Glycosylation of human proteinase-activated receptor-2 (hPAR2) role in cell surface expression and signalling
Glycosylation of human proteinase-activated receptor-2 (hPAR2) role in cell surface expression and signalling
复制标题
DOI：
10.1042/bj20020706
复制
发表时间：
2002-12-01
影响因子：
4.1
通讯作者：
Hollenberg, MD
中科院分区：
文献类型：
--
作者：
Compton, SJ;Sandhu, S;Hollenberg, MD
我们分析了n -链糖基化在调节人蛋白酶激活受体2 （hPAR(2)）表达和功能中的作用。表位标记的野生型hPAR(2) （wt-hPAR(2)）或在n端[hPAR(2)N30A (Asn(30) -> Ala)]，细胞外环2 [ECL2；hPAR(2)N222Q （Asn(222) -> Gln）或hPAR(2)N222A (Asn(222) -> Ala)]或两者(hPAR（2)N30A，N222A或hPAR(2)N30A,N222Q）在中国仓鼠卵巢（CHO）成纤维细胞系Pro5中表达。Western blot分析显示，成熟的wt-hPAR(2)分子量为55-100 kDa，经n -糖苷酶F去糖基化后分子量为33-48 kDa。对wt-hPAR(2)和PAR(2)突变细胞系的FACS分析和免疫细胞化学显示，去除两个糖基化序列降低了（50%的wt-hPAR(2)）细胞表面表达。Western blot分析表明，两个n -连锁位点都被糖基化。在功能研究中，hPAR(2)N30A对胰蛋白酶表现出选择性和显著的敏感性增加。有趣的是，hPAR(2)N222A对所有PAR(2)激动剂的敏感性均有所下降。然而，进一步的分析揭示了受体对该区域丙氨酸突变的敏感性，因为更保守的取代hPAR(2)N222Q对PAR 2激动剂的反应没有变化。hPAR(2)N30A、N222Q对胰蛋白酶的敏感性增加，但对胰蛋白酶和合成肽SLIGRL-NH2的敏感性下降，尽管这种对胰蛋白酶和SLIGRL-NH2的敏感性下降是继发于细胞表面表达的变化。最后，唾液酸缺乏wt-hPAR(2)在CHO Lec2糖基化缺陷突变细胞系中的表达，除了对胰蛋白酶的敏感性显着和选择性地增加外，还显示分子质量损失40 kDa。我们得出结论，hpa2 n -链糖基化和唾液酰化调节受体表达和/或信号传导。
We have analysed the role of N-linked glycosylation in regulating human proteinase-activated receptor-2 (hPAR(2)) expression and function. Epitope-tagged wild-type hPAR(2) (wt-hPAR(2)) or hPAR 2 that lacked glycosylation sequons (following site-directed mutagenesis) in either the N-terminus [hPAR(2)N30A (Asn(30) --> Ala)], extracellular loop 2 [ECL2; hPAR(2)N222Q (Asn(222) --> Gln) or hPAR(2)N222A (Asn(222) --> Ala)] or both (hPAR(2)N30A,N222A or hPAR(2)N30A,N222Q) were expressed in the Chinese-hamster ovary (CHO) fibroblast cell line, Pro5. Western blot analysis of wt-hPAR(2) showed mature wt-hPAR(2) to have a molecular mass of 55-100 kDa, and 33-48 kDa following N-glycosidase F deglycosylation. FACS analysis and immunocytochemistry of the wt-hPAR(2) and PAR(2) mutant cell lines revealed that removal of both glycosylation sequons decreases (50 % of wt-hPAR(2)) cell surface expression. Western blot analysis indicated that both N-linked sites are glycosylated. In functional studies, hPAR(2)N30A displayed a selective and significant increase in sensitivity towards tryptase. Interestingly, hPAR(2)N222A displayed a loss in sensitivity towards all PAR(2) agonists tested. However, further analysis revealed receptor sensitivity to alanine mutations in this domain, as the more conservative substitution hPAR(2)N222Q displayed no change in response to PAR 2 agonists. hPAR(2)N30A,N222Q displayed increased sensitivity towards tryptase, but a loss in sensitivity towards trypsin and the synthetic peptide SLIGRL-NH2, although this loss in sensitivity towards trypsin and SLIGRL-NH2, was secondary to changes in cell-surface expression. Finally, expression of sialic-acid-deficient wt-hPAR(2) in the CHO Lec2 glycosylation-deficient mutant cell line, showed a 40 kDa loss in molecular mass, in addition to a marked and selective increase in sensitivity towards tryptase. We conclude that hPAR 2 N-linked glycosylation and sialylation regulates receptor expression and/ or signalling.