June 2013
Volume 54, Issue 15
ARVO Annual Meeting Abstract  |   June 2013
Cloning and Characterization of a Carboxylesterase from Bovine Retinal Pigment Epithelium
Author Affiliations & Notes
  • Gennadiy Moiseyev
    Physiology, Univ of Oklahoma Hlth Sci Ctr, Oklahoma City, OK
  • Bill Wu
    Biochemistry & Molecular Biol, Medical Univ of South Carolina, Charleston, SC
  • Yusuke Takahashi
    Medicine-Endocrinology, Univ of Oklahoma Hlth Sci Ctr, Oklahoma City, OK
  • Ying Chen
    Physiology, Univ of Oklahoma Hlth Sci Ctr, Oklahoma City, OK
  • Andrew Tsin
    Biology, University of Texas San Antonio, San Antonio, TX
  • Rosalie Crouch
    Ophthalmology, Medical Univ of South Carolina, Charleston, SC
  • Jian-Xing Ma
    Physiology, Univ of Oklahoma Hlth Sci Ctr, Oklahoma City, OK
  • Footnotes
    Commercial Relationships Gennadiy Moiseyev, Charlesson LLC (E); Bill Wu, None; Yusuke Takahashi, None; Ying Chen, None; Andrew Tsin, None; Rosalie Crouch, None; Jian-Xing Ma, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 3760. doi:https://doi.org/
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      Gennadiy Moiseyev, Bill Wu, Yusuke Takahashi, Ying Chen, Andrew Tsin, Rosalie Crouch, Jian-Xing Ma; Cloning and Characterization of a Carboxylesterase from Bovine Retinal Pigment Epithelium. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3760. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: Retinyl esters are stored in lipid droplets in retinal pigment epithelium (RPE) and can serve as a substrate for RPE65 isomerohydrolase for generation of 11-cis retinol, the precursor rhodopsin chromophore. At present, RPE65 is the only known retinyl ester hydrolase in the eye. Some carboxylesterases of the liver and other tissues have been shown to hydrolyze retinyl esters. Carboxylesterases may also participate in xenobiotic, drug and lipid metabolism. However, little is known about carboxylesterases expression and function in the eye. The aim of this study was to examine whether RPE carboxylesterase has retinyl ester hydrolyzing activity or demonstrates other substrate preference.

Methods: Carboxylesterase (CES1) has been cloned from bovine RPE cDNA library by PCR methods. A 293A cell line stably expressing CES1 with His-tag using pcDNA6 vector was generated and used to express CES1. CES1 was purified from the culture medium using N-NTA resin. CES1 activity was determined spectrophotometrically in the reaction with various esters. Retinyl ester hydrolyzing activity was assayed by HPLC.

Results: The cloned bovine CES1 cDNA sequence contains 1695 bp (accession number AY369075 in GenBank). The deduced protein has a calculated molecular weight 61,780 Da and isoelectric point 6.79. CES1 mRNA was detected as a single transcript in the bovine RPE, kidney, liver and lung. No signal was detected in other bovine tissues analyzed, including the retina, pancreas, skeletal muscles, spleen, heart and brain. Immunohistochemistry showed the specific localization of CES1 in Bruch’s membrane. CES1 was purified to homogeneity from culture medium of 293A cell line that stably expressed this protein. CES1 cleaves various esters producing alcohol and acid. Kinetic parameters kcat and Km were determined for paranitrophenyl acetate, vinyl acetate, vinyl butyrate, vinyl laurate which were found to be substrates for CES1. Lipid esters triolein and retinyl palmitate were not hydrolyzed by CES1.

Conclusions: Bovine CES1 showed significant activity towards esters with a small alcohol group. However, CES1 did not cleave retinyl palmitate suggesting that it is not a retinyl ester hydrolase. We hypothesize that CES1 may have a physiological role as a detoxifying enzyme serving as a protector of RPE from various xenobiotic esters.

Keywords: 701 retinal pigment epithelium • 514 enzymes/enzyme inhibitors • 658 protein purification and characterization  

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