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  • Title: Asn-linked N-acetylglucosamine of the amylin receptor 2 extracellular domain enhances peptide ligand affinity.
    Author: Lee S.
    Journal: FEBS Open Bio; 2021 Jan; 11(1):195-206. PubMed ID: 33227824.
    Abstract:
    The calcitonin receptor (CTR) has a large extracellular domain (ECD) with multiple N-glycosylation sites. An asparagine (Asn)-linked N-acetylglucosamine (GlcNAc) of CTR ECD N130 was previously reported to enhance peptide hormone binding affinity for CTR ECD. CTR forms a complex with an accessory protein RAMP, and the RAMP:CTR complex gains affinity for peptide hormone amylin as the amylin receptor (AMY). Although N-glycosylation of AMY ECD was reported to enhance peptide hormone affinity, it remains underexplored which N-glycosites of AMY ECD are responsible for peptide affinity enhancement and it is unclear whether an Asn-linked GlcNAc of the N-glycosites plays a critical role. Here, I investigated the role of the Asn-linked GlcNAc of CTR N130 in the affinity of an antagonistic amylin analog (AC413) for AMY2 ECD (the RAMP2 ECD:CTR ECD complex). I used Endo H-treated CTR ECD in which N-glycans were trimmed to an Asn-linked GlcNAc on each of the N-glycosites. I incubated Endo H-treated CTR ECD with excess of glycan-free RAMP2 ECD to produce the RAMP2 ECD:CTR ECD complex. Using this coincubation system, I found that the RAMP2 ECD complex with Endo H-treated CTR ECD with N130D mutation showed a fourfold decrease in AC413 affinity compared with the RAMP2 ECD complex with Endo H-treated CTR ECD WT. In contrast, RAMP2 ECD N-glycosylation did not affect peptide binding affinity. These results indicate that the Asn-linked GlcNAc of CTR N130 is an important peptide affinity enhancer for AMY2 ECD and reveals a significant role of the Asn-linked GlcNAc in AMY2 function.
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