Cloning of an emopamil-binding protein (EBP)-like protein that lacks sterol delta8-delta7 isomerase activity.
EBP (emopamil-binding protein) is a high-affinity binding protein for [3H]emopamil and belongs to the family of so-called sigma receptors. Mutations that disrupt EBP's 3beta-hydroxysteroid sterol delta8-delta7 isomerase activity (EC 5.3.3.5) impair cholesterol biosynthesis and cause X-chromosomal dominant chondrodysplasia punctata. We identified a human cDNA for a novel EBPL (EBP-like protein) with a calculated mass of 23.2 kDa. Amino acid sequence alignments and phylogenetic analysis revealed that EBPL is distantly related to EBP (31% identity and 52% similarity) and found in animals but not in plants. EBPL is encoded by four exons on human chromosome 13q14.2 covering 30.7 kb, and a partially processed EBPL pseudogene was found on 16q21. The EBPL mRNA was expressed ubiquitously and most abundant in liver, lung and kidney. Upon heterologous expression in yeast EBPL had no detectable 3beta-hydroxysteroid sterol delta8-delta7 isomerase and sigma-ligand-binding activity. Nine out of ten amino acid residues essential for catalytic activity of EBP were conserved in EBPL. Replacement of the only differing residue (EBP-Y111W) reduced catalytic activity of EBP. Transfer of the divergent residue from EBP to EBPL (EBPL-W91Y) and chimaerization of EBP and EBPL at various positions failed to restore catalytic activity of EBPL. Chemical cross-linking induced homodimerization of EBPL and EBP. Whereas mevinolin increased the mRNA for EBP and DHCR7 (delta7-sterol reductase) in HepG2 cells, it had no effect on mRNAs for EBPL and sigma1 receptor, indicating that EBP and EBPL expression are not co-ordinated. We propose that EBPL has a yet-to-be-discovered function other than cholesterol biosynthesis.
Moebius
Fabian F
FF
Institut für Biochemische Pharmakologie, Peter-Mayr-Strasse 1, A-6020 Innsbruck, Austria. Fabian.Moebius@uibk.ac.at
Fitzky
Barbara U
BU
Wietzorrek
Georg
G
Haidekker
Alexander
A
Eder
Andrea
A
Glossmann
Hartmut
H
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002874:Chromosome Mapping; D002893:Chromosomes, Human, Pair 3; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005091:Exons; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D016415:Sequence Alignment; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D013251:Steroid Isomerases / Q000172:deficiency / Q000235:genetics*; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D018076:DNA, Complementary; D010446:Peptide Fragments; D012333:RNA, Messenger; D011994:Recombinant Proteins; D013251:Steroid Isomerases; C022669:delta(8)-delta(7)-sterol isomerase; C090445:EBP protein, human