去整合素样金属蛋白酶8的N-糖基化及其在神经胶质瘤细胞增殖和转移中的作用  

N-glycosylation of ADAM8 and its role in proliferation and metastasis of glioblastoma cells

作　　者：杨建权 艾尔帕提·买买提 王志涛 王国兵 张钶 戴永建 王增亮[1] YANG Jianquan;AIERPATI Maimaiti;WANG Zhitao;WANG Guobing;ZHANG Ke;DAI Yongjian;WANG Zengliang(Department of Neurosurgery,The First Affiliated Hospital of Xinjiang Medical University,Urumqi,Xinjiang Uygur Autonomous Region 830011,China;Department of Neurosurgery,Renmin Hospital,Hubei University of Medicine,Shiyan,Hubei 442000,China)

机构地区：[1]新疆医科大学第一附属医院神经外科,新疆维吾尔自治区乌鲁木齐830011 [2]湖北医药学院附属人民医院神经外科,湖北十堰442000

出　　处：《安徽医药》2024年第12期2391-2396,I0008,共7页Anhui Medical and Pharmaceutical Journal

基　　金：新疆维吾尔自治区自然科学基金项目(2021D01A02)。

摘　　要：目的阐明去整合素样金属蛋白酶8(ADAM8)的N-糖基化在胶质母细胞瘤增殖和转移中的作用。方法该研究起止时间为2023年6—9月。选用胶质母细胞瘤细胞株U251和U87进行实验。细胞分为两组:对照组(不处理)和处理组[使用N-糖苷酶F(PNGase F)和衣霉素破坏N-糖基化]。处理组通过点突变技术替换ADAM8的4个潜在N-糖基化位点的天冬酰胺(Asn)残基,构建突变体N67Q、N91Q、N436Q和N612Q,依次设为N67Q组、N91Q组、N436Q组和N612Q组。随后,利用蛋白质印迹法检测突变体的蛋白加工情况,并通过共聚焦显微镜观察其亚细胞定位。使用溶酶体抑制剂比较野生型和N436Q突变体的蛋白稳定性。最后,转染不同突变体至U87细胞,评估其对细胞存活、增殖、迁移和侵袭的影响。结果胶质母细胞瘤细胞中的ADAM8蛋白4个位点(Asn-67,Asn-91,Asn-436和Asn-612)经历N-糖基化修饰,并且这4个位点在细胞中具有功能重要性。ADAM8蛋白的N91Q和N612Q突变体会导致定位改变,而N67Q和N436Q突变体与野生型ADAM8类似,N436Q突变体中ADAM8蛋白的稳定性降低。与对照组(0.23±0.05、0.49±0.03、0.73±0.05、1.41±0.06)相比,N67Q组(0.21±0.04、0.43±0.02、0.61±0.03、0.93±0.04)、N91Q组(0.25±0.07、0.36±0.02、0.44±0.04、0.84±0.06)、N436Q组(0.23±0.04、0.32±0.04、0.35±0.06、0.51±0.07)、N612Q组(0.25±0.04、0.39±0.05、0.52±0.02、0.76±0.03)U87细胞0、24、48、72 h存活率均降低(均P<0.05)。与对照组[(280.38±17.29)个/微升]相比,N67Q组[(187.29±16.48)个/微升]、N91Q组[(142.18±17.04)个/微升]、N436Q组[(32.19±14.28)个/微升]、N612Q组[(146.63±18.25)个/微升]U87细胞增殖能力均降低(均P<0.05)。与对照组[(98.76±7.56)个/微升]相比,N67Q组[(83.19±7.19)个/微升]、N91Q组[(69.37±6.73)个/微升]、N436Q组[(63.27±6.35)个/微升]、N612Q组[(74.28±7.01)个/微升]U87细胞划痕能力均降低(均P<0.05)。与对照组[(102.19±7.34)个/微升]相比,N67Q组[(70.2Objective To elucidate the role of N-glycosylation of a desintegrin-like metalloproteinase 8(ADAM8)in the proliferation and metastasis of glioblastoma cells.Methods The study was conducted from June 2023 to September 2023.Glioblastoma cell lines U251 and U87 were selected and assigned into control group(no treatment)and treatment group[N-glycosylation was destroyed by pep-tide n-glycosidase F(PNGase F)and itamycin].In the treatment group,the Asparagine(Asn)residues at four potential N-glycoylation sites of ADAM8 were replaced by point mutation technology,and mutants N67Q,N91Q,N436Q and N612Q were constructed,which were successively set as N67Q group,N91Q group,N436Q group and N612Q group.Subsequently,the protein processing of the mu-tants was detected by Western blotting,and its subcellular localization was observed by confocal microscopy.Lysosome inhibitors were used to compare the protein stability between wild type and N436Q mutants.Finally,different mutants were transfected into U87 cells to evaluate their effects on cell survival,proliferation,migration,and invasion.Results Four sites of ADAM8 protein in glioblastoma cells(Asn-67,Asn-91,Asn-436 and Asn-612)underwent N-glycosylation modification and these four sites are of functional importance in the cell.The N91Q and N612Q mutants of the ADAM8 protein might lead to altered localization,while the N67Q and N436Q mu-tants were similar to the wild-type ADAM8,and the ADAM8 protein in the N436Q mutant was less stable.Compared with the control group[(0.23±0.05),(0.49±0.03),(0.73±0.05)and(1.41±0.06)],the survival rates of U87 cells in N67Q group[(0.21±0.04),(0.43±0.02),(0.61±0.03),(0.93±0.04)],N91Q group[(0.25±0.07),(0.36±0.02),(0.44±0.04),(0.84±0.06)],N436Q group[(0.23±0.04),(0.32±0.04),(0.35±0.06),(0.51±0.07)]and N612Q groups[(0.25±0.04),(0.39±0.05),(0.52±0.02),(0.76±0.03)]decreased at 0,24,48,72 h(P<0.05).Compared with the control group[(280.38±17.29)cells/μL],the proliferation capacities of U87 cells in N67Q group[(187.29±16.48)cells/μL]

关 键 词：胶质母细胞瘤 去整合素样金属蛋白酶8 N-糖基化 细胞增殖 细胞转移 

分 类 号：R739.4[医药卫生—肿瘤]