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Biologie

视黄醇运输的血浆视黄醇结合蛋白的膜受体的实时分析

Published: January 28, 2013
doi:
10.3791/50169
, ,
1Department of Physiology, Jules Stein Eye Institute and Howard Hughes Medical Institute,University of California, Los Angeles

Summary

在这里，我们描述了一个优化的技术，生产高品质的维生素A / RBP复杂的和实时监测技术研究维生素A运输STRA6，RBP受体。

Abstract

维生素A是必不可少的眼光和几乎所有的人体器官的生长/分化的。血浆视黄醇结合蛋白（RBP）的原则和具体的载体在血液中的维生素A。在这里，我们描述了一个优化的技术，生产和净化的holo-RBP和两个实时监测技术的研究维生素A高亲和力RBP受体STRA6的运输。第一种技术使得有可能产生大量的高品质的holo-RBP（100％-加载与视黄醇），维生素A运送检测。高品质的RBP功能分析是必不可少的，因为错误折叠的RBP版本维生素容易和细菌的污染在RBP准备的产生伪影。实时监控技术，如电的细胞膜传输方面的研究作出了重要的贡献。 RBP受体介导的视黄醇运输实时分析，直到最近。这里所描述的第二个技术是意图分析L-STRA6催化视黄醇释放或装载。第三种方法是STRA6催化视黄醇运输全息-RBP细胞视黄醇结合蛋白I（CRBP-I）的实时分析。这些技术提供了高灵敏度和分辨率，揭示RBP受体的维生素A的吸收机制。

Introduction

维生素A是一种有机分子，是人类生存和几乎所有的人体器官的正常运作至关重要。维生素A衍生物（视黄醇）参与不同的生物化学和细胞的活动，包括光检测视力1,2和调节胚胎发育过程中的基因表达和蛋白质的翻译和成人组织3-6。虽然维生素A有能力扩散全身，演变与血浆视黄醇结合蛋白，一个特定的载体蛋白，维生素在血液中的运输，以实现高效率和特异性，以避免毒性与随机扩散7-10。高亲和力受体结合的限制性商业惯例，并采取了维生素A的假设在20世纪70年代11-13。尽管RBP受体的存在14-31的三十年中积累的证据，受体假说争论多年的existence的全息-RBP一个不正确的定义。全息-RBP的正确定义是，它是维生素A和RBP高亲和1:1的复合物之间。重复提取的holo-RBP由有机溶剂是必要的产生的apo-RBP。这个定义所使用的几乎所有的实验室研究RBP的7,9,32-35或在RBP受体14-31,36-42。全息-RBP被用来反驳的RBP受体的存在是不正确的定义与APO-RBP的急性的免费视黄醇的混合物。由于RBP受体的功能的维生素A吸收全息-RBP-RBP全息释放视黄醇，RBP受体不发挥作用，在视黄醇摄入维生素A是开始（所提出的不正确的定义全息RBP）。

最近的研究发现RBP受体作为一个multitransmembrane的结构域蛋白称为STRA636和其功能的维生素A吸收从全息-RBP 36-43强烈反对的假设，即RBP不需要的受体提供维生素A的详细分析表明STRA6有9个跨膜结构域与位于细胞外的N-末端和C-末端位于细胞内40。位于跨膜6和图7之间是一个必不可少的RBP结合结构域39。 STRA6耦合都LRAT和CRBP-I中的维生素A吸收全息-RBP，但既不是LRAT也不是CRBP-I是绝对必要的，增强了STRA6活动41。 STRA6的能力，以促进维生素A的holo-RBP释放的关键是维生素A的吸收活动41。通过依靠STRA6释放其视黄醇，维生素A交付RBP可以运输维生素A的外周组织中的靶细胞，以高特异性和高效率。

RBP受体的存在，不仅历史辩论至关重要的holo-RBP的定义和准备说明，但最近三个相关的论文的基础上的holo-RBP定义不同ferent从原来的和正确的定义44-46。第一篇论文使用的holo-RBP定义被用来否决RBP受体的存在，研究RBP受体44。第二次和第三次的论文想出的holo-RBP与三分之一的定义，使得它更可能为视黄醇的研究，以形成适当的配合物与RBP 45,46。这些编写的研究报告3 H-retinol/RBP由混合的holo-RBP（APO-RBP），用3 H-视黄醇。由于该试验中没有3 H-retinol/RBP形成的，并没有除去过量的游离的3 H-视黄醇45,46，它是不测定3 H-视黄醇的吸收来自3 H-retinol/RBP，但是一个免费的的3 H-视黄醇扩散法。它已被证明先前STRA6不增强细胞摄取的免费视黄醇由38或LRAT CRBP-I 41。几乎所有的视黄醇被绑定到血液中的限制性商业惯例和有没有可检测频率Ë视黄醇。 RBP受体的主要功能是促进维生素A的释放维生素A摄取的holo-RBP在全息-RBP 41。如果人工释放或视黄醇是游离形式与45,46开始，RBP受体是不需要的。获得免费的视黄醇扩散法相比，分析的基础上正确处理的holo-RBP说明，正确的准备RBP显着不同的结果，它的功能分析是至关重要的。

RBP可以41从人血清中纯化的，但是该过程是复杂的和产率是低的。另一种方法是在大肠杆菌中产生RBP 大肠杆菌。因为E.大肠杆菌不正确折叠与多于一对RBP二硫键，如哺乳动物的分泌蛋白的能力，它是必不可少的正确折叠的蛋白质再折叠限制性商业惯例和纯化。错误折叠的蛋白质不仅有不同的行为矫正折叠RBP在不同的检测，但也导致蛋白质聚集在储存过程中。出于同样的原因，APO-RBP只生产高品质的holo-RBP。我们在这里介绍一种优化方案，以生产高品质的RBP与视黄醇通过细菌表达，复性和高效液相色谱（HPLC）分离，100％负载。高效液相色谱（HPLC）分离，不仅能消除错误折叠的RBP，但也明显受到细菌污染，可能会导致严重的文物，如果RBP使用的信号转导实验。我们还描述了两个敏感的实时监测技术研究视黄醇的运输STRA6。这两种技术都依赖于高品质的RBP。由于空间的限制，放射性维甲酸和HPLC-维生素A吸收分析的经典方法是没有描述在这里。

Protocol

1。生产，复性，HPLC纯化全新RBP 的pET3a载体，6X His标签上的N-末端的cDNA为人类RBP变换的BL-21cells与。生长转化BL-21细胞在40ml与羧苄青霉素的LB培养基在37℃下摇床直到OD在600nm处达到0.5。诱导RBP蛋白表达加入IPTG至1mm。长出的细菌在37℃下另外5小时。 RBP在大肠杆菌中产生的主要是存在于包涵体。为了丰富包涵体在10,000 xg离心20分钟，沉淀细胞。然后超声处理的细胞在5毫升的PBS在冰…

Representative Results

我们目前在这里代表的holo-RBP生产和净化高效液相色谱法（ 图1），视黄醇STRA6催化释放的holo-RBP和维生素A装到APO-RBP（ 图2）和实时分析的实时分析结果STRA6催化视黄醇运输全息-RBP EGFP-CRBP-I（ 图3）。 没有重折叠，RBP在细菌中产生的几乎完全是由于存在许多不正确的二硫键错折叠。因此，复性的配位体的存在下，视黄醇中是极为重要的，在…

Discussion

我们在这里分享一个优化的RBP生产协议，因为RBP生产和纯化过程是产生正确折叠RBP的关键。由于错误折叠RBP物种和即使在HPLC纯化的细菌产生的RBP的细菌蛋白质的存在下痕量的可能性，它是有帮助使用本机RBP从血清，确认有关RBP的结论。尿RBP，市场上可买到的，是一种复杂的混合物许多种RBP，包括APO-RBP和全息-RBP 48,49。

这里所描述的实时监测技术基础上的自身荧光的视…

Divulgations

The authors have nothing to disclose.

Acknowledgements

由美国国立卫生研究院授予R01EY018144支持。

Materials

Name of Reagent/Material Company Catalog Number Comments
guanidine hydrochloride EMD 5010
cystine Sigma C8755
cysteine Sigma C7352
EDTA Fisher BP118-500
Tris Fisher 7786-1
DTT EMD 3860
retinol Sigma R7632
carbenicillin Fisher BP2648-5
IPTG EMD 5810
PBS EMD 6508
NaCl Fisher BP358-10
Ni-NTA Qiagen 1018244
imidazole EMD 5720
heptane EMD HX0295-1
Blocker Casein Pierce 37528
Amicon Ultra 15 concentrator (MWCO 10 K) Millipore UFC901024
Microfluor-2 plate Fisher 14-245-177
Hamilton syringe Gastight #1710 Fisher 14-824-655
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References

  1. Crouch, R. K., Chader, G. J., Wiggert, B., Pepperberg, D. R. Retinoids and the visual process. Photochem. Photobiol. 64, 613-621 (1996).
  2. Travis, G. H., Golczak, M., Moise, A. R., Palczewski, K. Diseases caused by defects in the visual cycle: retinoids as potential therapeutic agents. Annu. Rev. Pharmacol. Toxicol. 47, 469-512 (2007).
  3. Napoli, J. L. Biochemical pathways of retinoid transport, metabolism, and signal transduction. Clin. Immunol. Immunopathol. 80, 52-62 (1996).
  4. Drager, U. C. Retinoic acid signaling in the functioning brain. Sci STKE. 2006, pe10 (2006).
  5. Maden, M. Retinoic acid in the development, regeneration and maintenance of the nervous system. Nat. Rev. Neurosci. 8, 755-765 (2007).
  6. Niederreither, K., Dolle, P. Retinoic acid in development: towards an integrated view. Nat. Rev. Genet. 9, 541-553 (2008).
  7. Goodman, D. S., Sporn, M. B., Boberts, A. B., Goodman, D. S. . The Retinoids. 2, 41-88 (1984).
  8. Blomhoff, R., Green, M. H., Berg, T., Norum, K. R. Transport and storage of vitamin A. Science. 250, 399-404 (1990).
  9. Newcomer, M. E., Ong, D. E. Plasma retinol binding protein: structure and function of the prototypic lipocalin. Biochim. Biophys. Acta. 1482, 57-64 (2000).
  10. Quadro, L., Hamberger, L., Colantuoni, V., Gottesman, M. E., Blaner, W. S. Understanding the physiological role of retinol-binding protein in vitamin A metabolism using transgenic and knockout mouse models. Mol. Aspects Med. 24, 421-430 (2003).
  11. Heller, J. Interactions of plasma retinol-binding protein with its receptor. Specific binding of bovine and human retinol-binding protein to pigment epithelium cells from bovine eyes. J. Biol. Chem. 250, 3613-3619 (1975).
  12. Bok, D., Heller, J. Transport of retinol from the blood to the retina: an autoradiographic study of the pigment epithelial cell surface receptor for plasma retinol-binding protein. Exp. Eye Res. 22, 395-402 (1976).
  13. Rask, L., Peterson, P. A. In vitro uptake of vitamin A from the retinol-binding plasma protein to mucosal epithelial cells from the monkey’s small intestine. J. Biol. Chem. 251, 6360-6366 (1976).
  14. Heller, M., Bok, D. A specific receptor for retinol binding protein as detected by the binding of human and bovine retinol binding protein to pigment epithelial cells. Am. J. Ophthalmol. 81, 93-97 (1976).
  15. Chen, C. C., Heller, J. Uptake of retinol and retinoic acid from serum retinol-binding protein by retinal pigment epithelial cells. J. Biol. Chem. 252, 5216-5221 (1977).
  16. Bhat, M. K., Cama, H. R. Gonadal cell surface receptor for plasma retinol-binding protein. A method for its radioassay and studies on its level during spermatogenesis. Biochim. Biophys. Acta. 587, 273-281 (1979).
  17. Rask, L., Geijer, C., Bill, A., Peterson, P. A. Vitamin A supply of the cornea. Exp. Eye Res. 31, 201-211 (1980).
  18. Torma, H., Vahlquist, A. Vitamin A uptake by human skin in. 276, 390-395 (1984).
  19. Torma, H., Vahlquist, A. Uptake of vitamin A and retinol-binding protein by human placenta in vitro. Placenta. 7, 295-305 (1986).
  20. Pfeffer, B. A., Clark, V. M., Flannery, J. G., Bok, D. Membrane receptors for retinol-binding protein in cultured human retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 27, 1031-1040 (1986).
  21. Eriksson, U., et al. Increased levels of several retinoid binding proteins resulting from retinoic acid-induced differentiation of F9 cells. Cancer Res. 46, 717-722 (1986).
  22. Ottonello, S., Petrucco, S., Maraini, G. Vitamin A uptake from retinol-binding protein in a cell-free system from pigment epithelial cells of bovine retina. Retinol transfer from plasma retinol-binding protein to cytoplasmic retinol-binding protein with retinyl-ester formation as the intermediate step. J. Biol. Chem. 262, 3975-3981 (1987).
  23. Sivaprasadarao, A., Findlay, J. B. The interaction of retinol-binding protein with its plasma-membrane receptor. Biochem. J. 255, 561-569 (1988).
  24. Sivaprasadarao, A., Findlay, J. B. The mechanism of uptake of retinol by plasma-membrane vesicles. Biochem. J. 255, 571-579 (1988).
  25. Shingleton, J. L., Skinner, M. K., Ong, D. E. Characteristics of retinol accumulation from serum retinol-binding protein by cultured Sertoli cells. Biochimie. 28, 9641-9647 (1989).
  26. Sivaprasadarao, A., Findlay, J. B. Structure-function studies on human retinol-binding protein using site-directed mutagenesis. Biochem. J. 300 (Pt. 2), 437-442 (1994).
  27. Melhus, H., Bavik, C. O., Rask, L., Peterson, P. A., Eriksson, U. Epitope mapping of a monoclonal antibody that blocks the binding of retinol-binding protein to its receptor. Biochem. Biophys. Res. Commun. 210, 105-112 (1995).
  28. Smeland, S., et al. Tissue distribution of the receptor for plasma retinol-binding protein. Biochem. J. 305 (Pt. 2), 419-424 (1995).
  29. Sundaram, M., Sivaprasadarao, A., DeSousa, M. M., Findlay, J. B. The transfer of retinol from serum retinol-binding protein to cellular retinol-binding protein is mediated by a membrane receptor. J. Biol. Chem. 273, 3336-3342 (1998).
  30. Vogel, S., et al. Retinol-binding protein-deficient mice: biochemical basis for impaired vision. Biochimie. 41, 15360-15368 (2002).
  31. Liden, M., Eriksson, U. Development of a versatile reporter assay for studies of retinol uptake and metabolism in vivo. Exp. Cell Res. 310, 401-408 (2005).
  32. Kanai, M., Raz, A., Goodman, D. S. Retinol-binding protein: the transport protein for vitamin A in human plasma. J. Clin. Invest. 47, 2025-2044 (1968).
  33. Rask, L., et al. The retinol-binding protein. Scand. J. Clin. Lab Invest. Suppl. 154, 45-61 (1980).
  34. Monaco, H. L., Rizzi, M., Coda, A. Structure of a complex of two plasma proteins: transthyretin and retinol-binding protein. Science. 268, 1039-1041 (1995).
  35. Zanotti, G., Berni, R. Plasma retinol-binding protein: structure and interactions with retinol, retinoids, and transthyretin. Vitam. Horm. 69, 271-295 (2004).
  36. Kawaguchi, R., et al. A membrane receptor for retinol binding protein mediates cellular uptake of vitamin A. Science. 315, 820-825 (2007).
  37. Isken, A., et al. RBP4 Disrupts Vitamin A Uptake Homeostasis in a STRA6-Deficient Animal Model for Matthew-Wood Syndrome. Cell Metab. 7, 258-268 (2008).
  38. Golczak, M., et al. Metabolic basis of visual cycle inhibition by retinoid and nonretinoid compounds in the vertebrate retina. J. Biol. Chem. 283, 9543-9554 (2008).
  39. Kawaguchi, R., Yu, J., Wiita, P., Honda, J., Sun, H. An essential ligand-binding domain in the membrane receptor for retinol-binding protein revealed by large-scale mutagenesis and a human polymorphism. J. Biol. Chem. 283, 15160-15168 (2008).
  40. Kawaguchi, R., Yu, J., Wiita, P., Ter-Stepanian, M., Sun, H. Mapping the membrane topology and extracellular ligand binding domains of the retinol binding protein receptor. Biochimie. 47, 5387-5395 (2008).
  41. Kawaguchi, R., et al. Receptor-mediated cellular uptake mechanism that couples to intracellular storage. ACS Chem. Biol. 6, 1041-1051 (2011).
  42. Kawaguchi, R., Zhong, M., Kassai, M., Ter-Stepanian, M., Sun, H. STRA6-Catalyzed Vitamin A Influx, Efflux and Exchange. J. Membr. Biol. 245, 731-745 (2012).
  43. Ruiz, A., et al. Retinoid content, visual responses and ocular morphology are compromised in the retinas of mice lacking the retinol-binding protein receptor, STRA6. Invest. Ophthalmol. Vis. Sci. 53, 3027-3039 (2012).
  44. Berry, D. C., Jin, H., Majumdar, A., Noy, N. Signaling by vitamin A and retinol-binding protein regulates gene expression to inhibit insulin responses. Proc. Natl. Acad. Sci. U.S.A. 108, 4340-4345 (2011).
  45. Berry, D. C., O’Byrne, S. M., Vreeland, A. C., Blaner, W. S., Noy, N. Cross Talk between Signaling and Vitamin A Transport by the Retinol-Binding Protein Receptor STRA6. Mol. Cell Biol. 32, 3164-3175 (2012).
  46. Berry, D. C., Croniger, C. M., Ghyselinck, N. B., Noy, N. Transthyretin blocks retinol uptake and cell signalling by the holo-retinol-binding protein receptor STRA6. Mol. Cell Biol. , (2012).
  47. Miyawaki, A., et al. Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature. 388, 882-887 (1997).
  48. Peterson, P. A., Berggard, I. Isolation and properties of a human retinol-transporting protein. J. Biol. Chem. 246, 25-33 (1971).
  49. Rask, L., Vahlquist, A., Peterson, P. A. Studies on two physiological forms of the human retinol-binding protein differing in vitamin A and arginine content. J. Biol. Chem. 246, 6638-6646 (1971).
  50. Koutalos, Y. Measurement of the mobility of all-trans-retinol with two-photon fluorescence recovery after photobleaching. Methods Mol. Biol. 652, 115-127 (2010).
  51. Koutalos, Y., Cornwall, M. C. Microfluorometric measurement of the formation of all-trans-retinol in the outer segments of single isolated vertebrate photoreceptors. Methods Mol. Biol. 652, 129-147 (2010).
  52. Peterson, P. A., Rask, L. Studies on the fluorescence of the human vitamin A-transporting plasma protein complex and its individual components. J. Biol. Chem. 246, 7544-7550 (1971).
  53. Futterman, S., Heller, J. The enhancement of fluorescence and the decreased susceptibility to enzymatic oxidation of retinol complexed with bovine serum albumin, beta-lactoglobulin, and the retinol-binding protein of human plasma. J. Biol. Chem. 247, 5168-5172 (1972).

Tags

Retinol TransportPlasma Retinol Binding ProteinHolo-RBPSTRA6Real-time AnalysisElectrophysiologyCRBP-IVitamin A Transport Mechanism
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Kawaguchi, R., Zhong, M., Sun, H. Real-time Analyses of Retinol Transport by the Membrane Receptor of Plasma Retinol Binding Protein. J. Vis. Exp. (71), e50169, doi:10.3791/50169 (2013).
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