Biosynthesis of oligosaccharides in intact Golgi preparations from rat liver. Analysis of N-linked glycans labeled by UDP-[6-3H]galactose, CMP-[9-3H]N-acetylneuraminic acid, and [acetyl-3H]acetyl-coenzyme A.

When a rat liver Golgi apparatus-enriched subcellular fraction is incubated with UDP-[3H]Gal, CMP-[3H] Neu5Ac, or [acetyl-3H]acetyl (Ac)-CoA, label is efficiently transferred to endogenous acceptors, which are resistant to added proteases, unless detergent is added at a sufficiently high concentration. Thus, the acceptors are within the lumen of intact compartments of correct topological orientation, which are likely to be similar to those of the Golgi apparatus in the intact cell. In each case, approximately 90% of the macromolecular radioactivity is specifically released by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase digestion, as labeled N-linked oligosaccharides. Label from UDP-[3H]Gal is transferred to several distinct N-linked oligosaccharides, and many of these carry sialic acid (Sia) residues. This amount increases if the transfer reaction is chased with CMP-Neu5Ac. A major fraction of the [3H]Gal is directly "covered" with Sia residues, indicating that at least a portion of the beta-galactosyltransferase(s) are co-localized with one or more sialyltransferases. The majority of the [3H]Gal is found in a beta 1,3-linkage, rather than the more common beta 1,4-linkage. The N-linked oligosaccharides labeled by CMP-[3H] Neu5Ac carry labeled Sia residues in either alpha 2,3 or alpha 2,6 linkage, and showed a range of charge distribution. The transferred [3H]Neu5Ac is not O-acetylated even when Ac-CoA is added at saturating concentrations, implying that the sialyltransferases and the O-acetyltransferase(s) are not functionally co-localized. However, approximately 20% of label released from N-linked oligosaccharides by sialidase does not co-migrate with authentic Neu5Ac in high performance liquid chromatography analysis, indicating that transferred [3H] Neu5Ac is modified by unknown enzymes in the Golgi. Most of the [3H]acetate transferred from [acetyl-3H] Ac-CoA to N-linked oligosaccharides is on Sia residues that are exclusively alpha 2,6-linked, and is enriched on tri- and tetra-antennary chains that do not appear to carry any 2,3-linked Sia residues. These data indicate a restricted substrate preference of the O-acetyltransferase(s). About one-quarter of the [3H]acetate transferred is sialidase-resistant, indicating either transfer to monosaccharides other than sialic acid, or to sialidase-resistant sialic acids. While most of these sialidase-resistant oligosaccharides remain negatively charged, about 10% are neutralized by sialidase, confirming transfer of [3H]acetate to monosaccharides other than sialic acid.