Abstract
Cytosolic α-mannosidase (Man2C1) trims free oligosaccharides in mammalian cells, and its down-regulation reportedly delays cancer growth by inducing mitotic arrest or apoptosis. However, the mechanism by which Man2C1 down-regulation induces apoptosis is unknown. Here, we demonstrated that silencing of Man2C1 via small hairpin RNAs induced mitochondria-dependent apoptosis in HeLa cells. Expression of CHOP (C/EBP homologous protein), a transcription factor critical to endoplasmic reticulum stress-induced apoptosis, was significantly up-regulated in Man2C1 knockdown cells. However, this enhanced CHOP expression was not caused by endoplasmic reticulum stress. Interestingly, Man2C1 catalytic activity was not required for this regulation of apoptosis; introduction of mutant, enzymatically inactive Man2C1 rescued apoptotic phenotypes of Man2C1 knockdown cells. These results show that Man2C1 has dual functions: one in glycan catabolism and another in apoptotic signaling.
Highlights
Man2C1 regulates apoptosis via an unknown mechanism
These results show that Man2C1 has dual functions: one in glycan catabolism and another in apoptotic signaling
Our results show that Man2C1 had a role in apoptosis that was independent of its enzymatic activity; this finding provides convincing evidence that Man2C1 has dual functions: one as a catabolic enzyme for the breakdown of free, cytosolic oligosaccharides and another as a modulator of apoptotic signaling
Summary
Man2C1 regulates apoptosis via an unknown mechanism. Results: Suppression of Man2C1 induces mitochondria-dependent apoptosis independently of its enzyme activity. Cytosolic ␣-mannosidase (Man2C1) trims free oligosaccharides in mammalian cells, and its down-regulation reportedly delays cancer growth by inducing mitotic arrest or apoptosis. Expression of CHOP (C/EBP homologous protein), a transcription factor critical to endoplasmic reticulum stress-induced apoptosis, was significantly up-regulated in Man2C1 knockdown cells. Our results show that Man2C1 had a role in apoptosis that was independent of its enzymatic activity; this finding provides convincing evidence that Man2C1 has dual functions: one as a catabolic enzyme for the breakdown of free, cytosolic oligosaccharides and another as a modulator of apoptotic signaling. Fractionation of Cytosol and Mitochondria—HeLa cells (1 ϫ 106) and Man2C1 knockdown HeLa cells were washed twice with PBS, harvested, and separated into cytosolic fractions and mitochondrial fractions using a Qproteome mitochondria isolation kit (Qiagen) according to the manufacturer’s protocol. The PA-Glc in the PA-glucose oligomer (TaKaRa Bio Inc.; 2 pmol/l) was used as a reference
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