Enhanced oligomerization of full-length RAGE by synergy of the interaction of its domains

Abstract

The pattern recognition receptor RAGE (receptor for advanced glycation end-products) transmits proinflammatory signals in several inflammation-related pathological states, including vascular diseases, cancer, neurodegeneration and diabetes. Its oligomerization is believed to be important in signal transduction, but RAGE oligomeric structures and stoichiometries remain unclear. Different oligomerization modes have been proposed in studies involving different truncated versions of the extracellular parts of RAGE. Here, we provide basic characterization of the oligomerization patterns of full-length RAGE (including the transmembrane (TM) and cytosolic regions) and compare the results with oligomerization modes of its four truncated fragments. For this purpose, we used native mass spectrometry, analytical ultracentrifugation, and size-exclusion chromatography coupled with multi-angle light scattering. Our results confirm known oligomerization tendencies of separate domains and highlight the enhanced oligomerization properties of full-length RAGE. Mutational analyses within the GxxxG motif of the TM region show sensitivity of oligomeric distributions to the TM sequence. Using hydrogen–deuterium exchange, we mapped regions involved in TM-dependent RAGE oligomerization. Our data provide experimental evidence for the major role of the C2 and TM domains in oligomerization, underscoring synergy among different oligomerization contact regions along the RAGE sequence. These results also explain the variability of obtained oligomerization modes in RAGE fragments.

A model of full-length RAGE in complex with S100B

“…A further 14 RAGE-RAGE intermolecular links in FL_RAGE oligomers and 13 in FL_RAGE-S100B complexes were observed among the following domains: V-V, V-C1, C1-C1, and CT-CT (Table S1; Figure 2B). The presence of multiple interaction contact surfaces demonstrates the participation of all domains in RAGE oligomerization, which is in agreement with previous results (Moysa et al, 2019). Also, it renders a rather complex picture of the oligomerization process.…”

“…Therefore, we purified and solubilized FL_RAGE in Triton X-100 detergent micelles at two times the critical micelle concentration for native MS analysis. The oligomeric species identified are identical with what has been observed previously (Moysa et al, 2019), ranging from monomeric up to tetrameric complexes (Figure 1D). The different oligomeric forms display a narrow charge state distribution, which indicates the native character of these complexes, suggesting that no substantial unfolding and/or dissociation occurred during the MS experiment.…”

“…The cryo-EM approach is more promising here, but despite spectacular improvement in the determination of three-dimensional (3D) structures of proteins, membrane proteins still represent a significant challenge for this technique (Cheng, 2018). In our previous paper, we showed that alternative methods of structure analysis may bring insight into the properties of FL-RAGE and its oligomeric distributions (Moysa et al, 2019). Therefore, in this work, we extended this study to analyze the structural properties of FL_RAGE-S100B complexes using a combination of mass spectrometry (MS)-based methods with molecular modeling.…”

An in Silico Approach Reveals the Potential Function of Cyanidin-3-o-glucoside of Red Rice in Inhibiting the Advanced Glycation End Products (AGES)-Receptor (RAGE) Signaling Pathway

“…RAGE is a transmembrane protein, which consists of three different extracellular domains (a V-type domain (residue 23-116), C1 domain (residue 124-221), and C2 domain (residue 227-317)), a transmembrane helix (residue 343-363), and a cytoplasmic tail (364-404) ( 18 ) including vascular diseases, cancer, neurodegeneration and diabetes. Its oligomerization is believed to be important in signal transduction, but RAGE oligomeric structures and stoichiometries remain unclear.…”

RAGE Signaling in Melanoma Tumors

“…In the C2 domain, a proline-rich region assembles into an external loop that may be critical for the formation of oligomers, as suggested in a hexameric model of RAGE [108]. Apart from oligomerization mediated by these domains, a recent study has correlated the oligomerization of the transmembrane domain to the presence of GxxxG motifs of these domains [110]. In general, the formation of dimers and higher order oligomers, such as tetramers and hexamers, are facilitated by electrostatic and hydrophobic interactions between multiple RAGE domain surfaces [104,108,111].…”