Positive regulation of myoblast differentiation by medaka Neu3b sialidase through gangliosides desialylation

• neu3b is found in the genome of Beloniformes, Perciformes and Cyprinodontiformes.
• Neu3b overexpression enhanced C2C12 cell differentiation and decreased cell proliferation.
• Decline of ERK and EGFR phosphorylation could be involved in the Neu3b-enhanced myogenesis.

Sialidase Neu3b is an unique enzyme conserved in medaka and tilapia, but not in mammals. Previous study revealed that medaka Neu3b is localized at cytosol and is a ganglioside-specific sialidase. Neu3b functions, however, have not been understood, while Neu3a sialidase, which is widely conserved from human to fish, is known as a regulator of neurite formation. Here, we investigated the biological function of Neu3b for C2C12 myoblast cell differentiation. Bioinformatics analysis using genome browser revealed the presence of neu3b gene in some orders of fish species such as Beloniformes, Perciformes and Cyprinodontiformes. With the treatment of 2% horse serum, Neu3b-overexpression accelerated myoblast cell differentiation to myotubes accompanied with up-regulation of myogenesis biomarkers mRNA, myod and myog. Neu3b altered ganglioside composition in C2C12 cells results showing a decrease in GM2, and the increase of Lac-Cer, while desialylation of glycoproteins were not detected. Contrary to cell differentiation, Neu3b cell proliferation was suppressed in normal growth medium. To understand the mechanism of the alteration of cell differentiation and proliferation, phosphorylation of signal molecules in EGFR/ERK pathway was investigated. Neu3b induced a decline in phosphorylation of ERK and EGFR. Surprisingly, immuno-blot and real-time PCR analysis revealed that down-regulation of egfr gene could be involved in the acceleration of cell differentiation by Neu3b. These results suggested that Neu3b sialidase is a positive regulator for myoblast differentiation, similar with mammalian cytosolic sialidase Neu2.