Abstract
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.
Highlights
From the Glycobwhgy Program, Cancer Center, and Diuision of Cellular and Molecular Medicine, University of Calijornia, San Diego, La Jolln, California92093
UDP-[3H]Gal, CMP-[3H]Neu5Ac, and [acetyl-3H]Ac-CoAto study the transfer of the terminal monosaccharides Gal and sialic acid, and the acetylation of oligosaccharides
Lack of 0-Acetylation of the PHlNeuSAc-labeled Oligosacchurides-We have recently shown that a large fraction (3040%) of the sialic acid residues on the N-linked oligosaccharides of rat liver membranes are 0-acetylated at the7- or 9positions [41].we have shown that, insimilarly prepared Golgi apparatus fractions, less than 3%of the total [3H]Neu5Actransferred from CMP-[3H]Neu5Acto total endogenous acceptors is 0-acetylated, even if Ac-CoA is added [23]
Summary
ANALYSIS OF N-LINKED GLYCANS LABELED BY UDP-[6-3H]GALACTOSE, CMP-[9-3H]NACETYLNEURAMINIC ACID,AND [uc~~~~-~H]ACETYL-COAE*NZYME (Received for publication, December 16, 1992,and in revised form, April 12, 1993). When a rat liver Golgi apparatus-enriched subcellu- dase-resistant sialic acids While most of these sialilar fraction isincubated with UDP-['HIGal, CMP-['HI dase-resistant oligosaccharides remain negatively. The reaction was chased by adding UDP-Gal to a final concentrationof 0.5 mM and theincubation continued for an EXPERIMENTALPROCEDURES additional 12 min. These final concentrations were chosen to be The dried samples were resuspended in 100 p1 of water, nonradioacgreater thanor equal tothe K,,, values of the sugar nucleotide tive Gal and Glc were added as internal standards, and analyzed by transporters, which have apparent K,,, values of 2-20 p M [21,22]. Sample 7was chased with 20 AX-5, HPLC column developed with a gradient of decreasing acetop~ CMP-Neu5Ac; sample 8 with 20 pM UDP-GlcNAc, 20 pM UDP- nitrile in water as described [17, 26].
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