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Jul 2019

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Site-specific Labeling of B Cell Receptor and Soluble Immunoglobulin
B细胞受体和可溶性免疫球蛋白的定位标记   

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Abstract

B lymphocyte activation is regulated by its membrane-bound B cell receptors (BCRs) upon recognizing diverse antigens. It is hypothesized that antigen binding would trigger conformational changes within BCRs, followed by a series of downstream signaling activation. To measure the BCR conformational changes in live cells, a fluorescent site-specific labeling technique is preferred. Genetically encoded fluorescent tags visualize the location of the target proteins. However, these fluorescent proteins are large (~30 kDa) and would potentially perturb the conformation of BCRs. Here, we describe the general procedures of utilizing short tag-based site-specific labeling methodologies combining with fluorescence resonance energy transfer (FRET) assay to monitor the conformational changes within BCR extracellular domains upon antigen engagement.

Keywords: B lymphocytes (B淋巴细胞), BCR (B细胞受体), Conformational changes (构象变化), Site-specific labeling (定位标记)

Background

B lymphocytes are responsible for the production of protective antibodies against pathology arising from the recognition of antigens by the cell membrane expressed B cell receptors (BCRs). BCR complex comprises a membrane-bound immunoglobulin (mIg) and a non-covalently linked heterodimer of Igα and Igβ. The mIg is composed of two surrogate light chains and two immunoglobulin heavy chains. The mIg heavy chain contains the extracellular domain, the transmembrane domain, and the intracellular domain. At the N-terminal domain of extracellular mIg, there are two variable antigen-binding motifs, following which are the constant domains (Reth, 1992). In terms of the heavy chain of IgM-BCR, it includes 4 domains, Cμ1, Cμ2, Cμ3, and Cμ4 (Figure 1). Structurally variable domains in the heavy and light chain polypeptides form an antigen-binding site unique to the antibody. For example, VRC01 broadly neutralizing antibodies (bnAbs) target the CD4-binding site (CD4BS) of the human immunodeficiency virus-1 (HIV-1) envelope glycoprotein 120 (gp120), neutralize over 90% of circulating HIV-1 isolates (Wu et al., 2010). BCRs expressing variable domains of VRC01 would recognize the gp120 and regulate B cell activation. In the BCR complex, disulfide-linked Igα/Igβ are the signaling subunits, contain immunoreceptor tyrosine activation motif (ITAM) in their intracellular domains. Antigen binding to BCR induces ITAM phosphorylation by tyrosine-protein kinase Lyn followed by subsequent recruitments and phosphorylation of the second kinase, spleen tyrosine kinase (Syk), resulting in the onset of several downstream signal transduction pathways altering gene expression to potentiate the immune mechanisms (Xu et al., 2014).

It is assumed that the antigen engagement induces conformational changes within the BCR complex to initiate B cell activation (Harwood and Batista, 2010; Pierce and Liu, 2010). However, it is technically difficult to capture the conformational information of BCR extracellular domains accurately during the transmembrane initiation of BCR activation. Fluorescence resonance energy transfer (FRET) is a powerful tool for analyzing the statics and dynamics of protein structures by introducing light-sensitive donor and acceptor. Usually, to acquire the location of target proteins in live cells, genetically encoded tags such as green fluorescent protein (GFP) are widely used. However, such fluorescent proteins are too large (~30 kDa) to show the conformational changes of BCRs (~150 kDa). Moreover, the presence of these proteins within BCR fragments may perturb the activity, localization, and functions of BCRs. To overcome this, we introduced short tag-based site-specific labeling techniques to label different domains of BCRs fluorescently (Shen et al., 2019). We constructed various BCR complexes with ybbR tag and tetracysteine tag inserted that permit targeted incorporation of pre-defined fluorophores. In terms of ybbR tag-based labeling, Sfp phosphopantetheinyl transferase covalently transfers 4′-phosphopantetheinyl (Ppant) groups from CoA to conserved serine residues on peptidyl carrier protein (PCP) and acyl carrier protein (ACP) domains (Lambalot et al., 1996). Sfp and AcpS have been widely used for site-specific protein labeling in cell lysates or on live-cell surfaces by fusing PCP or ACP at either the N or the C terminus of the target protein, and Sfp can enzymatically attach a small molecular probe, such as biotin, fluorophores, sugars, and peptides, to the PCP or ACP tag (George et al., 2004; Yin et al., 2004a and 2004b). Most recently, the ybbR tag, a short peptide (DSLEFIASKLA), was found to be an efficient substrate for Sfp-catalyzed protein labeling, thereby replacing the PCP or ACP domain for the construction of fusions of the target protein (Yin et al., 2006). And these fusions also can be labeled with probes by Sfp, further expanding the versatility of Sfp mediated site-special labeling system.

Another short tag site-specific labeling strategy used in this protocol is tetracysteine tag technology. This method is based on the binding of a small fluorescein derivative, fluorescein arsenical hairpin binder (FlAsH) to an optimized short peptide sequence (CCPGCC). Subsequently, flanking sequences were optimized and introduced as part of the tag (FLNCCPGCCMEP and HRWCCPGCCKTF) (Martin et al., 2005). FlAsH emits green-yellow fluorescence, while the color variant, resorufin arsenical hairpin binder (ReAsH) fluorescence peaks in the red (609 nm), would serve as a FRET acceptor for green fluorescent signal, such as Coenzyme A (CoA)-488. FlAsH and ReAsH are used as the nonfluorescent complex with ethanedithiol (EDT) and become fluorescent on binding to tetracysteine tag. Tetracysteine tag technology has been used in FRET studies, allowing the investigation of conformational changes in target proteins, such as AMPA receptors (Morishita et al., 2004), calmodulin (Chen et al., 2005), adrenergic receptor (Zürn et al., 2009), and HIV Gag proteins (Turville et al., 2008). In this chapter, we describe the procedures to label the BCRs and soluble Ig by inserting the ybbR tag and tetracysteine tag in BCR molecule, as well as the usage of necessary components to set up the site-special labeling system. This labeling protocol proceeds with high efficiency and can be easily carried out in living cells.

Materials and Reagents

  1. Glass coverslips (VWR International, catalog number: 16004-094 )
  2. 8-well chamber frame (Nunc Lab-Tek chamber, Thermo Fisher, catalog number: 155411PK )
  3. Six-well plate (NEST, catalog number: 703001 )
  4. Sfp phosphopantetheinyl transferase (NEB, catalog number: P9302S )
  5. Coenzyme A (CoA)-488 (NEB, catalog number: S9348 )
  6. 4,5-bis(1,3,2-dithiarsolan-2-yl)resorufin (ReAsH-EDT2, Santa Cruz, catalog number: sc-391916 )
  7. TCEP (tris(carboxyethyl) phosphine) (Sigma, catalog number: C4706 )
  8. X-tremeGENE HP DNA Transfection Reagent (Roche, catalog number: 0 6366236001 )
  9. MES (2-mercaptoethanesulfonate) (Macklin, catalog number: S818461 )
  10. BAL (2,3-dimercaptopropanol) (Macklin, catalog number: D807026 )
  11. Alexa Fluor 647 AffiniPure Fab Fragment Goat Anti-Human IgM, Fc5μ fragment specific (Jackson ImmunoResearch, catalog number: 109-607-043 )
  12. HBSS (Hank's Balanced Salt Solution) (Gibco, catalog number: 14025092 )
  13. Zeba Spin Desalting Columns (Thermo Fisher, catalog number: 89882 )
  14. SYLGARDTM 164 Silicone Elastomer Kit 210 ML KIT (Dow Corning, catalog number: 4028273 )

Equipment

  1. Fluoview FV1000 Laser Scanning Confocal Microscope (Olympus)

Procedure

  1. Site-specific labeling of VRC01-IgM-BCR expressed in 293T cell
    1. The constant region of human IgM heavy chain was fused with VRC01-specific variable region of heavy chain to construct mIg heavy chain of VRC01-mIgM-BCR (pHAGE-VRC01-H), while human Igκ constant region was fused with VRC01-specific variable region of light chain to construct mIg light chain of VRC01-mIgM-BCR (pHAGE-VRC01-L).
    2. A similar strategy was used for the construction of Ig heavy chain and Ig light chain of soluble VRC01-IgM. In addition, His6 tag was fused at C terminus of Ig heavy chain of soluble VRC01-IgM for protein purification (pHAGE-VRC01-H-His6).
    3. ybbR tag and tetracysteine tag were inserted in plasmid carrying mIg heavy chain of VRC01-IgM-BCR to construct dually tagged VRC01-mIgM-BCR (pHAGE-VRC01-H-Tag), while ybbR tag and tetracysteine tag were inserted in plasmid carrying Ig heavy chain of soluble VRC01-IgM to construct dually tagged soluble VRC01-IgM (pHAGE-VRC01-H-His6-Tag). Insertion sites for the two tags are indicated in bold in the following table (Table 1).
      All the plasmids are not commercially available. Please find the construction strategy illustrated in Figure 1. The DNA sequence of VRC01 variable region, ybbR tag, and tetracysteine tag are listed in Table 2. All the plasmid constructions were performed following Gibson assembly protocol (Gibson et al., 2009). Protein sequences are listed in Table 3.


      Figure 1. Schematic figures of the construction strategies on mIgM-BCR structure and tagged VRC01-mIgM-BCR/soluble VRC01-IgM construction

      Table 1. Insertion sites for the ybbR tag or tetracysteine tag in IgM-BCR


      Table 2. DNA sequences for plasmids construction


      Table 3. Amino acid sequences of the constructions


  2. An overall scheme of site-specific labeling procedures can be found in Figure 2.


    Figure 2. Overall scheme of site-specific labeling procedures for VRC01-IgM-BCR expressed in 293T cells

    1. Plate 293T cell in 6-well plate with DMEM containing 10% FBS and 1% penicillin-streptomycin reaching 50-60% confluency.
    2. For each 35-mm well in a six-well plate, prepare one vial of transfection medium containing 15 μl of X-tremGENE transfection reagent and 3.5 μg of the pHAGE-VRC01-H/pHAGE-VRC01-H-Tag, 2.5 μg pHAGE-VRC01-L, 2.5 μg pHAGE-CD79A and 2.5 μg pHAGE-CD79B plasmid diluted in 300 μl of opti-MEM (1×).
    3. Mix the transfection medium well and allow it to come to equilibrium for 30 min at room temperature.
    4. Incubate the cells with transfection medium for 5 h, and then incubate the cells in DMEM medium (1×) with penicillin and streptomycin for 24 h to allow time for protein expression.
    5. For ybbR tag labeling, detach the cells expressing dually tagged or untagged VRC01-IgM-BCR with trypsin and incubate them with 1 μM SFP Synthase, 2 μM CoA 488 and 10 mM MgCl2 in 500 μl HBSS for 20 min at room temperature, then wash the cells with HBSS.
    6. Labeling of tetracysteine tag in cells is adopted from published protocols (Hoffmann et al., 2010). Pre-incubate the cells in HBSS containing 5 mM MES and 0.5 mM TCEP for 20 min at room temperature, then wash with HBSS and stain 1 mM ReAsH-EDT2 and 25 mM 2,3-dimercapto-1-propanol in 500 μl HBSS for 10 min at 4 °C. After staining, wash the cells with HBSS containing 100 mM BAL.
    7. Stain the cells with 100 nM Alexa Fluor 647 AffiniPure Fab Fragment Goat Anti-Human IgM Fc5μ for 5 min at 4 °C in 500 μl HBSS, then wash twice with HBSS. Cells are ready for imaging.

  3. Site-specific labeling of soluble VRC01-IgM
    1. 293F cells were co-transfected with plasmid carrying dually tagged (pHAGE-VRC01-H-His6-Tag) or untagged Ig heavy chain of VRC01-IgM (pHAGE-VRC01-H-His6) and mIg light chain of VRC01-IgM (pHAGE-VRC01-L).
      Note: Please refer to B1 to B4 for transfection protocols.
    2. Ectopically express and purify the IgM protein in HEK293F cells according to standard protocols (Portolano et al., 2014).
    3. For ybbR tag labeling in soluble VRC01-IgM, purified proteins were incubated in HBSS containing 1 mM SFP synthase, 10 mM MgCl2, 5 mM CoA 488, 10 mM HEPES for 30 min at 37 °C. Then the labeled proteins were desalted using Zeba Spin Desalting Columns.
    4. For tetracysteine tag labeling, soluble VRC01-IgMs were treated with 1 mM ReAsH-EDT2 in HBSS at room temperature for 30 min and were desalted using Zeba Spin Desalting Columns according to standard protocol. In short, after removing the column’s bottom closure and loosen cap, centrifuge at 1,500 × g for 1 min to remove storage solution. Slowly apply 30-130 µl of the protein sample to the center of the compacted resin bed, followed with centrifuge at 1,500 × g for 2 min to collect the desalted sample.

  4. Imaging of labeled VRC01-IgM-BCR expressing 293T cells or labeled soluble VRC01-IgM
    1. Chamber coverslip was fabricated according to our previous publication (Wang et al., 2018). In short, the coverslip was pretreated with piranha buffer (H2SO4:H2O2 = 7:3) for overnight, after extensively washing with ddH2O, the coverslip was glued to the 8-well chamber frame with SYLGARDTM 164. After curing, the chamber coverslip was ready to use.
    2. Labeled 293T cells expressing dually tagged or untagged VRC01-IgM-BCR were resuspended in HBSS and then loaded on chamber coverslip.
    3. Labeled dually tagged or untagged soluble VRC01-IgMs were loaded on coverslip for 30 min at 4 °C to allow the adherence on the surface. Then soluble VRC01-IgMs were stained with 100 nM Alexa Fluor 647 AffiniPure Fab Fragment Goat Anti-Human IgM Fc5μ in HBSS for 10 min at 4 °C, followed by washing with HBSS.
    4. Confocal images were acquired by Fluoview FV1000 Laser Scanning Confocal Microscope with 60× 1.42 NA oil objective lens. Typical images were shown in Figure 3.
    5. All the images were analyzed with ImageJ.


      Figure 3. Site-specific labeling in mIg heavy chain of IgM-BCR. Representative confocal images of tagged (A) and untagged VRC01-IgM-BCR (B) expressed in 293T cells. ybbR tag and tetracysteine tag were labeled by CoA 488 and ReAsH, respectively. Alexa Fluor 647 (AF647) Fab fragment of goat anti-human IgM Fc5μ was used for IgM-BCR staining. Scale bars = 10 μm.

Acknowledgments

This work has been supported by funds from National Natural Science Foundation of China (81825010, 81730043, 81621002, 81961130394 and 31811540397). It is also supported by Beijing Advanced Innovation Center for Structural Biology, Center for Life Sciences, and Institute for Immunology at Tsinghua University. This protocol was derived from previous paper published in eLife (Shen et al., 2019).

Competing interests

The authors declare no financial or commercial conflict of interest.

References

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简介

[摘要 ] 识别各种抗原后,B淋巴细胞的活化受其膜结合B细胞受体(BCR)的调节。假设抗原结合将触发BCR内的构象变化,随后引发一系列下游信号激活。为了测量活细胞中的BCR构象变化,首选荧光位点特异性标记技术。遗传编码的荧光标签可视化目标蛋白的位置。但是,这些荧光蛋白很大(〜30 k Da ),可能会干扰BCR的构象。在这里,我们描述了利用基于短标签的位点特异性标记方法与荧光共振能量转移(FRET)分析相结合来监视BCR细胞外域内抗原结合时构象变化的一般程序。

[背景 ] B淋巴细胞是负责产生针对病理从识别由细胞膜抗原所产生的保护性抗体的表达的B细胞受体(BCRS)。BCR复合物包含膜结合免疫球蛋白(mIg )和Igα和Igβ的非共价连接的异二聚体。所述mIg的由两个替代轻链和两个免疫球蛋白重链。所述mIg的重链含有胞外结构域,跨膜结构域,和细胞内结构域。在胞外mIg 的N-末端结构域,有两个可变的抗原结合基序,其后是恒定结构域(Reth,1992)。就IgM-BCR的重链而言,它包含4个域,即Cμ1,Cμ2,Cμ3和Cμ4(图1)。重链和轻链多肽中的结构可变域形成了抗体特有的抗原结合位点。例如,VRC01广泛中和抗体(bnAbs )靶向人类免疫缺陷病毒1(HIV-1)包膜糖蛋白120(gp120)的CD4结合位点(CD4BS),中和超过90%的循环HIV-1分离株(Wu 等人,2010)。表达VRC01可变域的BCR将识别gp120并调节B细胞活化。在BCR复合物中,二硫键连接的Igα/Igβ是信号传导亚基,在其细胞内结构域中包含免疫受体酪氨酸激活基序(ITAM)。抗原与BCR结合会通过酪氨酸蛋白激酶Lyn诱导ITAM磷酸化,随后第二个激酶脾酪氨酸激酶(Syk )的募集和磷酸化,导致多个下游信号转导途径的发生,从而改变基因表达以增强免疫机制( Xu 等,2014)。

假定抗原参与在BCR复合物中诱导构象变化以启动B细胞活化(Harwood和Batista,2010; Pierce和Liu,2010)。但是,在BCR激活的跨膜启动过程中准确地捕获BCR细胞外域的构象信息在技术上是困难的。荧光共振能量转移(FRET)是通过引入光敏感的供体和受体来分析蛋白质结构的静态和动态的强大工具。通常,为了获得靶蛋白在活细胞中的位置,广泛使用了遗传编码的标签,例如绿色荧光蛋白(GFP)。但是,此类荧光蛋白太大(约30 kDa ),无法显示BCR的构象变化(约150 k Da )。而且,这些蛋白质在BCR片段中的存在可能会扰乱BCR的活性,定位和功能。为了克服这个问题,我们引入了基于短标签的位点特异性标记技术来荧光标记BCR的不同结构域(Shen et al。,2019)。我们构建了各种带有ybbR 标签和四半胱氨酸标签的BCR复合物,这些复合物允许定向掺入预定义的荧光团。就基于ybbR 标签的标记而言,Sfp 磷酸泛亚锡基转移酶将4'-磷酸泛亚锡基(Ppant )基团从CoA 共价转移至肽基载体蛋白(PCP)和酰基载体蛋白(ACP)域上的保守丝氨酸残基(Lambalot 等,1996)。 )。通过在目标蛋白的N或C末端融合PCP或ACP,Sfp 和AcpS 被广泛用于细胞裂解液或活细胞表面的位点特异性蛋白质标记,并且Sfp 可以酶促连接小分子探针,例如PCP或ACP标签的生物素,荧光团,糖和肽(George 等,2004; Yin 等,2004a 和2004b)。最近,发现ybbR 标签(一种短肽(DSLEFIASKLA))是Sfp 催化蛋白标记的有效底物,从而取代了PCP或ACP结构域以构建目标蛋白的融合体(Yin 等,2006)。这些融合物也可以用Sfp 标记探针,进一步扩展了Sfp 介导的位点特异性标记系统的多功能性。

此协议中使用的另一种短标签特定于站点的标记策略是四半胱氨酸标签技术。该方法基于小的荧光素衍生物荧光素砷发夹结合剂(FlAsH )与优化的短肽序列(CCPGCC)的结合。随后,优化侧翼序列并将其作为标签的一部分(FLNCCPGCCMEP和HRWCCPGCCKTF)引入(Martin 等,2005)。FlAsH 发出绿黄色荧光,而红色(609 nm)的变体,即间苯二酚砷发夹结合物(ReAsH )荧光峰,将用作绿色荧光信号的FRET受体,例如辅酶A (CoA)-488。FlAsH 和ReAsH 用作与乙二硫醇(EDT)的非荧光复合物,并在与四半胱氨酸标签结合后变为荧光。Tetracysteine 标签技术已用于FRET研究中,允许研究靶蛋白的构象变化,例如A MPA受体(Morishita 等,2004),钙调蛋白(Chen 等,2005),肾上腺素能受体(Zürn 等) 。,200 9 ),和HIV Gag的蛋白质(特维尔等人,2008) 。在本章中,我们描述了通过在BCR分子中插入ybbR 标签和四半胱氨酸标签来标记BCR 和可溶性Ig的步骤,以及使用必要的组分来建立特殊部位标记系统。该标记方案以高效率进行并且可以在活细胞中容易地进行。

关键字:B淋巴细胞, B细胞受体, 构象变化, 定位标记

材料和试剂
 
玻璃盖玻片(VWR International,目录号:16004-094) 8孔室框架(Nunc Lab-Tek室,Thermo Fisher,目录号:155411PK) 六孔板(NEST,目录号:703001) Sfp 磷酸泛肽基转移酶(NEB,目录号:P9302S) 辅酶A(CoA)-488(NEB,目录号:S9348) 4,5-双(1,3,2-二硫杂solan-2- 基)间苯二酚(ReAsH-EDT2,圣克鲁斯,目录号:sc-391916) TCEP(三(羧乙基)膦)(Sigma,目录号:C4706) X- tremeGENE HP DNA转染试剂(罗氏(Roche),目录号06366236001) MES(2-巯基乙磺酸盐)(Macklin,目录号:S818461) BAL(2,3-二巯基丙醇)(Macklin,目录号:D807026) Alexa Fluor 647 AffiniPure Fab片段山羊抗人IgM,Fc5μ片段特异性(Jackson ImmunoResearch ,目录号:109-607-043) HBSS(汉克平衡盐溶液)(Gibco,目录号:14025092) Zeba 旋转脱盐柱(Thermo Fisher,目录号:89882) SYLGARD TM 164硅胶弹性体套件210 ML KIT(Dow Corning,目录号:4028273)  
设备
 
Fluoview FV1000激光扫描共聚焦显微镜(奥林巴斯)  
程序
 
带标签的VRC01-mIgM-BCR或带标签的可溶性VRC01-IgM的构建 将人IgM重链恒定区与VRC01特异的重链融合,构建VR C01-mIgM-BCR(pHAGE-VRC01-H)的mIg 重链,而人Igκ 恒定区与VRC01特异融合。轻链可变区以构建VRC01-mIgM-BCR(pHAGE-VRC01-L)的mIg 轻链。 类似的策略被用于可溶性VRC01-IgM的Ig重链和Ig轻链的构建。另外,将His 6 标签融合在可溶性VRC01-IgM的Ig重链的C末端,用于蛋白质纯化(pHAGE-VRC01-H-His6)。 将ybbR 标签和四半胱氨酸标签插入带有VRC01-IgM-BCR的mIg 重链的质粒中,以构建双重标记的VRC01-mIgM-B CR(pHAGE-VRC01-H-Tag),而ybbR 标签和四半胱氨酸标签被插入具有VRC01-IgM-BCR的mIg 重链的质粒可溶性VRC01-IgM的Ig重链可构建双重标记的可溶性VRC01-IgM(pHAGE-VRC01-H-His6-Tag)。下表(表1)中以粗体显示了两个标签的插入位点。 所有质粒都不市售。请找到图1所示的构建策略。VRC01 可变区,ybbR 标签和四半胱氨酸标签的DNA序列列于表2。所有质粒构建均遵循Gibson组装规程(Gibson et al。,2009)。蛋白序列列于表3。
 
D:\ Reformatting \ 2020-8-3 \ 1902984--1523刘万里810635 \ Figs jpg \图1.jpg
图1. mIgM -BCR结构和标记的VRC01-mIgM-BCR /可溶性VRC01-IgM构建策略的示意图
 
表1. IgM-BCR中ybbR 标签或四半胱氨酸标签的插入位点
构造
氨基酸序列
VRC01-IgM-BCR或可溶性VRC01-IgM N末端的ybbR 标签
DSLEFIASKLA QVQLVQSGGQ
VRC01-IgM-BCR或可溶性VRC01- IgMCμ2 中的四半胱氨酸标签
VPPRDGFFGN FLNCCPGCCMEP PRKSKLICQA
 


表2. 用于质粒构建的DNA序列

DNA序列
ybbR 标签
GATTCTCTTGAATTTATTGCTAGTAAGCTTGCG
四半胱氨酸标签
TTTCTCAATTGTTGTCCTGGATGTTGTATGGAACCT
VRC01重链可变区
CAGGTGCAGCTGGTGCAGTCTGGGGGTCAGATGAAGAAGCCTGGCGAGTCGATGAGAATTTCTTGTCGGGCTTCTGGATATGAATTTATTGATTGTACGCTAAATTGGATTCGTCTGGCCCCCGGAAAAAGGCCTGAGTGGATGGGATGGCTGAAGCCTCGAGGAGGAGCCGTCAACTACGCACGTCCACTTCAGGGCAGAGTGACCATGACTCGAGACGTTTATTCCGACACAGCCTTTTTGGAGCTGCGCTCGTTGACAGTAGACGACACGGCCGTCTACTTTTGTACTAGGGGAAAAAACTGTGATTACAATTGGGACTTCGAACACTGGGGCCGGGGCACCCCGGTCATCGTCTCATCA
VRC01轻链可变区
GAAATTGTGTTGACACAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAACAGCCATCATCTCTTGTCGGACCAGTCAGTATGGTTCCTTAGCCTGGTATCAACAGAGGCCCGGCCAGGCCCCCAGGCTCGTCATCTATTCGGGCTCTACTCGGGCCGCTGGCATCCCAGACAGGTTCAGCGGCAGTCGGTGGGGGCCAGACTACAATCTCACCATCAGCAACCTGGAGTCGGGAGATTTTGGTGTTTATTATTGCCAGCAGTATGAATTTTTTGGCCAGGGGACCAAGGTCCAGGTCGACATTAAACGT
 
表3.结构的氨基酸序列
构造名称
氨基酸序列
pHAGE-VRC01-H
MDWTWRFLFVVAAATGVQSQVQLVQSGGQMKKPGESMRISCRASGYEFIDCTLNWIRLAPGKRPEWMGWLKPRGGAVNYARPLQGRVTMTRDVYSDTAFLELRSLTVDDTAVYFCTRGKNCDYNWDFEHWGRGTPVIVSSGSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTEGEVSADEEGFENLWATASTFIVLFLLSLFYSTTVTLFKVK
pHAGE-VRC01-L
METPAQLLFLLLLWLPDTTGEIVLTQSPGTLSLSPGETAIISCRTSQYGSLAWYQQRPGQAPRLVIYSGSTRAAGIPDRFSGSRWGPDYNLTISNLESGDFGVYYCQQYEFFGQGTKVQVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLYYKREAKVQQNSKSKYPSKQQNSQNS
pHAGE-VRC01-H-His6
MDWTWRFLFVVAAATGVQSQVQLVQSGGQMKKPGESMRISCRASGYEFIDCTLNWIRLAPGKRPEWMGWLKPRGGAVNYARPLQGRVTMTRDVYSDTAFLELRSLTVDDTAVYFCTRGKNCDYNWDFEHWGRGTPVIVSSGSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNA SSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTHHHHHH
pHAGE-VRC01-H-标签
MDWTWRFLFVVAAATGVQSDSLEFIASKLAQVQLVQSGGQMKKPGESMRISCRASGYEFIDCTLNWIRLAPGKRPEWMGWLKPRGGAVNYARPLQGRVTMTRDVYSDTAFLELRSLTVDDTAVYFCTRGKNCDYNWDFEHWGRGTPVIVSSGSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNFLNCCPGCCMEPPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTEGEVSADEEGFENLWATASTFIVLFLLSLFYSTTVTLFKVK
pHAGE-VRC01-H-His6-标签
MDWTWRFLFVVAAATGVQSDSLEFIASKLAQVQLVQSGGQMKKPGESMRISCRASGYEFIDCTLNWIRLAPGKRPEWMGWLKPRGGAVNYARPLQGRVTMTRDVYSDTAFLELRSLTVDDTAVYFCTRGKNCDYNWDFEHWGRGTPVIVSSGSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNFLNCCPGCCMEPPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTHHHHHH
 
在293T细胞中表达的VRC01-IgM-BCR的位点特异性标记 过度的位点特异性标记程序的所有方案,可以在图2中可以找到。
 
D:\ Reformatting \ 2020-8-3 \ 1902984--1523刘万里810635 \ Figs jpg \图2.jpg
图2. 在293T细胞中表达的VRC01-IgM-BCR 的位点特异性标记程序的总体方案
 
用含有10%FBS和1%青霉素-链霉素的DMEM在6孔板中培养293T细胞,达到50-60%融合度。 对于六孔板中的每个35毫米孔,准备一小瓶转染培养基,其中包含15 微升X- tremGENE 转染试剂和3.5 微克pHAGE-VRC01-H / pHAGE-VRC01-H-Tag,2.5 微克pHAGE -VRC01-L,2.5 微克噬菌体CD79A和2.5 微克噬菌体CD79B质粒稀释在300 微升的OPTI -MEM(1×)。 充分混合转染培养基,使其在室温下平衡30分钟。 用转染培养基孵育细胞5 h,然后将其在含有青霉素和链霉素的DMEM培养基(1x)中孵育24 h,以便有时间表达蛋白质。 对于ybbR 标签标记,分离细胞表达双重标记或未标记VRC01 IgM抗体-BCR用胰蛋白酶,并用1孵育他们μM SFP合酶,2 μM 辅酶A 488和10 毫摩尔MgCl 2 在500 微升HBSS为20分钟,在室温下,然后用HBSS洗涤细胞。 从公开的方案中采用细胞中四半胱氨酸标签的标记(Hoffmann 等,2010)。预孵育在HBSS含5mM MES和0.5mM TCEP细胞20分钟,在室温下,然后用HBSS和污渍洗涤1mM的ReAsH-EDT2和25mM 2,3-二巯基-1-丙醇在500 微升HBSS 为在4°C下10分钟。染色后,用含100 mM BAL的HBSS洗涤细胞。 在500° L HBSS中于4°C 用10 0 nM Alexa Fluor 647 AffiniPure Fab片段山羊抗人IgMFc5μl染色细胞5分钟,然后用HBSS洗涤两次。细胞已准备好成像。  
可溶性VRC01-IgM的位点特异性标记 293F细胞用带有双重标记的(pHAGE-VRC01-H-His6-Tag)或未标记的VRC01-IgM的Ig重链(pHAGE-VRC01-H-His6)和VRC01-IgM的mIg 轻链(pHAGE )共转染的质粒-VRC01-L)。 注意:有关转染方案,请参阅B1至B4。
根据标准方案(Portolano 等,2014)在HEK293F细胞中异位表达和纯化IgM蛋白。 对于ybbR 可溶性VRC01-IgM的标签标记,纯化的蛋白在含有1mM SFP合酶,HBSS中温育10毫摩尔MgCl 2 ,5mM的辅酶A 488,10mM HEPES的30分钟在37℃下。然后使用Zeba Spin脱盐柱对标记的蛋白质进行脱盐。 对于四半胱氨酸标签标记,将可溶性VRC01-IgMs在HBSS中于室温下用1 mM ReAsH-EDT2处理30分钟,并根据标准方案使用Zeba Spin脱盐柱进行脱盐。总之,在1去除塔的底部封闭和松开帽,离心后,500 ×g下进行1分钟,以去除存储解决方案。慢慢申请30-130 μ 升蛋白样品至压缩树脂床的中心,随后用离心机在1 ,500 ×g下2分钟以收集脱盐样品。  
表达标记的VRC01-IgM-BCR的293T细胞或标记的可溶性VRC01-IgM的成像 箱盖玻片是根据我们以前的出版物制作的(Wang 等,2018)。简而言之,将盖玻片用食人鱼缓冲液(H 2 SO 4 :H 2 O 2 = 7:3)预处理过夜,然后用ddH 2 O充分洗涤后,用SYLGARD 将盖玻片胶粘到8孔室框架上TM164 。固化后,即可使用腔室盖玻片。 表达双重标记或未标记的VRC01-IgM-BCR的标记293T细胞重悬于HBSS中,然后装在小室盖玻片上。 将标记的双标签或未标签的可溶性VRC01-IgMs在4°C下在盖玻片上放置30分钟,以使其粘附在表面上。然后将可溶性VRC01- IgMs用HBSS中的100 nM Alexa Fluor 647 AffiniPure Fab片段山羊抗人IgMFc5μ于4°C 染色10分钟,然后用HBSS洗涤。 通过具有60×1.42 NA油物镜的Fluoview FV1000激光扫描共聚焦显微镜获得共聚焦图像。典型图像如图3所示。 用ImageJ分析所有图像。  
D:\ Reformatting \ 2020-8-3 \ 1902984--1523刘万里810635 \ Figs jpg \图3.jpg
图3. IgM-BCR的mIg 重链中的位点特异性标记。在293T细胞中表达的标记的(A)和未标记的VRC01-IgM-BCR(B)的代表性共聚焦图像。ybbR 标签和TetR的acysteine 标签通过辅酶A 488和标记ReAsH 分别。使用山羊抗人IgMFc5μ的Alexa Fluor 647(AF647)Fab片段进行IgM-BCR染色。比例尺s = 10μm 。
 
致谢
 
这项工作得到了国家自然科学基金(81825010、81730043、81621002、81961130394和31811540397)的资助。它也得到了北京结构生物学高级创新中心,生命科学中心和清华大学免疫学研究所的支持。该协议源自eLife (Shen et al。,2019)上发表的先前论文。
 
利益争夺
 
作者声明没有经济或商业利益冲突。
 
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Copyright Wang et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Wang, Y., Shen, Z., Wan, Z. and Liu, W. (2020). Site-specific Labeling of B Cell Receptor and Soluble Immunoglobulin. Bio-protocol 10(18): e3767. DOI: 10.21769/BioProtoc.3767.
  2. Shen, Z., Liu, S., Li, X., Wan, Z., Mao, Y., Chen, C. and Liu, W. (2019). Conformational change within the extracellular domain of B cell receptor in B cell activation upon antigen binding. Elife 8: e42271.
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