THE BIOSYNTHESIS OF OLIGOSACCHARIDES IN INTACT GOLGI PREPARATIONS FROM RAT-LIVER - ANALYSIS OF N-LINKED AND O-LINKED GLYCANS LABELED BY UDP-6-H-3!N-ACETYLGALACTOSAMINE

Endogenous acceptors in a Golgi apparatus-enriched subcellular fractio n from rat liver were labeled with UDP-H-3!GalNAc. The great majority of these acceptors were protected from protease degradation in the ab sence of detergent. These molecules are therefore present in intact ve sicles of the correct topological orientation, which are likely to be similar to the Golgi compartments of the intact cell. Several distinct glycoproteins are labeled, but most are different from those labeled with UDP-H-3!GlcNAc. The enzyme peptide-N4(N-acetyl-beta-glucosiminyl )asparagine amidase releases label from a few specific proteins, indic ating that H-3!GalNAc is transferred to N-linked oligosaccharides. Bo th neutral and anionic N-linked oligosaccharides are found, the great majority of which do not bind to ConA-Sepharose. Most of the H-3!GalN Ac found in neutral oligosaccharides is terminal and beta-linked. The negative charge on the anionic molecules is due to sialic acid, and ph osphate. A major portion of the H-3! GalNAc in this fraction is acid labile, and is released with kinetics consistent with it being in a ph osphodiester linkage. These results show the existence of a whole new class of GalNAc-containing N-linked oligosaccharides, and demonstrates that this in vitro approach can detect previously undescribed structu res. O-Linked oligosaccharide biosynthesis was also studied in the sam e labeled rat liver Golgi apparatus preparations. Beta-elimination rel eases approximately 95% of the peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase (PNGase F)-resistant label which, in the absence of other added nucleotides, is almost exclusively H-3! GalNAcitol. If o ther unlabeled sugar nucleotides and adenosine 3'-phosphate,5'-phospho sulfate are added during the chase period two anionic O-linked oligosa ccharides are synthesized, indicating that the P-GalNAc:peptide-N-acet ylgalactosaminyltransferase is at least in part functionally co-locali zed with enzymes that extend and modify O-linked oligosaccharides.