Transcriptional regulation and the effects of sodium butyrate and glycosylation on catalytic activity of human germ cell alkaline phosphatase.
Human choriocarcinoma cells, the malignant trophoblasts, synthesize germ cell alkaline phosphatase (GCAP) which shares 98% sequence identity with the placental alkaline phosphatase (AP). The two isozymes are immunologically similar but react differentially toward inhibition by L-leucine or EDTA. Administration of sodium butyrate to choriocarcinoma cells greatly increased the transcription rate of the GCAP gene, resulting in an increase in mRNA expression and enzyme biosynthesis. The butyrate-modulated AP induction was blocked by cycloheximide, suggesting that a mediator protein may be involved. Protein sequence deduced from complementary DNA analysis suggests that GCAP contains two potential sites for asparagine (N)-linked glycosylation. The marked increase in GCAP expression by butyrate in choriocarcinoma cells allowed us to study the extent of N-linked glycosylation and its role on GCAP enzyme activity. After limited tunicamycin treatment, Mr 65,000 (fully processed), Mr 58,000 (nonglycosylated), and Mr 62,000 polypeptides were synthesized by these cells in the presence of butyrate. This suggests that the Mr 62,000 product may be the singly glycosylated GCAP monomer and that both sites are glycosylated in this phosphatase. The glycosylated and nonglycosylated GCAPs, synthesized by butyrate-treated choriocarcinoma cells in the absence or presence of tunicamycin, respectively, were similarly inhibited by L-leucine or EDTA. Moreover, the specific enzyme activity of glycosylated and nonglycosylated GCAP remained unchanged, indicating that AP lacking N-linked oligosaccharide side chains was catalytically active. This is supported by the finding that nonglycosylated GCAP incorporated inorganic phosphate which binds to the active site of AP. Since the active form of AP is a homodimer, our data indicate that the glycan moieties are not required for the dimerization and catalytic activity of GCAP.
Pan
C J
CJ
Human Genetics Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.
Sartwell
A D
AD
Chou
J Y
JY
D000469:Alkaline Phosphatase / Q000096:biosynthesis / Q000235:genetics*; D002087:Butyrates / Q000494:pharmacology*; D020148:Butyric Acid; D002822:Choriocarcinoma / Q000201:enzymology / Q000235:genetics*; D004790:Enzyme Induction / Q000187:drug effects; D005260:Female; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D006031:Glycosylation; D006801:Humans; D012333:RNA, Messenger / Q000096:biosynthesis*; D014158:Transcription, Genetic; D014415:Tunicamycin / Q000494:pharmacology; D014594:Uterine Neoplasms / Q000201:enzymology / Q000235:genetics*
D002087:Butyrates; D012333:RNA, Messenger; D020148:Butyric Acid; D014415:Tunicamycin; D000469:Alkaline Phosphatase