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Title: MICROSOMAL ACID RETINYL ESTER HYDROLASE ISOLATION, CHARACTERIZATION, SUBSTRATE AND TISSUE SPECIFICITY

Author
item Linke, Thomas
item Dawson, Harry
item Harrison, Earl

Submitted to: Journal of Biological Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/14/2005
Publication Date: 4/1/2005
Citation: Linke, T.N., Dawson, H.D., Harrison, E.H. 2005. Microsomal acid retinyl ester hydrolase isolation, characterization, substrate and tissue specificity. Journal of Biological Chemistry. 280:23287-23294.

Interpretive Summary: We are studying the liver enzymes that hydrolyze retinyl esters, the form in which newly absorbed dietary vitamin A is delivered to the liver. These enzymes (retinyl ester hydrolases or REHs) are important in both the uptake of dietary vitamin A by the liver and in the mobilization of stored vitamin A from the liver when dietary input is inadequate to meet the body's need for this essential nutrient. Although a number of liver enzymes can catalyze the hydrolysis of vitamin A esters, the identity and the physiological role of these enzymes is unclear. The aim of our research is to expand our knowledge in this important area of vitamin A biology. In the study reported here, we have isolated and characterized an acid retinyl ester hydrolase that is likely to involved in the initial uptake and storage of dietary vitamin A in liver. We have identified this protein as rat liver carboxylesterase ES-10. We have also demonstrated in this report that this protein is highly expressed in liver on both the gene and protein level. This work will enhance our understanding of vitamin A metabolism in humans and will benefit other research scientist and human nutritionists interested in vitamin A research.

Technical Abstract: Previous work in this laboratory demonstrated the presence of both acid and neutral, bile salt-independent retinyl ester hydrolase activities in rat liver homogenates. Here we present the purification, identification and characterization of an acid retinyl ester hydrolase activity from solubilized rat liver microsomes. Purification to apparent homogeneity was achieved by sequential chromatography using SP-Sepharose cation exchange, Phenyl-Sepharose hydrophobic interaction, Concanavalin A-Sepharose affinity and Superose 12 gel filtration chromatography. The isolated protein had a monomer molecular weight of approximately 62 kDa, as measured by mass spectrometry. Analytical gel filtration chromatography of the purified protein revealed a native molecular weight of approximately 172 kDa, indicating that the protein exists as a homotrimeric complex in solution. The purified protein was identified as carboxylesterase ES-10 (EC 3.1.1.1) by N-terminal Edman sequencing and extensive LC-MS/MS sequence analysis and cross reaction with an anti ES-10 antibody. Glycosylation analysis revealed that only one of two potential N-linked glycosylation sites is occupied by a high mannose-type carbohydrate structure. Using retinyl palmitate in a micellar assay system the enzyme was active over a broad pH range and displayed Michaelis-Menten kinetics with a Km of 86 'M. Substrate specificity studies showed that ES-10 is highly active toward retinyl palmitate, and is also able to catalyze hydrolysis of triolein to a minor degree. Cholesteryl oleate was not a substrate for ES-10 under these assay conditions. Real time RT-PCR and Western blot analysis revealed that ES-10 is highly expressed in liver and lung. Lower levels of ES-10 mRNA were also found in kidney, testis and heart. A comparison of mRNA expression levels in liver demonstrated that ES-10, ES-4 and ES-3 were expressed at a significantly higher level than ES-2, an enzyme previously thought to play a major role in retinyl ester metabolism in liver. Taken together these data indicate that carboxylesterase ES-10 plays a major role in the hydrolysis of newly-endocytosed, chylomicron retinyl esters in both neutral and acidic membrane compartments of liver cells, and may play a role in retinyl ester hydrolysis in other tissues as well.