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Published on: October 21, 2011
An inhibitor of O-glycosylation induces apoptosis in NIH3T3 cells and developing mouse embryonic mandibular tissues
E Tian1, Kelly G Ten Hagen, Lillian Shum
1Biological Chemistry Section, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA.
Abstract:
The family of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (ppGaNTases) is responsible for initiating mucin-type O-linked glycosylation in higher eukaryotes. To begin to examine the biological role of O-linked glycosylation, mammalian cells were treated with a small molecule inhibitor (designated 1-68A, Ref. 15) of ppGaNTase activity. NIH3T3 cells exposed to the inhibitor were shown to undergo a significant reduction in cell surface O-glycosylation as detected by staining with jacalin and peanut agglutinin lectins after 30 min of treatment; no reduction in staining using antibodies to O-linked N-acetylglucosamine or the lectin concanavalin A was detected. Apoptosis was also observed in treated cells after 45 min of exposure, ostensibly following the O-glycosylation reduction. Overexpression of several different ppGaNTase isoforms restored cell surface O-glycosylation and rescued inhibitor-induced apoptosis. Additionally, mouse embryonic mandibular organ cultures exposed to 1-68A developed abnormally, presumably because of epithelial and mesenchymal apoptosis that followed a reduction in jacalin and peanut agglutinin staining. Our studies suggest that mucin-type O-linked glycosylation may be required for normal development and that ppGaNTases may play a role in the regulation of apoptosis.
Insights
Inhibiting polypeptide N-acetylgalactosaminyltransferases (ppGaNTases) reduces O-linked glycosylation, triggering apoptosis in mammalian cells and abnormal development in embryonic organs. Restoring ppGaNTase activity rescues these effects, highlighting O-glycosylation
Area of Science:
- Biochemistry and Molecular Biology
- Cell Biology
- Developmental Biology
Background:
- Mucin-type O-linked glycosylation, initiated by UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (ppGaNTases), is crucial in higher eukaryotes.
- The biological significance of O-linked glycosylation remains incompletely understood.
Purpose of the Study:
- To investigate the biological role of O-linked glycosylation using a specific ppGaNTase inhibitor.
- To determine the impact of ppGaNTase inhibition on mammalian cell glycosylation and apoptosis.
- To assess the role of O-linked glycosylation in embryonic development.
Main Methods:
- Treatment of NIH3T3 cells and mouse embryonic mandibular organ cultures with a ppGaNTase inhibitor (1-68A).
- Detection of cell surface O-glycosylation using jacalin and peanut agglutinin lectins.
- Assessment of apoptosis in treated cells and organ cultures.
- Overexpression of ppGaNTase isoforms to evaluate rescue effects.
Main Results:
- Inhibitor treatment significantly reduced cell surface O-glycosylation in NIH3T3 cells within 30 minutes.
- Apoptosis was observed in treated cells after 45 minutes, correlating with reduced O-glycosylation.
- Overexpression of ppGaNTase isoforms restored O-glycosylation and prevented inhibitor-induced apoptosis.
- Inhibition of ppGaNTase activity led to abnormal development in mouse embryonic mandibular organ cultures due to apoptosis.
Conclusions:
- Mucin-type O-linked glycosylation is essential for normal mammalian development.
- ppGaNTases play a critical role in regulating apoptosis.
- Targeting ppGaNTase activity offers a potential avenue for studying glycosylation's biological functions.
