N-Glycosylation of Asparagine 130 in the Extracellular Domain of the Human Calcitonin Receptor Significantly Increases Peptide Hormone Affinity.
N-Glycosylation of Asparagine 130 in the Extracellular Domain of the Human Calcitonin Receptor Significantly Increases Peptide Hormone Affinity.
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DOI：
10.1021/acs.biochem.7b00256
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发表时间：
2017-07-05
期刊：
影响因子：
2.9
通讯作者：
Pioszak AA
中科院分区：
文献类型：
--
作者：
Lee SM;Booe JM;Gingell JJ;Sjoelund V;Hay DL;Pioszak AA
降钙素受体（CTR）是一种B类G蛋白偶联受体，可被肽激素降钙素和amylin激活。降钙素通过CTR调节骨重塑，而amylin通过激活CTR与受体活性修饰蛋白（RAMPs）复合物来调节血糖和食物摄入。这些受体是临床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胞外结构域（ECD）糖型、荧光极化/各向异性和等温滴定量热法肽结合测定来定义CTR n -糖基化在激素结合中的作用。大肠杆菌产生的无N-聚糖CTR ECD的肽亲和力比HEK293T细胞产生的CTR ECD的肽亲和力低10倍，HEK293S GnTI−细胞产生的CTR ECD产生复合N-聚糖（Man5GlcNAc2）。PNGase F催化去除CTR ECD中N73、N125和N130处的n -聚糖，使肽亲和力降低约10倍，而Endo h催化将n -聚糖修剪为单个GlcNAc残基，对肽结合没有影响。在胰淀素受体RAMP2-CTR ECD复合体中也观察到类似的结果。纯化的N→Q CTR ECD聚糖位点突变体结合PNGase F和Endo H处理策略对肽结合亲和力进行了表征，并用质谱法确定了聚糖种类，结果表明CTR N130上的单个GlcNAc残基是肽亲和力增强的原因。分子模拟表明，该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 -链糖基化在临床相关的B类GPCR的肽激素结合中的重要作用。
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 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 (Man5GlcNAc2). 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.
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影响因子：
2.9
作者：
Bisello, A;Greenberg, Z;Chorev, M
通讯作者：
Chorev, M
影响因子：
4.8
作者：
Lee, Sang-Min;Hay, Debbie L.;Pioszak, Augen A.
通讯作者：
Pioszak, Augen A.
影响因子：
4.1
作者：
MOSELEY, JM;FINDLAY, DM;MARTIN, TJ
通讯作者：
MARTIN, TJ
DOI：
10.1016/s1056-8719(00)00074-5
发表时间：
1999-12-01
影响因子：
1.9
作者：
Armour, SL;Foord, S;Chen, WJ
通讯作者：
Chen, WJ
DOI：
10.1107/s0907444906029799
发表时间：
2006-10-01
影响因子：
2.2
作者：
Aricescu, A. Radu;Lu, Weixian;Jones, E. Yvonne
通讯作者：
Jones, E. Yvonne