Characterization and functional expression of a cDNA encoding egasyn (esterase-22): the endoplasmic reticulum-targeting protein of beta-glucuronidase.
Egasyn (esterase-22), a member of the nonspecific carboxylesterase multigene family (E.C. 3.1.1.1), is the endoplasmic reticulum (ER)-targeting protein of beta-glucuronidase. We utilized the polymerase chain reaction (PCR) in the eventual isolation of murine egasyn cDNAs. PCR primers were based upon: (1) partial amino acid sequences derived from egasyn peptides and (2) a conserved active site region shared by carboxylesterases. The amino acid sequence deduced from the PCR product matched that obtained from egasyn protein. This product was utilized as a probe to screen a cDNA library. Two cDNAs whose composite sequence encoded an open reading frame of 562 amino acids were isolated. A message size of 1700-2000 bp was revealed by RNA blot hybridization analysis. S1 nuclease protection analyses detected mRNA in liver, kidney, lung, and submandibular gland, but not in spleen, brain, and testes. Genetic mapping confirmed the location of an egasyn cDNA fragment in cluster 1 of the esterase region on chromosome 8. Transfection of COS cells with the 2022-bp cDNA resulted in the expression of esterase activity, which comigrated on native gels with liver esterase-22. The features of the deduced amino acid sequence of the egasyn cDNA are compared with previously characterized carboxylesterases and with other lumenal ER proteins.
Ovnic
M
M
Department of Pediatrics, Children's Hospital Medical Center, Cincinnati, Ohio 45229.
Swank
R T
RT
Fletcher
C
C
Zhen
L
L
Novak
E K
EK
Baumann
H
H
Heintz
N
N
Ganschow
R E
RE
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002898:Chromosomes, Human, Pair 8; D003001:Cloning, Molecular; D004247:DNA / Q000302:isolation & purification; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D005260:Female; D015870:Gene Expression; D005966:Glucuronidase / Q000378:metabolism*; D006801:Humans; D008099:Liver / Q000201:enzymology; D008297:Male; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D009928:Organ Specificity / Q000235:genetics; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping; D016415:Sequence Alignment
D008562:Membrane Glycoproteins; D004247:DNA; D002265:Carboxylic Ester Hydrolases; C009751:egasyn; D005966:Glucuronidase