Lee Sang-Min, Booe Jason M, Gingell Joseph J, Sjoelund Virginie, Hay Debbie L, Pioszak Augen A
Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center , 975 NE 10th Street BRC 462B, Oklahoma City, Oklahoma 73104, United States.
Proteomics Division of the Laboratory for Molecular Biology and Cytometry Research, University of Oklahoma Health Sciences Center , 975 NE 10th Street, Oklahoma City, Oklahoma 73104, United States.
Biochemistry. 2017 Jul 5;56(26):3380-3393. doi: 10.1021/acs.biochem.7b00256. Epub 2017 Jun 26.
The calcitonin receptor (CTR) is a class B G protein-coupled receptor that is activated by the peptide hormones calcitonin and amylin. Calcitonin regulates bone remodeling through CTR, whereas amylin regulates blood glucose and food intake by activating CTR in complex with receptor activity-modifying proteins (RAMPs). These receptors are targeted clinically for the treatment of osteoporosis and diabetes. Here, we define the role of CTR N-glycosylation in hormone binding using purified calcitonin and amylin receptor extracellular domain (ECD) glycoforms and fluorescence polarization/anisotropy and isothermal titration calorimetry peptide-binding assays. N-Glycan-free CTR ECD produced in Escherichia coli exhibited ∼10-fold lower peptide affinity than CTR ECD produced in HEK293T cells, which yield complex N-glycans, or in HEK293S GnTI cells, which yield core N-glycans (ManGlcNAc). PNGase F-catalyzed removal of N-glycans at N73, N125, and N130 in the CTR ECD decreased peptide affinity ∼10-fold, whereas Endo H-catalyzed trimming of the N-glycans to single GlcNAc residues had no effect on peptide binding. Similar results were observed for an amylin receptor RAMP2-CTR ECD complex. Characterization of peptide-binding affinities of purified N → Q CTR ECD glycan site mutants combined with PNGase F and Endo H treatment strategies and mass spectrometry to define the glycan species indicated that a single GlcNAc residue at CTR N130 was responsible for the peptide affinity enhancement. Molecular modeling suggested that this GlcNAc functions through an allosteric mechanism rather than by directly contacting the peptide. These results reveal an important role for N-linked glycosylation in the peptide hormone binding of a clinically relevant class B GPCR.
降钙素受体（CTR）是一种B类G蛋白偶联受体，可被肽类激素降钙素和胰淀素激活。降钙素通过CTR调节骨重塑，而胰淀素则通过与受体活性修饰蛋白（RAMP）形成复合物激活CTR来调节血糖和食物摄入。这些受体是临床上治疗骨质疏松症和糖尿病的靶点。在这里，我们使用纯化的降钙素和胰淀素受体细胞外结构域（ECD）糖型以及荧光偏振/各向异性和等温滴定量热法肽结合试验，确定了CTR N-糖基化在激素结合中的作用。在大肠杆菌中产生的无N-聚糖CTR ECD与在产生复杂N-聚糖的HEK293T细胞或产生核心N-聚糖（ManGlcNAc）的HEK293S GnTI细胞中产生的CTR ECD相比，肽亲和力低约10倍。PNGase F催化去除CTR ECD中N73、N125和N130处的N-聚糖会使肽亲和力降低约10倍，而内切糖苷酶H催化将N-聚糖修剪为单个GlcNAc残基对肽结合没有影响。对于胰淀素受体RAMP2-CTR ECD复合物也观察到了类似的结果。结合PNGase F和内切糖苷酶H处理策略以及质谱对纯化的N→Q CTR ECD聚糖位点突变体的肽结合亲和力进行表征，以确定聚糖种类，结果表明CTR N130处的单个GlcNAc残基负责肽亲和力的增强。分子模型表明，这种GlcNAc通过变构机制起作用，而不是直接与肽接触。这些结果揭示了N-连接糖基化在临床相关B类GPCR的肽激素结合中的重要作用。
