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Jody Ann Summers, Angelica Harper, Hanke Van-Der-Wel, Marcela Hermann, Christopher M. West; Identification of apolipoprotein A-I as a novel retinoic acid binding protein. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2162.
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All-trans-retinoic acid (atRA) may be an important molecular signal in the postnatal control of eye size. Retinoids are closely associated with atRA binding proteins, which are important in regulating their transport, metabolism and biological activity. We and others (Mertz and Wallman, Exp. Eye Res. 2000) have previously identified a protein with an apparent Mr of 27 kD (p27) that represents the major secreted atRA binding protein in chick choroids. The goal of the current study was to identify p27 as we hypothesize that p27 may play a role in the regulation of atRA activity during visually guided ocular growth.
atRA binding proteins were initially identified from conditioned medium of chick ocular tissues by photoaffinity 3H-atRA labeling, SDS-PAGE and autoradiography. 3H-atRA binding proteins were purified using a combination of anion exchange (Q-sepharose), gel filtration (Superdex 200) columns and SDS-PAGE. Unlabeled samples were processed in parallel and peak fractions of unlabeled protein, corresponding to the elution position and mass of 3H-atRA-labelled protein were identified by mass spectrometry. The identify of p27 was confirmed using immunoprecipitation of 3H-atRA-labelled p27 from conditioned medium using anti-chick apolipoprotein A-I antibodies.
Following photoaffinity labeling of choroid and sclera conditioned medium, radiolabeled proteins migrating at 60 kD and 27 kD were detected by autoradiography. These proteins co-eluted from Q-sepharose and Superdex 200. Mass spectrometry analyses identified the 60 kD protein as serum albumin and the 27 kD protein as apolipoprotein A-I. Following immunoprecipitation of 3H-atRA labeled proteins from sclera conditioned medium with anti-chick apolipoprotein A-I, a single 27 kD band was detected on autoradiograms.
Apolipoprotein A-I is the 27 kD 3H-atRA-binding protein present in chick choroid and sclera conditioned medium. The expression of this protein may play a role in the regulation of atRA signaling in the choroid and sclera in postnatal ocular growth.
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