Identification of the sulfated monosaccharides of GlyCAM-1, an endothelial-derived ligand for L-selectin.
L-Selectin, a receptor bearing a C-type lectin domain, mediates the initial attachment of lymphocytes to high endothelial venules of lymph nodes. One of the endothelial-derived ligands for L-selectin is GlyCAM-1 (previously known as Sgp50), a mucin-like glycoprotein with sulfated, sialylated, and fucosylated O-linked oligosaccharide chains. Sialylation, sulfation, and fucosylation appear to be required for the avid interaction of this ligand with L-selectin, but the exact carbohydrate structures involved in recognition remain undefined. In this study, we examine the nature of the sulfate-modified carbohydrates of GlyCAM-1. GlyCAM-1 was metabolically labeled in lymph node organ culture with 35SO4 and a panel of tritiated carbohydrate precursors. Mild hydrolysis conditions were established that released sulfated oligosaccharides without cleavage of sulfate esters. Low molecular weight and singly charged fragments, obtained by a combination of gel filtration and anion-exchange chromatography, were analyzed. The structural identification of the fragments relied on the use of a variety of radiolabeled sugar precursors, further chemical and enzymatic hydrolysis, and high-pH anion-exchange chromatography analysis. Sulfated constituents of GlyCAM-1 were identified as Gal-6-SO4, GlcNAc-6-SO4, (SO4-6)Gal beta 1-->4GlcNAc, and Gal beta 1-->4(SO4-6)GlcNAc. In the accompanying paper [Hemmerich, S., & Rosen, S.D. (1994) Biochemistry 33, 4830-4835] evidence is presented that (SO4-6)Gal beta 1-->4GlcNAc forms the core of a sulfated sialyl Lewis x structure that may comprise a recognition determinant on GlyCAM-1.
Hemmerich
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S
Department of Anatomy, University of California, San Francisco 94143-0452.
Bertozzi
C R
CR
Leffler
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Rosen
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SD
D000117:Acetylglucosamine / Q000031:analogs & derivatives / Q000032:analysis; D000595:Amino Acid Sequence; D000818:Animals; D002240:Carbohydrate Sequence; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002853:Chromatography, Liquid; D005690:Galactose / Q000031:analogs & derivatives / Q000032:analysis; D006868:Hydrolysis; D019041:L-Selectin; D008198:Lymph Nodes / Q000737:chemistry; D051379:Mice; D008813:Mice, Inbred ICR; D008969:Molecular Sequence Data; D009077:Mucins / Q000737:chemistry*
D015815:Cell Adhesion Molecules; D009077:Mucins; C033137:N-acetylglucosamine-6-sulfate; D019041:L-Selectin; C075185:sulfated glycoprotein p50; C086947:galactose 6-sulfate; D000117:Acetylglucosamine; D005690:Galactose