N‐Glycans on the receptor for advanced glycation end products influence amphoterin binding and neurite outgrowth

Abstract

In this study we show that embryonic neurite growth-promoting protein amphoterin binds to carboxylated N-glycans previously identified on mammalian endothelial cells. Since amphoterin is a ligand for the receptor for advanced glycation end products (RAGE), and the ligand-binding V-domain of the receptor contains two potential N-glycosylation sites, we hypothesized that N-glycans on RAGE may mediate its interactions with amphoterin. In support of this, anti-carboxylate antibody mAbGB3.1 immunoprecipitates bovine RAGE, and PNGase F treatment reduces its molecular mass by 4.5 kDa, suggesting that the native receptor is a glycoprotein. The binding potential of amphoterin to RAGE decreases significantly in presence of soluble carboxylated glycans or when the receptor is deglycosylated. Oligosaccharide analysis shows that RAGE contains complex type anionic N-glycans with non-sialic acid carboxylate groups, but not the HNK-1 (3-sulfoglucuronyl b1-3 galactoside) epitope. Consistent with the functional localization of RAGE and amphoterin at the leading edges of developing neurons, mAbGB3.1 stains axons and growth cones of mouse embryonic cortical neurons, and inhibits neurite outgrowth on amphoterin matrix. The carboxylated glycans themselves promote neurite outgrowth in embryonic neurons and RAGE-transfected neuroblastoma cells. This outgrowth requires full-length, signalling-competent RAGE, as cells expressing cytoplasmic domain-deleted RAGE are unresponsive. These results indicate that carboxylated N-glycans on RAGE play an important functional role in amphoterin-RAGE-mediated signalling.

Amphoterin Stimulates Myogenesis and Counteracts the Antimyogenic Factors Basic Fibroblast Growth Factor and S100B via RAGE Binding

“…AGEs, ␤-amyloid, amphoterin, S100A12, S100B, and, recently, S100P have been shown to bind and activate RAGE in a variety of cell types with an affinity in the nanomolar range, thereby stimulating the activity of signaling pathways such as Ras-MEK-ERK, Cdc42-Rac-1, p38 MAPK, SAPK-JNK MAPK, and/or phosphatidylinositol 3-kinase-Akt (3, 20-23, 34, 48, 49, 54, 60, 63-65). While there is evidence that molecules recognized by RAGE might share a consensus sequence (26), and N-glycans on RAGE might play an important role in amphoterin-dependent activation of RAGE (53), the structural requirements of binding of individual ligands to RAGE are not fully elucidated at present. Interestingly, RAGE is expressed during development, repressed at completion of development, and reexpressed in adulthood in the course of certain pathological conditions (48,49 (6,27,41,46).…”

Purification and Characterization of Mouse Soluble Receptor for Advanced Glycation End Products (sRAGE)

“…4). Glycosylation of Mouse sRAGE-The importance of RAGE glycosylation was shown by Srikrishna et al (24) when they found a reduction in amphoterin-RAGE binding after RAGE deglycosylation. Presumably, the same is true for sRAGE because it has the same ligand specificity as RAGE.…”

Multiple levels of regulation determine the role of the receptor for AGE (RAGE) as common soil in inflammation, immune responses and diabetes mellitus and its complications

“…N-glycosylation of RAGE further modulates and enhances ligand recognition in two ways. While deglycosylation seems to sensitise RAGE to bind experimental AGE-modified proteins with an extremely high degree of modification by AGE (76-89% lysine residues modified) [20], carboxylated glycans on RAGE increase the affinity for S100-calcium binding protein (S100) and high-mobility group protein 1 (HMGB1) proteins [21,22]. The quaternary structure of the RAGE extracellular domain, but also post-translational modifications of its primary structure, might account for the diversity of ligand recognition.…”

“…Carboxylated N-glycans on RAGE facilitate binding of HMGB1 [21] and mediate binding of S100A8/A9 to subpopulation of RAGE on colon cancer cells [22], but increased ligand binding to RAGE can also be observed upon glycation of its ligands [42]. N ( -carboxymethyllysine (CML)-modifications of S100A8 and S100A9 occur in inflammatory bowel disease and enhance RAGE-mediated sustained inflammation [42].…”