Protein Labeling in Live Cells for Immunological Applications

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Review

Protein labeling in live cells for immunological applications Parisa Moghaddam-Taaheri, and Amy J. Karlsson Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00722 • Publication Date (Web): 22 Jan 2018 Downloaded from http://pubs.acs.org on January 23, 2018

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Bioconjugate Chemistry

Protein labeling in live cells for immunological applications Parisa Moghaddam-Taaheri‡ and Amy J. Karlsson†‡*

†

Department of Chemical and Biomolecular Engineering, University of Maryland, College Park

‡

Fischell Department of Bioengineering, University of Maryland, College Park

*

Correspondence:

Amy J. Karlsson Department of Chemical and Biomolecular Engineering, University of Maryland 2113 Chemical and Nuclear Engineering Building (#090) 4418 Stadium Drive College Park, MD 20742 USA Phone: 301-405-2610 Fax: 301- 405-0523 Email: [email protected]

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Abstract Protein labeling is often an important aspect of immunological experiments, as it allows observation of cellular processes, including protein synthesis and trafficking. Many protein labeling methods require permeabilization and fixation of cells, damaging the cells and preventing observation of processes in real time. However, a number of bioconjugation techniques allow protein labeling inside living cells to allow visualization of cellular processes as they occur and to facilitate retrieval of desired proteins. In this review, we describe bioconjugation methods that allow specific labeling of intracellular proteins of interest and discuss their applications to immunological studies. We focus on protein fusions, biotinylation, fluorescein arsenical helix binder (FlAsH) and resorufin arsenical helix binder (ReAsH) labeling, and tetrazine ligation.


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Introduction Labeling of cellular macromolecules provides valuable insight into the processes and potential changes occurring in cells following experimental treatment. Of particular interest is protein labeling, as changes in protein folding, expression levels, and trafficking can greatly affect cell phenotype and behavior. Many methods to assess these characteristics involve labeling proteins in a manner that requires damaging cells through permeabilization and fixation before staining the protein of interest (POI). One way to stain the POI is through non-covalent antibody-based staining, though protein staining is also commonly achieved through bioconjugation of the POI to a reactive compound via a stable covalent bond. Although methods that involve damaging cells are common, bioconjugation can also be performed in live cells. Labeling proteins on the surface of live cells is straightforward, as they are easy to access using bioorthogonal chemistries such as Staudinger ligation1-3 and strain promoted alkyne-azide cycloadditions4-6. Often more challenging is targeted bioconjugation of proteins inside cells. The ability to label proteins inside cells is beneficial as it allows for proteins to be observed in their native state and elucidate their role in specific biological events7. Through protein bioconjugation, protein expression and trafficking can be observed in real time. Tagged molecules can also be retrieved for quantification and analysis7. These capabilities can illuminate cellular processes at the molecular level and simplify isolation and purification of desired proteins. Bioconjugation in living cells presents a number of opportunities in immunological applications, allowing visualization of the role of proteins in cellular processes. One of the most common applications is labeling proteins to observe their interactions with one another and to follow the effect of those interactions on signaling pathways. Additionally, vaccine applications often benefit from labeling the antigen to observe its trafficking in cells, tissues, and organisms. In this review, we describe key techniques for bioconjugation in living cells and discuss examples and opportunities for their use in immunological applications. We focus on techniques where bioconjugation takes place intracellularly, as opposed to reactions that occur on the cell surface.



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Protein fusions One of the oldest and most common methods of bioconjugation of a protein is the expression of a protein fusion. In this method, the POI is covalently fused to another protein or peptide (fusion partner) that has desirable properties, such as fluorescence (e.g., fluorescent proteins) or affinity to an antibody or other molecule (e.g., a hexahistidine tag or FLAG tag). The fusion partner is expressed C- or N- terminally, with or without an interprotein linker, and in frame with the POI (Figure 1A). The gene fusion required for expression of the protein fusion is produced using standard methods for molecular cloning (e.g., see Green and Sambrook8). Perhaps the most commonly used fusion partner in immunology and other fields is green fluorescent protein (GFP), which emits a green color when excited. GFP can be used to visualize and quantify expression of the POI and to detect and track the cellular location of the POI. Additionally, modifications to the GFP gene have been made to produce other fluorescent proteins which emit different colors and can be used concurrently to visualize several POIs at once9. Fusions of POIs to fluorescent proteins have been widely used in immunology and vaccine studies, often in conjunction with single-molecule imaging techniques. For example, the tyrosine kinases Zap70 and Lck and the adaptor proteins SLP-76 and LAT10-15 have all been expressed as fusions to study T-cell receptor signaling. Douglass and Vale used a Lck-GFP fusion with total internal reflection fluorescence microscopy to image trafficking of Lck in T cells at high frame rates, allowing real–time, single-molecule imaging and quantitative information about tyrosine kinase diffusion (Figure 2A) and colocalization with other signaling molecules in T cells13. Additionally, recombinant production of antigen fused to GFP or a heat shock protein has facilitated studies in mice of antigen trafficking to lymph nodes and the spleen16 and antigen delivery to the major histocompatibility complex class I pathway17, respectively. Although fusion proteins are widely used, the method does present limitations. Molecular cloning or gene synthesis is required to create the genetic constructs to produce the fusion proteins. Additionally, generation of stable mammalian cell lines producing the fusions may be time-consuming. Fusion to a partner could also affect


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the expression or function of the POI or hinder cell growth. Despite these limitations, protein fusion is likely to remain a popular method for bioconjugation in living cells due to its proven utility in many applications.

Biotinylation Another method of protein labeling in live cells is biotinylation, which entails the addition of biotin to a recombinantly expressed protein. Biotin binds specifically to streptavidin or avidin reagents, which are widely available as conjugates to fluorescent molecules, enzymes, and magnetic beads. The biotin-streptavidin system is the strongest known noncovalent biological interaction, having a dissociation constant Kd of 10-14 M18,19, which has led to widespread use of the interaction for immobilizing or detecting biotinylated proteins. In some species, a native system for biotinylation is present, and this system can be harnessed for the in vivo biotinylation of recombinant proteins. Biotinylation is very specific since most organisms have fewer than five proteins that are natively biotinylated20. The most commonly used in vivo biotinylation system involves a biotin protein ligase and biotin acceptor protein from Escherichia coli. In E. coli, the biotin protein ligase BirA recognizes and biotinylates a specific lysine residue within the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase20,21 (Figure 1B). A POI can be expressed as a fusion to BCCP or to the smaller minimal acceptor peptide called the AviTag22,23. During expression of the POI-acceptor fusion, a reaction occurs that results in the formation of an amide linkage between the carboxyl group of biotin and the ε-amino group of the targeted lysine20. To achieve biotinylation in mammalian cells, E. coli BirA is often co-expressed in the mammalian cells with the POI fusion to a biotin acceptor sequence24-31, though biotinylation has also been achieved using an endogenous human biotin protein ligase32. Methods for biotinylating proteins in vivo in mammalian cells can be found in the literature cited above. In vivo biotinylation in E. coli has proven a useful tool in generating and immobilizing antibodies. Because biotinylation is specific and the streptavidin-biotin interaction is so strong, proteins can be efficiently



Figure 1. Approaches for bioconjugation of proteins in living cells. (A) Protein fusions are generated through a genetic fusion of the gene for the POI to another protein, such as GFP. (B) Biotinylation is achieved by fusing a biotin acceptor protein to the POI and expressing a biotin ligase to attach a biotin to the acceptor protein or peptide. (C) FlAsH bioconjugation occurs when FlAsH-EDT2 reacts with the tetracysteine motif in the FlAsH recognition sequence in a POI. ReAsH bioconjugation occurs in a similar manner. (D)Tetrazine ligation of proteins in living cells requires incorporation of an unnatural amino acid with an alkene or alkyne into the protein sequence via expression of an engineered tRNA synthetase and tRNA pair. A tetrazine derivative with a fluorescent label then modifies the unnatural amino acid.


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Figure 2. Examples of immunological applications of protein labeling methods. (A) Jurkat T cells were transiently transfected with Lck-GFP and imaged via total internal reflection fluorescence microscopy for single-molecule tracking. Trajectories of five individual Lck-GFP fusion molecules were tracked at 30 frames/s, and total elapsed time is shown. Trajectory color indicates the mobility of the molecule, with green indicating high mobility for that portion of the trajectory and red indicating low mobility. Scale bar = 2 µm. (Reprinted with permission from Douglas and Vale13. Copyright 2005 Elsevier.) (B) A glioma tumor expressing a biotinylated biotin acceptor protein fused to the transmembrane domain of platelet-derived growth factor receptor (left box) and a control tumor lacking biotinylation (right box) were imaged in mice 24 h after intravenous injection of streptavidin-Alexa 680 using fluorescence-mediated tomography. Color scale represents the level of streptavidin-conjugated fluorophore detected. Scale bar = 1 cm. (Adapted with permission from Tannous et al.32. Copyright 2006 Springer Nature.) (C) Dendritic cells infected with HIV containing a tetracysteine motif in the gag protein were labeled with FlAsH (green) and then with ReAsH (red) 4 h later (top) or 16 h later (bottom) to detect newly formed gag protein. Blue signal is DAPI staining, and overlaid signals are shown with and without differential interference contrast (DIC) images. Scale bar = 5 µm. (Adapted with permission from Turville et al.41. Copyright 2007 Springer Nature.) (D) HEK293 cells producing the engineered MbPyIRS/ tRNA pair were grown in the presence of a BCN-containing amino acid, and the BCN-functionalized amino acid was incorporated at the site of an amber stop codon in a fusion of the transcription factor jun to mCherry. Tetrazine ligation was performed by incubating cells with a tetrazinecarboxyfluorescein diacetate conjugate to react with the BCN moiety. Fluorescence microscopy was used to visualize mCherry (red) and fluorescein (green) and show colocalization of the dyes overlaid with a DIC image. (Adapted with permission from Lang et al.53. Copyright 2012 American Chemical Society.) ACS Paragon Plus Environment


purified from a bacterial cell lysate and immobilized on streptavidin-coated surfaces or beads in a single step33-35. Using in vivo biotinylation versus the more typical strategy of chemically modifying a protein with biotin allows site-specific biotinylation to enable oriented immobilization of the biotinylated protein. The approach has been used to immobilize antibody fragments on a surface for detection of antigens33,35 and to immobilize antigen on beads for immunization of mice34. Cull and Schatz provide detailed methods for producing recombinant proteins that are biotinylated by E. coli cells during protein production23. Biotinylation in mammalian cells has proven useful in labeling a number of proteins, including nuclear proteins, viral capsid proteins, transcription factors, RNA-binding proteins, and cell-surface proteins24-30,32. Tannous et al. used this approach for imaging tumor cells in mice32. A cell-surface receptor reporter protein was expressed as a fusion to an acceptor peptide and biotinylated by endogenous biotin ligase in mammalian cells. Tumors in mice expressing the biotinylated proteins were visualized using streptavidin-conjugated to magnetic nanoparticles (for magnetic resonance) or fluorochromes (for fluorescence tomographic imaging) (Figure 2B). This example, along with other instances of biotinylation of proteins in mammalian cells, illustrates the potential of using this approach to track the location of cells and proteins important in immunological studies. In vivo biotinylation relies on generating a fusion of the POI with a biotinylation tag. Thus, biotinylation has the same limitations as protein fusion expression in terms of requiring construction of a genetic fusion. Still, this technique provides the added capability of easily isolating the POI, which may be desirable in a number of applications.

FlAsH and ReAsH A method of protein labeling in live cells that is gaining popularity is the use of a fluorescent label that covalently binds to a specific amino acid sequence within an expressed protein. The fluorescein arsenical helix binder (FlAsH) and resorufin arsenical helix binder (ReAsH) reagents are two such compounds that are able to label a POI that contains a tetracysteine motif, CCXXCC36,37 (Figure 1C). Specifically, the reaction involves the


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trivalent organoarsenical compounds 4’,5’-bis(1,3,2-dithioarsolan-2-yl)fluorescein and 4,5-bis(1,3,2-dithiarsolan2-yl)-resorufin for FlAsH and ReAsH, respectively, which are prepared as the 1,2-ethanedithiol (EDT) adducts FlAsH-EDT2 and ReAsH-EDT2. The compounds bind to paired thiol groups of closely grouped cysteine pairs to form a covalent bond36,37. Importantly, FlAsH and ReAsH are non-fluorescent prior to binding the tetracysteine motif and undergo a structural change to become fluorescent (green for FlAsH, red for ReAsH) upon binding to the POI. The binding can be completely reversed by small vicinal dithiols such as 2,3-dimercaptopropanol or 1,2ethanedithiol (EDT), which successfully compete to bind to the organoarsenical compounds36-38. FlAsH and ReAsH are commercially available, and protocols for labeling are provided by the manufacturer (ThermoFisher Scientific). Because the organoarsenical compounds are membrane permeant, FlAsH and ReAsH bioconjugation can be used in live cells36-38. Optimization of the peptide binding tetracysteine motif revealed that the specific motifs HRWCCPGCCKTF and FLNCCCPGCCMEP show higher fluorescence, which allows improved contrast to be able to discern POI fluorescence over spontaneous background fluorescence or non-specific fluorescence36-38. By using two tetracysteine motifs—one containing KA in the XX position and one containing PG in the XX position—FlAsH and ReAsH can be used in combination to label two different POIs39. FlAsH and ReAsH have been used to study a variety of proteins and cellular processes in living cells and elucidate protein production and movement in immune cells. For example, Rudner et al. used ReAsH to label mature HIV virus Gag proteins and then used FlAsH as a secondary reagent to label newly synthesized Gag proteins40. They were able to observe recruitment and trafficking of the HIV virus Gag proteins to the plasma membrane in HeLa, Mel JuSo, and Jurkat T cells immediately after synthesis and at steady state. Later studies have shown that FlAsH-conjugated Gag proteins can be used to study the trafficking of HIV within dendritic cells and macrophages and between these cell types and T cells41-43. For example, by labeling HIV gag protein with FlAsH and pulse-chasing with ReAsH, Turville et al. differentiated between mature and newly synthesized virus particles within dendritic cells at different times41 (Figure 2C). This labeling method also allowed real-time virus trafficking, and mechanistic differences in transfer of virus between different immune cell types was observed41.



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Other applications of FlAsH and ReAsH include studying protein trafficking, signaling, and protein misfolding and conformational changes44-47, all of which could prove beneficial in immunological studies. Potential challenges in using FlAsH and ReAsH are focused mainly on the chemistry required for the labeling to occur. Similar to protein fusions and biotinylation, molecular cloning must be performed to insert the tetracysteine motif into the gene for the POI. Additionally, if the POI is rich in cysteine residues, the organoarsenical compounds may non-specifically bind at sites other than the target motif48. Finally, the FlAsH and ReAsH reagents are relatively costly, which may limit their utility in experiments where large quantities of protein need to be labeled.

Tetrazine ligation A number of bioorthogonal reactions are available for labeling proteins (reviewed by Lang and Chin49,50), but most of these reactions suffer from slow kinetics, toxic reagents, or an inability to label intracellular proteins. Tetrazine ligation, however, does not suffer from any of these limitations and is an important reaction for protein bioconjugation in living cells. In a tetrazine ligation, a fluorescently labeled tetrazine reacts with a strained alkene or alkyne, such as norbornene, trans-cyclooctene, or bicyclononyne, to form a stable bond51-55 (Figure 1D). The reaction is compatible with proteins and living cells and exhibits a very fast reactivity51-56. The fluorescence of the tetrazine-linked fluorescent probe is quenched prior to the reaction, but the cycloaddition to the alkene or alkyne “turns on” the fluorescence, increasing the fluorescence intensity of the probe up to 20-fold51. In order to use tetrazine ligation to label proteins in living cells, the bioorthogonal alkene or alkyne must be introduced into the cells where it can react with the tetrazine-linked probe. This is achieved by incorporating unnatural amino acids containing the alkene or alkyne into the POI52-54,57,58. An amber stop codon is introduced into the gene encoding for the POI, and cells producing the POI are supplied with the genes for an engineered tRNA and aminoacyl tRNA synthetase pair that recognizes and suppresses the amber stop codon by incorporating the unnatural amino acid. Unnatural amino acids containing a number of different moieties,


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including norbornene, cyclooctene, bicyclononyne, and cyclobutene, have been incorporated using this approach52-54,57,58. Methods detailed in the literature cited above can be adapted for labeling of most proteins. Tetrazine ligation has been used to label intracellular proteins in live bacterial and mammalian cells5254,57,58

. In one example, Lang et al. describe using a tetrazine ligation method to label intracellular proteins in

HEK293 cells53. An unnatural amino acid containing bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) was incorporated into a fusion of the transcription factor jun to mCherry using an engineered version of the Methanosarcina barkeri pyrrolysyl-tRNA synthetase (MbPyIRS) and tRNA pair optimized for mammalian-cell function. The HEK293 cells were grown in the presence of the BCN-containing amino acid, and the engineered MbPyIRS/ tRNA pair incorporated the BCN-functionalized amino acid at the site of an amber stop codon in the jun-mCherry fusion. The fusion protein was well-expressed and mCherry visualization showed that it localized in the nuclei, as expected (Figure 2D). To perform the tetrazine ligation, cells were incubated with a cell-permeable tetrazine-carboxyfluorescein diacetate conjugate. This resulted in green fluorescence that colocalized with the mCherry signal (Figure 2D). A similar set of experiments was also performed for epidermal growth factor receptor, resulting in successful labeling of the receptor on the cell surface53. In separate work, the same BCNcontaining amino acid was also used to label the cytoskeletal proteins β-actin and vimentin in HEK293T cells, which were subsequently imaged using super-resolution imaging58. Although direct immunological applications of tetrazine ligation have yet to be reported, these examples illustrate the potential of using tetrazine labeling in live cells. This bioconjugation approach will be useful in exploring the localization of signaling proteins, the presence of newly synthesized proteins, and protein-protein interactions that are important in develop an improved understanding of the role of various proteins in the immune system. While the bioorthogonal nature of tetrazine ligation has many advantages, it also introduces added complexity compared to the other methods mentioned. In addition to needing to introduce the amber stop codon into the POI, a tRNA synthetase/tRNA pair that can incorporate the desired unnatural amino acid must also be expressed by the cells producing the POI. However, the number of synthetase/tRNA pairs and the amino acids they can incorporate is steadily growing, which will facilitate future applications of this approach.



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Conclusion Bioconjugation of proteins in living cells is a powerful tool in immunology, enabling visualization of protein synthesis, trafficking, and function and allowing facile development of protein reagents. Protein and other macromolecule labeling also have potential in engineering the immune system. For example, expression of a protein antigen as a fusion to an expression partner may be useful in increasing retention time to modulate the immune response. The choice of bioconjugation technique requires consideration of the advantages each technique offers, along with the limitations and challenges in implementation. The techniques described here may require optimization to achieve ideal results, but they offer the opportunity to gain real-time, molecular-level insight into the function of the immune system and intracellular immunological molecules. As advancements and new techniques in labeling proteins in fixed cells and proteins on the cell surface emerge, the potential adaptation of these methods to labeling within live cells should be explored to expand the variety of chemistries and techniques available to achieve improved resolution and real-time imaging of important molecules and interactions in the immune system.

Acknowledgments This work was supported by a University of Maryland Tier 1 grant.

Abbreviations BCCP

biotin carboxyl carrier protein

BCN

bicyclo[6.1.0]non-4-yn-9-ylmethanol

FlAsH

fluorescein arsenical helix binder

GFP

Green fluorescent protein

MbPyIRS

Methanosarcina barkeri pyrrolysyl-tRNA synthetase


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POI

protein-of-interest

ReAsH

resorufin arsenical helix binder



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Protein Labeling in Live Cells for Immunological Applications

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