Figure 1.
Phylogenetic analysis of the Agpat1–Gpsm3 locus including Ager.
(A) The panel represents the structures of the Agpat1-Rnf5-Ager-Pbx2-Gpsm3 genes. The locus spans more than 1 Mbp in human chr 6. Ager (receptor for advanced glycation endproducts); Agpa1 (1-acylglycerol-3-phosphate O-acyltransferase 1); Gpsm3 (G-protein signaling modulator 3); Pbx2 (pre-B-cell leukemia homeobox 2); Rnf5 (ring finger protein 5). (B) A schematic phylogenetic tree with the main nodes. The presence of a gene is depicted by a green box, while the absence of the box indicates that the gene is not found in the specific phylogenetic group.
Figure 2.
Comparison of the RAGE, ALCAM, BCAM and MCAM protein sequences.
The multiple alignment was performed with CLUSTALW. Exons encoding a portion of the corresponding protein are highlighted alternatively in yellow and grey. Asterisks indicate conserved exon-intron boundaries; if conservation is among all four proteins, the asterisks are red, if conservation is between RAGE and at least one of the others the colour is blue. RAGE (receptor for advanced glycation endproducts); ALCAM (activated leukocyte cell adhesion molecule); BCAM (basal cell adhesion molecule); MCAM (melanoma cell adhesion molecule).
Figure 3.
Phylogenetic tree derived from 3D structural alignments.
The phylogenetic tree is based on the analysis of the closest 500 matches resulting from a search of the protein structure database using the structure of RAGE Ig domains V-C1 as a query. After filtering the results for a minimum length of alignment and removal of duplicates 27 different proteins were retrieved. Twenty four out of these 27 are cell adhesion molecules, only 2 belong to the T-cell receptor family and one protein represents the light chain of an antibody. RAGE V-C1 Ig domains group very closely with cell adhesion molecules BCAM and CD80. OCAM (olfactory cell adhesion molecule; pdb code 2jll); CEACAM-1 (Carcinoembryonic antigen-related cell adhesion molecule 1, CD 66a; pdb code 3R4D); PD-L1 (programmed death1 inhibitory receptor; pdb code 3BIS); CD80 (cluster of differentiation 80; pdb code 1DR9); RAGE (receptor for advanced glycation endproducts; pdb code 3CJJ); BCAM (Lutheran glycoprotein; basal cell adhesion molecule; pdb code 2PET); NECTIN-1 (pdb code 4FMF); NECTIN-2 (pdb code 4FMK); NECTIN-4 (pdb code 4FRW); CD155 (cluster of differentiation 155, poliovirus receptor; pdb code 3URO); CD2 (cluster of differentiation 2; pdb code 1HNG); MADCAM-1 (mucosal vascular addressin cell adhesion molecule 1; pdb code 1GSM); VCAM (vascular cell adhesion molecule; pdb code 1VSC); ICAM-1 (intercellular adhesion protein 1, pdb code 1IC1); ICAM-2 (intercellular adhesion protein 2; pdb code 1ZXQ); TCR-alpha (T-cell receptor a chain; pdb code 1NFD); TCR-gamma (T-cell receptor g chain; pdb code 1HXM); Fab-LC (antibody Fab fragment light chain; pdb code 3QNX); Neuroplastin (pdb code 2WV3); MUSK (pdb code 2IEP); NCAM-Ig-1–2 (neural cell adhesion molecule Ig domains 1 and 2; pdb code 1EPF); NCAM-Ig-2–3 (neural cell adhesion molecule Ig domains 1 and 2; pdb code 1QZ1); DSCAM (Down syndrom cell adhesion molecule; pdb code 3DMK); Hemolin (pdb code 1BIH); ROBO (Roundabout; pdb code 2VRA); ROBO-1 (Roundabout homolog 1; pdb code 2V9R); Contactin (protein tyrosine phosphatase z (PTPRZ); pdb code 3JXA); TAG-1 (axonin, pdb code 1CS6).
Figure 4.
Comparison of RAGE V-C1 structure with homophilic cell adhesion molecules BCAM, ALCAM and MCAM.
(A) The Ig domains V and C1 of RAGE which reside most distal from the cytoplasmic membrane (shown in blue) adopt a slightly bent structure. This spatial arrangement is very well conserved in the Ig domains 1 and 2 of its close homologue BCAM (red). Structural models of the corresponding Ig domains of ALCAM (green) and MCAM (magenta) suggest that these adopt as well a very similar structure that might be required for homophilic interaction. RAGE (receptor for advanced glycation endproducts); ALCAM (activated leukocyte cell adhesion molecule); BCAM (basal cell adhesion molecule); MCAM (melanoma cell adhesion molecule). (B) Model for RAGE-RAGE homophilic interaction mediating cell adhesion (derived from pdb code 4LP5) [44]. Left hand side: as observed in the crystal structure of sRAGE the extracellular domain adopts an extended conformation; alongside a cartoon representation is shown. Right hand side: in the crystal the V-domains (dark blue) form a large contact in trans orientation. This mode of interaction is conserved among different crystal structures suggesting that RAGE homophlic interaction occurs via the V-domain.
Figure 5.
RAGE expression enhances cell-matrix adhesion.
(A) Western blot analysis on rat lung lysate and R3/1 cells using three different antibodies recognizing RAGE extracellular (anti-RAGE N-term1 and anti-RAGE N-term2) or intracellular (anti-RAGE C-term) domains. Asterisk (*) indicates nonspecific bands. Forty µg of cell lysate were loaded and detection of GAPDH was used as loading control. (B) Western blot analysis on R3/1-pLXSN and R3/1-FL-RAGE cells using anti-RAGE N-term1 antibody. Forty µg of cell lysate were loaded and detection of GAPDH was used as loading control. (C, D) Cell-matrix adhesion assay. Adhesion of R3/1-pLXSN or R3/1-FL-RAGE cells onto culture dishes coated with 10 µg/ml ECM proteins. Adhesion to PBS, collagen I (Coll I), Fibronectin (FN), or Laminin (Lam) was assayed for 15 minutes (C) or 45 minutes (D). One representative experiment out of three is shown. Results from triplicate wells are displayed as means±SEM (ns, not significant; *, P<0.05; **, P<0.01).
Figure 6.
RAGE expression enhances cell spreading.
Cell spreading assay. (A) Spreading of R3/1-pLXSN or R3/1-FL-RAGE cells onto culture dishes coated with 10 µg/ml of ECM proteins (Coll I, FN, or Lam) or PBS was assessed at 90 minutes after seeding. Photographs were taken in phase contrast at 40× magnification. Bar corresponds to 20 µm. (B) Quantification of cell spreading based on cell surface area. Results are displayed as means±SEM (ns, not significant; *, P<0.05; **, P<0.01; ***, P<0.001).
Figure 7.
RAGE expression contributes to cell-cell adhesion.
(A) Western blot analysis on control HEK cells (HEK/pcDNA) or expressing FL-RAGE (HEK/FL-RAGE) using anti-RAGE N-term1 antibody. Forty µg of cell lysate were loaded and detection of GAPDH was used as loading control. (B) A dissociation assay was performed by applying mechanical force on monolayers of HEK/pcDNA or HEK/FL-RAGE cells. Photographs were taken at 40× magnification in phase contrast. Bar corresponds to 50 µm. The dissociation index is the means±SEM of three independent experiments (**, P<0.01). (C) Localization of FL-RAGE on HEK/pcDNA, HEK/FL-RAGE or a mix of both cell lines (orange). Nuclei are stained in blue. Red arrows indicate HEK/pcDNA cells in contact with HEK/FL-RAGE. Green arrows indicate HEK/FL-RAGE cells in contact with each other. Bar corresponds to 10 µm. (D) Localization of FL-RAGE on R3/1-pLXSN, R3/1-FL-RAGE or a mix of both cell lines (orange). Nuclei are stained in blue. Red arrows indicate R3/1-pLXSN cells in contact with R3/1-FL-RAGE. Green arrows indicates R3/1-FL-RAGE cells in contact with each other. Red arrows indicate cells not expressing RAGE in contact with cells expressing RAGE. Bar corresponds to 10 µm.
Figure 8.
Surface plasmon resonance analysis of homophilic interaction between soluble sRAGE and sRAGE immobilized on a Ni-NTA sensor chip. Soluble sRAGE was injected over the sensor chip at a flow rate of 10 µl/min. The arrows indicate the start and end of injections.
Figure 9.
RAGE expression mediates cell aggregation through homophilic interactions.
(A) Western blot analysis on control preB 300.19 cells (preB/pCAGS) or expressing FL-RAGE (preB/FL-RAGE) using αRAGE N-term1 antibody. Forty µg of cell lysate were loaded and detection of GAPDH was used as loading control. (B) Cell aggregation assay. preB/pCAGS or preB/FL-RAGE cells were cultured for 2, 6 or 24 hours before being photographed at 20× magnification in phase contrast (left panel). Bar corresponds to 50 µm. Quantification of cell aggregates area (right panel). Results are displayed as means±SEM (*, P<0.05; **, P<0.01; ***, P<0.0001). (C) Mixed cell aggregation assay. Red preB (Red-preB) or preB/FL-RAGE (expressing GFP; preB/FL-RAGE-GFP) cells were grown as single cell line suspension or mixed in equal number for 24 hours. Red-preB/FL-RAGE-GFP were mixed with preB/FL-RAGE-GFP for the same time. Bar corresponds to 50 µm.