De-N-glycosylation or G82S mutation of RAGE sensitizes its interaction with advanced glycation endproducts.
Interactions between advanced glycation endproducts (AGE) and the receptor for AGE (RAGE) have been implicated in the development of diabetic vascular complications. RAGE has two N-glycosylation sites in and near the AGE-binding domain, and G82S mutation in the second N-glycosylation motif was recently reported in human. In this study, we examined whether de-N-glycosylation or G82S of RAGE affect its ability to bind AGE and cellular response to AGE. Recombinant wild-type, de-N-glycosylation and G82S RAGE proteins were produced in COS-7 cells, purified and assayed for ligand-binding abilities. De-N-glycosylation at N81 and G82S mutation decreased Kd for glycolaldehyde-derived AGE to three orders of magnitude lower levels compared with wild-type. AGE-induced upregulation of VEGF mRNA was significantly augmented in endothelial cell-derived ECV304 cells expressing de-N-glycosylated and G82S RAGE when compared with wild-type expressor. Exposure to low glucose resulted in the appearance of RAGE proteins of deglycosylated size in wild-type RAGE-expressing cells and significantly enhanced glycolaldehyde-derived AGE-induced VEGF mRNA expression. De-N-glycosylation or G82S mutation of RAGE increases affinity for AGE ligands, and may sensitize cells or conditions with it to AGE.
Osawa
Mari
M
Department of Biochemistry and Molecular Vascular Biology, Kanazawa University Graduate School of Medical Science, 13-1 Takara-machi, Kanazawa 920-8640, Japan.
Yamamoto
Yasuhiko
Y
Munesue
Seiichi
S
Murakami
Naho
N
Sakurai
Shigeru
S
Watanabe
Takuo
T
Yonekura
Hideto
H
Uchigata
Yasuko
Y
Iwamoto
Yasuhiko
Y
Yamamoto
Hiroshi
H
D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D017127:Glycation End Products, Advanced / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020349:Surface Plasmon Resonance; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis
D017127:Glycation End Products, Advanced; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic; D042461:Vascular Endothelial Growth Factor A