Glycosylation sites in the atrial natriuretic peptide receptor: oligosaccharide structures are not required for hormone binding.
Glycosylation sites in the atrial natriuretic peptide receptor: oligosaccharide structures are not required for hormone binding.
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心房钠尿肽受体中的糖基化位点：激素结合不需要寡糖结构。
DOI：
10.1046/j.1432-1327.2000.01647.x
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发表时间：
2000
期刊：
影响因子：
--
通讯作者：
Misono,KS
中科院分区：
文献类型：
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作者：
Miyagi,M;Zhang,X;Misono,KS
心钠素(ANP)是一种通过利钠和扩血管作用参与心血管稳态的激素。介导这些作用的ANP受体是一种糖基化的跨膜蛋白，与鸟苷环化酶偶联。糖基化在受体信号转导中的作用仍未解决。在本研究中，我们通过高效液相-质谱仪和Edman测序相结合的方法，确定了在COS-1细胞中表达的大鼠心钠素受体(NPR-ECD)胞外区的糖基化位点。对NPR-ECD的胰酶消化液进行高效液相/质谱仪分析，确定了5个糖基化的多肽片段，然后通过Edman降解对这些片段进行测序以确定糖基化位点。数据显示，在六个潜在的位点中，有五个是ASN连接的糖基化。根据观察到的糖基化多肽的质量推断出每个位点上的寡糖结构类型如下：Asn13(高甘露糖)、Asn180(复合体)、Asn306(复合体)、Asn347(复合体)和Asn395(高甘露糖和杂交型)。Asn180和Asn347的糖基化是部分的。通过NPR-ECD的酶促脱糖反应和结合实验，研究了糖基在ANP结合中的作用。用内切糖苷酶 F2和 H脱糖后的NPR-ECD仍具有心钠素结合活性，与未处理的NPR-ECD具有相似的亲和力。内切酶处理COS-1细胞表达的全长ANP受体也没有检测到ANP结合的影响。这些结果表明，尽管新合成的ANP受体的折叠和转运可能需要糖基化，但寡糖部分本身并不参与激素结合。
Atrial natriuretic peptide (ANP) is a hormone involved in cardiovascular homeostasis through its natriuretic and vasodilator actions. The ANP receptor that mediates these actions is a glycosylated transmembrane protein coupled to guanylate cyclase. The role of glycosylation in receptor signaling remains unresolved. In this study, we determined, by a combination of HPLC/MS and Edman sequencing, the glycosylation sites in the extracellular domain of ANP receptor (NPR‐ECD) from rat expressed in COS‐1 cells. HPLC/MS analysis of a tryptic digest of NPR‐ECD identified five glycosylated peptide fragments, which were then sequenced by Edman degradation to determine the glycosylation sites. The data revealed Asn‐linked glycosylation at five of six potential sites. The type of oligosaccharide structure attached at each site was deduced from the observed masses of the glycosylated peptides as follows: Asn13 (high‐mannose), Asn180 (complex), Asn306 (complex), Asn347 (complex), and Asn395 (high‐mannose and hybrid types). Glycosylation at Asn180 and Asn347 was partial. The role of glycosyl moieties in ANP binding was examined by enzymatic deglycosylation of NPR‐ECD followed by binding assay. NPR‐ECD deglycosylated with endoglycosidase F2and endoglycosidase H retained ANP‐binding activity and showed an affinity for ANP similar to that of untreated NPR‐ECD. Endoglycosidase treatment of the full‐length ANP receptor expressed in COS‐1 cells also had no detectable effect on ANP binding. These results suggest that, although glycosylation may be required for folding and transport of the newly synthesized ANP receptor to the cell surface, the oligosaccharide moieties themselves are not involved in hormone binding.