previously equilibrated with buffer A containing 0.1% Lubrol PX, using a BPLC-600FC HPLC system (Yamazen Co., Japan). The enzyme was eluted from the column with increasing concentrations of NaCl up to 1 M at a flow rate of 5 ml/min. The active fractions were pooled (129 ml), and half of this sample was applied to a HiTrap Blue column (1.0 ml, Amersham Biosciences, Inc.) equilibrated with buffer A, containing 1.0 M NaCl. The enzyme was eluted from the column with decreasing concentrations of NaCl down to 0 M at a flow rate of 1 ml/min.

Protein Assay and SDS-PAGE-- The amount of protein was measured by the bicinchoninic acid protein assay (Pierce) with bovine serum albumin as the standard. SDS-PAGE was carried out according to the method of Laemmli (32). The proteins on SDS-PAGE gel were visualized with a silver-staining solution (33) and quantified by a Shimadzu CS-9300 chromatoscanner with the reflection mode set at 540 nm.

Amino Acid Microsequencing-- The purified SCDase was concentrated with a Y-shaped gel (160 × 160 × 2 mm) modified from a funnel-shaped one (34). After concentration, the protein band (75 kDa) localized with Coomassie Brilliant Blue was cut out, reduced with dithiothreitol, and loaded again on the well of a SDS-PAGE gel. After electrophoresis, the gel was blotted onto a polyvinylidene difluoride membrane (Immobilon-P, Millipore) and stained with Coomassie Brilliant Blue. The SCDase band was cut out and treated in situ with lysylendopeptidase AP-1 (Wako Pure Chemical Industries, Japan). Peptides released from the membrane were fractionated by reversed-phase HPLC using a C8 column (RP-300, 1.0 × 100 mm, Applied Biosystems) and sequenced with a pulse-liquid phase protein sequencer (Procise 492 cLc, Applied Biosystems).

Molecular Cloning and DNA Sequencing-- General cloning techniques were used, essentially as described by Sambrook et al. (35). Genomic DNA of S. alga G8 was isolated by the method of Saito and Miura (36). Nucleotide sequences were determined by the dideoxynucleotide chain termination method with a BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems) and a DNA sequencer (model 377A, Applied Biosystems).

PCR Amplification-- PCR with degenerate oligonucleotides was used to amplify a DNA fragment encoding the SCDase. Sense and antisense oligonucleotide primers were designed using the N-terminal amino acid sequence of C-603 and the internal amino acid sequence of C-606. PCR was performed with the G8 genomic DNA as a template in a GeneAmp PCR System 2400 (Applied Biosystems) for 35 cycles (each consisting of denaturation at 95 °C for 20 s, annealing at 52 °C for 30 s, and extension at 72 °C for 1.5 min) using AmpliTaq Gold (Applied Biosystems). Amplified PCR products were cloned into the pGEM-T Easy vector (Promega), and their DNA was sequenced. One PCR product (named probe 1) contained the deduced amino acid sequence found in the purified SCDase.

Isolation of a Genomic DNA Clone Encoding SCDase-- Genomic DNA (3 µg) was digested with various restriction endonucleases, and the digests were fractionated by 0.7% agarose gel electrophoresis by the standard method (35). DNA was transferred from agarose gels onto nylon membranes (Hybond N+, Amersham Biosciences) according to the protocol of the manufacturer. Probe 1 was labeled with [-³²P]dCTP using a Ready-To-Go DNA labeling kit (Amersham Biosciences) and used for hybridization, which was performed in 0.5 M sodium phosphate buffer, pH 7.0, containing 1 mM EDTA and 7% SDS at 65 °C for 16 h. After hybridization, the membrane was washed three times with 40 mM sodium phosphate buffer, pH 7.0, containing 1% SDS at 65 °C and exposed on an imaging plate, which was then analyzed with a BAS1500 imaging analyzer (Fuji Film, Japan). Judging from the Southern blot of the EcoRV digest obtained using probe 1, the 4.5-kbp fragment contained the SCDase gene. To clone the gene, an EcoRV digest was prepared using 10 µg of G8 genomic DNA. Restriction fragments of the DNA were fractionated by preparative 1.0-% agarose gel electrophoresis, and 4.5-kbp fragments were extracted from the gel. The EcoRV fragments were ligated to the EcoRV site of pBluescript II SK (Stratagene). The recombinant plasmids thus obtained were used to transform Escherichia coli DH5, which were employed for the preparation of a DNA library enriched with the SCDase gene. Colony hybridization was performed by the standard procedure using probe 1. One clone was selected, and the plasmid in the clone was designated pSCD1.

Construction of an Expression Plasmid with the SCDase Gene-- The following primers were used for PCR; UN1 (5'-GAC GCT AGC ATG AAA AAG CTA ATC GGA CAT-3'), and LC2 (5'-CTT GAG GCT GGA CTT CCA CTT CTG-3'). UN1 contained a NheI site (underlined) and LC2 contained a XhoI restriction site (double underlined). PCR was performed in a GeneAmp PCR System 9600 (Applied Biosystems) for 30 cycles (each consisting of denaturation at 98 °C for 10 s and extension at 68 °C for 3.5 min) using Pyrobest DNA polymerase and pSCD1 as a template. After gel purification, the amplified products were digested with NheI and XhoI. The NheI/XhoI fragments were cloned into the NheI/XhoI-digested pET23b. The recombinant plasmid was designated pETSCD-T.

In Vitro Translation of SCDase and Its Deletion Mutant-- The following primers were used for PCR; UN1ERV (5'-GCT GAT ATC ATG AAA AAG CTA ATC GGA CAT-3'), LSCD2125HisXho (5'-AAA TCA ATG ATG ATG ATG ATG ATG GTG GGC TTC TGC GCG CTC CCA-3'), and LSCD2955HisXho (5'-AAA TCA ATG ATG ATG ATG ATG ATG GAG GCT GGA CTT CCA CTT CTG). UN1ERV contained an EcoRV restriction site (underlined), and LSCD2125HisXho and LSCD2955HisXho contained an XhoI restriction site (double underlined). PCRs were performed in a T personal PCR System (Biometra, Germany) for 30 cycles (each consisting of denaturation at 98 °C for 10 s and extension at 68 °C for 3 min) using Pyrobest DNA polymerase, pSCD1 as a template, and the following sets of primers: UN1ERV-LSCD2955HisXho for the construction of pEUSCD-T (encoding wild type SCDase) and UN1ERV-LSCD2125HisXho for the construction of pEUSCD-del (encoding SCDase deletion mutant). After gel purification, the amplified products were digested with EcoRV and XhoI. The EcoRV-XhoI fragments were cloned into the EcoRV/XhoIdigested pEU-NII vector. mRNA encoding the wild type or deletion mutant of SCDase was prepared from pEUSCD-T or pEUSCD-del using T7 RNA polymerase. In vitro translation was performed using a PROTEIOS wheat germ cell-free protein synthesis kit (TOYOBO, Japan) according to the manufacturer's protocol.

Expression of Recombinant SCDase-- Escherichia coli BL21(DE3)pLysS cells transformed with pETSCD-T were grown at 37 °C in Luria-Bertani medium containing 100 µg/ml ampicillin until the optical density at 600 nm reached about 1.0. Then, isopropyl-1-thio--D-galactopyranoside (IPTG) was added to the culture at a final concentration 1 mM, and cultivation was continued for an additional 2 h at 37 °C. Cells were harvested by centrifugation, suspended in extraction buffer (10 mM Tris-HCl buffer, pH 7.5, containing 0.5 mM 4-(2-aminoethyl)-benzensulfonyl fluoride hydrochloride and 1% Triton X-100), and sonicated. After sonication, the solution was centrifuged at 15,000 rpm for 3 min, and the supernatant obtained was used as the crude enzyme solution.

Other Methods-- The nucleotide and amino acid sequences were evaluated using the DNASIS computer program developed by Hitachi Software Engineering (Japan).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Purification of SCDase-- The newly isolated SCDase-producing bacterium G8 was assigned to S. alga based on its morphological, physiological, and biochemical characteristics and on the results of a phylogenetic analysis using 16 S rDNA. G8 released SCDase into the culture medium (12 units/liter of culture) when cultivated at 25 °C for 20-24 h in a PYG medium. SCDase was then purified from 4.6 liters of the culture supernatant by Ultrafiltration and sequential chromatography using Butyl-Toyopearl 650M and DEAE-Sepharose FF, as described under "Experimental Procedures." SCDase was finally purified 1600-fold with an overall yield of 24% (Table I).

Purity and Molecular Mass of SCDase-- The final preparation of G8 SCDase was completely free from the following enzymes: - and -galactosidases, - and -glucosidases, - and -mannosidases, -N-acetylhexosaminidase, -N-acetylgalactosaminidase, -N-acetylglucosaminidase, and -L-fucosidase, as confirmed by activity determination using 0.1 mU of the purified SCDase for each assay and an appropriate substrate for 16 h. The purified enzyme gave one major protein band having a molecular mass of 75 kDa on SDS-PAGE after staining with a silver-staining solution under reducing conditions (Fig. 1A). To examine whether or not the 75-kDa band is SCDase per se, the SCDase preparation after DEAE-Sepharose FF chromatography was further applied to a column of HiTrap Blue, and an aliquot of the eluent was subjected to an assay for SCDase activity as well as SDS-PAGE. The elution profile of SCDase activity on the HiTrap Blue column was found to exactly coincide with that of the 75-kDa band on SDS-PAGE (Fig. 1B), indicating that the 75-kDa protein is likely to be SCDase.

View larger version (29K): [in this window] [in a new window]   Fig. 1.   Identification of SCDase on SDS-PAGE. A, SDS-PAGE of G8 SCDase under reducing conditions. SCDase, SCDase from DEAE-Sepharose FF. STD, standard proteins (molecular masses in parentheses) were: phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20 kDa), and lysozyme (14 kDa). Proteins were stained with silver staining solution. B, elution profiles of SCDase activity and 75-kDa protein from HiTrap Blue chromatography. Aliquots of each fraction were subjected to SCDase assay and SDS-PAGE. Proteins were stained with silver staining solution, and the content of the 75-kDa protein was determined using a Shimadzu CS-9300 chromatoscanner with the reflectance mode set at 540 nm. SCDase activity was determined using GM1a as a substrate by the method described under "Experimental Procedures." , activity for SCDase; , elution of the 75-kDa protein (A at 540 nm).

Molecular Cloning of the SCDase Gene-- Four peptides (C-599, C-606, C-614, and C-616) were obtained from the purified 75-kDa SCDase after digestion with lysylendopeptidase and subjected to amino acid microsequencing. The N-terminal amino acid sequence was also determined using the purified native enzyme (C-603). Finally, 59 amino acid residues of the SCDase were determined. PCR was performed using one set of primers (SS2a, 5'-CTNGCNCARCARTGYTT-3'; SA3, 5'-CCNSAYTGNACRTCDAT-3') and G8 genomic DNA as a template. A 997-bp PCR product was obtained (probe 1), which contained DNA sequences encoding the peptide sequences of C-603, C-599, and C-606 (Fig. 2A). Southern blotting using -³²P-labeled probe 1 showed a 4.5-kbp band in the G8 genomic DNA digested with EcoRV. A clone, designated as pSCD1, containing a 4.5-kbp insert was isolated from a DNA library enriched with the SCDase gene. The nucleotide sequence of pSCD1 was determined, and a putative open reading frame was found. The amino acid sequences of C-603, C-599, C-606, C-614, and C-616 were found in the deduced amino acid sequence of the open reading frame (Fig. 2A). We sequenced 4434 nucleotides of the clone pSCD1, which includes an open reading frame of position 319 through 3294. 

View larger version (60K): [in this window] [in a new window]   Fig. 2.   DNA and deduced amino acid sequences (A) and hydropathy plot (B) of the SCDase. A, the deduced amino acid sequence is shown as a one-letter code below the nucleotide sequence. Amino acids determined by peptide sequencing are underlined (C-606, C-603, C-599, C-614, and C-616). The amino acid residues are numbered beginning with the first methionine. The termination codon is denoted by an asterisk. A DNA sequence with bold under line represents the probe 1 used for the colony hybridization. B, hydropathy analysis of the coding region based upon the deduced amino acids according to Kyte and Doolittle (48). Amino acid residues are numbered beginning with the first methionine.

DNA and Deduced Amino Acid Sequences of SCDase-- The open reading frame, 2976 bp long with 992 codons, encoded a signal sequence of 35 residues and a mature protein of 957 amino acid residues (Fig. 2A). The molecular weight of the SCDase was estimated to be 109,843 from the deduced amino acid sequences, which was quite different from that of the SCDase (75 kDa) purified from S. alga G8. The predicted pI was 5.34. All of the peptide sequences determined and a hydrophobic motif composed of 35 amino acid residues starting with ATG were found in the deduced amino acid sequence (Fig. 2A). The presence of the hydrophobic motif was also clearly indicated by hydrophobicity plot analysis (Fig. 2B). The finding of a signal peptide sequence was consistent with the observation that the SCDase was secreted into the culture medium.

Expression of Recombinant SCDase-- The expression plasmid, pETSCD-T, was constructed by inserting a fragment of the coding sequence with a putative signal sequence between the NheI and XhoI sites of the plasmid pET23b. In pETSCD-T, transcription of the recombinant gene is controlled by the T7 promoter and can be induced by IPTG. The 106-kDa protein was detected with the anti-histidine tag (C-term) monoclonal antibody (Invitrogen) on SDS-PAGE (Fig. 3A), which was consistent with the molecular mass deduced from the SCDase gene. Cell lysates from transfectants containing pETSCD-T hydrolyzed [¹⁴C]SM (labeled at the choline residue) to produce [¹⁴C]SPC whereas those of mock transfectants did not, indicating that the recombinant protein possesses the SCDase activity (Fig. 3B). The extent of hydrolysis of [¹⁴C]SM by the recombinant SCDase was time-dependent and reached a plateau after incubation for 120 min under the conditions used (Fig. 3C). The reaction of TK4 SCDase is a reversible one in which the amide linkage of ceramide is either cleaved or synthesized (30). The recombinant G8 enzyme also catalyzed the reverse hydrolysis reaction (condensation), i.e., [¹⁴C]stearic acid was condensed with SPC to generate [¹⁴C]SM (Fig. 3D). The synthesis of SM by the reverse hydrolysis reaction increased during incubation and reached a plateau after 120 min (Fig. 3E). Cell lysates of mock transfectants showed no activity for the reverse reaction (Fig. 3D).

View larger version (29K): [in this window] [in a new window]   Fig. 3.   Expression and activity of recombinant G8 SCDase. A, Western blotting of the recombinant G8 SCDase. E. coli BL21(DE3)pLysS cells transformed with pETSCD-T were cultured at 37 °C for 2 h after addition of IPTG. The cell lysate was analyzed by 7.5% SDS-PAGE and transferred onto nitrocellulose membrane for detection with anti-His tag (C-term) monoclonal antibody (Invitrogen). B, time course for hydrolysis of [14C]SM by the recombinant G8 SCDase. The reaction mixture contained 100 pmol of [14C]SM (labeled at the choline residue), 900 pmol of unlabeled SM, and 60 µU of SCDase (cell lysate of E. coli BL21(DE3)pLysS transformed with pETSCD-T) in 20 µl of 25 mM sodium acetate buffer, pH 6.0, containing 0.1% Triton X-100. Mock represents the incubation of substrate with cell lysate of E. coli BL21(DE3)pLysS transformed with pET23b vector without SCDase gene. After incubation at 37 °C for the periods indicated, [14C]SPC was separated from [14C]SM using TLC and analyzed by BAS1500 as described under "Experimental Procedures." C, quantification of B by BAS1500. D, time course for the generation of [14C]SM by the reverse hydrolysis reaction of the recombinant SCDase. The reaction mixture contained 100 pmol of [14C]stearic acid, 900 pmol of unlabeled stearic acid, 2 nmol of D-erythro-SPC, and 60 µU of the recombinant G8 enzyme in 20 µl of 25 mM sodium acetate buffer, pH 6.0, containing 0.1% Triton X-100. After incubation at 37 °C for the periods indicated, [14C]SM was separated from [14C]stearic acid using TLC and analyzed by BAS1500 as described under "Experimental Procedures." E, quantification of D by BAS1500.

Specificity and Properties of the Recombinant SCDase-- Recombinant G8 SCDase hydrolyzed various gangliosides, including GT1b (89.3%, value is the extent of hydrolysis of 10 nmol of substrate after incubation with 2 mU of the enzyme at 37 °C for 16 h), GD1b (82.9%), GM3 (14.5%), neutral GSLs such as Gb4Cer (16.6%), asialoGM1 (27.0%), LacCer (28.5%), GlcCer (27.2%), GalCer (28.5%), and SM (31%). However, free ceramide was quite resistant to hydrolysis by the enzyme (less than 5%). These results are very consistent with the TK4 enzyme. However, clear differences in specificity were found in the hydrolysis of C12-NBD-GalCer, in which dodecanoic acid at the -position was labeled with fluorescent dye NBD, and that of 2P5N-Glc. As shown in Fig. 4A, C12-NBD-GalCer was efficiently hydrolyzed by the G8 recombinant enzyme, whereas the fluorescent substrate is quite resistant to hydrolysis by the TK4 enzyme. In contrast, 2P5N-Glc was only hydrolyzed by the TK4 enzyme (Fig. 4B). The metal-ion requirement of the recombinant G8 enzyme was almost the same as that of the native enzyme but somewhat different from that of the TK4 enzyme; EDTA at 5 mM strongly inhibited the activities of G8 enzymes but not the TK4 enzyme, and Ca²⁺, Mn²⁺, and Mg²⁺ activated the G8 enzymes at 5 mM (Fig. 4C) but not the TK4 enzyme. The optimum pH for G8 and TK4 enzymes was found to be 5.5-6.5 when the activity was determined using [¹⁴C]GM1a as a substrate in the presence of Triton X-100 at 0.1% (Fig. 4D). The effects of Triton X-100 on both G8 and TK4 enzymes were almost the same (Fig. 4E), whereas those of taurodeoxycholate (TDC) differed between the enzymes (Fig. 4F); the TK4 enzyme was gradually activated by increasing the concentration of TDC up to 2%, whereas the optimum concentration of TDC for G8 enzymes was found to be 0.5%, with a gradual decrease beyond this. In summary, the substrate specificity and general properties of the recombinant G8 enzyme are somewhat different from those of the TK4 enzyme.

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Comparison of the properties of the G8 with those of TK4 enzymes. A, hydrolysis of NBD-GalCer by SCDase. The substrate (100 pmol) was incubated with different amounts of enzyme in 20 µl of 25 mM sodium acetate buffer, pH 6.0, containing 0.1% Triton X-100 and incubated at 37 °C for 30 min. The extent of hydrolysis of the substrate was evaluated by the method described under "Experimental Procedures." B, hydrolysis of 2P5N-Glc by SCDase. The substrate (100 nmol) was incubated with different amounts of enzyme in 200 µl of 25 mM sodium acetate buffer, pH 6.0, containing 0.8% Triton X-100 and incubated at 37 °C for 30 min. The extent of hydrolysis of the substrate was evaluated by the method described under "Experimental Procedures." C, effects of divalent cations and EDTA on SCDase. The activity was assayed by the standard method using 50 µU of SCDase and GM1a as a substrate as described under "Experimental Procedures" except that the reaction was performed in the presence of 5 mM metal ions or EDTA. D, effects of pH on SCDase. The activity was measured using 50 µU of SCDase and GM1a as a substrate by the standard method except that 300 mM GTA buffer was used instead of 25 mM sodium acetate buffer. D and E, effects of detergents on SCDase. The activity was measured using 50 µU of SCDase and GM1a as a substrate by the standard method except that Triton X-100 (D) or TDC (E) was used at the indicated concentrations.

Significance of C-terminal Processing of SCDase-- The molecular wieght of the mature SCDase was deduced to be 105,994 from the DNA sequence of the gene encoding SCDase. This value is very consistent with the molecular mass of recombinant SCDase expressed in E. coli (106 kDa) but quite different from that of the native SCDase (75 kDa) purified from S. alga. This discrepancy may stem from the post-translational processing of the enzyme in S. alga. The processing is likely to occur in the C-terminal region, because the N-terminal sequence of the purified 75-kDa SCDase was followed only by the putative signal sequence. Thus, we have designed two constructs; one contains the full-length DNA encoding the 106-kDa SCDase (pEUSCD-T) and the other contains the DNA with a C-terminal deletion encoding the 75-kDa enzyme (pEUSCD-del). In both cases the C-terminal end was tagged with polyhistidine (Fig. 5A). As expected, 106-kDa and 75-kDa proteins were generated by in vitro translation using wheat germ extracts and visualized by Western blotting using anti-His tag antibody (Fig. 5B). The activities of the 106-kDa protein (WT) and the 75-kDa protein (DEL) were determined using [¹⁴C]GM1a as a substrate. Both proteins hydrolyzed [¹⁴C]GM1a to generate [¹⁴C]stearic acid whereas wheat germ extract itself (WGE) did not (Fig. 5C). Interestingly, the extent of hydrolysis of [¹⁴C]GM1a by the 75-kDa protein was found to be much greater than that by the 106-kDa enzyme (Fig. 5D). This was confirmed by the experiment using different concentrations of the substrate (Fig. 5E). These results strongly suggest that C-terminal processing is necessary for a fully active SCDase. It is noteworthy that the 30-kDa C-terminal region is homologous to Lumbricus terrestris lectin (Fig. 5A), although the physiological significance of this lectin-like domain is unknown.

View larger version (23K): [in this window] [in a new window]   Fig. 5.   SCDase activity of the deletion mutant. A, expression vectors for in vitro translation. Details are described under "Experimental Procedures." B, Western blotting of the SCDase. The wild type (pEUSCD-T, 106 kDa) or deletion mutant (pEUSCD-del, 75 kDa) was subjected to in vitro translation using Proteios wheat germ extract. Then, an aliquot of the reaction mixture (18 µg as protein) was subjected to 10% SDS-PAGE and transferred onto a polyvinylidene difluoride membrane for detection with anti-His tag (C-term) monoclonal antibody (Invitrogen). C, 10 µl (18 µg as protein) of wheat germ extract (WGE), wheat germ extract containing the 106-kDa wild-type SCDase (WT) or wheat germ extract containing the 75-kDa mutant SCDase (DEL) was incubated with substrate solution (100 pmol of [14C]GM1a in 10 µl of 50 mM sodium acetate buffer, pH 6.0, containing 0.2% Triton X-100) at 37 °C for the periods indicated. [14C]Stearic acid was separated from [14C]GM1 using TLC and analyzed by BAS1500 as described under "Experimental Procedures." D, quantification of C by BAS 1500. E, 100, 200, or 500 pmol of [14C]GM1a (each corresponds to 5, 10, and 25 µM, respectively) was incubated with 18 µg (as protein) of WGE, WT, or DEL at 37 °C for 6 h in 20 µl of 25 mM sodium acetate buffer, pH 6.0, containing 0.1% Triton X-100. The extent of hydrolysis was determined by the method described in C.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Lyso-sphingolipids are present at only low levels in normal tissues but are abnormally accumulated in various lysosomal storage diseases. Several possible mechanisms for the formation of lyso-sphingolipids in vivo have been proposed: 1) sphingosine could be utilized as an acceptor substrate instead of ceramide by UDP-galactose:ceramide:galactosyltransferase to generate galactosylsphingosine (37), 2) acidic ceramidase catalyzes the N-deacylation of not only ceramide but also GlcCer to generate glucosylsphingosine (38), and 3) SCDase or a SCDase-like enzyme participates in the enzymatic N-deacylation of various GSLs and SM. However, the molecular mechanism for lyso-sphingolipid formation in vivo remains to be elucidated.

The first description of enzymatic N-deacylation of GSLs was made by Hirabayashi and co-workers (16) who observed the formation of lyso-GM2 from GM2 by Nocardia cell lysates. To date, four microbial SCDases or SCDase-like enzymes have been reported, including the present study. Among them, the enzymes from Pseudomonas sp. TK4 (17) and S. alga G8 (this work) were purified to be apparently homogenous. The G8 enzyme cloned in this study seems to be novel judging from its molecular weight, substrate specificity, and metal-ion and detergent requirements. The molecular masses of the G8 and TK4 enzymes were estimated by SDS-PAGE to be 75 kDa (106 kDa for recombinant enzyme expressed in E. coli) and 52 kDa, respectively, and those from other origins have not yet been determined. Very recently, Higuchi et al. reported that the activity of a non-microbial SCDase-like enzyme was abnormally elevated in the skin of a patient with atopic dermatitis (11). The enzyme hydrolyzed GlcCer as well as SM. They argued that the hydrolysis of SM and GlcCer by the enzyme might cause the decrease of ceramide in atopic skin, because SM and GlcCer are thought to be precursors of ceramide in epidermis (39, 40). A decrease of ceramide was actually observed in atopic skin and seems to be related to some etiologic aspects of atopic dermatitis, i.e. a decrease of ceramide causes a change in epidermal permeability, facilitating invasion by allergens or irritants (41). The purification and molecular cloning of this non-microbial SCDase should provide further information.

The molecular cloning of a SCDase gene has been performed for the first time in this study. The deduced amino acid sequence of G8 SCDase showed no significant sequence similarity with other known proteins, including other sphingolipid-degrading enzymes such as ceramidases (19, 20), sphingomyelinases (42), and endoglycoceramidases (43-45). However, we found a sequence homologous to the G8 SCDase in a hypothetical protein of Streptomyces fradiae Y59 (46) and L. terrestris galactose-binding lectin (47). Streptomyces and Lumbricus proteins showed 35.9 and 25% identities at the amino acid level to the Shewanella SCDase. Actually, Ashida et al. (18) reported the presence of SCDase activity in a strain of Streptomyces sp. Although TK4 SCDase has not been cloned, its primary structure is probably different from that of G8, because probe 1 (Fig. 2), which is used for the colony hybridization of G8 SCDase, did not hybridize the genomic DNA of TK4 after digestion with several restriction enzymes under several different conditions. It is also worth noting that no homologous sequences for SCDase were found in human and other mammalian DNA data bases when searched by BLAST and FASTA.

Herein we report the first isolation of a full-length DNA encoding a lyso-sphingolipid-generating enzyme, SCDase. Identification of the DNA encoding SCDase will make possible the isolation of novel SCDase genes and could be useful in generating model cells/animals in which the SCDase gene is knocked in. The availability of such information would facilitate further development of lyso-sphingolipid biology and pathophysiology.

	ACKNOWLEDGEMENTS

We thank Dr. M. Sano and H. Izu for a generous gift of 2P5N-Glc and valuable suggestions for preparing it. We acknowledge the encouragement of Prof. Dr. T. Nakamura of Kyushu University during the course of this study.

	FOOTNOTES

^* This work was supported by grants-in-aid for Scientific Research of Priority Area (B) (1240204) and Research on (B) (13460044) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence reported in this paper has been submitted to the DDBJ/GenBank^TM/EBI Data Bank with accession number AB079849.

^¶ To whom correspondence should be addressed. Tel.: 81-92-642-2898; Fax: 81-92-642-2907; E-mail: makotoi@agr.kyushu-u.ac.jp.

Published, JBC Papers in Press, February 4, 2002, DOI 10.1074/jbc.M110688200

	ABBREVIATIONS

The abbreviations used are: GSLs, glycosphingolipids; GalCer, galactosylceramide; GlcCer, glucosylceramide; LacCer, lactosylceramide; NBD, nitrobenz-2-oxa-1,3-diazole; 2P5N-Glc, 2-palmitoyl-5-nitrophenyl--D-glucoside; SCDase, sphingolipid ceramide N-deacylase; SM, sphingomyelin; SPC, sphingosylphosphorylcholine; HPLC, high performance liquid chromatography; IPTG, isopropyl-1-thio--D-galactopyranoside; TDC, taurodeoxycholate; GD1a, NeuAc2,3Gal1,3GalNAc1,4(NeuAc2,3)Gal1,4Glc1,1'-Cer; GM1a, Gal1,3GalNAc1,4(NeuAc2,3)Gal1,4G1c1,1'-Cer; GM2, GalNAc1,4(NeuAc2,3)Gal1,4G1c1,1'-Cer; GM3, NeuAc2,3Gal1,4Glc1,1'-Cer; Gb4Cer, GalNAc1,3Gal1,4Gal1,4G1c1,1Cer.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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