Comparison of distinct protein isoforms of the receptor for advanced glycation end-products expressed in murine tissues and cell lines.
The receptor for advanced glycation end-products (RAGE) is thought to be expressed ubiquitously as various protein isoforms. Our objective was to use Northern blotting, immunoblotting, and sensitivity to N-glycanase digestion to survey RAGE isoforms expressed in cell lines and mouse tissues in order to obtain a more comprehensive view of the RAGE expressome. Pulmonary RAGE mRNA (1.4 kb) was smaller than cell-line and tissue RAGE mRNA (6 kb-10 kb). Three anti-RAGE antibodies that recognized three distinct RAGE epitopes were used for protein studies (N-16, H-300, and alphaES). Lung expressed three predominant protein isoforms with apparent molecular masses of 45.1, 52.6, and 57.4 kDa (N-16/H-300) and four isoforms at 25.0, 46.9, 52.5, and 54.2 kDa (alphaES). These isoforms were expressed exclusively in lung. Heart, ileum, and kidney expressed a 44.0-kDa isoform (N-16), whereas aorta and pancreas expressed a 53.3-kDa isoform (alphaES). Each of these isoforms were absent in tissue extracts prepared from RAGE(-/-) mice. Cell lines expressed a 70.0-kDa isoform, and a subset expressed a 30.0-kDa isoform (alphaES). Lung RAGE appeared to contain two N-linked glycans. Tissue and cell-line RAGE isoforms were completely insensitive to PNGase F digestion. Thus, numerous RAGE protein isoforms are detectable in tissues and cell lines. Canonical transmembrane and soluble RAGE appear to be expressed solely in lung (N-16/H-300). Non-pulmonary tissues and cell lines, regardless of the source tissue, both express distinct RAGE protein isoforms containing the N-terminal N-16 epitope or the alphaES RAGE epitope encoded by alternate exon 9, but lacking the H-300 epitope.
Department of Critical Care Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.
Julia V
JV
Gefter
Angel L
AL
Shaufl
Mitchell P
MP
Fink
Russell L
RL
Delude
D000818:Animals; D001011:Aorta / Q000378:metabolism; D002460:Cell Line; D000939:Epitopes / Q000276:immunology; D006031:Glycosylation; D006801:Humans; D007082:Ileum / Q000378:metabolism; D007668:Kidney / Q000378:metabolism; D008168:Lung / Q000378:metabolism; D008297:Male; D051379:Mice; D009206:Myocardium / Q000378:metabolism; D010179:Pancreas / Q000378:metabolism; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D020033:Protein Isoforms / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D051381:Rats; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*
D000939:Epitopes; D020033:Protein Isoforms; D012333:RNA, Messenger; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase