title: Soluble expression in E. coli of a functional interphotoreceptor retinoid-binding protein module fused to thioredoxin: correlation of vitamin A binding regions with conserved domains of C-terminal processing proteases.
abstract: The exchange of all-trans retinol and 11-cis retinal between the photoreceptors and retinal pigmented epithelium is mediated by interphotoreceptor retinoid-binding protein (IRBP). IRBP contains binding sites for retinoids, docosahexaenoic acid and probably cell surface and matrix receptors. IRBP arose through the quadruplication of an ancient protein, represented by its carboxy-terminal module (module 4 in amphibians and mammals). Module 4 has retinol binding activity and is composed of regions coded for by each of IRBP's four exons. Determining the function of the exons has been hampered by insoluble expression of module 4 in Escherichia coli. Here, we found that module 4 of Xenopus IRBP (X4IRBP), as well as its exon segments, can be expressed in a soluble form as thioredoxin fusion proteins. The recombinant proteins were purified by ion exchange and arsenical-based affinity chromatography. Liquid chromatography/mass spectrometry confirmed that the sequence of X4IRBP is correct. All-trans retinol binding was characterized by monitoring enhancement of retinol fluorescence, quenching of intrinsic protein fluorescence, and transfer of energy to the bound retinol. Retinol bound to X4IRBP at 2.20+/-0.29 sites with a KD=1.25+/-0.39. One of the two sites was localized to Exons(2+3) and had a KD=0.26+/-0.13 micron. This site, which supported protein quenching and energy transfer, probably contains at least one of the two conserved tryptophans present in this segment. The second site was localized to Exon 4. This site supported the enhancement of retinol fluorescence but not protein quenching or energy transfer and had a KD=1.94+/-0.20 micron. Exon 1 had no retinol binding activity. The location of the retinol binding regions correlated with the distribution of domains conserved between IRBPs and the newly recognized family of C-terminal processing proteases (CtpAs), proteins which bind and cleave non-polar carboxy termini.
authors.affiliation: Department of Ophthalmology, University of Virginia Health Center, Charlottesville, Virginia 22908, USA.
authors.forename: C A
authors.initials: CA
authors.lastname: Baer
authors.forename: J D
authors.initials: JD
authors.lastname: Retief
authors.forename: E
authors.initials: E
authors.lastname: Van Niel
authors.forename: M S
authors.initials: MS
authors.lastname: Braiman
authors.forename: F
authors.initials: F
authors.lastname: Gonzalez-Fernandez
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017124:Conserved Sequence; D010450:Endopeptidases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D005091:Exons; D005136:Eye Proteins; D008956:Models, Chemical; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D012177:Retinol-Binding Proteins / Q000235:genetics*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013879:Thioredoxins / Q000235:genetics*; D014801:Vitamin A / Q000378:metabolism*; D014981:Xenopus; D000099214:Retinol-Binding Proteins, Interstitial
chemical_list: D010450:Endopeptidases; D005136:Eye Proteins; D011993:Recombinant Fusion Proteins; D012177:Retinol-Binding Proteins; D013879:Thioredoxins; D014801:Vitamin A; D000099214:Retinol-Binding Proteins, Interstitial