N -Glycans on the receptor for advanced glycation end products influence amphoterin binding and neurite outgrowth.
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 beta1-3 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.
Srikrishna
Geetha
G
The Burnham Institute, La Jolla, California 92037, USA. hudson@burnham.org
Huttunen
Henri J
HJ
Johansson
Lena
L
Weigle
Bernd
B
Yamaguchi
Yu
Y
Rauvala
Heikki
H
Freeze
Hudson H
HH
D000818:Animals; D000906:Antibodies / Q000378:metabolism / Q000494:pharmacology; D002417:Cattle; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D024243:HMGB1 Protein / Q000378:metabolism*; D006801:Humans; D008168:Lung / Q000737:chemistry; D051379:Mice; D016501:Neurites / Q000187:drug effects / Q000502:physiology*; D009447:Neuroblastoma / Q000737:chemistry / Q000378:metabolism; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic / Q000737:chemistry* / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology
D000906:Antibodies; D024243:HMGB1 Protein; D009844:Oligosaccharides; D011134:Polysaccharides; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic