| 杨建权,艾尔帕提 ·买买提,王志涛,等.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[J].安徽医药,2024,28(12):2391-2396. |
| 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 |
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| DOI：10.3969/j.issn.1009-6469.2024.12.012 |
| 中文关键词: 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 |
| 英文关键词: Glioblastoma Desintegrin-like metalloproteinase |
| 基金项目:新疆维吾尔自治区自然科学基金项目( 2021D01A02) |
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| 中文摘要: |
| 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［( 70.21±7.13)个 /微升］、 N91Q组［( 49.07±6.16)个 /微升］、 N436Q组［( 16.28±3.24)个 /微升］、 N612Q组［( 39.71±4.37)个 /微升］ U87细胞侵袭能力均降低(均 P<0.05)。转染 N-糖基化缺陷的 ADAM8突变体的 U87细胞表现出波形蛋白和紧密连接蛋白 1(Claudin-1)蛋白水平的下降，而上皮钙黏素( E-cadherin)上升。结论 ADAM8的 N-糖基化促进了胶质母细胞瘤细胞的增殖和转移。 N-糖基化位点突变抑制了细胞的恶性表型。 |
| 英文摘要: |
| Objective To elucidate the role of N-glycosylation of a desintegrin-like metalloproteinase 8 (ADAM8) in the proliferationand metastasis of glioblastoma cells. Methods The study was conducted from June 2023 to September 2023. Glioblastoma cell linesU251 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-glycoylationsites of ADAM8 were replaced by point mutation technology, and mutants N67Q, N91Q, N436Q and N612Q were constructed, whichwere 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 wereused 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 importancein 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 controlgroup [(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], N91Q group [(142.18±17.04) cells/μL], N436Q group [(32.19±14.28) cells/μL] and N612Q group [(146.63±18.25)cells/μL] were decreased (all P<0.05). Compared with the control group [(98.76±7.56) cells/μL], the scratching capacities of U87 cellsin N67Q group [(83.19±7.19) cells/μL], N91Q group [(69.37±6.73) cells/μL], N436Q group [(63.27±6.35) cells/μL] and N612Q group[(74.28±7.01) cells/μL] were decreased (all P<0.05). Compared with the control group [(102.19±7.34) cells/μL], the invasion capacitiesof U87 cells in N67Q group [(70.21±7.13) cells/μL], N91Q group [(49.07±6.16) cells/μL], N436Q group [(16.28±3.24) cells/μL] andN612Q group [(39.71±4.37) cells/μL] were decreased (all P<0.05). U87 cells in ADAM8 mutants transfected with N-glycosylation de-fects showed decreased vimentin and Claudin-1 protein levels and increased E-cadherin.Conclusions N-glycosylation of ADAM8 pro-motes the proliferation and metastasis of glioblastoma cells. N-glycosylation site mutation inhibits the malignant phenotype of cells. |
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