STRUCTURAL-ANALYSIS AND FUNCTIONAL-ROLE OF THE CARBOHYDRATE COMPONENTOF GLYCINE TRANSPORTER

The sodium- and chloride-coupled glycine transporter from pig brain st em has been shown to be a 100-kDa glycoprotein (Lopez-Corcuera, B., Va zquez, J., and Aragon, C. (1991) J. Biol. Chem. 266, 24809-24814). To structurally identify the carbohydrate components of glycine transport er, 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-glyca nase did not affect the electrophoretic mobility of the protein, and n euraminidase produced a slight reduction of its apparent mass. The eff ect 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 antise rum (Lopez-Corcuera, B., Alcantara, R., Vazquez, J., and Aragon, C. (1 993) J. Biol. Chem. 268, 2239-2243), suggesting that the epitopes are in the peptidic part of the glycoprotein. These and other results sugg est that glycine transporter-linked carbohydrates are predominantly tr i- or tetra- antennary complex N-linked oligosaccharides containing si alic acid residues. To investigate the functional role of the carbohyd rate moiety, liposomes reconstituted with purified glycine transporter were subjected to PNGaseF and neuraminidase treatments, and the effec t on specific glycine transport activity was tested. Whereas neuramini dase did not affect the activity of the transporter, PNGaseF treatment produced a drastic reduction of transport activity. This treatment pr oduced two different deglycosylated glycine transporter species, sugge sting that two N-glycosylation sites would be occupied in the native p rotein. These studies arise as a first evidence supporting the notion that N-linked carbohydrates play a relevant role in glycine transporte r functionality.