Molecular cloning and characterization of a human mitochondrial ceramidase

We have recently purified a rat brain membrane-bound nonlysosomal ceramidas e (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 Arabidosp is thaliana, Mycobacterium tuberculosis, and Dictyostelium discoideum, Seve ral blocks of amino acids were highly conserved in all of these proteins. A nalysis 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 en zyme. 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 m ajor band on the blot indicated that the enzyme is ubiquitously expressed w ith higher levels in kidney, skeletal muscle, and heart. The enzyme was the n 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-fol d, respectively. Next, the enzyme was characterized using lysate of overexp ressing cells. The results confirmed that the enzyme catalyzes the hydrolys is of ceramide in the neutral alkaline range and is independent of cations, Finally, a green fluorescent protein-ceramidase fusion protein was constru cted to investigate the localization of this enzyme. The results showed tha t the green fluorescent protein-ceramidase fusion protein presented a mitoc hondrial localization pattern and colocalized with mitochondrial specific p robes. These results demonstrate that this novel ceramidase is a mitochondr ial enzyme, and they suggest the existence of a topologically restricted pa thways of sphingolipid metabolism.