Biochimica et Biophysica Acta (BBA) - General Subjects
De-N-glycosylation or G82S mutation of RAGE sensitizes its interaction with advanced glycation endproducts
Introduction
Interactions between advanced glycation endproducts (AGE) and the receptor for AGE (RAGE) have been implicated in the development of diabetic vascular complications [1], [2], [3]. Recent in vivo studies with gene-manipulated animals in this laboratory and others have shown that when RAGE expression is upregulated, the indices of diabetic vascular complications are aggravated [4], [5], [6], [7], [8], and that when mice are made deficient for RAGE, macro- and micro-vascular complications of diabetes are ameliorated [9], [10], [11]. Up- and down-regulation of human RAGE gene expression may, therefore, make diabetic patients susceptible or resistant to the development of vascular complications. This has prompted us to learn how human RAGE gene expression is regulated to develop remedies for overcoming this disease. Tanaka et al. [12] revealed transcriptional regulation of human RAGE gene; extracellular signals that cause an increase in RAGE mRNA, cis-acting elements of genomic RAGE that respond to those inducers, and relevant trans-acting transcription factors have been identified. Through analysis of polysomes from human vascular cells, Yonekura et al. [13] revealed regulation of RAGE gene at the step of RNA splicing and identified endogenous secretory RAGE (esRAGE) as one of the major splice variants.
Since the cDNA coding for RAGE was isolated by the group of Stern and Schmidt [14], [15] N-glycosylation motifs N–X–S/T have been noticed in the deduced amino acid sequence. There are two putative N-glycosylation sites, one is located in the amino terminus adjacent to the V-region-like domain, and the other is located within the V domain, which was subsequently revealed to be engaged by AGE ligands [13]. This suggests the presence of additional regulation in the expression and function of human RAGE at the step of post-translational modification. The present study was designed to examine whether the state of glycosylation may affect the ability of RAGE to bind AGE ligands and cellular response to AGE. For this, we mutagenized the residues required for N-glycosylation, and compared the resultant mutants with wild-type proteins from these aspects. Further, human subjects who G82S substitution within the second N-glycosylation site have been described [16], [17]. The effect of this mutation on RAGE function was also tested in the present study.
Section snippets
Construction of expression vectors
To construct expression vectors to yield wild-type and mutant RAGE proteins, full-length cDNA coding for the membrane-bound RAGE (Ref. 13: Gene Bank accession number AB036432) cloned in plasmid Bluescript SK(−) (Stratagene, La Jolla, CA) was used as the stating template. Four mutant cDNAs were constructed using the template and primer sets shown in Table 1. To obtain 1Q, wild-type cDNA in SK(−) was amplified with 5′ATCCGGGCTGTGATTTGTTGAGC3′ and M13 forward primer, and the amplified DNA
Results
RAGE has two N-glycosylation sites in and near the AGE-binding domain [15], and G82S mutation in the second N-glycosylation motif was reported in human [16], [17]. As shown in Fig. 1A, native human RAGE proteins had the size of 55 kDa as previously reported [13]. When treated with glycopeptidase-F, the RAGE proteins migrated faster to the position at approximately 50 kDa, indicating that RAGE proteins have glycopeptidase-F-sensitive N-glycosylation. In this study, we examined whether de-N-
Discussion
The present study has demonstrated for the first time that N-glycosylation state of RAGE can critically affect its ability to bind AGE ligands. In particular, RAGE proteins that lacked glycosylation in the V domain showed three orders of magnitude higher affinity to glycolaldehyde-derived AGE (Fig. 2A). Since glycolaldehyde-derived AGE are one of the major AGE fraction present in human subjects [32], the finding seems to be of significance for our understanding of roles of the AGE-RAGE system
Acknowledgments
The authors thank S. Matsudaira and R. Kitamura for their assistance. This work was supported in part by the “Research for the Future” Program of the Japan Society for the Promotion of Science (Grant 97L00805), by a grants-in-aid from the Ministry of Education, Science, Sports, Culture and Technology, Japan (no. 13670113) and for scientific research from Japan Society for the Promotion of Sciences (nos. 16790183, 17590241, 16570113), and from the Japan Diabetes Foundation.
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