Molecular cloning and expression of cDNA for rat pancreatic cholesterol esterase.
A full-length cDNA complementary to the rat pancreatic cholesterol esterase mRNA was isolated by screening a rat pancreatic cDNA expression library in lambda gt11 vector with antibodies against the porcine pancreatic cholesterol esterase. The isolated cholesterol esterase cDNA is 2050 bp in length and contains an open reading frame coding for a protein of 612 amino acids. A 20-amino acid hydrophobic leader sequence is predicted, based on the position of the first ATG initiation codon upstream from the sequenced amino terminus of the isolated cholesterol esterase. The cholesterol esterase cDNA was subcloned into a mammalian expression vector, pSVL, for transfection studies. Expression of the cDNA in COS cells resulted in the production of bile salt-stimulated cholesterol esterase. Comparison of the cholesterol esterase cDNA sequence with other proteins revealed that the pancreatic cholesterol esterase is identical to rat pancreatic lysophospholipase. The primary structure of cholesterol esterase displayed no significant homology with other lipases, although the putative lipid interfacial recognition site of G-X-S-X-G is present in the cholesterol esterase sequence. However, the cholesterol esterase sequence revealed a 63-amino-acid domain which is highly homologous to the active site domain of other serine esterases. These data suggest that cholesterol esterase may be a member of the serine esterase supergene family. Analysis of the cholesterol esterase structure also revealed a repetitive sequence enriched with Pro, Asp, Glu, Ser, and Thr residues at the C-terminal end of the protein. This sequence is reminiscent of the PEST-rich sequences in short-lived proteins, suggesting that cholesterol esterase may have a short half-life in vivo. Northern blot hybridization showed that the bile salt-stimulated cholesterol esterase mRNA is present in liver suggesting that this protein may also be synthesized by liver cells.
Kissel
J A
JA
Department of Pathology, University of Cincinnati, College of Medicine, OH 45267-0529.
Fontaine
R N
RN
Turck
C W
CW
Brockman
H L
HL
Hui
D Y
DY
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002265:Carboxylic Ester Hydrolases / Q000235:genetics*; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D015342:DNA Probes; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D010179:Pancreas / Q000201:enzymology*; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D015183:Restriction Mapping; D002787:Sterol Esterase / Q000235:genetics*; D014162:Transfection
D015342:DNA Probes; D012333:RNA, Messenger; D004247:DNA; D002265:Carboxylic Ester Hydrolases; D002787:Sterol Esterase