Functional characterization of three naturally occurring single nucleotide polymorphisms in the CES2 gene encoding carboxylesterase 2 (HCE-2).
Twelve single nucleotide polymorphisms (SNPs) in the human CES2 gene, which encodes a carboxylesterase, hCE-2 [human carboxylesterase 2 (EC 3.1.1.1)], have been reported in the Japanese. In this report, we have examined functional alterations of three SNPs, a nonsynonymous SNP (100C>T, R34W), an SNP at the splice acceptor site in intron 8 (IVS8-2A>G), and one newly discovered nonsynonymous SNP (424G>A, V142M). For the two nonsynonymous SNPs, the corresponding variant cDNAs were expressed in COS-1 cells. Both the R34W and V142M variants showed little esterase activities toward the anticancer agent irinotecan and two typical carboxylesterase substrates, p-nitrophenol acetate and 4-methylumbelliferyl acetate, although increased levels of cDNA-mediated protein expression were observed by Western blotting as compared with the wild type. To investigate a possible splicing aberration in IVS8-2A>G, an in vitro splicing assay was utilized and transcripts derived from CES2 gene fragments of the wild type and IVS8-2A>G were compared. Sequence analysis of the cloned transcripts revealed that IVS8-2A>G yielded mostly aberrantly spliced transcripts, including a deleted exon or a 32-bp deletion proximal to the 5' end of exon 9, which resulted in truncated hCE-2 proteins. These results suggested that 100C>T (R34W), 424G>A (V142M), and IVS8-2A>G are functionally deficient SNPs.
Kubo
Takashi
T
Division of Pharmacology, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.
Kim
Su-Ryang
SR
Sai
Kimie
K
Saito
Yoshiro
Y
Nakajima
Toshiharu
T
Matsumoto
Kenji
K
Saito
Hirohisa
H
Shirao
Kuniaki
K
Yamamoto
Noboru
N
Minami
Hironobu
H
Ohtsu
Atsushi
A
Yoshida
Teruhiko
T
Saijo
Nagahiro
N
Ohno
Yasuo
Y
Ozawa
Shogo
S
Sawada
Jun-Ichi
J
D000818:Animals; D044466:Asian People / Q000235:genetics; D019556:COS Cells; D002166:Camptothecin / Q000031:analogs & derivatives / Q000378:metabolism; D043182:Carboxylesterase / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D006801:Humans; D000077146:Irinotecan; D009596:Nitrophenols / Q000378:metabolism; D020641:Polymorphism, Single Nucleotide; D012326:RNA Splicing; D014468:Umbelliferones / Q000378:metabolism
D009596:Nitrophenols; D014468:Umbelliferones; C032078:4-methylumbelliferyl acetate; D000077146:Irinotecan; C008642:4-nitrophenyl acetate; D043182:Carboxylesterase; D002166:Camptothecin