UDP galactose:ceramide galactosyltransferase and glutamate/aspartate transporter. Copurification, separation and characterization of the two glycoproteins.

Abstract

The oligodendrocyte-specific UDP-galactose:ceramide galactosyltransferase (CGT) is the key enzyme involved in the biosynthesis of the oligodendrocyte- and myelin-specific cerebrosides. The galactosyltransferase was isolated and purified to homogeneity from Triton-X-100-solubilized rat brain microsomes by ion exchange, dye ligand and lectin affinity chromatography as a 64-kDa protein homogenous in SDS/PAGE. It copurified with the brain-specific Na(+)-dependent high-affinity L-glutamate/aspartate neurotransmitter transporter (GLAST-1) of the central nervous system. Differential lentil lectin affinity chromatography led to the separation of two glycoproteins with very similar physical properties. CGT was identified as a high-mannose glycoprotein and GLAST-1 as a hybrid glycoprotein, both with a molecular mass of 64 kDa. Deglycosylation reduced the molecular mass of the two proteins to 59 kDa. A 70-kDa isoform of GLAST-1 was isolated from whole brain by wheat germ lectin affinity chromatography. Deglycosylation again reduced the molecular mass to 59 kDa. Therefore the 70-kDa isoform differs only in the degree of glycosylation from the 64-kDa GLAST-1 isoform. The two isoproteins form homodimers of 130 and 140 kDa, respectively. They were isolated and characterized with protein-chemical and immunological methods. Oligonucleotides derived from respective peptide sequences of CGT and GLAST-1 were successfully applied to the cloning of CGT and the first high-affinity glutamate neurotransmitter transporter (GLAST-1) in glia of the central nervous system as well.