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Gennadiy P Moiseyev, Yusuke Takahashi, Jian-Xing (Jay) Ma; Human carboxylesterase 1 is expressed in retinal pigment epithelium and hydrolyzes retinyl ester. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5506.
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Retinyl esters are stored in retinosomes in retinal pigment epithelium (RPE) and serve as a substrate for RPE65 to generate 11-cis-retinol which is further oxidized and used to regenerate rhodopsin. Currently, RPE65 is the only known retinyl ester cleaving enzyme found in RPE. Several carboxylesterases from liver and intestine were shown to hydrolyze retinyl esters and to release vitamin A into the circulation. However, it is not known whether carboxylesterases are expressed in the RPE and contribute to retinoid metabolism in the eye. The aim of this study was to examine whether human carboxylesterase 1 (CES1) has retinyl ester hydrolyzing activity and is expressed in human RPE.
The distribution of the carboxylesterase-1 (CES1) mRNA in human eye was analyzed by RT-PCR. Expression levels of CES1 protein in the RPE and retina were analyzed by western blotting. CES1 was cloned from human RPE cDNA by PCR. 293A cells expressing CES1 with an N-terminal His-tag by adenovirus vector were used in the study. CES1 was purified from 293A cells infected with adenovirus using Ni-NTA resin. Retinyl ester hydrolyzing activity was assayed by HPLC
The cloned human CES1 cDNA sequence contains 1704 bps (accession number NM_001025194 in GenBank). The CES1 mRNA was detected as a single transcript in the human RPE, retina and choroid. CES1 was purified to homogeneity from culture medium of 293A cells infected with adenoviral vector at MOI 100. Western blot analysis demonstrated that CES1 is abundantly expressed in human RPE/choroid and at significantly lower levels in the retina. Purified CES1 efficiently cleaved retinyl ester generating all-trans-retinol. Kinetic parameters (Vmax = 24.7±4.0 nmol/mg/h and Km = 2.2±0.6 mM) of CES1 were determined for retinyl palmitate substrate.
Human CES1 showed significant activity towards retinyl palmitate suggesting that it is a retinyl ester hydrolase in the RPE. We hypothesize that CES1 may have a physiological role in mobilization of retinol from the RPE lipid droplets during vitamin A deficiency.
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