Abstract
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.
Highlights
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
Acid CDase in lysosomes plays a crucial role in the catabolism of ceramide, and an inherited deficiency of this enzyme leads to the accumulation of ceramide in lysosomes that is known as Farber disease [4]
Genes encoding isoforms of CDase showing neutral to alkaline pH optima have not yet been cloned these enzymes could function in sphingosine-mediated signal transduction pathways in eukaryotic cells (8 –10)
Summary
(Received for publication, June 16, 1999, and in revised form, September 18, 1999). Nozomu Okino, Sachiyo Ichinose‡, Akira Omori‡, Shuhei Imayama§, Takashi Nakamura, and Makoto Ito¶. We report the molecular cloning, sequencing, and expression of the gene encoding the ceramidase of this strain. Expression of the ceramidase gene in Escherichia coli, resulted in production of a soluble enzyme with the identical N-terminal amino acid sequence. Genes encoding isoforms of CDase showing neutral to alkaline pH optima have not yet been cloned these enzymes could function in sphingosine-mediated signal transduction pathways in eukaryotic cells (8 –10). We purified and characterized a CDase from the Gram-negative bacterium Pseudomonas aeruginosa, strain AN17, isolated from a patient with atopic dermatitis [21] This CDase, the first reported from a prokaryote, showed the highest activity around pH 8.5 and required calcium ions for its. We report the molecular cloning, sequencing, and expression of the gene encoding the alkaline CDase from P. aeruginosa. We describe the cloning and expression of a CDase homologue in Mycobacterium tuberculosis which is known to cause tuberculosis
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