Cloning of an emopamil-binding protein (EBP)-like protein that lacks sterol Δ8-Δ7 isomerase activity

Abstract

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.

ABotrytis cinereaEmopamil Binding Domain Protein, Required for Full Virulence, Belongs to a Eukaryotic Superfamily Which Has Expanded in Euascomycetes

“…6). The monophyly of the EBP-like clade, previously identified by Moebius and colleagues (47), is strongly supported, and fungal EBDPs encoded by single-copy genes were related to this clade (Fig. 6).…”

“…In euascomycetes, four EBD-encoding genes were found in the genome of B. cinerea, three were found in each of the genomes of Sclerotinia sclerotiorum, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus oryzae, Fusarium graminearum, and Magnaporthe grisea, and two were found in each of the genomes of Neurospora crassa and Stagonospora nodorum. Two EBD-encoding genes in each of the vertebrate genomes were also identified, as previously reported (47). Finally, a single EBDencoding gene was detected in the genomes of the basidiomycete species Ustilago maydis, Phanerochaete chrysosporium, Coprinus cinereus, and Laccaria bicolor and of the plants Arabidopsis thaliana, Zea mays, and Oryza sativa.…”

“…Third, there is a similar example of a protein which, despite sharing basic structural features with EBPs, has no detectable ⌬ 8 -⌬ 7 isomerase or sigma ligand binding activity: the human EBP-like protein (47). The absence of both activities in the EBP-like protein is in agreement with the hypothesis of Jbilo and colleagues (31), who suggested that the sterol binding pocket present in the SI constitutes the sigma ligand binding site, regardless of the protein in which this pocket is found.…”

“…SIs (EC 5.3.3.5) act at one of the last steps of the postsqualene sterol biosynthesis pathway. Interestingly, the EBD is also present in another type of proteins, called EBP-like proteins, which have been identified in vertebrates (47). The only studied EBP-like protein, the human EBP-like protein, lacks both the SI and the sigma ligand binding activities, despite sharing basic structural features with EBPs.…”

“…The only studied EBP-like protein, the human EBP-like protein, lacks both the SI and the sigma ligand binding activities, despite sharing basic structural features with EBPs. Its function is still unknown (47).…”