Cholesteryl ester hydrolase in human monocyte/macrophage: cloning, sequencing, and expression of full-length cDNA.
The sensitive technique of RT-PCR was used to identify cholesteryl ester hydrolase (CEH) expressed in human macrophages. This enzyme is thought to regulate the availability of intracellular free cholesterol for efflux. The expected 667-bp product was obtained starting with RNA from human peripheral blood and THP-1 monocytes and macrophages. The cDNA for human macrophage CEH was then cloned by PCR-based screening of a lambda-gt11 cDNA library. The full-length cDNA was sequenced and found to exhibit 76% homology (at the nucleotide and conceptually translated protein level) to hepatic CEH, an enzyme shown to be involved in hepatic cholesterol homeostasis and regulated by cholesterol at the transcription level via sterol response elements in the proximal promoter. Identification of the conserved catalytic triad (Ser(221</SUP >, His(468), and Glu354)) and the SEDCLY motif places human macrophage CEH in the family of carboxylesterases. A greater than 20-fold increase in CEH activity was observed when COS-1 and COS-7 cells were transiently transfected with an eukaryotic expression vector, pcDNA3.1/V5/His-TOPO, containing the cDNA for human macrophage CEH. Using this full-length cDNA as a probe, a 2.2-kb transcript was identified by Northern blot analysis of total RNA from human peripheral blood and THP -1 macrophages. Overexpression of human macrophage CEH resulted in an impairment of upregulation of low-density lipoprotein (LDL) receptor mRNA in Chinese hamster ovary (CHO-K1) cells grown in cholesterol-deficient environment. These data identify the human macrophage CEH, demonstrate its expression in human peripheral blood macrophage and human macrophage cell line, THP-1, and suggest its role in the intracellular cholesteryl ester metabolism.
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Department of Internal Medicine, Pulmonary/Critical Care Division, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, Virginia 23298-0050, USA. shobha@hsc.vcu.edu
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D016466:CHO Cells; D019556:COS Cells; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D005786:Gene Expression Regulation; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D008264:Macrophages / Q000166:cytology / Q000201:enzymology*; D008969:Molecular Sequence Data; D009000:Monocytes / Q000166:cytology / Q000201:enzymology*; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011973:Receptors, LDL / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D002787:Sterol Esterase / Q000235:genetics*; D014162:Transfection
D018076:DNA, Complementary; D004274:DNA, Recombinant; D012333:RNA, Messenger; D011973:Receptors, LDL; D002787:Sterol Esterase