A 58-kDa resident protein of the cis Golgi cisterna is not terminally glycosylated.
A 58-kDa Golgi protein (gp58) was previously identified and found to be concentrated in cis Golgi cisternae in several cell types (Saraste, J., Palade, G.E., and Farquhar, M.G. (1987) J. Cell Biol. 105, 2021-2029). In this study the protein was partially purified from rat pancreas and mouse myeloma cells in order to characterize its oligosaccharides. It migrated on sodium dodecyl sulfate-polyacrylamide gels as a 57-58-kDa doublet under reducing conditions or as a single approximately 116-kDa band under nonreducing conditions. Pancreatic gp58 was susceptible to alpha-N-acetylgalactosaminidase digestion and it bound concanavalin A, Helix pomatia, Dolichos biflorus, soybean agglutinin, and Bauhinia purpurea lectins, but not Ricinus communis agglutinin or lectins from Griffonia simplicifolia-1, Arachis hypogaea, and Limulus polyphemus. It bound Ricinus communis agglutinin after galactosylation with GlcNAc galactosyltransferase. These data demonstrate that pancreatic p58 contains immature N-linked moieties with nonreducing terminal GlcNAc residues as well as the initiating GalNAc of O-linked glycoproteins. Myeloma gp58 was sensitive to endo-beta-N-acetylglucosaminidase H, and oligosaccharide analysis of its [3H]glucosamine-labeled glycopeptides indicated that it also contained immature N-linked glycans. Some of the latter consist of high mannose chains (high affinity for concanavalin A, endo-beta-N-acetylglucosaminidase H-sensitive), but the predominant (95%) species are neutral tri- or tetraantennary N-linked chains containing GlcNAc (no binding to concanavalin A). Glycopeptides from biosynthetically labeled myeloma cells did not contain detectable base labile oligosaccharides, indicating that unlike pancreatic p58, myeloma gp58 may not be an O-linked glycoprotein. Neither pancreatic nor myeloma gp58 contained terminally processed oligosaccharides, indicating that gp58 has not been modified by trans-Golgi glycosyltransferases. Thus, the oligosaccharide content of gp58 is consistent with the assumption that this protein is retained in the cis Golgi cisternae during biosynthesis instead of being transported across the Golgi stacks and targeted back to the cis Golgi from the trans side.
Hendricks
L C
LC
Division of Cellular and Molecular Medicine, University of California, San Diego, La Jolla 92093-0651.
Gabel
C A
CA
Suh
K
K
Farquhar
M G
MG
D000818:Animals; D001692:Biological Transport; D002846:Chromatography, Affinity; D006023:Glycoproteins / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism*; D015151:Immunoblotting; D007124:Immunoenzyme Techniques; D051379:Mice; D009844:Oligosaccharides / Q000378:metabolism; D010179:Pancreas / Q000378:metabolism; D051381:Rats; D014407:Tumor Cells, Cultured
D006023:Glycoproteins; D009844:Oligosaccharides; D006026:Glycoside Hydrolases