In situ subcellular localization of epitope‐tagged human and rabbit carboxylesterases

Abstract

Carboxylesterases are a ubiquitous class of enzymes thought to be involved in xenobiotic metabolism and detoxification. Primary amino acid sequence data suggest that these proteins localize to the endoplasmic reticulum. However, since this family of proteins is highly homologous, the generation of specific reagents to monitor expression and subcellular localization has been unsuccessful. To accomplish in situ detection of a human alveolar macrophage carboxylesterase and a rabbit liver carboxylesterase, we constructed plasmids that expressed recombinant proteins containing an 11 amino acid influenza hemagglutinin tag near the C-terminus. These proteins retained carboxylesterase activity as determined by the conversion of o-nitrophenol acetate to o-nitrophenol. Following transfection of plasmids encoding these proteins into mammalian cells, cells were analyzed by both fluorescence and electron microscopy. The tagged enzymes were localized to the endoplasmic reticulum of both Cos7 monkey kidney cells and Rh30 human rhabdomyosarcoma cells. No tagged protein was detectable in the culture media. Hence, epitope tagging allowed the analysis of expression and localization of specific carboxylesterases. The methods described in this paper are, therefore, applicable to any protein, including those that are highly homologous to other candidate molecules.

Decreased Serum Levels of Carboxylesterase-2 in Patients with Ovarian Cancer

“…It is converted in vivo to SN-38 (7ethyl-10-hydroxy-camptothecin), the active metabolite that causes cell death 7 , by human liver CES-2. The process of conversion is very important, since SN-38 is 300-2000 times more potent than irinotecan in vitro and in vivo [13][14][15][16][17][18] . It has been reported that patients with high CES-2 mRNA expression levels in peripheral blood mononuclear cells present a higher activation ratio of irinotecan to SN38 7 .…”