Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation

Jun Imai; Mayu Otani; Takahiro Sakai; Shinichi Hatta

doi:10.3791/55949

JoVE Journal > Immunology and Infection

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İmmünoloji ve Enfeksiyon

纯化膜隔间跨演示文稿中的外源抗原内质网关联降解

Published: August 21, 2017

doi:

10.3791/55949

Jun Imai¹, Mayu Otani¹, Takahiro Sakai¹, Shinichi Hatta¹

¹Laboratory of Physiological Chemistry, Faculty of Pharmacy,Takasaki University of Health and Welfare

Özet

这里介绍的方法是一种新的囊泡隔离协议，允许纯化细胞的车厢外源性抗原处理由内质网关联退化跨演示文稿中的地方。

Abstract

树突状细胞 (Dc) 是高度能够处理和后主要组织相容性类 (MHC) 的内化外源抗原呈递我分子也被称为跨演示文稿 (CP)。CP 扮演重要的角色，不仅在天真 CD8 的刺激⁺ T 细胞和 CD8 记忆⁺ T 细胞的传染性，而且在自立式灭活肿瘤免疫 t 细胞的 T 细胞无能或 T 细胞删除。虽然 CP 的关键分子机制仍有待澄清，越来越多的证据表明外源抗原进行处理通过内质网关联降解 (ERAD) 后从非古典出口内吞隔间。直到最近，刻画这些细胞内吞的车厢都有限，因为有没有除了外源性抗原的特异分子标记。这里介绍的方法是一种新的囊泡隔离协议，允许纯化这些细胞内吞的车厢。我们使用此纯化的微粒，重组 ERAD 像运输、泛素化和处理的外源性抗原体外，暗示泛素-蛋白酶体系统后从这出口加工外源性抗原细胞的隔间。该协议可以进一步应用于其他细胞类型以澄清 CP 的分子机制。

Introduction

MHC 我用内源性抗原，由泛素-蛋白酶体系统在胞浆¹处理短抗原肽表达上所有的有核细胞表面的分子。经处理后，抗原肽被送入内质网 (ER) 腔通过肽转运蛋白水龙头。在 ER 腔，一系列特定的分子伴侣的协助肽加载和正确折叠的 MHC I 复合。这一系列的分子称为肽加载复杂 (PLC)，指示 ER 车厢中部为肽加载在 MHC²。后加载，多肽 MHC 我分子被运送到细胞表面，在发挥关键作用自适应免疫系统自我标记，以及使 CD8⁺细胞毒性 T 淋巴细胞 (Ctl) 来检测癌细胞或传染性病原体的抗原从非自我蛋白质³肽。

在抗原提呈细胞 (APC)，从外源抗原的抗原肽也出示 MHC 我⁴^，^，⁵⁶^，⁷^，⁸ 通过CP，其中主要进行由 DCs⁹^，^，¹⁰¹¹。CP 是必不可少的两个为激活天真 CD8⁺ T 细胞和 CD8 记忆⁺ T 细胞抗感染和抗肿瘤 Ctl¹²^，¹³，和在维持免疫耐受的失活自署理幼稚 T 细胞¹⁴^，¹⁵。CP 许多重要作用中自适应的免疫系统，然而 CP 的分子机制尚未予以详细说明。以前对 CP 的研究揭示了外源性抗原被本地化在急诊室和内体和通过 ERAD，暗示外源抗原从内体运到 ERAD 样加工和肽加载^{16 ER 进行处理}.然而，越来越多的证据表明 CP 的肽加载进行不在急诊室，而在于非经典的内吞车厢，也有特色的 ER (图 1)¹⁷^，¹⁸ ^，¹⁹^，^，²⁰²¹。通过来避免退化的抗原肽前体肽氨基肽酶活性较高²²胞液、加工和多肽在 CP 中加载发生在近端地区的这些非经典的内吞车厢 (图 1)。虽然这些细胞内吞的车厢的刻画是有争议的没有现有的特定分子，除了这个包间的外源性抗原。

ERAD 是细胞通路，专门从急诊室中移除错误折叠的蛋白质。ERAD 通路中错误折叠的蛋白是逆行通过对细胞质 ER 膜运输和处理由泛素-蛋白酶体系统²³^，^，²⁴²⁵。大分子，比如蛋白质，运输时通过脂质双分子层，这些分子通过分子的装置称为解，如 Sec61 复杂和德林在 ER²⁶，和汤姆复杂复杂和蒂姆在复杂线粒体²⁷。当外部添加抗原是通过内质网膜，他们必须穿透脂质双分子层中复杂的 translocons，例如 Sec61 复杂。这里介绍的方法通过利用这些膜穿透的分子标志物在细胞内吞的隔间作为纯化有针对性的囊泡。

这里介绍的方法是一种新的使用作为外源性抗原的树突状细胞线 DC2.4²⁸和生物素化的卵清蛋白（宝物）的囊泡净化协议。内吞的车厢进行纯化的链霉亲和 (SA)-磁珠作为制造商使用膜穿透宝物。在这纯净的微粒，一些外部添加宝物仍保持膜组分中但被运到外面的微粒体，然后泛素化和处理体外²⁹。这纯净的微粒体载不仅吞隔间特异性蛋白二村居民蛋白质 ERAD 和肽加载复杂;建议细胞隔间 CP²⁹前瞻性吞车厢。本议定书依赖外源性抗原，这种并不是也适用于其他 DC 亚群和其他细胞类型，如巨噬细胞、 B 细胞和内皮细胞，以澄清精通 CP DCs 的精确分子机制。

Protocol

1.生长细胞与外源性抗原另外准备宝物使用生物素蛋白标记试剂盒随着制造商 ' s 协议。注意：通常，宝物平均包含 2 M 生物素每 1 M OVA。成长 DC2.4 细胞在 RPMI 1640 辅以 2 毫米 L-谷氨酰胺、丙酮酸钠 1 毫米、 0.1 毫米非必需氨基酸，100 U/mL 青霉素-链霉素、 2-巯基乙醇 55 毫米，10 毫米复 (pH 7.5)，10%胎牛血清 (以下简称 RPMI) 在 37 ° C 在 5%CO 2 在湿的孵化器（以下简称?…

Representative Results

为了阐明 CP 的分子机制，是有必要确定细胞的隔间，外源抗原在哪里接受 ERAD 样运输和加工。虽然通过免疫荧光显微镜或电子显微镜的观察确定外源抗原在那里积累16，17，，1819，细胞室 30，，31<s…

Discussion

在以往研究中的 CP，注册外源抗原在禁区的晚体膜或 ER 通过免疫荧光显微镜¹⁶^，³⁰^，^，³¹³²积累。据估计在这些专业领域的 ER 或晚体膜进行 ERAD 样运输和加工的外源性抗原细胞室是由蔗糖或碘密度梯度离心法分离使用标识泛素化作为一种 ERAD 样处理标记的宝物。后刺激他们先天免疫，CP 效率显著提高，…

Açıklamalar

The authors have nothing to disclose.

Acknowledgements

这项工作被支持由高崎大学健康和福利。

Materials

RPMI 1640	gibco by life technologies	11875-093
Fetal bovine serum	Equitech bio	SFB30
Sodium pyruvate	gibco by life technologies	11360-070
MEM non-essential amino acids	gibco by life technologies	11140-050
HEPES	gibco by life technologies	15630-080
2-mercaptoethanol	gibco by life technologies	21985-023
L-glutamine	gibco by life technologies	25030-164
Penisicillin-Sreptomycin	gibco by life technologies	15140-122
DMEM	gibco by life technologies	12100-46
OVA	SIGMA	A5503
Biotin-protein labelling kit	Thermo Fisher Scientific	F6347
MG-132	Santa Cruz Biotechnology	201270
lactacystin	SIGMA	L6785
Dounce homogenizer	IUCHI	131703
protease inhibitor cocktails	SIGMA	P8340
iodixanol	Cosmo bio	1114542
SA-magnetic beads	New England Biolabs	201270
control magnetic beads	Chemagen	M-PVA012
magnetic stand	BD Biosciences	552311
BCA protein assay kit	Thermo Fisher Scientific	23225
silver staining kits	Cosmo bio	423413
Reticulocyte Lysate	Promega	1730714
Flag-tagged ubiquitin	SIGMA	U5382
anti-ovalbumin (OVA,mouse)	Antibody Shop	HYB 094-06
ant-multi-ubiquitin (mouse)	MBL	D058−3
anti-Flag (mouse)	SIGMA	F3165
trypsin	SIGMA	85450C

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Etiketler

Membrane Compartment Endoplasmic Reticulum-associated Degradation (ERAD)Cross-presentation Exogenous Antigen Dendritic Cells Biotinylated Ovalbumin (bOVA)Vesicle Isolation Microsome Ubiquitination

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Imai, J., Otani, M., Sakai, T., Hatta, S. Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation. J. Vis. Exp. (126), e55949, doi:10.3791/55949 (2017).