Molecular cloning and characterization of a secretory neutral ceramidase of Drosophila melanogaster

We report here the molecular cloning and characterization of the Drosophila neutral ceramidase (CDase). Using the BLAST program, a neutral CDase homologue (AE003774) was found in the Drosophila GenBank and cloned from a cDNA library of Drosophila imaginal discs. The open reading frame of 2,112 nucleotides encoded a polypeptide of 704 amino acids having five putative N-glycosylation sites and a putative signal sequence composed of 23 residues. When a His-tagged CDase was overexpressed in D. melanogaster Schneider's line 2 (S2) cells, the enzyme was continuously secreted into the medium through a vesicular transport system. Treatment of the secretory 86.3-kDa CDase with glycopeptidase F resulted in the generation of a 79.3-kDa protein, indicating that the enzyme is actually glycosylated with N-glycans. The enzyme hydrolyzed various N-acylsphingosines but not galactosylceramide, GM1a or sphingomyelin, and exhibited a peak of activity at pH 6.5-7.5, and thus was classified as a neutral CDase. RNAi for the enzyme remarkably decreased the CDase activity in a cell lysate as well as a culture supernatant of S2 cells mostly at neutral pH, indicating that both activities were derived from the same gene product.     

Molecular cloning and functional characterization of zebrafish ATM

Ataxia-telangiectasia mutated (ATM) is the gene product mutated in ataxia-telangiectasia (A-T), which is an autosomal recessive disorder with symptoms including neurodegeneration, cancer predisposition and premature aging. ATM is thought to play a pivotal role in signal transduction in response to genotoxic DNA damage. To study the physiological and developmental functions of ATM using the zebrafish model system, we cloned the zebrafish homolog cDNA of human ATM (hATM), zebrafish ATM (zATM), analyzed the expression pattern of zATM during early development, and further developed the system to study loss of zATM function in zebrafish embryos. Employing information available from the zebrafish genomic database, we utilized a PCR-based approach to isolate zATM cDNA clones. Sequence analysis of zATM showed a high level homology in the functional domains of hATM. The putative FAT, phosphoinositide 3-kinase-like, and FATC domains of zATM, which regulate ATM kinase activity and functions, were the most highly conserved regions, exhibiting 64-94% amino acid identity to the corresponding domains in hATM, while exhibiting approximately 50% amino acid identity outside these domains. The zATM gene is expected to consist of 62 coding exons, and we have identified at least 55 exons encompassing more than 100kb of nucleotide sequence, which encodes about 9 kb of cDNA. By in situ hybridization, zATM mRNA was detected ubiquitously with a dramatic increase at the 18-somite stage, then more specifically in the eye, brain, trunk, and tail at later stages. To inhibit zATM expression and function, we designed and synthesized splice-blocking antisense-morpholino oligonucleotides targeting the phosphoinositide 3-kinase-like domain. We demonstrated that this knockdown of zATM caused abnormal development upon ionizing radiation-induced DNA damage. Our data suggest that the ATM gene is structurally and functionally conserved in vertebrates from zebrafish to human.     

Molecular cloning and characterization of neutral ceramidase homologue from the red flour beetle, Tribolium castaneum

Ceramidase plays an important role in regulating the metabolism of sphingolipids, such as ceramide, sphingosine (SPH), and sphingosine-1-phosphate (S1P), by controlling the hydrolysis of ceramide. Here we report the cloning and biochemical characterization of a neutral ceramidase from the red flour beetle Tribolium castaneum which is an important storage pest. The Tribolium castaneum neutral ceramidase (Tncer) is a protein of 696 amino acids. It shares a high degree of similarity in protein sequence to neutral ceramidases from various species. Tncer mRNA levels are higher in the adult stage than in pre-adult stages, and they are higher in the reproductive organs than in head, thorax, and midgut. The mature ovary has higher mRNA levels than the immature ovary. Tncer is localized to the plasma membrane. It uses various ceramides (D-erythro-C₆, C₁₂, C₁₆, C_18:1, and C_24:1-ceramide) as substrates and has an abroad pH optimum for its in vitro activity. Tncer has an optimal temperature of 37 °C for its in vitro activity. Its activity is inhibited by Fe²⁺. These results suggest that Tncer has distinct biochemical properties from neutral ceramidases from other species.     

Molecular cloning,regulation, and functional analysis of two GHS-R genes in zebrafish

Mammalian ghrelin is derived from stomach and regulates growth hormone release and appetite by modulating GHS-R (Growth hormone secretagogue receptor) activity. Zebrafish has been developed as a forward genetic screening model system and previous screening identified a number of genes involved in multiple signaling pathways. In this system, ghrelin has been identified and its function and regulation have been shown to be highly conserved to that of mammals.     

Molecular cloning and characterization of a human mitochondrial ceramidase

We have recently purified a rat brain membrane-bound nonlysosomal ceramidase (El Bawab, S., Bielawska, A., and Y. A. Hannun (1999) J. Biol. Chem. 274, 27948-27955). Using peptide sequences obtained from the purified rat brain enzyme, we report here the cloning of the human isoform. The deduced amino acid sequence of the protein did not show any similarity with proteins of known function but was homologous to three putative proteins from Arabidospis thaliana, Mycobacterium tuberculosis, and Dictyostelium discoideum. Several blocks of amino acids were highly conserved in all of these proteins. Analysis of the protein sequence revealed the presence at the N terminus of a signal peptide followed by a putative myristoylation site and a putative mitochondrial targeting sequence. The predicted molecular mass was 84 kDa, and the isoelectric point was 6.69, in agreement with rat brain purified enzyme. Northern blot analysis of multiple human tissues showed the presence of a major band corresponding to a size of 3.5 kilobase. Analysis of this major band on the blot indicated that the enzyme is ubiquitously expressed with higher levels in kidney, skeletal muscle, and heart. The enzyme was then overexpressed in HEK 293 and MCF7 cells using the pcDNA3. 1/His-ceramidase construct, and ceramidase activity (at pH 9.5) increased by 50- and 12-fold, respectively. Next, the enzyme was characterized using lysate of overexpressing cells. The results confirmed that the enzyme catalyzes the hydrolysis of ceramide in the neutral alkaline range and is independent of cations. Finally, a green fluorescent protein-ceramidase fusion protein was constructed to investigate the localization of this enzyme. The results showed that the green fluorescent protein-ceramidase fusion protein presented a mitochondrial localization pattern and colocalized with mitochondrial specific probes. These results demonstrate that this novel ceramidase is a mitochondrial enzyme, and they suggest the existence of a topologically restricted pathways of sphingolipid metabolism.     

Molecular cloning, functional analysis, and RNA expression analysis of connexin45.6: a zebrafish cardiovascular connexin

In the vertebrate cardiovascular system, gap junctions function in intercellular communication essential for both the coordinated propagation of the heartbeat and the control of vasomotor responses in the vascular system. Connexins, the protein subunits of gap junctions, are coded by a multigene family. In this study, a connexin gene (zfCx45.6), which exhibits 53% amino acid identity to chick Cx42, was cloned from zebrafish genomic DNA. With the use of the LN54 radiation hybrid panel, zfCx45.6 was mapped to zebrafish linkage group 9. Northern blots and RT-PCR revealed the presence of zfCx45.6 mRNA in the embryo before 2 h postfertilization (hpf) and then again beginning at about 12 hpf, after which time no major changes in relative expression levels were detected. In the adult, zfCx45.6 mRNA continued to be detected in the heart, as well as the brain, liver, and ovary, but not the lens. Whole mount in situ hybridization revealed zfCx45.6 mRNA was expressed at high levels in the major vessels of the entire embryo and in both the atrium and ventricle of the adult heart. Expression of zfCx45.6 channels in paired Xenopus oocytes produced high levels of intercellular coupling that was voltage sensitive. With the previous isolation of zebrafish Cx43 and Cx43.4, zebrafish orthologues have now been isolated for three of the four connexins expressed in the mammalian cardiovascular system.     

Molecular cloning of the full-length cDNA encoding mouse neutral ceramidase. A novel but highly conserved gene family of neutral/alkaline ceramidases

We report here the molecular cloning, sequencing, and expression of the gene encoding the mouse neutral ceramidase, which has been proposed to function in sphingolipid signaling. A full-length cDNA encoding the neutral ceramidase was cloned from a cDNA library of mouse liver using the partial amino acid sequences of the purified mouse liver ceramidase. The open reading frame of 2,268 nucleotides encoded a polypeptide of 756 amino acids having nine putative N-glycosylation sites. Northern blot analysis revealed that the mRNA of the ceramidase was expressed widely in mouse tissues, with especially strong signals found in the liver and kidney. The ceramidase activity of lysates of CHOP cells increased more than 900-fold when the cells were transformed with a plasmid containing the cDNA encoding ceramidase. We also cloned the ceramidase homologue from the cDNA library of mouse brain and found that the sequence of the open reading frame, but not the 5'-noncoding region, was identical to that of the liver. Interestingly, phylogenetic analysis of various ceramidases clearly indicated that neutral/alkaline ceramidases form a novel but highly conserved gene family that is evolutionarily different from lysosomal acid ceramidases.     

Molecular cloning,expression, and functional characterization of a novel zebrafish cytosolic sulfotransferase

By searching the zebrafish expressed sequence tag (EST) database, we have identified a cDNA clone encoding a putative zebrafish cytosolic sulfotransferase (ST). This cDNA was isolated and subjected to nucleotide sequencing. Analysis of the sequence data revealed that this novel zebrafish ST displays 32-35% amino acid sequence identity to members of all major cytosolic ST gene families. Therefore, this zebrafish ST, while belonging to the cytosolic ST gene superfamily, appears to be independent from all known constituent ST gene families. Recombinant zebrafish ST, expressed using the pET23c prokaryotic expression vector and purified from transformed Escherichia coli cells, migrated as a 34-kDa protein upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified zebrafish ST displayed sulfating activities toward dopamine and thyroid hormones (T(3) and T(4)), with a pH optimum spanning 7-9. The enzyme also exhibited activities toward a number of xenobiotics including some flavonoids, isoflavonoids, and other phenolic compounds. A thermostability experiment revealed the enzyme to be relatively stable over a temperature range between 20 and 48 degrees C. Among 10 divalent metal cations tested, Fe(++), Hg(++), Co(++), Zn(++), Cu(++), and Cd(++) exhibited dramatic inhibitory effects on the activity of the enzyme. These results constitute a first study on the cloning, expression, and characterization of a zebrafish cytosolic ST.     

Molecular cloning and characterization of OsCDase, a ceramidase enzyme from rice

SUMMARY: Sphingolipids are a structurally diverse group of molecules based on long-chain sphingoid bases that are found in animal, fungal and plant cells. In contrast to the situation in animals and yeast, much less is known about the spectrum of sphingolipid species in plants and the roles they play in mediating cellular processes. Here, we report the cloning and characterization of a plant ceramidase from rice (Oryza sativa spp. Japonica cv. Nipponbare). Sequence analysis suggests that the rice ceramidase (OsCDase) is similar to mammalian neutral ceramidases. We demonstrate that OsCDase is a bona fide ceramidase by heterologous expression in the yeast double knockout mutant Deltaypc1Deltaydc1 that lacks the yeast ceramidases YPC1p and YDC1p. Biochemical characterization of OsCDase showed that it exhibited classical Michaelis-Menten kinetics, with optimum activity between pH 5.7 and 6.0. OsCDase activity was enhanced in the presence of Ca(2+), Mg(2+), Mn(2+) and Zn(2+), but inhibited in the presence of Fe(2+). OsCDase appears to use ceramide instead of phytoceramide as a substrate. Subcellular localization showed that OsCDase is localized to the endoplasmic reticulum and Golgi, suggesting that these organelles are sites of ceramide metabolism in plants.     

Genomic structure and promoter analysis of the mouse neutral ceramidase gene

We report here the molecular cloning of the mouse neutral ceramidase gene and its promoter analysis. The gene, composed of 27 exons ranging in size from 40 to 292 bp, spans more than 70 kb. Analysis of the 5(')-flanking region of the ceramidase genes revealed that the first exon of the gene of mouse liver was exactly the same as that of mouse kidney and Swiss 3T3 fibroblasts but completely different from that of mouse brain. The putative promoter regions of liver and brain ceramidase genes contained several well-characterized promoter elements such as GATA-2, C/EBP, and HNF3beta but lacked TATA and CAAT boxes, a typical feature of a housekeeping gene, although the expression is regulated in a tissue-specific manner. Interestingly, a GC box was exclusively found in the putative promoter of mouse liver whereas potential AP1 and AP4 binding sites were present in that of mouse brain. By a luciferase reporter gene assay, it was shown that the GC-rich region, which exists just upstream of the first exon, conferred the promoter activity in Swiss 3T3 cells.     

Molecular cloning and characterization of the alkaline ceramidase from Pseudomonas aeruginosa PA01

Ceramidase (CDase) hydrolyzes the amide bond in ceramides to yield free fatty acid and sphingosine. From a 3-L Pseudomonas aeruginosa PA01 culture, 70 microg of extracellular alkaline, Ca(2+)-dependent CDase, was purified to homogeneity, the N-terminal sequence was determined, and the CDase gene was cloned. The CDase gene encodes a 670 amino acid protein with a 26 amino acid signal peptide. CDase was expressed in five prokaryotic and eukaryotic expression systems. Small amounts of recombinant active extracellular CDase were expressed by Pseudomonas putida KT2440. In Pichia pastoris GS115 low amounts of recombinant extracellular glycosylated CDase were expressed. High levels of intracellular CDase were expressed by Escherichia coli DH5alpha and E. coli BL21 cells under control of the lac-promoter and T7-promoter, respectively. From a 3-L E. coli DH5alpha culture, 280 microg of pure CDase was obtained after a three-step purification protocol. Under control of the T7-promotor CDase, without its signal peptide, was produced in inclusion bodies in E. coli BL21 cells. After refolding, 1.8 mg of pure active CDase was obtained from a 2.4-L culture after ammonium sulfate precipitation and gel filtration. Both the recombinant and wild-type CDases have a pH optimum of 8.5. The recombinant enzyme was partially characterized. This is the first report of a high yield CDase production system allowing detailed characterization of the enzyme at the molecular level.     

Purification, characterization, molecular cloning, and subcellular distribution of neutral ceramidase of rat kidney

Previously, we reported two types of neutral ceramidase in mice, one solubilized by freeze-thawing and one not. The former was purified as a 94-kDa protein from mouse liver, and cloned (Tani, M., Okino, N., Mori, K., Tanigawa, T., Izu, H., and Ito, M. (2000) J. Biol. Chem. 275, 11229--11234). In this paper, we describe the purification, molecular cloning, and subcellular distribution of a 112-kDa membrane-bound neutral ceramidase of rat kidney, which was completely insoluble by freeze-thawing. The open reading frame of the enzyme encoded a polypeptide of 761 amino acids having nine putative N-glycosylation sites and one possible transmembrane domain. In the ceramidase overexpressing HEK293 cells, 133-kDa (Golgi-form) and 113-kDa (endoplasmic reticulum-form) Myc-tagged ceramidases were detected, whereas these two proteins were converted to a 87-kDa protein concomitantly with loss of activity when expressed in the presence of tunicamycin, indicating that the N-glycosylation process is indispensable for the expression of the enzyme activity. Immunohistochemical analysis clearly showed that the ceramidase was mainly localized at the apical membrane of proximal tubules, distal tubules, and collecting ducts in rat kidney, while in liver the enzyme was distributed with endosome-like organelles in hepatocytes. Interestingly, the kidney ceramidase was found to be enriched in the raft microdomains with cholesterol and GM1 ganglioside.     

Purification and characterization of human intestinal neutral ceramidase

Sphingolipids are degraded by sphingomyelinase and ceramidase in the gut to ceramide and sphingosine, which may inhibit cell proliferation and induce apoptosis, and thus have anti-tumour effects in the gut. Although previous rodent studies including experiments on knockout mice indicate a role of neutral ceramidase in ceramide digestion, the human enzyme has never been purified and characterized in its purified form. We here report the purification and characterization of neutral ceramidase from human ileostomy content, using octanoyl-[(14)C]sphingosine as substrate. After four chromatographic steps, a homogeneous protein band with 116kDa was obtained. MALDI mass spectrometry identified 16 peptide masses similar to human ceramidase previously cloned by El Bawab et al. [Molecular cloning and characterization of a human mitochondrial ceramidase, J. Biol. Chem. 275 (2000) 21508-21513] and Hwang et al. [Subcellular localization of human neutral ceramidase expressed in HEK293 cells, Biochem. Biophys. Res. Commun. 331 (2005) 37-42]. By RT-PCR and 5'-RACE methods, a predicted partial nucleotide sequence of neutral ceramidase was obtained from a human duodenum biopsy sample, which was homologous to that of known neutral/alkaline ceramidases. The enzyme has neutral pH optimum and catalyses both hydrolysis and formation of ceramide without distinct bile salt dependence. It is inhibited by Cu(2+) and Zn(2+) ions and by low concentrations of cholesterol. The enzyme is a glycoprotein but deglycosylation does not affect its activity. Our study indicates that neutral ceramidase is expressed in human intestine, released in the intestinal lumen and plays a major role in ceramide metabolism in the human gut.     

Molecular cloning and functional analysis of the MutY homolog of Deinococcus radiodurans

The mutY homolog gene (mutY(Dr)) from Deinococcus radiodurans encodes a 39.4-kDa protein consisting of 363 amino acids that displays 35% identity to the Escherichia coli MutY (MutY(Ec)) protein. Expressed MutY(Dr) is able to complement E. coli mutY mutants but not mutM mutants to reduce the mutation frequency. The glycosylase and binding activities of MutY(Dr) with an A/G-containing substrate are more sensitive to high salt and EDTA concentrations than the activities with an A/7,8-dihydro-8-oxoguanine (GO)-containing substrate are. Like the MutY(Ec) protein, purified recombinant MutY(Dr) expressed in E. coli has adenine glycosylase activity with A/G, A/C, and A/GO mismatches and weak guanine glycosylase activity with a G/GO mismatch. However, MutY(Dr) exhibits limited apurinic/apyrimidinic lyase activity and can form only weak covalent protein-DNA complexes in the presence of sodium borohydride. This may be due to an arginine residue that is present in MutY(Dr) at the position corresponding to the position of MutY(Ec) Lys142, which forms the Schiff base with DNA. The kinetic parameters of MutY(Dr) are similar to those of MutY(Ec). Although MutY(Dr) has similar substrate specificity and a binding preference for an A/GO mismatch over an A/G mismatch, as MutY(Ec) does, the binding affinities for both mismatches are slightly lower for MutY(Dr) than for MutY(Ec). Thus, MutY(Dr) can protect the cell from GO mutational effects caused by ionizing radiation and oxidative stress.     

Molecular cloning and functional analysis of three subunits of yeast proteasome.

The genes encoding three subunits of Saccharomyces cerevisiae proteasome were cloned and sequenced. The deduced amino acid sequences were homologous not only to each other (30 to 40% identity) but also to those of rat and Drosophila proteasomes (25 to 65% identity). However, none of these sequences showed any similarity to any other known sequences, including various proteases, suggesting that these proteasome subunits may constitute a unique gene family. Gene disruption analyses revealed that two of the three subunits (subunits Y7 and Y8) are essential for growth, indicating that the proteasome and its individual subunits play an indispensable role in fundamental biological processes. On the other hand, subunit Y13 is not essential; haploid cells with a disrupted Y13 gene can proliferate, although the doubling time is longer than that of cells with nondisrupted genes. In addition, biochemical analysis revealed that proteasome prepared from the Y13 disrupted cells contains tryptic and chymotryptic activities equivalent to those of nondisrupted cells, indicating that the Y13 subunit is not essential for tryptic or chymotryptic activity. However, the chymotryptic activity of the Y13 disrupted cells is not dependent on sodium dodecyl sulfate (SDS), an activator of proteasome, since nearly full activity was observed in the absence of SDS. Thus, the activity in proteasome of the Y13 disrupted cells might result in unregulated intracellular proteolysis, thus leading to the prolonged cell cycle. These results indicate that cloned proteasome subunits having similar sequences to the yeast Y13 subunit are structural, but not catalytic, components of proteasome. It is also suggested that two subunits (Y7 and Y8) might occupy positions essential to proteasome structure or activity, whereas subunit Y13 is in a nonessential but important position.