Structural analysis and functional role of the carbohydrate component of glycine transporter

The sodium- and chloride-coupled glycine transporter from pig brain stem has been shown to be a 100-kDa glycoprotein (López-Corcuera, B., Vázquez, J., and Aragón, C. (1991) J. Biol. Chem. 266, 24809-24814). To structurally identify the carbohydrate components of glycine transporter, the purified and radioiodinated protein was subjected to specific glycosidase treatments. When the glycine transporter was treated with peptide-N-glycosidase F (PNGaseF) to remove N-linked oligosaccharides, a significant reduction of the apparent molecular mass of the protein was observed. However, incubations with endoglycosidase F and O-glycanase did not affect the electrophoretic mobility of the protein, and neuraminidase produced a slight reduction of its apparent mass. The effect of PNGaseF indicates that sugar chains represent about 30% of the mass of this heavily glycosylated transporter. The deglycosylated form is recognized by previously characterized anti-100-kDa protein antiserum (López-Corcuera, B., Alcántara, R., Vázquez, J., and Aragón, C. (1993) J. Biol. Chem. 268, 2239-2243), suggesting that the epitopes are in the peptidic part of the glycoprotein. These and other results suggest that glycine transporter-linked carbohydrates are predominantly tri- or tetra- antennary complex N-linked oligosaccharides containing sialic acid residues. To investigate the functional role of the carbohydrate moiety, liposomes reconstituted with purified glycine transporter were subjected to PNGaseF and neuraminidase treatments, and the effect on specific glycine transport activity was tested. Whereas neuraminidase did not affect the activity of the transporter, PNGaseF treatment produced a drastic reduction of transport activity. This treatment produced two different deglycosylated glycine transporter species, suggesting that two N-glycosylation sites would be occupied in the native protein. These studies arise as a first evidence supporting the notion that N-linked carbohydrates play a relevant role in glycine transporter functionality.