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J. C. Saari, G. G. Garwin, M. Nawrot; Release of Retinal From CRALBP by Glycerophospholipids. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3518.
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To examine binding of cellular retinaldehyde-binding protein (CRALBP) to membrane lipids and to determine whether lipids release retinoid from CRALBP. CRALBP is a retinoid-binding protein necessary for efficient regeneration of visual pigments.
Lipids were from natural sources (Avanti Polar Lipids, Inc): PA, phosphatidic acid; PC, phosphatidylcholine: PE, phosphatidylethanolamine; PS, phosphatidylserine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PI(4)P, phosphatidylinositol 4-phosphate; SphM, sphingomyelin. Binding of CRALBP to membrane lipids was measured by incubating CRALBP with nitrocellulose strips dotted with lipids (Echelon Biosciences, Inc.; our fabrication) and immunostaining with anti-CRALBP. Release of retinoids from CRALBP by small unilamellar vesicles (SUVs) of PC doped with various lipids was assessed with spectral and HPLC assays.
Incubation of CRALBP·11-cis-retinal with lipids absorbed on nitrocellulose revealed binding to some anionic lipids [PA > PS > PI(4,5)P2 > PI(3,4,5)P3] and little or no binding to PC, PG, SphM, PG, or PI(4)P. SUVs of PC doped with PA (50 mol%) produced a rapid release of 9-cis-retinal from rCRALBP (his-tagged) and a slower release of 11-cis-retinal from CRALBP (mole ratio CRALBP:total lipid, 1:15). PC SUVs had little effect on the release from either form of CRALBP. The efficacy for release of 11-cis-retinal from CRALBP by phospholipid-doped SUVs generally paralleled that of binding affinity (PA>PS>PI>PC).
Our results describe the first interaction of CRALBP with a physiologic substance that resulted in the release of bound 11-cis-retinal. PA is a minor membrane lipid but it can be generated in response to various signal transduction pathways in the cytoplasmic leaflet of the plasma membrane, where it could interact with cytosolic CRALBP. PA may be more efficient in releasing retinal from rCRALBP because the anionic lipid is locally concentrated by binding to the oligo-his sequence. The mechanism for release of retinal from CRALBP remains to be determined but could involve displacement of retinal from its binding site by PA or binding of CRALBP to PA in SUVs via a positive patch on the protein surface. These results open a new chapter in our understanding of how CRALBP functions in the regeneration of visual pigments.
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