TY - JOUR
T1 - Soluble expression in E. coli of a functional interphotoreceptor retinoid-binding protein module fused to thioredoxin
T2 - Correlation of vitamin A binding regions with conserved domains of C-terminal processing proteases
AU - Baer, C. A.
AU - Retief, J. D.
AU - Van Niel, E.
AU - Braiman, M. S.
AU - Gonzalez-Fernandez, F.
N1 - Funding Information:
The authors thank Dr Vladimir Kalashnikov and Dr Yongbe Bao of the University of Virginia Biomedical Research Facility for determining the concentration of the purified recombinant IRBP solutions by amino acid analysis, and confirming the sequence of the expression plasmids by DNA sequencing. The Biomedical Research Facility is funded the University of Virginia Pratt Committee. The LC-MS and capillary LC-tandem mass spectrometry analysis was performed by Dr Michael Kinter at the University of Virginia W. M. Keck Biomedical Mass Spectrometry Laboratory. Our study was supported by National Institutes of Health grants EY09412 (F.G-F.); The Thomas F. Jeffress and Kate Miller Memorial Trust (F.G.-F.); Medical Scientist Training Program of the NIH GM 07267 (C.A.B.); National Science Foundation instrumentation and instrument development grants BIR-901677 and BIR-9216996 to the Department of Biochemistry; and an unrestricted grant from Research to Prevent Blindness to the Department of Ophthalmology at the University of Virginia Health Sciences Center.
PY - 1998/2
Y1 - 1998/2
N2 - 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 K(D) = 1.25 ± 0.39. One of the two sites was localized to Exons(2 + 3) and had a K(D) = 0.26 ± 0.13 μM. 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 K(D) = 1.94 ± 0.20 μM. 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.
AB - 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 K(D) = 1.25 ± 0.39. One of the two sites was localized to Exons(2 + 3) and had a K(D) = 0.26 ± 0.13 μM. 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 K(D) = 1.94 ± 0.20 μM. 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.
KW - Carboxy-terminal processing protease
KW - Interphotoreceptor retinoid-binding protein
KW - Retina
KW - Retinoid-binding proteins
KW - Thioredoxin fusion protein
KW - Vitamin A
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U2 - 10.1006/exer.1997.0418
DO - 10.1006/exer.1997.0418
M3 - Article
C2 - 9533851
AN - SCOPUS:0031913261
SN - 0014-4835
VL - 66
SP - 249
EP - 262
JO - Experimental Eye Research
JF - Experimental Eye Research
IS - 2
ER -