Biosynthesis of GlyCAM-1, a mucin-like ligand for L-selectin.
L-selectin, a member of the selectin family of leukocyte-endothelial adhesion proteins, mediates the initial attachment of lymphocytes to lymph node high endothelial venules during lymphocyte recirculation. One of the endothelial-associated ligands for L-selectin is GlyCAM-1, a mucin-like glycoprotein, which presents novel sulfated, sialylated and fucosylated O-glycans. In order to understand the generation of these glycans, we have examined the biosynthesis of GlyCAM-1 in lymph node organ culture. Using peptide-specific antibodies, lectins, and recombinant L-selectin, we detected the following species of GlyCAM-1: unglycosylated (< 28 kDa); modified with GalNAc only (28-33 kDa); modified with sialic acid, fucose, and sulfate but lacking L-selectin reactivity (40-50 kDa); and mature (L-selectin-reactive) ligand (50-60 kDa). Pulse-chase labeling at 15 degrees C suggested that GalNAc is added in a pre-Golgi compartment. Treatment with brefeldin A almost completely blocked sulfation, indicating that this modification occurs in the trans-Golgi network. Two distinct sialylation events occurred in the presence of brefeldin A, while fucosylation was partially blocked. We conclude that sialylation precedes both fucosylation and sulfation during biosynthesis. This ordering will help to identify the critical acceptor structures recognized by lymph node glycosyltransferases and sulfotransferases.
Crommie
D
D
Department of Anatomy and Program in Immunology, University of California, San Francisco 94143-0452, USA.
Rosen
S D
SD
D000818:Animals; D020126:Brefeldin A; D002240:Carbohydrate Sequence; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D003080:Cold Temperature; D003517:Cyclopentanes / Q000494:pharmacology; D006031:Glycosylation; D066298:In Vitro Techniques; D019041:L-Selectin; D008024:Ligands; D008198:Lymph Nodes; D051379:Mice; D008969:Molecular Sequence Data; D009077:Mucins / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D009844:Oligosaccharides / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D013431:Sulfates / Q000378:metabolism
D015815:Cell Adhesion Molecules; D003517:Cyclopentanes; D008024:Ligands; D009077:Mucins; D009844:Oligosaccharides; D013431:Sulfates; D019041:L-Selectin; C075185:sulfated glycoprotein p50; D020126:Brefeldin A