Cloning, sequencing, and expression of cDNA for human beta-glucuronidase.
We report here the cDNA sequence for human placental beta-glucuronidase (beta-D-glucuronoside glucuronosohydrolase, EC 3.2.1.31) and demonstrate expression of the human enzyme in transfected COS cells. We also sequenced a partial cDNA clone from human fibroblasts that contained a 153-base-pair deletion within the coding sequence and found a second type of cDNA clone from placenta that contained the same deletion. Nuclease S1 mapping studies demonstrated two types of mRNAs in human placenta that corresponded to the two types of cDNA clones isolated. The NH2-terminal amino acid sequence determined for human spleen beta-glucuronidase agreed with that inferred from the DNA sequence of the two placental clones, beginning at amino acid 23, suggesting a cleaved signal sequence of 22 amino acids. When transfected into COS cells, plasmids containing either placental clone expressed an immunoprecipitable protein that contained N-linked oligosaccharides as evidenced by sensitivity to endoglycosidase F. However, only transfection with the clone containing the 153-base-pair segment led to expression of human beta-glucuronidase activity. These studies provide the sequence for the full-length cDNA for human beta-glucuronidase, demonstrate the existence of two populations of mRNA for beta-glucuronidase in human placenta, only one of which specifies a catalytically active enzyme, and illustrate the importance of expression studies in verifying that a cDNA is functionally full-length.
Oshima
A
A
Kyle
J W
JW
Miller
R D
RD
Hoffmann
J W
JW
Powell
P P
PP
Grubb
J H
JH
Sly
W S
WS
Tropak
M
M
Guise
K S
KS
Gravel
R A
RA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D005260:Female; D005966:Glucuronidase / Q000235:genetics*; D006801:Humans; D010920:Placenta / Q000201:enzymology; D010957:Plasmids; D011247:Pregnancy; D013154:Spleen / Q000201:enzymology; D014158:Transcription, Genetic
D004247:DNA; D005966:Glucuronidase