Enhanced interaction of L-selectin with the high endothelial venule ligand via selectively oxidized sialic acids.

Abstract

The selectins are adhesion receptors that play key roles in leukocyte trafficking. Each has an N-terminal C-type lectin domain that binds to specific carbohydrates in a calcium-dependent manner. L-selectin recognizes sulfated, sialylated ligands on lymph node high endothelial venules. This recognition is abolished by strong periodate oxidation under conditions that destroy oligosaccharides. In contrast, mild periodate oxidation, which selectively oxidizes the side chain of sialic acid residues without affecting the underlying oligosaccharide, markedly enhances this interaction. The enhancement is calcium dependent, indicating that lectin recognition is maintained. Reduction of the sialic acid aldehydes generated by mild periodate to alcohol groups abolishes this effect. Covalent cross-linking of the oxidized ligand to L-selectin can be demonstrated, suggesting Schiff base formation between lysine residues of the selectin and the newly formed aldehydes. Such selectively oxidized sialylated ligands could be used to probe the lectin domains of the selectins and to identify lysine residues near the binding site. Also, this approach could be used to design drugs for disrupting leukocyte-endothelial interactions leading to pathological inflammation.The selectins are a family of adhesion receptors involved in leukocyte migration into lymphoid tissues and areas of inflammation (1-7). Each selectin has an N-terminal C-type lectin domain that binds specific carbohydrate ligands in a calcium-dependent manner. These domains also contain 10-14 lysine residues (8), which may be involved in ligand binding. Naturally occurring selectin ligands contain sialylated fucosylated lactosamine-type oligosaccharides (9)(10)(11). Sulfation of these and/or other oligosaccharides may also be involved in binding (7,9,(12)(13)(14)(15). Two specific ligands have been more precisely identified. The L-selectin binds certain sulfated and sialylated glycoproteins from lymph node high endothelial venules (HEVs) (13, 16), and P-selectin binds a minor 120-kDa neutrophil sialoglycoprotein (17). In both instances, sialic acids are absolutely required for binding.Periodate oxidation has been used to destroy carbohydrates and to demonstrate biological functions, including lymphocyte-endothelium recognition (18). Such studies used strong periodate oxidation (e.g., 50 mM; pH 5.0), which causes cleavage between cis-diols throughout oligosaccharides and destroys them. In contrast, mild periodate oxidation (e.g., 1-2 mM; pH 7.0) selectively oxidizes only the side chain of terminal sialic acids (19)(20)(21)(22), leaving the rest of the oligosaccharide intact. Conditions can be adjusted to selectively eliminate only the C9 carbon or both the C8 and C9 carbons (Fig. 1, structures B and C). The resulting aldehyde can be reduced, leaving a truncated side chain with a primary alcohol group (Fig. 1, structures D and G). These reactions have been used to explore the importance of the sialic acid side chains in interactions involving viral hemagglutinins, lec...

Binding Specificities of the Sialoadhesin Family of I-type Lectins

“…5). These data are consistent with prior studies on the sensitivity of sialoadhesin and CD22 binding to modifications of the sialic acid residue (6, 33, 46 -48) and contrast with studies on selectins, in which extensive modifications of sialic acids have no effect (12,14,49). In fact, substitution of the entire sialic acid (e.g.…”

The interaction ofTrypanosoma congolensewith endothelial cells

“…Cover-slips were rinsed extensively in PBS, then stored in 0-16 M ethanolamine, pH 8-3, for at least 24 h at 4 °C in order to saturate free aldehyde groups. After fixation in 1 % paraformaldehyde for 20 min at 4 °C, ethanolamine treatment was not necessary (Norgard et al 1993, data not shown).…”

Distinct Selectin Ligands on Colon Carcinoma Mucins Can Mediate Pathological Interactions among Platelets, Leukocytes, and Endothelium

“…51 We also used EDTA-containing buffer to confirm that the interactions are calciumdependent and adjusted salt concentrations to more closely approximate the physiological state. 53 Although some staining for selectin ligands was also seen in goblet cells of normal colonic epithelium, the polarity and fate of these cells (eventually shed into the lumen of the gut) assures that these ligands would never be exposed to the vascular compartment in the normal situation.…”

“…Recombinant selectin immunoglobulin-Fc fusion proteins (selectin-Igs) were prepared and purified as reported. 19,51 For some experiments, we used selectin-Ig proteins incorporating the FLAG epitope tag (Eastman Kodak; details of these constructs will be reported elsewhere). The ␣2-3 linkage specific sialidase L was a kind gift from Dr. Y.-T. Lee of Tulane University (New Orleans, LA).…”