Identification of mouse orthologue of endogenous secretory receptor for advanced glycation end-products: structure, function and expression

Abstract

The cell-surface RAGE [receptor for AGE (advanced glycation end-products)] is associated with the development of diabetic vascular complications, neurodegenerative disorders and inflammation. Recently, we isolated a human RAGE splice variant, which can work as a decoy receptor for RAGE ligands, and named it esRAGE (endogenous secretory RAGE). In the present study, we have isolated the murine equivalent of esRAGE from brain polysomal poly(A)+ (polyadenylated) RNA by RT (reverse transcription)-PCR cloning. The mRNA was generated by alternative splicing, and it encoded a 334-amino-acid protein with a signal sequence, but lacking the transmembrane domain. A transfection experiment revealed that the mRNA was actually translated as deduced to yield the secretory protein working as a decoy in AGE-induced NF-kappaB (nuclear factor kappaB) activation. RT-PCR and immunoblotting detected esRAGE mRNA and protein in the brain, lung, kidney and small intestine of wild-type mice, but not of RAGE-null mice. The esRAGE expression was increased in the kidney of diabetic wild-type mice. The present study has thus provided an animal orthologue of esRAGE for clarification of its roles in health and disease.

Figure 1. Isolation and structure of mouse esRAGE

(A) Schematic representation of the mouse RAGE gene and esRAGE mRNA. Open and shaded boxes indicate exons and introns respectively. Arrows indicate the positions of the primers used in RT–PCR: F1 and R1 in exons 1 and 11 respectively; F2 and R2 in exon 1–2 boundary and intron 9 respectively; F3 and R3 in exons 9 and 11 respectively. TM, transmembrane region. (B) Amplification of mouse esRAGE cDNA from brain polysomal poly(A)⁺ RNA. Sizes are indicated in bp. (C) Alignment of the deduced amino acid sequences of the mouse esRAGE, mouse full-length membrane-bound RAGE and human esRAGE. Amino acid residues are numbered starting from the first methionine residue. The putative signal sequence and transmembrane region are underlined and boxed respectively. Asterisks indicate identical amino acids. The arrowhead indicates a proteolytic site for generation of sRAGE protein [11].

Figure 2. Recombinant expression of RAGE cDNAs in COS-7 cells

Cell lysates (10 μg) and conditioned media (30 μl) of the transfected COS-7 cells were analysed by Western blotting as described in the Experimental section. es, esRAGE cDNA-expressing-vector-transfected cells; Full, full-length RAGE cDNA-transfected cells; sRAGE, mouse sRAGE (equivalent to the proteolytically cleaved form [11])-encoding cDNA-transfected cells. The mouse-esRAGE-specific antibody recognized only mouse esRAGE at 48 kDa. The human-esRAGE-specific antibody recognized only human esRAGE at 50 kDa. A polyclonal anti-(RAGE extracellular domain) antibody (Anti-RAGE) [8,9] recognized all of these human and mouse RAGE variant proteins. Molecular-mass sizes are indicated in kDa.

Figure 3. Binding of mouse esRAGE to AGE

(A) Estimation of a concentration of mouse esRAGE secreted into the culture medium by Western blotting using a polyclonal anti-(RAGE extracellular domain) antibody. Purified recombinant mouse sRAGE was used as a standard. The conditioned medium (0.4 μl) was equivalent to approx. 4.8 ng of sRAGE. (B) SPR assay was performed as described in the Experimental section. Non-glycated BSA (BSA) or glyceraldehyde-derived AGE–BSA (AGE) (150 μg/ml) [8] was passed over a CM5 sensorchip of BIAcore 2000, on which secreted mouse esRAGE protein had been immobilized. On a control chip, equivalent volumes of control non-transfected COS-7 cell media were immobilized. Immobilization of mouse esRAGE on a sensorchip was confirmed by a positive response when 10 μg/ml anti-(mouse esRAGE) antibody or an anti-(RAGE extracellular domain) antibody was injected. Positive response was detected on injection of AGE, but not of control BSA. The white arrow indicates the injection point. At 15 s after the injection (white arrowhead), the mobile phase was changed back to the buffer alone.

Figure 4. Inhibitory effect of mouse esRAGE on AGE-induced NF-κB activation

C6 glioma cells were stably transfected with an NF-κB-responsive cis-reporter gene construct together with a full-length RAGE expression vector as described in the Experimental section. The cells were stimulated by no addition (control), or with 50 μg/ml BSA (BSA), 50 μg/ml glyceraldehyde-derived AGE–BSA alone (AGE), 50 μg/ml AGE–BSA plus additives: 1 or 2 μl of concentrated conditioned media from mock-transfected cells (Mock 2 μl) or mouse esRAGE cDNA-transfected cells (Mouse esRAGE 1 μl and 2 μl, corresponding to ∼0.9 and ∼1.8 μg of esRAGE proteins respectively), or 20 μg of sRAGE (sRAGE) (kindly provided by Mitsubishi Pharma Corporation). Relative luciferase activity is shown. Results are means±S.E.M. (n=3). *P<0.04, **P<0.02; statistical analysis was performed by ANOVA combined with a multiple comparison test (Scheffe's type).

Figure 5. Detection of esRAGE mRNA and protein in mouse tissues

(A) Total RNA (100 ng) from brain, lung, kidney and small intestine of wild-type (+/+) or RAGE-null (−/−) mice (C57BL/6J) at 16 weeks of age was analysed by RT–PCR as described in the Experimental section. (B) Determination of relative abundance of esRAGE and full-length RAGE mRNAs. Polysomal poly(A)⁺ RNAs were analysed with primers common to both esRAGE and full-length RAGE mRNA as described in the Experimental section. All bands were sequence-verified: the 161 bp band (open arrowhead) represents the full-length RAGE mRNA, while the 278 and 364 bp bands (black arrows) represent the esRAGE mRNA. (C) Immunoblotting for esRAGE protein in C57BL/6J mouse tissues. Proteins were extracted from the brain, lung, kidney and small intestine of wild-type (RAGE+/+) or RAGE-null (RAGE−/−) mice at 16 weeks of age, and were analysed by Western blotting using a polyclonal anti-(mouse esRAGE) antibody. The amounts of protein loaded for the Western blotting were: brain, 8 μg; lung, 15 μg; kidney, 10 μg; small intestine, 5 μg. Molecular-mass sizes are indicated in kDa.