A neutral ceramidase homologue from Dictyostelium discoideum exhibits an acidic pH optimum.
The nucleotide sequence reported for the Dictyostelium discoideum ceramidase is available on the DNA Data Bank of Japan (DDBJ). Ceramidases (CDases) are currently classified into three categories (acid, neutral and alkaline) based on their optimal pHs and primary structures. Here, we report the first exception to this rule. We cloned the CDase cDNA, consisting of 2142 nucleotides encoding 714 amino-acid residues, from the slime mould, Dictyostelium discoideum. The putative amino-acid sequence indicates 32-42% identity with various neutral CDases, but does not show any similarity to the acid and alkaline CDases, indicating the enzyme should be classified as a neutral CDase. However, overexpression of the cDNA in D. discoideum resulted in increased CDase activity at an acidic, but not a neutral pH range. Knockout of the gene in slime mould eliminated CDase activity at acidic pH. The recombinant enzyme expressed in the slime mould was purified and then characterized. Consequently, the purified CDase was found to exhibit the maximal activity at approx. pH 3.0. The singular pH dependency of slime mould CDase is not derived from the specific post-translational modification in the slime mould, because the enzyme showed an acidic pH optimum even when expressed in Chinese hamster ovary cells, whereas rat neutral-CDase exhibited a neutral pH optimum when expressed in slime mould.
Monjusho
Hatsumi
H
Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.
Okino
Nozomu
N
Tani
Motohiro
M
Maeda
Mineko
M
Yoshida
Motonobu
M
Ito
Makoto
M
D000581:Amidohydrolases / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D055572:Ceramidases; D003001:Cloning, Molecular; D004023:Dictyostelium / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D055576:Neutral Ceramidase; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013379:Substrate Specificity
D000581:Amidohydrolases; C526748:Asah2 protein, rat; D055572:Ceramidases; D055576:Neutral Ceramidase