Liver carboxylesterase 1
carboxylesterase activity
Glycosylation-dependent activity of baculovirus-expressed human liver carboxylesterases: cDNA cloning and characterization of two highly similar enzyme forms.
A cDNA, designated hCE, encoding the entire sequence of a carboxylesterase, was isolated from a human liver lambda gt11 library. The hCE-deduced protein sequence contained 568 amino acids, including an 18 amino acid signal peptide sequence, and had a calculated molecular mass of the mature protein of 60,609 Da. A second cDNA, designated hCEv, was isolated from the same lambda gt11 library and contained a 3-bp deletion resulting in the loss of the final amino acid in the signal peptide sequence (Ala-1) and a second 3-bp deletion leading to an in-frame loss of Gln345. Expression of mRNA corresponding to both hCE and hCEv was detected in eight adult human liver samples, with individual levels varying 5-fold (hCE) and 12-fold (hCEv). A single immunoreactive protein was detected in 13 adult human liver samples when probed with antibody directed against a rat carboxylesterase. Based on allele-specific oligonucleotide hybridizations, we believe that the hCE and hCEv cDNAs represent two distinct members of the carboxylesterase family. The carboxylesterase genes were localized to human chromosome 16 using a somatic cell hybrid mapping strategy. Baculovirus expression of hCE in Sf9 cells produced a protein with an estimated molecular mass of 59,000 Da. This enzyme was able to hydrolyze aromatic and aliphatic esters but possessed no catalytic activity toward amides or a fatty acyl CoA ester. Baculovirus-mediated expression of the hCEv cDNA yielded a second protein of 56,000 Da resulting from inefficient N-glycosylation of the hCEv protein. Although the substrate specificity for the hCEv protein was identical to that of expressed hCE for any given substrate, the specific activity for the hCE protein was always higher than that for the hCEv protein. Tunicamycin inhibition studies provided the first evidence that N-glycosylation of these luminal enzymes is essential for maximal catalytic activity.