Rat liver beta-glucuronidase. cDNA cloning, sequence comparisons and expression of a chimeric protein in COS cells.
A cDNA for rat liver beta-glucuronidase was isolated, its sequence determined and its expression after transfection into COS cells studied. The deduced amino acid sequence of the rat liver clone showed 77% homology with that from the cDNA for human placental beta-glucuronidase and 47% homology with that deduced from the cDNA for Escherichia coli beta-glucuronidase. Several differences were found between the cDNA from rat liver and that previously reported from rat preputial gland. Only one change leads to an amino acid difference in the mature enzyme. A chimeric clone was constructed by using a fragment encoding the first 18 amino acid residues of the signal sequence from the human placental cDNA clone and a fragment from the rat clone encoding four amino acid residues of the signal sequence, all 626 amino acid residues of the mature rat enzyme, and all of the 3' untranslated region. After transfection into COS cells the chimeric clone expressed beta-glucuronidase activity that was specifically immunoprecipitated by antibody to rat beta-glucuronidase. The Mr value of 76,000 of the expressed gene product was characteristic of the glycosylated rat enzyme. It was proteolytically processed in COS cells to Mr 75,000 6 h after metabolic labelling. At least 50% of the expressed enzyme was secreted at 60 h post-transfection, but the secreted enzyme did not undergo proteolytic processing. These results provide evidence that the partial cDNA isolated from a rat liver library contains the complete coding sequence for the mature rat liver enzyme and that the chimeric signal sequence allows normal biosynthesis and processing of the transfected rat liver enzyme in COS cells.
Powell
P P
PP
E.A. Doisy Department of Biochemistry, St. Louis University Medical Center, MO 63104.
Kyle
J W
JW
Miller
R D
RD
Pantano
J
J
Grubb
J H
JH
Sly
W S
WS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002678:Chimera; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D005786:Gene Expression Regulation; D005966:Glucuronidase / Q000235:genetics*; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics; D051381:Rats; D014162:Transfection
D011506:Proteins; D004247:DNA; D005966:Glucuronidase