N-glycosylation of the Human κ opioid receptor enhances its stability but slows its trafficking along the biosynthesis pathway
N-glycosylation of the Human κ opioid receptor enhances its stability but slows its trafficking along the biosynthesis pathway
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DOI：
10.1021/bi700443j
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发表时间：
2007-09-25
期刊：
影响因子：
2.9
通讯作者：
Liu-Chen, Lee-Yuan
中科院分区：
文献类型：
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作者：
Li, Jian-Guo;Chen, Chongauang;Liu-Chen, Lee-Yuan
我们检测了在CHO细胞中表达的FLAG-hKOR的糖基化，并确定了它的功能意义。免疫印迹显示FLAG-hKOR为一条弥漫的55 kDa宽带和一条弥散较少的45 kDa条带，表明该受体是糖基化的。内切糖苷酶H将45 kDa的条带切割成类似38 kDa的条带，但不改变55 kDa的条带，表明45 kDa的条带是N-糖基化的高甘露糖或杂交型糖链。肽-N-糖苷酶F经溶解的hKOR消化或与衣霉素孵育细胞后，分别产生43和38 kDa两种酶带，表明43 kDa的条带是O-糖基化的。神经氨酸酶和O-糖苷酶将FLAG-hKOR还原为较低的M-r条带，表明hKOR含有O-连接的糖链。N-末端的Asn25或Asn39突变为G1n，使M-r降低约5 kDa，表明两个残基都是糖基化的。双突变体hKOR-N25/39Q经鉴定为一条43 kDa的成熟条带和一条38 kDa的中间型条带。在瞬时表达时，hKOR-N25/39Q的表达水平低于野生型。在稳定表达hKOR-N25/39Q的CHO细胞中，脉冲追逐实验表明，中间和成熟形式的周转速率常数(k(E))接近野生型的3倍。此外，43 kDa的hKOR-N25/39Q的成熟速率常数(k(A))是野生型的6倍。HKOR-N25/39Q突变体表现出激动剂诱导的受体磷酸化、脱敏、内化和下调，而不改变配体结合亲和力或受体-G蛋白偶联。因此，hKOR的N-糖基化在受体蛋白的生物合成途径的稳定和运输以及激动剂诱导的受体调节中起着重要的作用。
We examined glycosylation of FLAG-hKOR expressed in CHO cells and determined its functional significance. FLAG-hKOR was resolved as a broad and diffuse 55-kDa band and a less diffuse 45-kDa band by immunoblotting, indicating that the receptor is glycosylated. Endoglycosidase H cleaved the 45-kDa band to similar to 38 kDa but did not change the 55-kDa band, demonstrating that the 45-kDa band is N-glycosylated with high-mannose or hybrid-type glycan. Peptide-N-glycosidase F digestion of solubilized hKOR or incubation of cells with tunicamycin resulted in two species of 43 and 38 kDa, suggesting that the 43-kDa band is O-glycosylated. FLAG-hKOR was reduced to lower M-r bands by neuraminidase and O-glycosidase, indicating that the hKOR contains O-linked glycan. Mutation of Asn25 or Asn39 to G1n in the N-terminal domain reduced the M-r by similar to 5 kDa, indicating that both residues were glycosylated. The double mutant hKOR-N25/39Q was resolved as a 43-kDa (mature form) and a 38-kDa (intermediate form) band. When transiently expressed, hKOR-N25/39Q had a lower expression level than the wild type. In CHO cells stably expressing the hKOR-N25/39Q, pulse-chase experiments revealed that the turnover rate constants (k(e)) of the intermediate and mature forms were similar to 3 times those of the wild type. In addition, the maturation rate constant (k(a)) of the 43-kDa form of hKOR-N25/39Q was 6 times that of the mature form of the wild type. The hKOR-N25/39Q mutant showed increased agonist-induced receptor phosphorylation, desensitization, internalization, and downregulation, without changing ligand binding affinity or receptor-G protein coupling. Thus, N-glycosylation of the hKOR plays important roles in stability and trafficking along the biosynthesis pathway of the receptor protein as well as agonist-induced receptor regulation.