Molecular Cloning and Characterization of a Human Mitochondrial Ceramidase

Samer El Bawab,Patrick Roddy,Ting Qian,Alicja Bielawska,John J Lemasters,Yusuf A Hannun

doi:10.1074/jbc.m002522200

Abstract

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.

Highlights

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF 250847
Using peptide sequences obtained from the purified rat brain enzyme, we report here the cloning of the human isoform
The results showed that the green fluorescent protein-ceramidase fusion protein presented a mitochondrial localization pattern and colocalized with mitochondrial specific probes

Summary

Introduction

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF 250847. Recent studies are beginning to suggest a role for ceramidases in regulating the net levels of ceramide in response to stimuli. It has been shown in rat hepatocytes that interleukin 1␤ at low concentration activates sphingomyelinases and ceramidases, resulting in the formation of sphingosine, whereas high concentrations of interleukin-1␤, stimulated only sphingomyelinases resulting in the accumulation of ceramide (3). Ceramidases have been shown to be activated in response to platelet-derived growth factor in rat glomerular mesangial cells (6) These studies underscore a key role for ceramidases in regulating cell death and proliferation, in response to various stimuli and in different cell types. These results demonstrate significant compartmentation of sphingolipid metabolism and raise important possibilities on direct interaction between ceramide and mitochondria

Methods

Results

Conclusion