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.     

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.     

Subcellular localization of human neutral ceramidase expressed in HEK293 cells

We previously reported that rat and mouse neutral ceramidases were mainly localized to plasma membranes as a type II integral membrane protein and partly detached from the cells via processing of the N-terminal/anchor sequence when expressed in HEK293 cells [M. Tani, H. Iida, M. Ito, O-glycosylation of mucin-like domain retains the neutral ceramidase on the plasma membranes as a type II integral membrane protein, J. Biol. Chem. 278 (2003) 10523-10530]. In contrast, the human homologue was exclusively detected in mitochondria when expressed in HEK293 and MCF7 cells as a fusion protein with green fluorescent protein at the N-terminal of the enzyme [S.E. Bawab, P. Roddy, T. Quian, A. Bielawska, J.J. Lemasters, Y.A. Hannun, Molecular cloning and characterization of a human mitochondrial ceramidase, J. Biol. Chem. 275 (2000) 21508-21513]. Given this discrepancy, we decided to clone the neutral ceramidase from human kidney cDNA and re-examine the intracellular localization of the enzyme when expressed in HEK293 cells. The putative amino acid sequence of the newly cloned enzyme was identical to that reported for human neutral ceramidase except at the N-terminal; the new protein was 19 amino acids longer at the N-terminal. We found that the putative full-length human neutral ceramidase was transported to plasma membranes, but not to mitochondria, possibly via a classical ER/Golgi pathway and localized mainly in plasma membranes when expressed in HEK293 cells. The N-terminal-truncated mutant, previously reported as a human mitochondrial ceramidase, was also weakly expressed in HEK293 cells but mainly released into the medium possibly due to the insufficient signal/anchor sequence.     

Molecular cloning, sequencing, and expression of the gene encoding alkaline ceramidase from Pseudomonas aeruginosa. Cloning of a ceramidase homologue from Mycobacterium tuberculosis

We previously reported the purification and characterization of a novel type of alkaline ceramidase from Pseudomonas aeruginosa strain AN17 (Okino, N., Tani, M., Imayama, S., and Ito, M. (1998) J. Biol. Chem. 273, 14368-14373). Here, we report the molecular cloning, sequencing, and expression of the gene encoding the ceramidase of this strain. Specific oligonucleotide primers were synthesized using the peptide sequences of the purified ceramidase obtained by digestion with lysylendopeptidase and used for polymerase chain reaction. DNA fragments thus amplified were used as probes to clone the gene encoding the ceramidase from a genomic library of strain AN17. The open reading frame of 2,010 nucleotides encoded a polypeptide of 670 amino acids including a signal sequence of 24 residues, 64 residues of which matched the amino acid sequence determined for the purified enzyme. The molecular weight of the mature enzyme was estimated to be 70,767 from the deduced amino acid sequence. Expression of the ceramidase gene in Escherichia coli, resulted in production of a soluble enzyme with the identical N-terminal amino acid sequence. Recombinant ceramidase was purified to homogeneity from the lysate of E. coli cells and confirmed to be identical to the Pseudomonas enzyme in its specificity and other enzymatic properties. No significant sequence similarities were found in other known functional proteins including human acid ceramidase. However, we found a sequence homologous to the ceramidase in hypothetical proteins encoded in Mycobacterium tuberculosis, Dictyostelium discoideum, and Arabidopsis thaliana. The homologue of the ceramidase gene was thus cloned from an M. tuberculosis cosmid and expressed in E. coli, and the gene was demonstrated to encode an alkaline ceramidase. This is the first report for the cloning of an alkaline ceramidase.     

Purification and characterization of a neutral ceramidase from mouse liver. A single protein catalyzes the reversible reaction in which ceramide is both hydrolyzed and synthesized

We report here a novel ceramidase that was purified more than 150, 000-fold from the membrane fraction of mouse liver. The enzyme was a monomeric polypeptide having a molecular mass of 94 kDa and was highly glycosylated with N-glycans. The amino acid sequence of a fragment obtained from the purified enzyme was homologous to those deduced from the genes encoding an alkaline ceramidase of Pseudomonas aeruginosa and a hypotheical protein of the slime mold Dictyostelium discoideum. However, no significant sequence similarities were found in other known functional proteins including acid ceramidases of humans and mice. The enzyme hydrolyzed various N-acylsphingosines but not galactosylceramide, sulfatide, GM1a, or sphingomyelin. The enzyme exhibited the highest activity around pH 7.5 and was thus identified as a type of neutral ceramidase. The apparent K(m) and V(max) values for C12-4-nitrobenzo-2-oxa-1, 3-diazole-ceramide and C16-(14)C-ceramide were 22.3 microM and 29.1 micromol/min/mg and 72.4 microM and 3.6 micromol/min/mg, respectively. This study also clearly demonstrated that the purified 94-kDa ceramidase catalyzed the condensation of fatty acid to sphingosine to generate ceramide, but did not catalyze acyl-CoA-dependent acyl-transfer reaction.     

Molecular cloning and expression of aminopeptidase A isoforms from rat hippocampus

The full-length cDNA encoding aminopeptidase A (APAL) was cloned from a rat hippocampus cDNA library. A short variant aminopeptidase A (APAS), produced by deletion, was also cloned. In the case of APAL, the longest open reading frame encodes 945 amino acid residues with a calculated molecular mass of 108 kDa, and the deduced amino acid sequence shows 76, 86 and 78% identity with its human, murine and porcine counterparts, respectively. Rat aminopeptidase A mRNAs were detected in the kidney, liver, heart and brain by Northern blot analysis. When overexpressed in COS-1 cells, APAL shows apparent aminopeptidase A activity, whereas APAS does not.     

Molecular cloning and functional analysis of zebrafish neutral ceramidase

Almost all observations on the functions of neutral ceramidase have been carried out at cellular levels but not at an individual level. Here, we report the molecular cloning of zebrafish neutral ceramidase (znCD) and its functional analysis during embryogenesis. We isolated a cDNA clone encoding znCD by 5' and 3' rapid amplification of cDNA ends-PCR. It possessed an open reading frame of 2,229 base pairs encoding 743 amino acids. A possible signal/anchor sequence near the N terminus and four potential O-glycosylation and eight potential N-glycosylation sites were found in the putative sequence. The enzyme activity at neutral pH increased markedly after transformation of Chinese hamster CHOP and zebrafish BRF41 cells with the cDNA. The overexpressed enzyme was found to be distributed in endoplasmic reticulum/Golgi compartments as well as the plasma membranes. The antisense morpholino oligonucleotide (AMO), which was designed based on the sequence of znCD mRNA, successfully blocked the translation of znCD in a wheat germ in vitro translation system. The knockdown of znCD with AMO led to an increase in the number of zebrafish embryos with severe morphological and cellular abnormalities such as abnormal morphogenesis in the head and tail, pericardiac edema, defect of blood cell circulation, and an increase of apoptotic cells, especially in the head and neural tube regions, at 36 h post-fertilization. The ceramide level in AMO-injected embryos increased significantly compared with that in control embryos. Simultaneous injection of both AMO and synthetic znCD mRNA into one-cell-stage embryos rescued znCD activity and blood cell circulation. These results indicate that znCD is essential for the metabolism of ceramide and the early development of zebrafish.     

Specific and sensitive assay for alkaline and neutral ceramidases involving C12-NBD-ceramide

A fluorescent analogue of ceramide, C12-NBD-ceramide, was found to be hydrolyzed much faster than 14C-labeled ceramide by alkaline ceramidase from Pseudomonas aeruginosa and neutral ceramidase from mouse liver, while this substrate was relatively resistant to acid ceramidase from plasma of the horseshoe crab. The radioactive substrate was used more preferentially by the acid ceramidase. It should be noted that C6-NBD-ceramide, which is usually used for ceramidase assays, was hardly hydrolyzed by any of the enzymes examined, compared to C12-NBD-ceramide. For the alkaline and neutral enzymes, the Vmax and k (Vmax/Km) with C12-NBD-ceramide were much higher than those with 14C-ceramide. In contrast, for the acid enzyme these parameters with C12-NBD-ceramide were less than half those with the radioisotope-labeled substrate. It is noteworthy that the labeling of ceramide with NBD did not itself reduce the Km of the alkaline enzyme, but did that of the neutral enzyme. It was also found that C12-NBD-ceramide was preferentially hydrolyzed by the alkaline and neutral enzymes, but not the acid one, in several mammalian cell lines. This study clearly shows that the attachment of NBD, but not dansyl, increases the susceptibility of ceramide to alkaline and neutral enzyme, and decreases that to acid enzymes. Thus the use of this substrate provides a specific and sensitive assay for alkaline and neutral ceramidases.     

Molecular cloning and sequence analysis of the cDNA for the mouse counterpart to adult hamster liver purified growth inhibitory factor

A cDNA clone encoding the mouse counterpart to adult hamster liver purified growth inhibitory factor (PGIF) was isolated from a mouse liver cDNA library by using antibodies raised against PGIF and sequenced. It contained a single open reading frame with a coding capacity for a 323 amino acid protein. Sequence analysis showed that it shared high homology with rat- and human liver arginases: the cDNA clone was 92% identical for rat arginase at the nucleotide level and was 93% identical to it at the deduced amino acid level. These results suggest that PGIF derived from adult hamster liver was identical or closely related to an isoform of hamster liver arginases.     

Molecular cloning and sequence analysis of full-length cDNA coding for mouse contrapsin

Four overlapping cDNA clones encoding contrapsin were isolated from a mouse liver cDNA library constructed in the expression vector, lambda gt11. M13 vector sequence analysis revealed that contrapsin cDNA contained an open reading frame of 1,254 bases encoding 418 amino acids. The N-terminal amino acid sequence of the isolated contrapsin matched residues 30 to 48 of the sequence deduced on nucleotide analysis. One clone, which had the longest 3' untranslated region, contained two sets of tandem polyadenylation signals, AATACA and AATAAA, which were located 497 bases apart, while the remaining three clones terminated at the first signal. The entire reading frame sequence of contrapsin cDNA showed 64% homology with that of human alpha-1-antichymotrypsin.     

Molecular diversity in the adenylylcyclase family. Evidence for eight forms of the enzyme and cloning of type VI.

The conservation of amino acid sequence among types I-IV adenylylcyclase has made it possible to apply the polymerase chain reaction to examine the extent of the molecular diversity within this family of enzymes. cDNA templates from rat heart, liver, kidney, guinea pig brain and testes, and mouse skeletal muscle were amplified with primers specific to adenylylcyclase sequences. Evidence was obtained for a total of eight distinct gene products divisible into five subfamilies. Five of the products correspond to regions from cloned forms of adenylylcyclase, while three are previously unidentified. As many as seven different adenylylcyclases are expressed in rat heart, liver, and kidney based on this analysis. Two newly identified polymerase chain reaction (PCR) products were utilized to screen a rat cDNA library from H35 Reuber hepatoma cells. A 6080-nucleotide cDNA contains an open reading frame encoding the 1166-amino acid type VI protein which has a predicted topography similar to that of other adenylylcyclases. The type VI message is abundantly expressed in rat heart, kidney, and brain. Human embryonal kidney cells stably expressing the cDNA showed an enhanced response to isoproterenol that could be inhibited by carbachol in intact cells. Increases in intracellular Ca2+ contribute to the inhibitory effect of carbachol.     

Sequencing and heterologous expression in Saccharomyces cerevisiae of a Cryptococcus neoformans cDNA encoding a plasma membrane H(+)-ATPase

A cDNA containing an open reading frame encoding a putative plasma membrane H(+)-ATPase in the human pathogenic basidiomycetous yeast Cryptococcus neoformans was cloned and sequenced by means of PCR and cDNA library hybridization. The cloned cDNA is 3475 bp in length, containing a 2994 bp open reading frame encoding a polypeptide of 997 amino acids. As in the case of another basidiomycetous fungus (Uromyces fabae), the deduced amino acid sequence of CnPMA1 was found to be more homologous to those of P-type H(+)-ATPases from higher plants than to those from ascomycetous fungi. In order to prove the sequenced cDNA corresponds to a H(+)-ATPase, it was expressed in Saccharomyces cerevisiae and found to functionally replace its own H(+)-ATPase. Kinetic studies of CnPMA1 compared to ScPMA1 show differences in V(max) values and H(+)-pumping in reconstituted vesicles. The pH optimum and K(m) values are similar in both enzymes.     

Molecular cloning of complementary DNA for human medullasin: an inflammatory serine protease in bone marrow cells

Medullasin, an inflammatory serine protease in bone marrow cells, modifies the functions of natural killer cells, monocytes, and granulocytes. We have cloned a medullasin cDNA from a human acute promyelocytic cell (ML3) cDNA library using oligonucleotide probes synthesized from the information of N-terminal amino acid sequence of natural medullasin. The cDNA contained a long open reading frame encoding 237 amino acid residues beginning from the second amino acid of natural meduallasin. The deduced amino acid sequence of medullasin shows a typical serine protease structure, with 41% homology with pig elastase 1.     

Molecular cloning and expression of a second glucuronyltransferase involved in the biosynthesis of the HNK-1 carbohydrate epitope

A cDNA encoding a novel glucuronyltransferase was cloned from a rat brain cDNA library. The cDNA sequence contained an open reading frame encoding 324 amino acids, with type II transmembrane topology. The amino acid sequence revealed 49% homology to rat GlcAT-P, a glucuronyltransferase involved in the biosynthesis of the HNK-1 carbohydrate epitope of glycoproteins, [Terayama et al. (1997) Proc. Natl. Acad. Sci. USA 94, 6093-6098] and the highest sequence homology was found in the catalytic region. Northern blot analysis indicated that this newly cloned glucuronyltransferase is expressed in the nervous system, consistent with the selective localization of the HNK-1 carbohydrate epitope in the nervous system. Transfection of this cDNA into COS-1 cells induced the expression of the HNK-1 carbohydrate epitope on cell surfaces, and induced the morphological changes in these cells. These results indicated that this newly cloned cDNA is a second glucuronyltransferase involved in the biosynthesis of the HNK-1 carbohydrate epitope.     

Molecular cloning and characterization of a ras p21-like GTP-binding protein (24KG) from rat liver.

We have isolated cDNA clones from a rat liver cDNA library that encode a ras p21-like small GTP-binding protein (24KG) which was purified from the microsomes-Golgi complex fraction of the rat liver. The cloning was accomplished using polymerase chain reaction amplified with a set of oligonucleotide primers which were designed from the partial amino acid sequences for 24KG. The cDNA contained an open reading frame encoding a 216 amino acid protein with a calculated Mr weight of 24,397. This Mr weight was similar to that of the purified 24KG estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The sequence analysis of 24KG revealed that a 24KG cDNA is the rat counterpart of a rab11 cDNA cloned from a Madin-Darby canine kidney cell cDNA library. The 1.0-kilobase 24KG mRNA corresponding to the isolated cDNA was also detected in various rat tissues, such as brain, testis, spleen, and heart.     

Molecular cloning and characterization of phosphatidylcholine transfer protein-like protein gene expressed in murine haploid germ cells

We have isolated a cDNA clone specifically expressed in spermiogenesis from a subtracted cDNA library of mouse testis. The cDNA consisted of 1392 nucleotides and had an open reading frame of 873 nucleotides encoding a protein of 291 amino acid residues. Computer-mediated homology search revealed that the nucleotide sequence was unique but the deduced amino acid sequence had similarity to mouse phosphatidylcholine transfer protein (PCTP). We named this newly isolated gene PCTP-like protein. Northern blot analysis revealed a 1.4-kilobase mRNA expressed in the testis, kidney, liver, and intestine with the highest level in the testis. Messenger RNA expression in the testis was detected first on Day 23 in postnatal development and then increased up to adulthood. The protein, having a molecular weight of approximately 40 000, was encoded by the mRNA and was detected at the tail of the elongated spermatids and sperm by immunohistochemical staining.     

柽柳(Tamarix androssowii)Tadir基因的克隆及分析

在柽柳cDNA文库测序中获得了Tadir基因的全长cDNA序列,去除PolyA后,该基因全长724bp。其中5′非翻译区26bp,3′非翻译区143bp,开放阅读框(ORF)长555bp,编码184个氨基酸。基因编码蛋白的分子量为19.69kD,理论等电点为6.96。疏水性分析表明,蛋白的前41个氨基酸为亲水性的。该基因的Genbank登录号为DQ462418(基因),ABE73781(蛋白)。实时荧光定量PCR分析结果表明,0.4mol·L-1NaCl和NaHCO3胁迫后该基因表达量发生变化,其可能与柽柳的耐盐性有关。     

Activity of neutral and alkaline ceramidases on fluorogenic N-acylated coumarin-containing aminodiols

Ceramidases catalyze the cleavage of ceramides into sphingosine and fatty acids. Previously, we reported on the use of the RBM14 fluorogenic ceramide analogs to determine acidic ceramidase activity. In this work, we investigated the activity of other amidohydrolases on RBM14 compounds. Both bacterial and human purified neutral ceramidases (NCs), as well as ectopically expressed mouse neutral ceramidase hydrolyzed RBM14 with different selectivity, depending on the N-acyl chain length. On the other hand, microsomes from alkaline ceramidase (ACER)3 knockdown cells were less competent at hydrolyzing RBM14C12, RBM12C14, and RBM14C16 than controls, while microsomes from ACER2 and ACER3 overexpressing cells showed no activity toward the RBM14 substrates. Conversely, N-acylethanolamine-hydrolyzing acid amidase (NAAA) overexpressing cells hydrolyzed RBM14C14 and RBM14C16 at acidic pH. Overall, NC, ACER3, and, to a lesser extent, NAAA hydrolyze fluorogenic RBM14 compounds. Although the selectivity of the substrates toward ceramidases can be modulated by the length of the N-acyl chain, none of them was specific for a particular enzyme. Despite the lack of specificity, these substrates should prove useful in library screening programs aimed at identifying potent and selective inhibitors for NC and ACER3.     

Gene note. Cloning of a cDNA encoding the thaumatin-like protein of winter rye (Secale cereale L. Musketeer) and its functional characterization

A cDNA clone, WRTLP2, encoding an open reading frame of 173 amino acids, was recovered from a cDNA library of winter rye (Secale cereale L. Musketeer). The amino acid sequence deduced from the cloned cDNA exhibits very high sequence similarity (70-95%) with those of extracellular and low molecular weight thaumatin-like proteins of other cereals. It was possible to overexpress this isolated cDNA in Escherichia coli and it was found that the encoded protein of this clone exhibited antifungal activities against several fungal strains.