Disulfide Bonds within the C2 Domain of RAGE Play Key Roles in Its Dimerization and Biogenesis

<div><h3>Background</h3><p>The receptor for advanced glycation end products (RAGE) on the cell surface transmits inflammatory signals. A member of the immunoglobulin superfamily, RAGE possesses the V, C1, and C2 ectodomains that collectively constitute the receptor's extracellular structure. However, the molecular mechanism of RAGE biogenesis remains unclear, impeding efforts to control RAGE signaling through cellular regulation.</p> <h3>Methodology and Result</h3><p>We used co-immunoprecipitation and crossing-linking to study RAGE oligomerization and found that RAGE forms dimer-based oligomers. Via non-reducing SDS-polyacrylamide gel electrophoresis and mutagenesis, we found that cysteines 259 and 301 within the C2 domain form intermolecular disulfide bonds. Using a modified tripartite split GFP complementation strategy and confocal microscopy, we also found that RAGE dimerization occurs in the endoplasmic reticulum (ER), and that RAGE mutant molecules without the double disulfide bridges are unstable, and are subjected to the ER-associated degradation.</p> <h3>Conclusion</h3><p>Disulfide bond-mediated RAGE dimerization in the ER is the critical step of RAGE biogenesis. Without formation of intermolecular disulfide bonds in the C2 region, RAGE fails to reach cell surface.</p> <h3>Significance</h3><p>This is the first report of RAGE intermolecular disulfide bond.</p> </div

Design of tripartite split GFP complementation to study RAGE dimerization in the ER.

<p>(<b>A</b>) Illustration of general tripartite split GFP complementation strategy. GFP s10 and s11 are used to tag test proteins whereas GFPs1-9 functions as a detector. When tagged test proteins interact with each other to bring s10 and s11 sufficiently close that they interact with s1-9 to generate green fluorescence. (<b>B</b>) Illustration of tripartite split GFP complementation to detect RAGE dimerization in the ER. GFPs1-9 is targeted to the ER with RAGE signal peptide (black bar). Upon entering the ER, the signal peptide is cleaved and GFPs1-9 is glycosylated (magenta chain), and complementation occurs only when s10 and s11-tagged RAGE molecules dimerise. Double disulfide bridge-linked RAGE dimers then leave the ER-Golgi for the cell surface. (<b>C</b>) Targeting GFPs1-9 to the ER. Glycosylation of GFPs1-9 confirms that GFPs1-9 is localized in the ER.</p

Cell surface expression of RAGE and RAGE cysteine-to-serine mutants.

<p>FLAG-tagged RAGE and RAGE mutants were transfected to CHO-CD14 cells. After overnight incubation, the transfected cells (10⁶) were stained with anti-FLAG antibodies and subjected to flow cytometry analyses. Non-transfected cells with same staining were used as negative controls. All values were expressed as mean ± SEM, and the data were from independent transfections (<i>n</i> = 3). The <i>p</i> value for presented data is <0.01 (ANOVA).</p

Unstable RAGE dimers are subjected to the ERAD pathway.

<p>Ubiquitination assays of RAGE (WT) and RAGE cysteine-to-serine mutants. CHO-CD14 cells were co-transfected with HA-ubiquitin and FLAG-RAGE/RAGE mutants. The transfected cells were then lysed and unfractionated membrane extracts were prepared. The lysates were IPed with anti-FLAG antibodies, and IBed with anti-HA (HRP conjugates) antibodies to demonstrate ubiquitination of RAGE mutants. Anti-FLAG (HRP conjugates) antibodies IB showed the expression of RAGE and RAGE mutants.</p

RAGE(C259S/C301S) is unstable and deglycosylated.

<p>(<b>A</b>) Cyclohaximide chase and IB to compare the protein decay of RAGE(WT) and RAGE(C259S/C301S) in the cells. For FLAG-RAGE transfected cells, 5 µg of lysates were used; whereas for FLAG-RAGE(C259S/C301S) transfceted cells, 10 µg of lysates were used due to the lower expression. After IB with anti-FLAG antibodies, the blot was striped, and reprobed with anti-β-actin antibodies as a loading control. CHX: cyclohaximide. (<b>B</b>) Intracellular decay rate of RAGE and RAGE(C259S/C301S) calculated from two CHX chase experiments. The blot intensity was measured with a Kodak Gel Logic 2200 Imaging System and processed with molecular imaging software. The starting point was used as 100% and blot intensity from each time point was calculated relative to the 0 time point. The intensity value of each point was expressed as mean ± SEM, and d_1/2 was calculated when 50% of the protein I decayed. <i>C</i>, RAGE cysteine-to-serine mutants are deglycosylated. Cell lysates from FLAG-tagged RAGE and RAGE cysteine-to-serine mutants were treated with PNGase F, as described, and resolved on a SDS 4–12% NuPAGE gel.</p

RAGE from mouse lung also exhibits disulfide-bond mediated dimeric structure.

<p>The lungs were isolated from both wild-type and RAGE(KO) mice and crude membrane fraction was prepared. The extracted membrane protein lysates (15 µg) were then resolved on SDS-PAGE (4–12% gradient gel) under reducing and non-reducing conditions followed with immunoblotting by anti-RAGE antibodies. ns, major non-specific protein species. Monomeric and dimeric forms are marked.</p

Intermolecular disulfide bonds contribute to the formation of RAGE dimers.

<p>(<b>A</b>) Schematic drawing of RAGE domains. aa: amino acids; * indicates C₂₅₉ and C₃₀₁; TM: transmembrane helix. (<b>B</b>) Identification of RAGE domain that is responsible for covalent-linked dimerization. About 5 µg of total membrane extracts was loaded. * indicate dimers of RAGE (WT) and deletion mutants. (<b>C</b>) RAGe C2 domain exhibits disulfide bond-mediated dimerization. * indicate dimers of RAGE(WT) and RAGE(C2). (<b>D</b>) Testing whether C₂₅₉ and C₃₀₁ are responsible for covalent-linked dimerization of RAGE. About 7.5 µg of total membrane extracts was loaded.</p

Intracellular localization of RAGE and RAGE cysteine-to-serine mutants.

<p>FLAG-tagged RAGE and mutants were transfected to HeLa cells and intracellular immunocytochemistry was performed. Scale bars: 50 µM for all images. (<b>A</b>) Localization of RAGE (WT). Blue: DAPI (stain nucleus); green: anti-calnexin (as the ER marker); red: anti-FLAG. Co-localization is demonstrated by the yellow color of the merged image. (<b>B</b>) Co-localization of RAGE (C259S/C301S) with the ER marker calnexin. (<b>C</b>) Co-localization of RAGE (C259S) with calnexin. (<b>D</b>) Co-localization of RAGE (C301S) with calnexin.</p