Release of 11-cis-retinal from cellular retinaldehyde-binding protein by acidic lipids

[摘要] Purpose: To determine molecularmechanisms for the release of 11-cis-retinal from the bindingpocket of cellular retinaldehyde-binding protein (CRALBP). Methods: Binding of CRALBP to lipidsurfaces was assessed with a lipid-immunoblot assay. Lipids werepresented to CRALBP as small unilamellar vesicles (SUVs) consisting ofphosphatidylcholine (PC) plus other lipids. Release of 9-cis-retinalor 11-cis-retinal from CRALBP was measured with spectral andhigh performance liquid chromatography (HPLC) assays based on theprotection of the protein-bound retinal carbonyl group from reactionwith NH2OH. The electrostatic surface potential of CRALBPwas calculated from a model of its structure using the program CCP4mg. Results: Incubation of CRALBP·11-cis-retinalwith lipids absorbed on nitrocellulose revealed binding to the acidiclipids, phosphatidic acid (PA)>phosphatidylinositol3,4,5-trisphosphate [PI(3,4,5)P3]>phosphatidylserine(PS)> PI(4,5)P2 and little or no binding to PC,phosphatidylethanolamine (PE), or PI(4)P. 11-cis-retinal wasreleased during incubation of CRALBP with SUVs consisting of PC plus 50mol% PA but not during incubation with those composed of 100 mol% PC.The efficacy of release of 9-cis-retinal or 11-cis-retinalfrom CRALBP by phospholipid-containing SUVs generally paralleled thatof the binding of CRALBP to the lipids (PA>PS>PI>>PC).Examination of the electrostatic surface potential of the proteinstructure revealed a basic recess on one face of the protein, which maybind acidic lipids. Conclusions: Our results identify thefirst physiologic substances that release 11-cis-retinal fromCRALBP. PA and PS are relatively minor membrane lipids that can begenerated in the cytoplasmic leaflet of the plasma membrane in responseto various signal transduction pathways, where they could interact withcytosolic CRALBP. The mechanism for release of retinal from CRALBP byacidic lipids remains to be determined but could involve binding of theacidic lipid in the 11-cis-retinal binding site or to thepositive basic recess on the protein surface. These results open a newfacet in our understanding of how CRALBP functions in the regenerationof visual pigments.

[学科分类] 生物化学/生物物理