Targeting of neutral cholesterol ester hydrolase to the endoplasmic reticulum via its N-terminal sequence.
Neutral cholesterol ester hydrolase (NCEH) accounts for a large part of the nCEH activity in macrophage foam cells, a hallmark of atherosclerosis, but its subcellular localization and structure-function relationship are unknown. Here, we determined subcellular localization, glycosylation, and nCEH activity of a series of NCEH mutants expressed in macrophages. NCEH is a single-membrane-spanning type II membrane protein comprising three domains: N-terminal, catalytic, and lipid-binding domains. The N-terminal domain serves as a type II signal anchor sequence to recruit NCEH to the endoplasmic reticulum (ER) with its catalytic domain within the lumen. All of the putative N-linked glycosylation sites (Asn(270), Asn(367), and Asn(389)) of NCEH are glycosylated. Glycosylation at Asn(270), which is located closest to the catalytic serine motif, is important for the enzymatic activity. Cholesterol loading by incubation with acetyl-LDL does not change the ER localization of NCEH. In conclusion, NCEH is targeted to the ER of macrophages, where it hydrolyzes CE to deliver cholesterol for efflux out of the cells.
Igarashi
Masaki
M
Department of Metabolic Diseases, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan.
Osuga
Jun-Ichi
J
Isshiki
Masashi
M
Sekiya
Motohiro
M
Okazaki
Hiroaki
H
Takase
Satoru
S
Takanashi
Mikio
M
Ohta
Keisuke
K
Kumagai
Masayoshi
M
Nishi
Makiko
M
Fujita
Toshiro
T
Nagai
Ryozo
R
Kadowaki
Takashi
T
Ishibashi
Shun
S
D000818:Animals; D055162:Biocatalysis; D020134:Catalytic Domain; D002417:Cattle; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D042541:Intracellular Space / Q000378:metabolism; D050356:Lipid Metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D021381:Protein Transport; D002787:Sterol Esterase / Q000737:chemistry* / Q000378:metabolism*
D002787:Sterol Esterase; D005947:Glucose