Study on the influence of G82S RAGE polymorphism on RAGE-Amyloid interaction in AD pathology

Receptor for advanced glycation end products (RAGE) has been implicated in the pathophysiology of AD due to its ability to bind amyloid-beta and mediate inflammatory response. G82S RAGE polymorphism is associated with AD but the molecular mechanism for this association is not understood. Our previous in silico study indicated a higher binding affinity for mutated G82S RAGE, which could be caused due to changes in N linked glycosylation at residue N81. To confirm this hypothesis, in the present study molecular dynamics (MD) simulations were used to simulate the wild type (WT) and G82S glycosylated structures of RAGE to identify the global structural changes and to find the binding efficiency with Aβ42 peptide. Binding pocket analysis of the MD trajectory showed that cavity/binding pocket in mutant G82S glycosylated RAGE variants is more exposed and accessible to external ligands compared to WT RAGE, which can enhance the affinity of RAGE for Aβ. To validate the above concept, an in vitro binding study was carried using SHSY5Y cell line expressing recombinant WT and mutated RAGE variant individually to which HiLyte Fluor labeled Aβ42 was incubated at different concentrations. Saturated binding kinetics method was adopted to determine the Kd values for Aβ42 binding to RAGE. The Kd value for Aβ42-WT and Aβ42-mutant RAGE binding were 92±40 nM (95% CI-52 to 152nM; R2-0.92) and 45±20 nM (95% CI −29 to 64nM; R2-0.93), respectively. The Kd value of <100nM observed for both variants implicates RAGE as a high-affinity receptor for Aβ42 and mutant RAGE has higher affinity compared to WT. The alteration in binding affinity is responsible for activation of the inflammatory pathway as implicated by enhanced expression of TNFα and IL6 in mutant RAGE expressing cell line which gives a mechanistic view for the G82S RAGE association with AD.


54
Receptor for Advanced Glycation End-products (RAGE) belongs to the immunoglobulin 55 superfamily, which interacts with various ligands and plays an important role in several 56 pathological conditions [1]. Due to alternative splicing, various isoforms are generated such as Initiation of signal transduction upon the interaction of RAGE with its specific ligands helps 67 in physiological processes such as chemotaxis, angiogenesis, inflammation, apoptosis, and 68 proliferation [1,4]. The interaction of the same ligand with RAGE has different effects specific 69 to the cell physiology where the activation of NF-kB helps in the survival of some cells and 70 apoptosis of other cells [5]. As a multiligand receptor, fRAGE binds to the ligands like 71 advanced glycosylation end products (AGEs), s100/calgranulins, amyloid-beta (Aβ) and demonstrated that N25 carries complex N-glycans while N81 may be unmodified or partially 97 glycosylated with hybrid or high mannose glycans. G82S polymorphism also shown to affect 98 glycosylation patterns in RAGE which could alter binding affinity to its ligands [16]. It is 99 essential to understand the interaction of RAGE and Aβ, which would provide insight into its 100 role in AD pathology and also to understand the molecular mechanism for the association of 101 G82S RAGE polymorphism with AD.

102
The current study is designed to find RAGE-Aβ interaction scenario by comparing WT RAGE 103 and G82S mutant RAGE through in silico and in vitro studies and to get a clear mechanistic 104 view on the influence of glycosylation pattern on ligand binding affinity.

106
Structures 107 X-ray crystallographic structure of monomeric RAGE ectodomain (PDB ID human 3cjj) is 108 used in this study. G82S mutation was created and homology modeling of this RAGE variant 109 was done using SCWRL4 with 3cjj as the template. All molecular structures were generated 110 using Pymol. The glycans were virtually attached to the protein structure using the glycoprotein    The expected product size is 1215 base pairs. PCR was performed and the product was purified  confirm the inserted sequence changes and also to ensure that no other mutations were created.

169
The created G82S mutation was confirmed by restriction profiling of fRAGE gene PCR product The experiment was performed to analyze the specific binding of Aβ to WT and mutant RAGE. 218 We have used only Aβ42 since it is more pathogenic than other forms. To determine the optimal 219 concentration for Aβ treatment, recombinant SHSY5Y cells were incubated with varying concentrations of Aβ42. The data were analyzed using Image J software as described in 226 Priesnitz et al. [17].

Quantification of RAGE variants and inflammatory markers 228
To study the RAGE expression and inflammatory pathway activation due to RAGE-Aβ  Primers used for qPCR for quantification of total RAGE (fRAGE), sRAGE, TNFα, and IL6 241 were represented in  volumes were generated with an MD pocket using a grid spacing of 1 Å. It has been found that 283 the cavity/binding pocket in the polymorphic variant of glycosylated RAGE (G82S) is more 284 exposed /accessible to external ligands compared to WT RAGE which suggests that G82S 285 polymorphism enhances the ligand-binding affinity of RAGE (Fig 2D).

286
In the present modeling study, G82S RAGE glycosylated at N81 and N25 showed a more   The gradient PCR with gene specific primers was used for amplification of fRAGE gene. The

308
PCR product was electrophoresed in 1% agarose gel. Expected product size of 1215bp 309 corresponding to fRAGE was observed in annealing temperature of 69 -72°C (Fig 3A). The   438 The RAGE receptor which binds to a variety of proinflammatory ligands transmits the signal 439 from the ligand to NFκB regulated cytokines production. To confirm the inflammatory pathway 440 activation due to RAGE-Aβ interaction, the cells were exposed to Aβ42 and tested for In our study, we report that G82S polymorphism stabilizes RAGE glycosylation at Asn81 457 suggesting that the increase in flexibility of the V-domain caused by the global destabilization 458 effect of G82S mutation might be the cause for more exposed binding cavity in polymorphic 459 glycosylated RAGE which enhances the ligand-binding affinity of RAGE towards Aβ.

460
Our study suggests that expression of RAGE is increased at sites of Aβ42 accumulation and