6'-sulfated sialyl Lewis x is a major capping group of GlyCAM-1

The binding of L-selectin to the HEV-derived ligand GlyCAM-1 bears a strict requirement for oligosaccharide sulfation. In the companion study [Hemmerich, S., Bertozzi, C.R., Leffler, H., & Rosen, S. D. (1994) Biochemistry 33, 4820-4829], we identified the major sulfated mono- and disaccharides of GlyCAM-1 as Gal-6-SO4, GlcNAc-6-SO4, (SO4-6)Gal beta 1-->4GlcNAc, and Gal beta 1-->4(SO4-6)GlcNAc. Sialic acid and fucose are also critical to the recognition determinants on GlyCAM-1. However, the hydrolysis conditions employed in the previous study resulted in cleavage of these moieties, precluding their positional assignment. Here, we employ lectins of defined specificity in conjunction with specific exoglycosidases to identify a major GlyCAM-1 capping structure that includes all three critical elements. The complementary reactivity of Maackia amurensis agglutinin with fully sialylated, undersulfated GlyCAM-1 and Sambucus nigra agglutinin/Trichosanthes japonica agglutinin with desialylated but normally sulfated GlyCAM-1 indicates the presence of terminal 6'-sulfated sialyllactosamine. alpha (1-->3/4)Fucosidase removes fucose almost quantitatively from asialo-GlyCAM-1 while substantially enhancing its binding to Lycopersican esculentum agglutinin (specific for beta 1-->4-linked GlcNAc), indicating the presence of Fuc in an alpha 1-->3 linkage to GlcNAc. The strict requirement for desialylation to achieve defucosylation indicates a proximal location of Fuc with respect to terminal sialic acid. The nature of the capping group was further defined by studying the effects of sulfation, sialylation, and fucosylation on the ability of exo-beta(1-->4)galactosidase to release [3H]Gal from GlyCAM-1.(ABSTRACT TRUNCATED AT 250 WORDS)