Minimal Sulfated Carbohydrates for Recognition by L-selectin and the MECA-79 Antibody

Abstract

Sulfated forms of sialyl-LeX containing Gal-6-SO 4 or GlcNAc-6-SO 4 have been implicated as potential recognition determinants on high endothelial venule ligands for L-selectin. The optimal configuration of sulfate esters on the N-acetyllactosamine (Gal␤134GlcNAc) core of sulfosialyl-Le X , however, remains unsettled. Using a panel of sulfated lactose (Gal␤134Glc) neoglycolipids as substrates in direct binding assays, we found that 6,6-disulfolactose was the preferred structure for L-selectin, although significant binding to 6-and 6-sulfolactose was observed as well. Binding was EDTA-sensitive and blocked by L-selectin-specific monoclonal antibodies. Surprisingly, 6,6-disulfolactose was poorly recognized by MECA-79, a carbohydrate-and sulfate-dependent monoclonal antibody that binds competitively to L-selectin ligands. Instead, MECA-79 bound preferentially to 6-sulfolactose. The difference in preferred substrates between L-selectin and MECA-79 may explain the variable activity of MECA-79 as an inhibitor of lymphocyte adhesion to high endothelial venules in lymphoid organs. Our results suggest that both Gal-6-SO 4 and GlcNAc-6-SO 4 may contribute to L-selectin recognition, either as components of sulfosialyl-Le X capping groups or in internal structures. By contrast, only GlcNAc-6-SO 4 appears to contribute to MECA-79 binding. L-selectin, the "leukocyte selectin," mediates the tethering and rolling of lymphocytes along high endothelial venules (HEVs) 1 in peripheral lymph nodes, a prerequisite for extravasation of the lymphocytes (1-3). By virtue of a calcium-type lectin domain at its amino terminus, L-selectin functions as a calcium-dependent, carbohydrate-binding receptor that recognizes a set of discrete counter-receptors (generally termed ligands) displayed on the luminal aspect of HEVs (reviewed in Refs. 4 and 5). Several HEV-expressed, L-selectin-reactive ligands (all of which possess mucin-like domains) have been identified as potential physiological ligands for L-selectin (reviewed in Ref. 6). These include GlyCAM-1 (7), CD34 (8, 9), and podocalyxin (10).A large body of evidence has established that the optimal interaction between L-selectin and these HEV-expressed ligands requires sialylation, fucosylation, and carbohydrate sulfation (11)(12)(13)(14)(15)(16)(17). In an attempt to rationalize these requirements in terms of carbohydrate structures, a detailed analysis of the O-linked oligosaccharide side chains of GlyCAM-1 was conducted (18 -20). Sulfation analysis of acid-hydrolyzed glycans revealed monosulfated monosaccharides and disaccharides (Nacetyllactosamine) with equivalent levels of Gal-6-SO 4 and GlcNAc-6-SO 4 . Analysis of the simplest oligosaccharide side chains identified equivalent levels of two novel sulfated isomers of sialyl-Le X (sLe X ), 6Ј-sulfo-sLe X , containing Gal-6-SO 4 , and 6-sulfo-sLe X , containing GlcNAc-6-SO 4 (see Fig. 1), as capping groups of core-2 branched oligosaccharides. The finding of a sLe X motif was significant because this tetrasaccharide binds to all three sel...

N-Acetylglucosamine 6-O-Sulfotransferase-1 Regulates Expression of L-Selectin Ligands and Lymphocyte Homing

“…The requirement of sialyl 6-sulfo Lewis X for L-selectin binding has been established by the finding that G152 blocks the binding of lymphocytes to HEV with absolute dependence on GlcNAc-6-O-sulfation (14,17). The importance of GlcNAc-6-O-sulfation is reinforced by the finding that the MECA-79 epitope requires this modification (17,18) and that the full MECA-79 epitope consists of an extended core 1 O-glycan capped by Gal␤1,4(SO 4 -6)GlcNAc ( Fig. 1) (19).…”

Core 2 Branching β1,6-N-Acetylglucosaminyltransferase and High Endothelial Venule-restricted Sulfotransferase Collaboratively Control Lymphocyte Homing

“…Isolation of GlyCAM-1-GlyCAM-1 was prepared from sera of wildtype and LSST ⌬ mice as described previously (27). The samples were subjected to SDS-PAGE, blotted to a nitrocellulose membrane and reacted with anti-GlyCAM-1 antibodies that had been prepared as described previously (27), or MECA-79 antibody followed by goat antirabbit immunoglobulins and goat anti-rat IgM antibodies, respectively.…”

“…The samples were subjected to SDS-PAGE, blotted to a nitrocellulose membrane and reacted with anti-GlyCAM-1 antibodies that had been prepared as described previously (27), or MECA-79 antibody followed by goat antirabbit immunoglobulins and goat anti-rat IgM antibodies, respectively. The bound antibodies were visualized by ECL (Amersham Biosciences).…”

“…GlyCAM-1 was purified from the condition medium using a column of anti-GlyCAM-1 antibodies conjugated to UltraLink Biosupport Medium (Pierce), as described previously (27).…”

Expression of potential lymphocyte trafficking mediator molecules in the mammary gland

“…Sgp90 was characterized as sialomucin CD34 and podocalyxin-like protein (PCLP) [5,43], however Sgp200 is not yet characterized. L-selectin recognizes a specific sulfated form of these molecules and basic structure was reported [13,26].…”