用GFP标记TMEM184A构建肝素受体身份确认
Summary
与羧基末端设计用于真核表达GFP的标签的构建编码TMEM184A，在旨在证实TMEM184A的鉴定如在血管细胞肝素受体测定中使用。
Abstract
当新的蛋白质通过基于亲和力的分离和生物信息学分析发现，他们往往在很大程度上未鉴定。针对预测序列中的特定肽抗体允许一些本地化实验。然而，与抗体的其他可能的相互作用往往不能排除。这种情况提供给开发一套依赖于蛋白质序列测定的机会。具体地，得到含有耦合到在蛋白质的C末端的绿色荧光蛋白编码序列的基因序列的构建体和用于这些目的。实验以表征本地化，配体亲和力和功能的获得最初设计和执行，以确认TMEM184A的鉴定为肝素受体1。此外，该构建体可用于研究，处理膜拓扑问题，并详细蛋白质 – 配体相互作用。本报告介绍AR的基础上在血管细胞中表达的GFP-TMEM184A构造，可以很容易地适用于其他的新的蛋白质实验方案法兰。
Introduction
对于新功能的候选蛋白的鉴定常常取决于基于亲和力的分离方法并进行部分序列测定。新发现的蛋白质的最近的例子包括跨膜蛋白184A（TMEM184A），之后肝素亲和互动1确定了肝素受体，并TgPH1，结合磷酸肌醇PI（3,5）P 2 2 PH结构域蛋白。其他新的蛋白质鉴定涉及肽的直接序列分析，例如由维生素等。谁用跨膜肽识别蛋白产品之前未基因3。同样地，新的蛋白质序列的鉴定可以通过使用生物信息学的先前特征的蛋白质家族的搜索，如新4TM蛋白4的识别来完成的。水通道蛋白家族基因序列的考试都有人所以产生了新成员的身份与新功能5。鉴定后，蛋白质功能的分析通常是有时可以使用蛋白质功能的一个特定的测定法，诸如在水通道壳体被检查的下一个步骤。
如果可能的话，一个新发现的蛋白质的功能，可以与特定的酶或类似的体外检测功能进行检查。因为新的蛋白质的许多功能取决于只发生在完整细胞或生物体， 在体外测定法复杂的相互作用并不总是有效的。然而， 在体内测定，必须以这样的方式，它们依赖于基因序列来设计。在细胞培养和/或简单的模式生物，击倒可以提供证据为蛋白质/功能鉴定6。与如上所述确定的新的蛋白质，它通常是不足以简单地击倒的蛋白质进行确认的功能，一个d 在体内依赖于基因序列功能测定法的设计成为新的蛋白质的表征重要。
最近TMEM184A鉴定为肝素受体使用亲和层析和MALDI MS 1（其调节血管平滑肌和内皮细胞的炎性应答的增殖），7提供给开发实验的集合的机会后击倒得到的结果与识别相一致。最近的综述证实肝素与许多生长因子，它们的受体，细胞外基质组分，细胞粘附受体，和其它蛋白质8特异性相互作用。在血管系统，肝素和硫酸乙酰肝素蛋白多糖（含有在结构上与肝素类似的硫酸乙酰肝素链）与几百蛋白9相互作用。在功能上证实塔ŧTMEM184A参与了肝素吸收和结合，即用于TMEM184A基因构建技术开发的。本报告包括实验的基础上GFP-TMEM184A集合构建用于确认TMEM184A的身份肝素受体。
Protocol
1. GFP蛋白质构造的设计购买或设计和建造，根据有关的蛋白质有GFP标记的结构。 注：对于购买构建体，标准载体可从商业实验室，包括部分或全部的以下建议：对于膜蛋白，选择的GFP的C-末端的位置，因为它不太可能与膜蛋白质运输到干扰。考虑的感兴趣的基因，如果有理由相信有关蛋白质的C-末端被紧凑地折叠成蛋白质的结构域将GFP之间的延伸。选择一般的真核细胞表达的概念，但…
Representative Results
而，在理论上，任何DNA构建体转染到细胞中可以与亲脂转染试剂来完成，以前的报道显示GFP的更有效的转染构建成利用电12内皮细胞。这里提供的协议典型地实现了使用的主要来源的内皮细胞和平滑肌细胞的大于80％的GFP构建表达。使用时可能会迅速提供这种构造的商用系统所采用的结构设计。主要用途集中于位置的问题，因此，正确传递到膜，具有最佳…
Discussion
此处所报告的协议被设计成提供用于鉴定TMEM184A作为在血管细胞1肝素受体确证证据。击倒技术被常规用作一种机制以确认新的蛋白质的鉴定。然而，击倒后，一些功能损失通常不是足以证明候选蛋白实际上是正确的受体（或其他功能蛋白）。同样重要的是要有证据表明该候选蛋白实际上具有的功能。用人有标签GFP的新基因的构建实现功能的实验增益和促进基于绿色荧光蛋白的亲?…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
Research in the Lowe-Krentz lab is supported by research grant HL54269 from the National Institutes of Health to LLK.
Materials
| GFP-TMEM184A construct | OriGene | RG213192 | |
| Rhodamine-Heparin | Creative PEGWorks | HP-204 | Light Sensitive |
| Fluorescein-Heparin | Creative PEGWorks | HP-201 | Light Sensitive |
| Mowiol | EMD Millipore | 475904-100GM | |
| Paraformaldehyde (methanol free) | Thermo Sci Pierce Biotech, available through Fisher Scientific | PI28908 at Fisher | Use in Fume Hood |
| Reacti-bind neutravidin plates (Avidin coated black 96 well dishes) | Thermo Sci Pierce Biotech, through Fisher Scientific | PI15510 at Fisher | Pay attention to shelf-life |
| Black 96 well plates | Corning Life Sciences Plastic, purchased through Fisher Scientific | 064432 at Fisher | |
| A7r5 vascular smooth muscle cell line | ATCC | CRL 1444 | Can be exchanged into MEM medium1 |
| BAOEC bovine aortic endothelial cells | Cell Applications, Inc. | B304-05 | Culture as recommended initially, can be exchanged into MEM medium for continuing culture1,7 |
| BAOSMC bovine aortic smooth muscle cells | Cell Applications, Inc. | B354-05 | Culture as recommended initially, can be exchanged into MEM medium for continuing culture1 |
| RAOEC rat aortic endothelial cells | Cell Applications, Inc. | R304-05a | Culture as recommended initially, can be exchanged into MEM medium for continuing culture7 |
| Biotinylated anti-GFP | Thermo Sci Pierce Biotech, through Fisher Scientific | MA5-15256-BTIN | |
| Streptavidin-coated beads | Sigma | S1638 | |
| HeBS | Available from Bio-Rad | Can be prepared in the lab. The pH is 6.8 | |
| TMEM184A antibody to the N-terminus | Santa Cruz Biotechnology | sc292006 | Only known TMEM184A antibody to N-terminal region. |
| TMEM184A antibody to the C-terminus | Obtained from ProSci Inc, Poway, CA | Pro Sci 5681 | ProSci used in figure 1 |
| GFP antibodies | Santa Cruz Biotechnology | sc9996 | Used in figures 5 |
| Secondary antibodies, labeled with TRITC or Cy3 | Jackson ImmunoResearch Laboratories, Inc, West Grove, PA | 711 025 152 (donkey anti-rabbit, TRITC) 715 165 150 (donkey anti-mouse, Cy3) |
Minimal cross-reactivity to minimize any non-specific staining. |
| CHAPS | Purchased from Sigma | C5849 | Note that this specific catalog number has been discontinued. Supplier will provide information regarding replacement. |
| Live imaging 35 mm dishes | MatTek (Ashland MA) | P35G-1.0 – 20 mm – C | |
| Confocal Microscope | Zeiss | LSM 510 Meta with a 63X oil-immersion lens | Used for images and live-imaging in Figures 1, 2 and 3 |
| Confocal Microscope | Nikon | C2+ confocal with a 60X oil-immersion lens | Used for images in Figure 5 |
| Confocal Microscope | Zeiss | Zeiss LSM 880 with a 63X oil-immersion lens | Used for images in Figure 2C |
| Electroporation equipment | Bio-Rad | Gene Pulser X-Cell System | |
| Electroporation cuvettes | Available from MidSci | EC2L | Can also be obtained from equipment supplier |
| Plate reader | TECAN | TECAN Infinite® m200 Pro plate reader | Readings in the middle of the wells rather than at the surface. |
| Computer program for measuring staining intensity | Image J | https://imagej.nih.gov/ij/ Program and information available on-line |
Any appropriate program can be used. See https://theolb.readthedocs.io/en/latest/imaging/measuring-cell-fluorescence-using-imagej.html for additional detail |
| Cell Culture trypsin solution | Sigma | T4174 | purchased as a 10X solution |
Referenzen
- Pugh, R. J., et al. Transmembrane Protein 184A Is a Receptor Required for Vascular Smooth Muscle Cell Responses to Heparin. J Biol Chem. 291, 5326-5341 (2016).
- Daher, W., et al. Identification of Toxoplasma TgPH1, a pleckstrin homology domain-containing protein that binds to the phosphoinositide PI(3,5)P. Mol Biochem Parasitol. , (2016).
- Vit, O., et al. Large-scale identification of membrane proteins based on analysis of trypsin-protected transmembrane segments. J Proteomics. , (2016).
- Attwood, M. M., et al. Topology based identification and comprehensive classification of four-transmembrane helix containing proteins (4TMs) in the human genome. BMC genomics. 17, 268 (2016).
- Zou, Z., et al. Genome-Wide Identification of Jatropha curcas Aquaporin Genes and the Comparative Analysis Provides Insights into the Gene Family Expansion and Evolution in Hevea brasiliensis. Front Plant Sci. 7, 395 (2016).
- Gilotti, A. C., et al. Heparin responses in vascular smooth muscle cells involve cGMP-dependent protein kinase (PKG). J Cell Physiol. 229, 2142-2152 (2014).
- Farwell, S. L., et al. Heparin Decreases in Tumor Necrosis Factor alpha (TNFalpha)-induced Endothelial Stress Responses Require Transmembrane Protein 184A and Induction of Dual Specificity Phosphatase 1. J Biol Chem. 291, 5342-5354 (2016).
- Xu, D., Esko, J. D. Demystifying heparan sulfate-protein interactions. Annu Rev Biochem. 83, 129-157 (2014).
- Chiodelli, P., Bugatti, A., Urbinati, C., Rusnati, M. Heparin/Heparan sulfate proteoglycans glycomic interactome in angiogenesis: biological implications and therapeutical use. Molecules. 20, 6342-6388 (2015).
- Slee, J. B., Lowe-Krentz, L. J. Actin realignment and cofilin regulation are essential for barrier integrity during shear stress. J Cell Biochem. 114, 782-795 (2013).
- Patton, W. A., et al. Identification of a heparin-binding protein using monoclonal antibodies that block heparin binding to porcine aortic endothelial cells. The Biochemical journal. 311, 461-469 (1995).
- Doggett, T. M., Breslin, J. W. Study of the actin cytoskeleton in live endothelial cells expressing GFP-actin. J Vis Exp. , (2011).
- Skalamera, D., et al. Generation of a genome scale lentiviral vector library for EF1alpha promoter-driven expression of human ORFs and identification of human genes affecting viral titer. PloS one. 7, 51733 (2012).
- Castro, M., Nikolaev, V. O., Palm, D., Lohse, M. J., Vilardaga, J. P. Turn-on switch in parathyroid hormone receptor by a two-step parathyroid hormone binding mechanism. Proc Natl Acad Sci U S A. 102, 16084-16089 (2005).
- Albertazzi, L., Arosio, D., Marchetti, L., Ricci, F., Beltram, F. Quantitative FRET analysis with the EGFP-mCherry fluorescent protein pair. Photochem Photobiol. 85, 287-297 (2009).
- Wang, S., et al. Domain organization of the ATP-sensitive potassium channel complex examined by fluorescence resonance energy transfer. J Biol Chem. 288, 4378-4388 (2013).
- Christiansen, E., Hudson, B. D., Hansen, A. H., Milligan, G., Ulven, T. Development and Characterization of a Potent Free Fatty Acid Receptor 1 (FFA1) Fluorescent Tracer. J Med Chem. 59, 4849-4858 (2016).
- Chiang, C. F., Okou, D. T., Griffin, T. B., Verret, C. R., Green Williams, M. N. fluorescent protein rendered susceptible to proteolysis: positions for protease-sensitive insertions. Arch Biochem Biophys. 394, 229-235 (2001).
Diesen Artikel zitieren
Farwell, S. L. N., Slee, J. B., Li, Y., Lowe-Krentz, L. J. Using a GFP-tagged TMEM184A Construct for Confirmation of Heparin Receptor Identity. J. Vis. Exp. (120), e55053, doi:10.3791/55053 (2017).