Articles Cite This: ACS Chem. Biol. XXXX, XXX, XXX−XXX
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Internalization of Influenza Virus and Cell Surface Proteins Monitored by Site-Specific Conjugation of Protease-Sensitive Probes Ross W. Cheloha,† Zeyang Li,†,‡ Djenet Bousbaine,†,‡ Andrew W. Woodham,† Priscillia Perrin,‡ Jana Volaric,́ † and Hidde L. Ploegh*,† †
Boston Children’s Hospital and Harvard Medical School, 1 Blackfan Circle, Boston, Massachusetts 02115, United States Massachusetts Institute of Technology, 455 Main St, Cambridge, Massachusetts 02142, United States
‡
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S Supporting Information *
ABSTRACT: Commonly used methods to monitor internalization of cell surface structures involve application of fluorescently or otherwise labeled antibodies against the target of interest. Genetic modification of the protein of interest, for example through creation of fusions with fluorescent or enzymatically active protein domains, is another approach to follow trafficking behavior. The former approach requires indirect methods, such as multiple rounds of cell staining, to distinguish between a target that remains surface-disposed and an internalized and/or recycled species. The latter approach necessitates the creation of fusions whose behavior may not accurately reflect that of their unmodified counterparts. Here, we report a method for the characterization of protein internalization in real time through sortase-mediated, site-specific labeling of single-domain antibodies or viral proteins with a newly developed, cathepsin-sensitive quenched-fluorophore probe. Quenched probes of this type have been used to measure enzyme activity in complex environments and for different cell types, but not as a sensor of protein movement into living cells. This approach allows a quantitative assessment of the movement of proteins into proteasecontaining endosomes in real time in living cells. We demonstrate considerable variation in the rate of endosomal delivery for different cell surface receptors. We were also able to characterize the kinetics of influenza virus delivery to cathepsin-positive compartments, showing highly coordinated arrival in endosomal compartments. This approach should be useful for identifying proteins expressed on cells of interest for targeted endosomal delivery of payloads, such as antibody−drug conjugates or antigens that require processing.
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challenge. In addition, recycling of endocytosed membrane proteins is common and can occur rapidly,7 yet many methods do not differentiate between nonendocytosed and recycled surface-resident proteins. Such approaches rely on indirect methods to account for recycling.6 Furthermore, the act of labeling with conventional (bivalent) antibodies can cause cross-linking of surface proteins that can itself induce protein relocalization and internalization.8 As an alternative approach, a recombinantly expressed single-domain (monovalent) antibody (VHH or nanobody) that binds green fluorescent protein (GFP) was used to track the movement of cell membrane proteins expressed as GFP fusions.9 While the use of nanobodies eliminates possible artifacts induced by crosslinking, the fusion with GFP can affect its properties, including trafficking.10 The use of monovalent nanobody probes that target the protein of interest directly and can discriminate between the cell surface and the intracellular environment would therefore be preferable.
he characterization of protein movement within and across cell membranes is an important aspect of cell biology. Cellular functions depend on regulated trafficking of proteins to and from the cell surface. This occurs via characteristic intermediate sites such as the Golgi apparatus and endocytic structures, both as a biosynthetic event and to deliver receptor-bound ligands into the cell interior for degradation.1 Characterization of endocytosis and endosomal trafficking is especially critical for understanding antigen processing,2 neurotransmission,3 and G-protein-coupled receptor signaling.4 Methods to study endocytosis and subsequent trafficking often rely on labeling with antibodies functionalized with fluorophores or other tags visualized either directly or indirectly.5,6 This approach requires that microscopy, or some other approach such as sequential labeling with biotinylated antibodies and fluorophore-labeled streptavidin, be used to distinguish membrane proteins that have been internalized from those that remain at the cell surface over the observation period. Such approaches have a number of drawbacks. In cells with small volumes of cytoplasm such as lymphocytes, distinguishing between an internalized protein and its counterpart that remained at the cell surface poses a © XXXX American Chemical Society
Received: June 19, 2019 Accepted: July 12, 2019
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DOI: 10.1021/acschembio.9b00493 ACS Chem. Biol. XXXX, XXX, XXX−XXX
Articles
ACS Chemical Biology
Figure 1. Design of sortase-compatible, protease-sensitive quenched fluorophore probes. (a) Mechanism by which proteolysis relieves quenching and restores fluorescence. (b) Structures of sortase-compatible quenched probes prepared for this work. Molecular masses were measured using QTof mass spectrometry as described in the Methods section. (c) General structure of quenched probe functionalized conjugates used in these studies. Camelid single domain antibodies (VHHs) that targets MHC-II, CD36, CD45, CD47, and Ig-κ.
The site-specific attachment of a protease-sensitive fluorophore−quencher pair to a protein of interest offers a way to directly monitor the entry of that given protein or its binding partner, to protease-containing endosomal compartments. Peptide-based conjugation using the bacteria-derived enzyme sortase A, or sortagging, offers a convenient method for making such conjugates.19 The present work characterizes protein conjugates functionalized with quencher−fluorophore pairs via sortagging. We then use these conjugates to monitor the internalization of the modified protein and its binding partner, where applicable, in real time. We present here the first examples of conjugates consisting of nanobodies sitespecifically functionalized with fluorogenic probes. These novel conjugates are used to monitor processes difficult to characterize by other means such as the spontaneous internalization of cell surface proteins and the entry of infectious influenza particles into cells in real time using lattice light sheet microscopy.
Other methods to report on internalization of proteins rely on the high local concentration of proteolytic enzymes, such as the cathepsins, in the endosomal pathway. This requires that once the protein of interest reaches a protease-containing compartment, some detectable change occurs.11 Early versions of this approach made use of soluble proteins that were randomly and heavily labeled, using amine-reactive fluorophores. Labeling was performed at a density of fluorophore such that self-quenching rendered these conjugates essentially nonfluorescent. Dequenching required internalization and proteolysis.12 Indeed, similarly prepared quenched fluorophore-antibody conjugates that recognize surface proteins showed relatively low levels of fluorescence prior to internalization13 and yielded a fluorescent signal upon internalization.14 However, such conjugates suffered from only modest enhancement in fluorescence upon proteolysis. Moreover, a determination of their intracellular distribution proved imprecise because of uncontrolled, heterogeneous labeling. Fluorescence quenching can also be imposed by the placement of fluorophores adjacent to nonfluorescent FRET partners (fluorescence quenchers).15 Random conjugation of both fluorophore and quencher to monoclonal antibodies improved the imaging properties of the resulting conjugates relative to earlier versions labeled only with fluorophore.15 However, this approach still used random labeling and yielded a heterogeneous preparation of labeled antibodies. Site-specific labeling of proteins is often preferable to random labeling. Extensive random labeling can impair the binding of antibodies to their targets16 or alter their biological distribution in vivo.14 With few exceptions, site-specific methods to attach dyes or other small molecules to antibodies or other proteins allow attachment of only a single type of label.17 The fusion of proteins of interest with domains that selectively react with small organic functional groups, such as HaloTag and SnapTag, offer another method for site-specific attachment of reporters;18 however, such approaches still require engineering to provide the desired fusion proteins.
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RESULTS AND DISCUSSION Synthesis of Sortase-Compatible Quencher-Fluorophore Probes and Conjugation. Sortase A and its variants provide a versatile tool to generate protein conjugates. Sortase A recognizes the LPXTGG motif on a protein (where X is any amino acid except proline) and cleaves between the threonine and glycine residues to form a thioester intermediate, which is then resolved by attack of the N-terminal amine from a peptide containing an N-terminal oligo-glycine motif.20 Sortasemediated ligation, or sortagging, has proven a robust and versatile method to attach a probe of choice to target proteins with an LPXTGG tag near the C-terminus.19,21,22 We developed sortase-compatible probes that contain a fluorophore and a quencher, linked via a protease-sensitive sequence, to label proteins that carry the LPXTGG motif (Figure 1). Such self-quenched peptides have been used to monitor protease activity in solution,23 in phagocytic cell types,24 and on tissue sections25 and have potential for intraoperative use in B
DOI: 10.1021/acschembio.9b00493 ACS Chem. Biol. XXXX, XXX, XXX−XXX
Articles
ACS Chemical Biology surgical interventions.26 As an extension of this approach for cell biological applications, we chose to attach self-quenched probes site-specifically to proteins of interest, using sortase. First, we produced the peptide backbone functionalized with fluorophore by solid-phase peptide synthesis and attachment of tetramethyl rhodamine to a lysine side chain via on-resin deprotection of the 4-methyltrityl protecting group, subsequent coupling with 5,6-carboxytetramethylrhodamine, and purification by high-performance liquid chromatography (HPLC). Following purification, the fluorescence quencher (QSY7) was attached using cysteine-maleimide chemistry, and purified again by HPLC (Supporting Information Figure 1). We also prepared an analogue of this probe in which the VVR cathepsin-sensitive linker (qP-VVR)27 was replaced with a protease-resistant (β-Ala)3 linker (qP-β-Ala).28 We confirmed the high purity of these conjugates using HPLC (Supporting Information Figure 1). Purified preparations of these probes exhibited negligible fluorescence in the absence of added protease (data not shown). The addition of trypsin or cathepsin S resulted in the time-dependent emergence of fluorescence for the VVR quenched probe, but not for the (β-Ala)3 probe, consistent with the predicted protease susceptibilities of these linkers (Figure 2a). The concentration of cathepsin S used for this
20 nM cathepsin S was approximately 60% complete after an hour (Figure 2a). The rate of dequenching in the presence of 20 nM cathepsin S greatly underestimates the rate of dequenching that would be observed in cells, as the actual concentration of cathepsins in endolysosomal compartments can approach 1 mM.29 The internalization of cell membrane proteins is thought to occur over time frames ranging from 10 min to a few hours. If indeed cathepsins reach endolysosomal concentrations reported in the literature, we conclude that internalization, not proteolysis, is rate limiting. We used higher concentrations of cathepsin S or cathepsin B and observed more rapid dequenching (Figure 2b). We complemented these findings by assessing Michaelis−Menten kinetic parameters for the cleavage of qP-VVR by cathepsin S, cathepsin B, and trypsin (Supporting Information Figure 2). Each of these enzymes efficiently cleaved qP-VVR, indicating that several different enzymes can cleave this probe in the context of a live cell. We performed control experiments to test whether proteases released from cells upon lysis, or the high concentrations of reducing agents found in endosomes, could dequench these probes. Incubation of qP-VVR, but not qP-β-Ala, in freshly prepared lysates of a murine B-cell lymphoma line (A20 cells) resulted in a slow increase in fluorescence (Supporting Information Figure 2). Thioether linkages produced through thiol-maleimide chemistry can be cleaved by free thiols.30 The addition of a supraphysiological concentration of reduced glutathione to solutions of the quenched probes did not increase the fluorescence signal above baseline levels (Supporting Information Figure 3). These findings support the mechanism of fluorescence dequenching depicted in Figure 1. We then tested whether these probes could be conjugated to an antibody fragment using sortase, without a loss of proteaseinducible fluorescence or a loss of antigen recognition by the antibody. For this purpose, we used recombinantly expressed variable domains of heavy chain-only antibodies (VHHs) from camelids, also known as nanobodies or single-domain antibodies. These small proteins (12−15 kDa) are the smallest antibody fragments that retain antigen recognition properties, and they can be expressed in, and purified from, bacteria in high yield. In many cases, VHHs do not require disulfide bonds for folding or function.31,32 They are monomeric, monovalent, and cannot cross-link their targets at the cell surface. VHHs are available that target a variety of proteins, including many expressed at the cell surface.31,32 By means of sortagging, these VHHs can be readily equipped with a variety of probes, such as antigenic peptides, radio-metal chelators, or fluorophores.22,33 We used sortagging to functionalize VHHs with quenched fluorophore probes. Standard sortagging reactions were used, with slight modifications. The quenched fluorophore probes were hydrophobic, so we performed conjugations reactions in buffer containing 20% (v/v) DMSO. A VHH that binds to mouse class II MHC (VHHMHC‑II or anti-MHC-II, previously named VHH7),22 was conjugated to quenched probes. We added the purified products at 0 °C to A20 cells, which express class II MHC. Flow cytometry showed excellent staining with VHHMHC‑II sortagged with tetramethylrhodamine (Anti-MHCII-TMR), but not with VHH that binds GFP (VHHGFP or antiGFP, previously named enhancer)34 sortagged with TMR (Anti-GFP-TMR); cells stained with VHHMHC‑II functionalized with quenched probes (Anti-MHC-II-qP-VVR or Anti-
Figure 2. Assessment of fluorophore dequenching by exogenous protease. (a) Purified quenched peptide probes (5 μM concentration) were incubated with either cathepsin S (CatS, 20 nM) or trypsin (500 nM) for the indicated durations using conditions described in the Methods section. Fluorescence (excitation 555 nm, emission 575 nm) was recorded and normalized to the maximal fluorescence observed upon exposure to 500 nM trypsin, which was normalized to 100%. Error bars represent SD. (b) Dequenching reactions were performed as in panel a but with different concentrations of qP-VVR (6.3 μM) and proteases (CatS at 33 nM, CatB at 13 nM, trypsin at 43 nM), and fluorescence readings were not normalized. (c) Either qP-VVR or qPβ-Ala was conjugated to either VHHMHC‑II or VHHGFP using sortagging and the purified conjugates added to a suspension of A20 cells for 1 h at 0 °C, followed by washing. Cells were then analyzed by flow cytometry in the presence or absence of 0.004% (w/ v) trypsin solution.
assay was relatively low as endosomal proteases can exhibit reduced stability at higher concentrations. Trypsin was used at a high concentration (500 nM) to drive dequenching rapidly to completion. In the presence of 500 nM trypsin, dequenching was >80% complete at the first time point tested (
