靶向半胱氨酸硫用于体外重组蛋白的特异糖基化
Summary
糖化蛋白的生物化学和结构分析需要相对较大数量的均质样品。在这里, 我们提出了一种有效的化学方法的 site-specific 糖基化的重组蛋白纯化细菌的靶向活性胱氨酸硫。
Abstract
基质相互作用 molecule-1 (STIM1) 是一种 I 型跨膜蛋白, 位于内质网 (ER) 和细胞膜 (PM)。ER 驻留 STIM1 调节 PM Orai1 通道的活动, 该过程称为存储操作钙 (ca2 +), 它是主要的 ca2 +信号处理程序, 它可驱动免疫应答。STIM1 经历修饰N-糖基化在两个腔 Asn 网站内的 Ca2 +传感领域的分子。然而, 直到最近, 由于无法轻易获得高水平的均质性蛋白质, 糖化 STIM1 的生物化学、生物物理和结构生物学效应都没有得到很好的理解. 在这里, 我们描述了一个体外化学方法的实现, 它将葡萄糖基附加到特定的蛋白质部位, 以了解N-糖基化对蛋白质结构和机制的潜在影响。使用溶液核磁共振波谱法, 我们评估的效率的修改, 以及结构的后果, 葡萄糖附件与单一样本。这种方法可以很容易地适应研究在自然界中发现的无数糖化蛋白。
Introduction
存储操作钙 (ca2 +) 条目 (SOCE) 是免疫细胞从细胞外空间进入胞的主要途径.t 淋巴细胞, t 细胞受体位于细胞膜 (PM) 结合抗原, 激活蛋白酪氨酸激酶 (在1,2,3中进行了审查。磷酸化级联导致磷脂酶γ (PLCγ) 的活化, 随后将膜醇 45–1,6-(PIP2) 的水解转化为甘油和肌醇 14, 5-trisphosphate (IP3).ip3是一个小的扩散信使, 它绑定到内质网 (er) 上的 ip3受体 (ip3R), 从而打开这个受体通道, 并允许 Ca2 +从 ER 流下来的浓度梯度。流明到胞 (在4中进行了审阅)。受体信号从 G 蛋白耦合和酪氨酸激酶受体在各种其他易激动和不可的细胞类型导致相同的生产 ip3和激活 ip3Rs。
由于 ER 的有限 Ca2 +存储容量, IP3介导的释放和胞浆 ca2 +的结果增加只是暂时的;但是, er 腔 Ca2 +的这一损耗深刻影响了基质相互作用 molecule-1 (STIM1), 一种主要在 er 膜上发现的 I 型跨膜 (TM) 蛋白, 5,6,7。STIM1 包含一个面向流明的 Ca2 +传感域由一个 EF 手对和无菌α-主题 (EFSAM) 组成。三由单 TM 域与 EFSAM 分离的胞内定向盘绕线圈域 (在8中进行了评审)。当 ER 腔 Ca2 +耗尽时, EFSAM 经历了一个不稳定耦合的齐聚, 7,9 , 这将导致 TM 和盘绕线圈域的结构重10。这些结构的变化最终导致 STIM1 在 ER pm 路口的陷印11,12,13,14通过与 pm phosphoinositides 15的交互, 16和 Orai1 子单元17,18。Orai1 蛋白质是组装成 Ca2 +通道19、20、21、22的 PM 子单元。STIM1-Orai1 交互在 ER PM 连接促进开放的 ca2 +释放激活的 ca2 + (裂纹) 通道构象, 使 ca2 +的移动到胞从高浓度的细胞外空间。在免疫细胞中, 通过裂纹通道的持续胞浆钙离子 2 +的升高, 诱发了 ca 的2 +-钙调素/磷酸依赖性磷酸的核因子的活化 T 细胞, 随后进入细胞核和开始转录调控基因促进 T 细胞激活1,3。裂纹通道激活的过程由 STIM1 23,24通过激动剂诱发 ER 腔 ca2 +耗尽和结果持续的胞浆 ca2 +提升统称为 SOCE 25。SOCE 在 T 细胞中的重要作用是显而易见的研究表明, STIM1 和 Orai1 可遗传突变会导致严重的联合免疫缺陷综合症3,19,26, 27. EFSAM 启动 SOCE 后, 传感 ER 腔 ca2 +损耗通过 ca 的损失2 +协调在规范 EF 手, 最终导致不稳定耦合 self-association 7, 28,29。
糖基化是通过 ER 和高尔基体中的各种生物合成步骤 (在30,32,33) 中对寡糖结构的共价键连接和处理, 也称为糖。在真核生物中有两种主要的糖基化类型: N-链接和O链接, 具体取决于特定的氨基酸和原子桥接连接。在N-糖基化, 糖附着在 Asn 的侧链酰胺上, 在大多数情况下, 当多肽链移动到流明34时, 在 ER 中发生起始步骤。n的第一步-糖基化是由葡萄糖 (Glc)、甘露糖 (人) 和N-乙酰 (GlcNAc) (即 Glc3人9GlcNAc2) 从 ER膜脂由 oligosaccharyltransferase 35,36。进一步的步骤, 如分裂或转移葡萄糖残留, 是催化在 ER 由特定的糖苷和转移。一些蛋白质, 离开 ER 和移动到高尔基可以进一步处理37。O-糖基化指的是添加糖, 通常对侧链羟基的 Ser 或的残留物, 这一修改完全发生在高尔基复合体33,34。有几个O-糖结构, 可以由N-乙酰, 藻, 半乳糖, 和唾液酸, 每个单糖添加顺序33。
虽然没有任何特定的序列被确定为许多类型的O-糖基化的先决条件, 但一个共同的共识序列已经与N链接的修改相关联: Asn x Ser/胱氨酸, 其中 x 可以是任何氨基酸除了 Pro 33。STIM1 EFSAM 包含其中的两个共识N-糖基化的网站: Asn131-Trp132-Thr133 和 Asn171-Thr172-Thr173。事实上, 以前的研究表明, EFSAM 可以是N-糖化在哺乳动物细胞在 Asn131 和 Asn171 38,39,40,41。然而, 以前的研究的后果, N-糖基化对 SOCE 已不, 建议抑制, 强化或没有效果, 这修饰修改 SOCE 激活38,= “xref” > 39,40,41。因此, 对 EFSAM N-糖基化的基础生物物理、生物化学和结构后果的研究对于理解这种修改的调节作用至关重要。由于在这些体外实验中对高水平的均质蛋白的要求, 采用了一种以现场选择的方法将葡萄糖基共价附着在 EFSAM 上。有趣的是, Asn131 和 Asn171 糖基化导致了在 EFSAM 核心内聚集的结构变化, 并增强了促进 STIM1-mediated SOCE 42的生物物理特性。
糖组对胱氨酸硫的化学附着已被一项开创性工作所证实, 这一研究首次证明了这种无酶方法的效用, 以了解糖基化对蛋白质功能的 site-specific 作用43,44. 最近和关于 STIM1, Asn131 和 Asn171 残留物被变异了对胱氨酸和葡萄糖-5-(methanethiosulfonate) [葡萄糖 5-(MTS)] 被用共价键连接葡萄糖到自由的硫42。在这里, 我们描述了这种方法, 不仅使用诱变, 以纳入现场特定的胱氨酸残留物的修改, 但也应用解决方案核磁共振 (NMR) 光谱学, 以快速评估的修改效率和结构由于糖基化而引起的扰动。值得注意的是, 这种通用的方法很容易适应于研究任何 recombinantly 产生的蛋白质的O或N-糖基化的影响。
Protocol
Representative Results
Discussion
蛋白质糖基化是一种修饰的修饰, 糖通过与氨基酸侧链的连接, 以共价键的多肽为主。多达50% 的哺乳动物蛋白质是糖化54, 其中糖化蛋白可以有不同范围的影响, 改变生物分子结合亲和力, 影响蛋白质折叠, 改变渠道活动, 目标降解和细胞贩运的分子, 命名为少数 (在33中进行了审查)。糖基化在哺乳动物生理学中的重要作用是由数百种蛋白质进化而来的, 以构建哺乳?…
Acknowledgements
这项研究得到了加拿大自然科学和工程研究理事会 (05239 至 P.B.S.)、加拿大创新基金会/安大略省研究基金 (P.B.S.)、前列腺癌防治基金会–科学爸爸 (P.B.S.) 和安大略的支持。研究生奖学金 (Y.J.C. 和生理盐水)。
Materials
| Phusion DNA Polymerase | Thermo Fisher Scientific | F530S | Use in step 1.3. |
| Generuler 1kb DNA Ladder | Thermo Fisher Scientific | FERSM1163 | Use in step 1.6. |
| DpnI Restriction Enzyme | New England Biolabs, Inc. | R0176 | Use in step 1.8. |
| Presto Mini Plasmid Kit | GeneAid, Inc. | PDH300 | Use in step 1.16. |
| BL21 DE3 codon (+) E. coli | Agilent Technologies, Inc. | 230280 | Use in step 2.1. |
| DH5a E. coli | Invitrogen, Inc. | 18265017 | Use in step 1.9. |
| 0.22 mm Syringe Filter | Millipore, Inc. | SLGV033RS | Use in step 2.3. |
| HisPur Ni2+-NTA Agarose Resin | Thermo Fisher Scientific | 88221 | Use in step 3.3. |
| 3,500 Da MWCO Dialysis Tubing | BioDesign, Inc. | D306 | Use in step 3.8, 3.16, 4.2, 4.5 and 4.6. |
| Bovine Thrombin | BioPharm Laboratories, Inc. | SKU91-055 | Use in step 3.9. |
| 5 mL HiTrap Q FF Anion Exchange Column | GE Healthcare, Inc. | 17-5156-01 | Use in step 3.11. |
| Glucose-5-MTS | Toronto Research Chemicals, Inc. | G441000 | Use in step 4.1. |
| Vivaspin 20 Ultrafiltration Centrifugal Concentrators | Sartorius, Inc. | VS2001 | Use in step 3.11, 4.2, 4.5 and 4.6. |
| PageRuler Unstained Broad Protein Ladder | Thermo Fisher Scientific | 26630 | Use in step 3.7, 3.10 and 3.15 |
| HiTrap Q FF Anion Exchange Column | GE Healthcare, Inc. | 17-5053-01 | Use in step 3.12. |
| AKTA Pure Fast Protein Liquid Chromatrography System | GE Healthcare, Inc. | 29018224 | Use in step 3.14. |
| 600 MHz Varian Inova NMR Spectrometer | Agilent Technologies, Inc. | Use in step 5.2 and 5.5. |
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