Nongenetically Encoded and Erasable Imaging Strategy for Receptor

Feb 1, 2019 - Furthermore, the donor-equipped ligand can be flexibly competed with an unlabeled compound, leading to an efficient erasure of donor ...
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Cite This: Anal. Chem. XXXX, XXX, XXX−XXX

Nongenetically Encoded and Erasable Imaging Strategy for Receptor-Specific Glycans on Live Cells Nan Li,† Weifei Zhang,† Ling Lin,*,‡ Syed Niaz Ali Shah,† Yuxuan Li,† and Jin-Ming Lin*,† †

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Department of Chemistry, Beijing Key Laboratory of Microanalytical Methods and Instrumentation, MOE Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Tsinghua University, Beijing 100084, China ‡ CAS Key Laboratory of Standardization and Measurement for Nanotechnology, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology, Beijing 100190, China S Supporting Information *

ABSTRACT: Glycans on specific receptors play a crucial role in regulating receptor functions and indicating cell pathological states. For a detailed glycosylation regulatory mechanism, live cell imaging of receptor-specific glycans becomes significantly important. In this work, we present a nongenetically encoded labeling strategy to specifically install a fluorescence resonance energy transfer (FRET) pair onto the receptor of interest by ligand−receptor binding and metabolic glycan engineering. This method breaks the limitation that the receptors have to possess an extracellular terminus which can be used to attach a fluorescent tag, avoiding the undesired effects introduced by inserting amino acids into proteins. Furthermore, the donor-equipped ligand can be flexibly competed with an unlabeled compound, leading to an efficient erasure of donor fluorescence signal. We envision that this strategy will have the potential to sequentially identify and characterize multiple receptor-specific glycans on the live cell surface through multiple cycles including labeled ligand binding, FRET-induced fluorescence imaging, and the unlabeled compound competing for fluorescence erasure. Besides, this in situ glycan profiling strategy will have wide applications in molecular diagnosis and cellular targeted therapies.

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inserting amino acids into proteins may introduce undesired effects and compromise the structural and functional integrity receptors.18,19 It had previously been reported that fluorescence donors for time-resolved FRET can be conjugated to cell-surfaces via metabolic glycan labeling and click chemistry, followed by receptor labeling with acceptor-equipped ligands.19 We sought to expand upon this approach by conjugating a FRET acceptor to cell surface glycans and labeling a receptor of interest with a FRET donor though a receptor-specific ligand. Although the acceptor was attached not only to the receptor of interest but also to other cell surface proteins, only the receptor of interest was site-specifically equipped with a complete FRET pair. Because of the high density of acceptors on the cell membrane, it was important to avoid the direct excitation of the acceptor at the excitation wavelength of the donor. Therefore, we required a donor−acceptor pair with well-separated excitation/ emission maxima and chose Alexa Fluor 488 and Alexa Fluor 647, respectively (Figure S1). As a proof of concept, we applied our strategy to image the sialylated glycoforms of Interleukin-36 receptor (IL-36R) on the cell surface (Figure 1). IL-36R is a member of interleukin-1

ssentially most eukaryotic cell surface receptors are posttranslationally modified with glycans which, as one of the basic components of cells, are covalent assemblies of sugar.1,2 These attached glycans can profoundly regulate the dynamics and function of the receptors, such as modulating ligand− receptor binding, receptor dimerization, endocytosis, and degradation.3−5 On the other hand, the expression of glycans are often related to some cell pathological states,6,7 and indepth analyses indicate that these pathological phenotypes are caused by the variation of the glycans on certain specific receptors instead of overall glycans, which can be regarded as hallmarks of diseases.8,9 Therefore, in situ dynamic observation of a receptor-specific glycan would be of great importance to understand glycosylation mechanisms and also provide a promising tool for the development of diagnosis and therapy. To date, fluorescence resonance energy transfer (FRET) has been used to visualize receptor-specific glycans on the cell surface by labeling receptor and glycans with FRET donors and acceptors.10−12 Specifically, acceptors are anchored to the cell surface by incorporating a reactive non-natural sugar and subsequent bio-orthogonal ligation,13,14 while donors are attached to a receptor of interest by genetic engineering to produce fluorescent fusion proteins or to insert binding sequences for labeling.15 However, for this approach to be successful, receptors have to have an extracellular terminus which can be used to attach a fluorescent tag.16,17 Moreover, © XXXX American Chemical Society

Received: November 15, 2018 Accepted: January 31, 2019 Published: February 1, 2019 A

DOI: 10.1021/acs.analchem.8b05292 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry

Figure 1. Schematic of the nongenetically encoded and erasable strategy for imaging the receptor-specific glycans on live cells. The global cellsurface glycans were metabolically labeled with a FRET acceptor (Alexa Fluor 647), while the receptors of interest were site-specifically bound to the ligand containing a FRET donor (Alexa Fluor 488), thus only the receptor-specific glycans were excited through intramolecular FRET within the range of 1−10 nm. After imaging, the ligands were competed and removed by an unlabeled compound IL-36Ra with stronger binding affinity.

receptor superfamily and linked to inflammation-associated diseases. Upon binding of IL-36 ligand, IL-36R recruits a coreceptor subunit, Interleukin-1 receptor accessory protein (IL-1RAcP), to form a heteromeric complex, regulating the downstream signal transduction.20,21 IL-36R possesses eight Nlinked glycosylation sites on the extracellular domain. The glycosylation has been found to play an important role in regulating the function of receptor,22 yet the visualization of glycans on IL-36R has not been realized on live cells. To develop the assay, HEK 293T cells were transfected to express recombinant IL-36R and simultaneously incubated with N-azidoacetylmannosamine-tetraacylated (Ac4ManNAz) for 48 h. Afterward, we sequentially bound the FRET pair onto the target receptor. First, the azidosugar-incorporated cell surface were reacted with a FRET acceptor, azadibenzocyclooctyne (DBCO)-Fluor 647 via click chemistry.23 Second, a recombinant IL-36γ ligand labeled with Fluor 488 specifically recognized and bound to IL-36R as a FRET donor. The dually labeled live cells were imaged by confocal laser scanning microscopy (CLSM) using three-channel method, and the images of 488 fluorescence, 647 fluorescence and FRETinduced fluorescence were shown (Figure 2A). Since the acceptor must be excited by the donor within a distance range of 1−10 nm, consequently, the FRET signal specifically indicated the sialylated glycan functionalized on IL-36R. Additionally, we investigated whether labeled ligand would arouse any variation in its ability to bind to the receptor on the cell surface. Herein, we detected the release of downstream cytokine of IL-36 signaling pathway, CXCL1, using an ELISA kit. The results showed no difference in the aspect of labeled IL-36γ inducing cytokine release in comparison with unlabeled ligand (Figure S2). To further verify the energy transfer process, two negative controls were conducted. Obviously, in the absence of the DBCO-Fluor 647, an increase in donor emission was observed, which could be explained that the lack of acceptors reduced the consumption of donor emission, and no FRET-induced fluorescence signal was generated (Figure 2B). Likewise, in the absence of IL-36γ-Fluor 488, the DBCOFluor 647 labeled cells exhibited no FRET-induced fluorescence signal, either, indicating no direct excitation of acceptors even when they were in large excess (Figure 2C). Thus, these results verified that the fluorescence signal was induced by FRET. To confirm that the donor signal resulted

Figure 2. In situ imaging of receptor-specific glycans on live cells via a nongenetically encoded and erasable strategy. (A) HEK 293T cells were transfected with IL-36R and incubated with Ac4ManNAz for 48 h, followed by acceptor DBCO-Fluor 647 labeling and ligand IL-36γFluor 488 binding. (B−D) Negative control in which DBCO-Fluor 647 was absent (B), IL-36γ-Fluor 488 was absent (C), and HEK 293T cells were not transfected with IL-36R (D). 488 fluorescence (excitation, 488 nm/collected imaging, 503−600 nm); 647 fluorescence (excitation, 633 nm/collected imaging, 648−700 nm); FITC-induced FRET (excitation, 488 nm/collected imaging, 648− 700 nm). Scale bar: 20 μm.

from the selective combination of IL-36γ-488 ligand to IL-36R receptor, a control experiment introducing IL-36γ-Fluor 488 to HEK 293T cells without transfection of IL-36R plasmid was carried out. As expected, negligible fluorescence signal was recorded by the donor channel owing to the lack of innate immune IL-36R expression in HEK 293T cells, which confirmed the specificity of this ligand−receptor binding assay (Figure 2D). Furthermore, we sought to exclude the possibility that the observed FRET signal came from glycans on other adjacent receptors. First, the cells that expressed IL-36R were treated B

DOI: 10.1021/acs.analchem.8b05292 Anal. Chem. XXXX, XXX, XXX−XXX

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contact with ligands,22 we deduced that glycosylation was presumably necessary to maintain the stability of receptor and could affect IL-36R trafficking to the cell surface. To verify this speculation, we investigated the receptor expression level under various concentrations of tunicamycin via Western blotting assay. The results suggested that the cell surface localization of IL-36R was indeed suppressed by the tunicamycin inhibitor in a dose-dependent manner (Figure 3D and Figure S4). Considering the multiplicity and complexity of the glycans on cell surface, it would be of great importance to image multiple receptor-specific glycans on a given live cell. However, methods for multiple imaging are largely lacking due to the difficulty in seeking interference-free single-excitation-multiplexed-emission FRET pairs. We envisioned that our strategy could effectively avoid this obstacle by using an unlabeled compound to compete with the labeled ligand and erase the original donor signal, then sequentially imaging another receptor-specific glycans with the same FRET pair. To conduct this speculation, first, the cells were engineered with metabolic oligosaccharide and incorporated with DBCO-Fluor 647 on the whole cell surface. Then, a labeled agonist IL-36γ-Fluor 488 was introduced to specifically combine with IL-36R with a KD = 1800 ± 200 nM, the FRET signal indicated the glycans on IL-36R. After that, an unlabeled antagonist IL-36Ra with stronger binding affinity (KD = 5.8 ± 0.3 nM) was introduced to compete with the IL-36γ-Fluor 488, binding to IL-36R which was either newly synthesized or dissociated from the complex IL-36γ-Fluor 488/IL-36R. It has been demonstrated that IL-36γ had fast-on and fast-off kinetics to IL-36R; in contrast, IL-36Ra exhibited a much slower off-rate, which was in favor of the competition occurrence of IL-36Ra.25 Apparently, a pretty weak IL-36γ-Fluor 488 signal remained after a 20-min competition with 70 μg/mL IL-36Ra (Figure 4A). This phenomenon was consistent with the previous

with N-azidoacetylgalactosamine-tetraacylated (Ac4GalNAz), an azido sugars predominantly used to label the O-linked glycosylation. Since the IL-36R possessed only N-linked glycosylation site, thus this O-linked experiments could serve as a good control. Theoretically, no FRET-induced signal would be detected if intermolecular FRET was negligible. As expected, strong donor and acceptor signals were observed; however, no intermolecular FRET-induced signal was detected, which indicated the negligible contribution from the intermolecular FRET owing to the far distance between donors on IL-36 ligand and acceptors on the O-glycosylation site (Figure 3A). Next, the receptor-specific glycan signals were

Figure 3. Verification of the intramolecular FRET-induced fluorescence. (A) The cells were treated with Ac4GalNAz which were predominantly used to label the O-linked glycosylation. (B) The cells were treated with Ac4ManNAz and incubated with 10 μg/mL tunicamycin to inhibit the N-linked glycosylation. (C) A control where the cells were treated with Ac4ManNAz which were predominantly used to label the N-linked glycosylation. Scale bar: 20 μm. (D) The expression of cell surface localization IL-36R under the treatment of various Tunicalycin concentration (0, 1, 2, 4, 6, 8, 10 μg/mL) was analyzed by Western blot assay.

investigated by tunicamycin inhibition experiment, which was used to block N-linked glycosylation at a concentration of 10 μg/mL. As might be seen, the acceptor fluorescence signal became significantly weaker (Figure 3B) in contrast to the untreated cells (Figure 3C) due to the lessened glycan attachment. Quantitative analysis of the FRET-induced fluorescence intensity was shown in Figure S3, where the FRET-induced fluorescence/488 fluorescence ratios were calculated to normalize the expression of receptor-specific glycan. The ratio of tunicamycin inhibition group apparently decreased compared with the control group, which also supported that the FRET-induced signal was specifically generated by intramolecular FRET. Additionally, we found that the donor fluorescence signal also suffered from attenuation. Previous research has reported that glycosylation could regulate the function of receptors by changing the ligand binding or the expression of receptors.24 Since most of the glycosylation sites of IL-36R were not involved in direct

Figure 4. Erasure of the IL-36 ligand fluorescence signal via a competitive binding of an unlabeled compound (antagonist IL, 36Ra). (A) Imaging of FRET-induced fluorescence signal when IL-36γ-Fluor 488 bound to IL-36R (left) and when IL-36γ-Fluor 488 was in competition and removed by the unlabeled compound, IL-36Ra (right). (B) The dose−response curve of the FRET ratio under the competition of IL-36Ra. The donor signal at various concentrations (a−g) was also exhibited. Scale bar: 20 μm.

report that a 10-fold concentration of antagonist IL-36Ra was sufficient to inhibit the effects of IL-36γ.26 The cytotoxicity assay indicated that a 20-min incubation of IL-36Ra with a series of concentrations had negligible impact on HEK 293T cells viability (Figure S5). A dose−response curve about the FRET ratio was shown in Figure 4B, demonstrating the C

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2017YFC0906800), and the China Postdoctoral Science Foundation (Grant 2018M630137).

competition response was dose dependent on the competitors. The residual IL-36γ-Fluor 488 signals after different concentrations of IL-36Ra competition were also exhibited. These data demonstrated that our strategy had the ability to flexibly erase the ligand signal by appropriate competitions. The CCK8 cell proliferation assay indicated that this sequential modification and competition had negligible impact on cell viability (Table S1). Besides, we also investigated the dose− response curve of an unlabeled IL-36γ, and the FRET-ratio curve suggested that its ability to compete with the labeled IL36γ-Fluor 488 was much weaker than IL-36Ra (Figure S6). In conclusion, a nongenetically encoded FRET-based labeling method was developed for in situ imaging receptorspecific glycans. This strategy could attach the FRET donor onto any cell-surface specific receptor beyond the restriction that the receptors have to have an extracellular terminus which is used to attach a fluorescent tag. Furthermore, our strategy had the potential to profile multiple receptor-specific glycans on live cell surfaces by continuous cycles of the labeled ligand binding, FRET-induced fluorescence imaging, and the unlabeled compound competing for fluorescence erasure. Although this labeling strategy was based on the previous ideas of time-resolved FRET,18,19 the compelling aspect here is that this approach could be imaged and quantified by microscopy and also allows the target localization. In our future work, we will further explore and optimize the cell culture conditions27 to improve the cell viability after multiple times of labeling and erasure on a given cell surface for uncovering the complex glycan-regulated mechanism. We believe this in situ receptor-specific glycan profiling strategy will have wide applications in molecular diagnosis and cellular targeted therapies.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.8b05292. Experimental methods, evaluation of the cytotoxicity of the labeling and competing procedures, measure of CXCL1 assay, inhibition of N-linked glycosylation by tunicamycin treatment, real-time RT-PCR, Western blotting assay; statistical analysis, and supplementary figures (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Nan Li: 0000-0003-4246-2994 Weifei Zhang: 0000-0002-1214-6782 Syed Niaz Ali Shah: 0000-0001-9902-857X Jin-Ming Lin: 0000-0001-8891-0655 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by National Natural Science Foundation of China (Grants 21435002, 21727814, 21621003), National Key R&D Program of China (Grant D

DOI: 10.1021/acs.analchem.8b05292 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry (27) Li, X.; Valadez, A. V.; Zuo, P.; Nie, Z. Bioanalysis 2012, 4, 1509−1525.

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DOI: 10.1021/acs.analchem.8b05292 Anal. Chem. XXXX, XXX, XXX−XXX