A cleavable affinity biotinylating agent reveals a retinoid binding role for RPE65.
Retinal pigment epithelial (RPE) membranes contain the full biochemical apparatus capable of processing all-trans-retinol (vitamin A) into 11-cis-retinal, the visual chromophore. As many of these proteins are integral membrane proteins and resistant to traditional methods of identification, alternate methods of identifying these proteins are sought. The approach described here involves affinity biotinylation with alkali cleavable linkers. A vitamin A containing affinity-labeling haloacetate is described which facilitates the identification of retinoid binding proteins (RBPs). Treatment of crude bovine RPE membranes with (3R)-3-[boc-lys(biotinyl)-O]-all-trans-retinol chloroacetate 1 in the low micromolar range led to the specific labeling of RPE65 and lecithin retinol acyltransferase (LRAT). Only RPE65 is labeled at 5 microM 1 at 4 degrees C. Labeled RPE65 was readily isolated by binding the labeled protein to avidin-containing beads, followed by cleavage of the protein from the beads at pH 11. Trypsin digestion of RPE65 modified by 1, followed by mass spectrometry, demonstrates that C231 and C448 are alkylated by 1. These studies validate the approach that was used, and furthermore demonstrate that RPE65, a major membrane-associated protein of the RPE, is a RBP.
Jahng
Wan Jin
WJ
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 45 Shattuck Street, Boston, Massachusetts 02115, USA.
David
Charles
C
Nesnas
Nasri
N
Nakanishi
Koji
K
Rando
Robert R
RR
D000217:Acyltransferases / Q000378:metabolism; D000345:Affinity Labels / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001710:Biotin / Q000737:chemistry; D002417:Cattle; D005136:Eye Proteins; D066298:In Vitro Techniques; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010857:Pigment Epithelium of Eye / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012176:Retinoids / Q000737:chemistry / Q000378:metabolism*; D012177:Retinol-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D000345:Affinity Labels; D005136:Eye Proteins; D011506:Proteins; D012176:Retinoids; D012177:Retinol-Binding Proteins; D001710:Biotin; D000217:Acyltransferases; C056942:lecithin-retinol acyltransferase