Cell-permeable probe for identification and imaging of sialidases

Charng-Sheng Tsaia,b, Hsin-Yung Yena,c, Meng-I Lina, Tsung-I Tsaia, Shi-Yun Wanga, Wen-I Huanga, Tsui-Ling Hsua, Yih-Shyun E. Chenga, Jim-Min Fanga,d, and Chi-Huey Wonga,b,1

aGenomics Research Center, Academia Sinica, Taipei 115, Taiwan; bDepartment of Chemistry, The Scripps Research Institute, La Jolla, CA 92037; and cInstitute of Biochemical Sciences, and dDepartment of Chemistry, National Taiwan University, Taipei 106, Taiwan

Contributed by Chi-Huey Wong, December 22, 2012 (sent for review November 5, 2012) Alkyne-hinged 3-fluorosialyl fluoride (DFSA) containing an alkyne sialylglycoconjugates and development of progressive neurosomatic group was shown to be a mechanism-based target-specific irre- manifestations (13). versible inhibitor of sialidases. The ester-protected analog DFSA Activity-based protein profiling (ABPP) is a functional proteo- (PDFSA) is a membrane-permeable precursor of DFSA designed to mic technology that uses chemical probes for specific be used in living cells, and it was shown to form covalent adducts (14). An ABPP probe typically is composed of two elements: a with virus, bacteria, and human sialidases. The fluorosialyl– reactive group and a tag. The reactive group is designed based on adduct can be ligated with an azide-annexed biotin via click reac- the catalytic mechanism of the target enzyme, and it usually con- tion and detected by the streptavidin-specific reporting signals. tains an electrophile that can react with a nucleophilic residue in Liquid chromatography-mass spectrometry/mass spectrometry anal- the enzyme active site to form a covalent adduct. The tag may be ysis on the tryptic peptide fragments indicates that the 3-fluorosialyl either a reporter, such as a fluorophore, or an affinity label, such as moiety modifies tyrosine residues of the sialidases. DFSA was used biotin. The tag may incorporate a moiety, such as an alkyne or to demonstrate influenza infection and the diagnosis of the viral azide, for subsequent modification, such as by the Cu(I)-catalyzed susceptibility to the anti-influenza drug oseltamivir acid, whereas azide–alkyne [3+2] cycloaddition (CuAAC), to introduce a re- PDFSA was used for in situ imaging of the changes of sialidase ac- porter (15, 16). ABPP probes have been developed to monitor tivity in live cells. changes of specific enzymes associated with certain biological states, including serine (17, 18), cysteine proteases (19– ABPP probe | click chemistry | imaging agents | proteomics 22), protein phosphatases (23–25), oxidoreductases (26), histone deacetylases (27), kinases (28, 29), metalloproteases (30–32), and ialidase, also called (NA), is an exoglycosidase glycosidases (33–36). Sthat catalyzes the hydrolysis of terminal residues Two types of sialidase ABPP probes, the quinone methide and from the oligosaccharides of glycoconjugates. Sialidases are widely the photoaffinity labeling probes, have been reported. These expressed for various functions (1). Many pathogens, such as probes often have problems in nonspecific labeling when used in viruses, bacteria, and protozoa, produce sialidases for invasion, complex protein samples, such as cell lysates (37, 38). In addition, nutrition, detachment, and immunological escape (2). Mammal these probes cannot be applied to in situ labeling experiments sialidases also have been implicated in many biological processes, because they are impermeable to cell membranes. For sialidase including regulation of cell proliferation/differentiation, modula- profiling under physiological conditions, preparation of a target- tion of cell adhesion, metabolism, and immunological functions specific and cell-permeable ABPP probe is needed to study sia- (3, 4). Four types of sialidases have been identified and charac- lidase changes in living cells. terized in mammals. These sialidases are encoded by different Introducing a fluorine at C-3 of sialic acid has been reported to and expressed at different intracellular localizations as ly- antagonize sialic acid biosynthesis or modify sialylation (39–47). sosomal (Neu1), cytosolic (Neu2), plasma-membrane (Neu3), and Because of the strong electron-withdrawing nature, the fluorine mitochondrial/lysosomal (Neu4) enzymes. Although these enzymes can destabilize the formation of positive charge within the car- share a common catalytic mechanism, they have little overlapping bohydrate ring to inhibit the catalytic activity of sialidases. 3- functions, probably because of differences in subcellular distribu- Fluorosialyl fluoride was used as an effective inhibitor against fi tion, pH optimum, kinetic properties, and substrate speci cities sialidase (40) or Trypanosoma cruzi transsialidase (TcTs) (46, 47), (5). The regulation and detailed functions of these enzymes are and these works have prompted us to design an ABPP probe fi largely unde ned (6). for sialidases by using an alkyne-hinged 3-fluorosialyl fluoride Alterations in sialidase activities have been implicated in dif- (DFSA) that is expected to form a covalent adduct with sialidases ferent diseases. For example, elevated sialidase activities have (Fig. 1). In this study, DFSA is shown to be a mechanism-based been reported in BHK-transformed cells and in human breast/ irreversible inhibitor by trapping the 3-fluorosialyl–enzyme in- colon cancer tissues (7, 8). Animal studies also suggest the roles termediate, which can be ligated with an azide-annexed biotin of sialidases in tumorigenic transformation and tumor invasion. (azido-biotin) via CuAAC for isolation and identification of sia- Biochemical characterizations of mammalian sialidases suggest lidases. We also developed an ester-protected DFSA (PDFSA) as that increases in Neu3 are involved in colon, renal, and prostate the cell-permeable precursor of DFSA to allow cell uptake (48, cancers. Transfection of the Neu3 into cancer cells leads to 49), identification, and in situ imaging of sialidase activities under protection against apoptosis by increased Bcl-2 expression and physiological conditions. decreased activity of caspase-3/-9 (9). Furthermore, Neu3 over- expression increases cell motility and invasion by modulation of EGF receptor phosphorylation and Ras activation (10, 11). In Author contributions: C.-S.T., T.-L.H., Y.-S.E.C., J.-M.F., and C.-H.W. designed research; C.-S.T., contrast to the apparent Neu3 promotion in cancer progressions, H.-Y.Y., M.-I.L., T.-I.T., S.-Y.W., and W.-I.H. performed research; C.-S.T. and H.-Y.Y. analyzed other sialidases play roles in cancer reduction through acceler- data; and C.-S.T., H.-Y.Y., T.-L.H., Y.-S.E.C., and C.-H.W. wrote the paper. ated cell apoptosis, differentiation, and suppression of cell in- The authors declare no conflict of interest. vasion (12). In other aspects, deficiency of the lysosomal sialidase 1To whom correspondence should be addressed. E-mail: [email protected]. (Neu1) is considered a major cause of , an inherited This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. lysosomal storage disease resulting in excessive accumulation of 1073/pnas.1222183110/-/DCSupplemental.

2466–2471 | PNAS | February 12, 2013 | vol. 110 | no. 7 www.pnas.org/cgi/doi/10.1073/pnas.1222183110 Downloaded by guest on September 25, 2021 DFSA Labeling of Sialidases and Characterization. To examine the feasibility of DFSA as an activity-based probe, we evaluated the inhibition of various sialidases by DFSA, 3-fluorosialyl fluoride, 2-deoxy-2,3-didehydro-N- acetylneuraminic acid (DANA), and PDFSA using 2-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (MUNANA) as the substrate. The sialidases used in this study are from a variety of species, including influenza virus (NA), bacteria (nanA, nanB, nanC, nanJ, nanI, and nanH), and human (Neu1, Neu2, Neu3, and Neu4) sialidases. Similar to DANA, all sialidases were sensitive to both DFSA and 3-fluorosialyl fluoride with IC50 values at micro- to submicromolar levels (Table S1). In comparison with 3-fluorosialyl fluoride, DFSA slightly attenuated the inhibition for most sialidases. However, enhanced inhibitory effects were observed in Neu1, nanB, and nanC, probably as a result of certain subtle structural differences. The ability to in- hibit all these sialidases suggested that DFSA might be a potent activity-based probe for these enzymes. In contrast to the sensitive inhibition by DFSA, the ester-protected analog PDFSA did not inhibit these sialidases, suggesting that esterification of the hy- droxy and carboxy groups in PDFSA prevents binding to the sialidase active sites. To validate the sialidase labeling by DFSA, we examined the formation of the fluorosialyl–enzyme adducts by SDS/PAGE analyses. All the bacterial sialidases tested in this study formed an adduct that was captured by azido-biotin and detected by the streptavidin-specific reporting signal (Fig. 2A). The influenza NA located at the surface of influenza virus (A/WSN/1933/H1N1) also formed a DFSA adduct that could be outcompeted by the NA inhibitor oseltamivir acid (OS), suggesting that DFSA inter- acted with NA at the active site (Fig. 2B). To detect the specific DFSA labeling in cells, we overexpressed four human sialidases, Fig. 1. Identification and imaging of sialidase with activity changes using Neu1–4 in 293T cells. It has been reported that mature Neu1 and these activity-based sialidase probes. Neu4 proteins can be processed further at N-termini (50–53). To ensure positive anti-FLAG staining, we constructed the expres-

sion plasmids with the sequences encoding FLAG tags at both 5′- CHEMISTRY Results and 3′-ends of Neu1 and Neu4 genes. Fig. 2C shows that DFSA Synthesis of PDFSA and DFSA. As shown in Scheme 1, the reaction positively labels all the human sialidases in the protein extracts of of N-(pent-4-ynoyl)-mannosamine (1)with3-fluoropyruvic acid transfected 293T cells. We also found that the DFSA labeling of (as the sodium salt) was carried out under the catalysis of human sialidases was pH sensitive: Neu1 and Neu3 were poorly N-acetylneuraminic acid aldolase (Neu5Ac aldolase, EC 4.1.3.3) to labeled at neutral to alkaline pH (Fig. S1). fi yield adduct 2 as a mixture of C-3 diastereomers (axial/equatorial = To identify DFSA-labeled sites on sialidase, puri ed sialidases 7:1 ∼3:1). The adduct was subjected to esterification, acetylation, were used for labeling with DFSA and digested with trypsin for 3 R liquid chromatography-mass spectrometry/mass spectrometry BIOCHEMISTRY and chromatographic isolation to afford ester having the 3 fi fi fl fi (LC-MS/MS). The identi ed peptides modi ed by 3- uorosialyl con guration. Selective deacetylation at the anomeric position moiety are shown in Fig. S2 and Table S2. Except for nanB, there 4 was achieved by using hydrazine acetate to give , which was was only one tyrosine residue covalently modified by 3-fluorosialyl fl treated with diethylaminosulfur tri uoride to give PDFSA moiety in each sialidase. NanB was labeled at an additional ad- (α-anomer, 60%) and its β-anomer (30%). Final deprotection of jacent tyrosine on the same peptide. Based on the protein struc- PDFSA under alkaline conditions produced DFSA in 85% yield tures of nanA, nanB, and nanI (54–56), it was revealed that DFSA after purification on a reverse-phase column. labeled the tyrosine residue located in the activity sites of these

Scheme 1. Synthesis of PDFSA and DFSA.

Tsai et al. PNAS | February 12, 2013 | vol. 110 | no. 7 | 2467 Downloaded by guest on September 25, 2021 Fig. 2. Identification of sialidases by DFSA adduct formation. (A) Recombinant sialidases produced in Escherichia coli were treated briefly with DFSA, separated in SDS/PAGE, and transferred to PVDF membranes (Left and Center) that were reacted with the click reaction reagent azido-biotin to ligate the biotin moiety to the alkyne group of the enzyme conjugate. The biotin-modified sialidases present in the washed membrane were detected through the streptavidin-conjugated HRP reporting system. These sialidase adducts also were shown by Coomassie blue staining (Right). (B) Detection of influenza NA was conducted after incubating influenza virus (A/WSN/1933/H1N1) samples with DFSA with or without addition of the specific inhibitor OS to compete with DFSA for binding to the active site (Left). These total lysates also were shown by Coomassie blue staining (Right). (C) Human sialidase samples present in the lysates of 293T transfected or untransfected cells (Mock) were treated with or without DFSA before SDS/PAGE analyses. The sialidases also were detected by im- munoblot analyses of the FLAG epitope presented in Neu1, Neu2, Neu3, and Neu4. (D) Labeling of human sialidases also was conducted by incubating PDFSA with sialidase-expressing 293T cells and processed for adduct detection similarly. The sialidases also were detected by immunoblot analyses of the FLAG epitope presented in Neu1, Neu2, Neu3, and Neu4.

sialidases. By sequence alignment, the orthologous tyrosine resi- (51–53), which may be a result of the addition of FLAG tags. due in the catalytic center also was labeled by DFSA in nanC, Here, the analyses of sialidase activity by PDFSA and anti- nanJ, and nanH (Table S3). It was strongly suggested that DFSA FLAG staining showed very high colocalization ratios in all the specifically labeled the catalytic tyrosine residue in sialidases. overexpressed human sialidases, suggesting specific PDFSA la- beling of the sialidases in live cells. In Situ Labeling of Intracellular Sialidases by PDFSA Treatment of 293T Cells Overexpressing Sialidases. For the profiling of in- Profiling of Sialidase Changes Using the DFSA/PDFSA Labeling System. tracellular sialidases, the probe needs to be cell permeable, but Sialidosis is an inherited lysosomal storage disease usually caused DFSA is poorly permeable to cells. To enhance the cellular by Neu1 deficiency. We examined Neu1 activity differences in the uptake, we used the ester-protected probe, PDFSA, to test the fibroblasts of a normal person and sialidosis patients by live cell labeling of intracellular sialidases overexpressed in 293T cells. In labeling using PDFSA. We found that the sialidase labeling was comparison with the sialidase labeling of cell extracts with DFSA significantly reduced in the more severe sialidosis (GM02921) (Fig. 2C), we observed similar results of PDFSA labeling after fibroblast cells compared with the milder sialidosis (GM02922) incubation of live cells overexpressed with FLAG-tagged Neu1, cells (Fig. 4A) (57). By blotting with anti-Neu1 antibody (Fig. Neu2, Neu3, or Neu4 (Fig. 2D). The success in sialidase labeling 4B), results showed a correlation between the expression level of using PDFSA prompted us to determine the cellular local- Neu1 and PDFSA-mediated sialidase labeling (Fig. 4A). The izations of the expressed sialidase activities in live cells with differences in sialidase activity observed by PDFSA labeling are PDFSA and to examine the cellular location of the sialidase consistent with the conventional activity measurement of the cell adducts in fixed and permeated cells (Fig. 3). The sialidase ac- extracts using MUNANA as the substrate (Fig. 4C). In contrast, tivities were detected as green signals through the PDFSA-me- no sialidase activities were found in cells treated with PDFSA, diated sialidase labeling; the sialidase proteins also were suggesting that the intracellular sialidases of treated cells were detected as red signals by staining with anti-FLAG antibody. effectively modified by the adduct formation. Consistent with previous reports (5), Fig. 3 shows that the siali- DFSA also successfully detected the NA expressed on in- dase signals are located in for Neu1, cytosol for Neu2, fluenza virus–infected cells by microscopy (Fig. 5). Furthermore, and plasma membrane for Neu3. The cytosolic location of Neu4, DFSA was shown to bind at the active site of influenza NA, and as detected by both PDFSA labeling and anti-FLAG staining, the binding can be competitively inhibited by OS (Fig. S3). We was different from the lysosomal location reported previously expect that OS can inhibit the DFSA labeling to OS-sensitive

2468 | www.pnas.org/cgi/doi/10.1073/pnas.1222183110 Tsai et al. Downloaded by guest on September 25, 2021 Fig. 3. Imaging analyses of sialidase-expressing 293T cells labeled by PDFSA. Live sialidase-expressing 293T cells were treated with PDFSA at 0.2 mM for 15 h. Cells were fixed, permeated, and biotin tagged for confocal microscopic analyses. PDFSA-mediated sialidase labeling is shown in green, and FLAG labeling is shown in red. (Scale bars: 10 mm.)

(OSs) viruses competitively because both compounds bind the the mutant NA and should not inhibit the DFSA binding to in- active site of NA. However, for OS-resistant (OSr)influenza fluenza. Indeed, both OSs and OSr H1N1 influenza viruses were r viruses that have been the prevailing clinical isolates for H1N1 labeled by DFSA in the absence of OS, but only the OS virus since 2008 (58, 59), OS cannot effectively bind the active site of was detectable by DFSA labeling in the presence of OS, sug- gesting the possibility of using a DFSA probe to detect drug- resistant influenza strains. Discussion

We have designed an activity-based sialidase probe, DFSA, by CHEMISTRY using 3-fluorosialyl fluoride as the mechanism-based inhibitor and by incorporating an alkyne group for reporter ligation. DFSA is shown to be an active-site inactivator of all tested sialidases. Bio- chemical analyses of the DFSA-inactivated sialidases by LC-MS/ MS analysis showed that tyrosine residues in the enzyme active site were specifically labeled by DFSA. Our study also demonstrates that the DFSA probe may be used not only for the detection of influenza infections but also for diagnosis of oseltamivir suscepti- BIOCHEMISTRY bility. The ability of DFSA to label sialidases from viral, bacterial, and human enzymes suggests that DFSA may be used as a general sialidase probe for various applications. DFSA is advantageous as a general ABPP probe because of its small size. We further introduce the ester-protected PDFSA to enhance cell-permeable properties and allow the profiling of in- tracellular sialidases. The ability of PDFSA to probe intracellular sialidases using living cells has an added advantage over the methods using cell lysates containing vulnerable sialidases. The sialidase adducts formed by live cell labeling using PDFSA record the status of sialidase activity under physiological conditions. After the enzyme adducts are formed in live cells, analysis of the sialidase adducts may be applied even in harsh conditions. We also have illustrated the use of these probes to study sialidase changes involved in different biological systems. Because sialidase is known to be involved in various diseases, these probes may be Fig. 4. Profiling of sialidase changes in the fibroblasts of sialidosis patients. (A) Fibroblast cells derived from normal (D551) or sialidosis patients (GM02921 and used to study cellular localization changes of sialidases and the GM02922) were cultured for in situ sialidase labeling with PDFSA (10 μM). The differences in sialidase expression in normal and disease states. relevant sialidase labeling signals are marked with stars (Left). These total lysates also were shown by DB71 staining (Right). (B) Fibroblast cells derived Materials and Methods from normal (D551) or sialidosis patients (GM02921 and GM02922) were ana- Membrane Click Reaction. The PVDF membranes were blocked with blocking lyzed by anti-Neu1 antibody. (C) Cellular sialidase activities were measured buffer, 5% (wt/vol) BSA/Phosphate Buffered saline with Tween 20 (PBST) using MUNANA as the substrate and compared with the sialidase activities in [0.1% (vol/vol) Tween 20/PBS]. The membranes were washed with PBS for extracts of cells cultured with or without prior incubation with PDFSA. Values 5 min two times. The protein side of the PVDF membrane was faced down to are means ± SEM of three independent experiments. immerse in click reaction mixture [25 μM azido-biotin (3-azidopropanyl

Tsai et al. PNAS | February 12, 2013 | vol. 110 | no. 7 | 2469 Downloaded by guest on September 25, 2021 In Situ Labeling of Sialidase Expressing Cells with PDFSA. Sialidase transfectant 293T cells (obtained from ATCC) and normal (D551, obtained from Bio- resource Collection and Research Center, Taiwan) and sialidosis fibroblasts (GM02921 and GM02922, obtained from Coriell Cell Repositories) were in- cubated with PDFSA (10 μM) at 37 °C for 24 h. Cells were lysed by NuPAGE LDS Sample Buffer (Invitrogen, 80 mM DTT) and then heated at 90 °C for 15 min. For each sample, 20 μg total lysate was loaded and separated on 4–12% NuPAGE (Invitrogen). After transferring proteins onto the PVDF membrane (Millipore), membrane click reaction was performed and labeling signal was analyzed by chemiluminescence detector. For confocal microscopic analysis, sialidase transfectant 293T cells were seeded onto four-well chamber slices (3 × 105/mL per well), and were cultivated in penicillin/ streptomycin-containing 10% FBS/DMEM. Growth medium was supplemented with PDSFA (0.2 mM) and cultured for 15 h. Cells were fixed with 4% paraformaldehyde for 15 min, permeabilized in 0.5% Triton X-100 for 10 min at room temperature, and subjected to the Fig. 5. Visualization of influenza-infected cells using DFSA labeling. Fluo- probe labeling reaction consisting of 0.1 mM azide-biotin probe/0.1 mM rescence image of influenza-infected cells that were treated with 30 μM DFSA, biotin tagged, and stained with FITC-tagged streptavidin. The in- Tris-triazole ligand/1 mM CuSO4/2 mM sodium ascorbate, in PBS, at room fi fluenza NA is shown in green, and influenza nucleoprotein (NP) is shown in temperature for 1 h. Subsequently, the xed and labeled cells were rinsed – μ red after anti-NP monoclonal antibody staining. Cell nuclei are shown in with PBS and stained with DyLight 488 conjugated streptavidin (2.5 g/mL blue by 4′6-diamidino-2-phenylindole (DAPI) staining. MOCK, noninfected in 0.5% BSA/PBS) at room temperature for 30 min. Recombinant sialidases cells. (Scale bars: 20 μm.) were detected by Alexa Fluor 594–conjugated anti-FLAG antibody (5 μg/mL in 0.5% BSA/PBS). Fluorescent images were captured by Leica TCS-SP5-MP- SMD. All the cell lines were obtained with informed consent and approval biotin), 0.1 mM Tris-triazoleamine catalyst (60), 1 mM CuSO4, and 2 mM of institutional review board of Academia Sinica. sodium ascorbate, with 1 mL for a blot of mini-gel size] and incubated at room temperature for 1 h. After washing with PBST twice, the membrane ACKNOWLEDGMENTS. We thank Mr. Chein-Hung Chen for technical support was probed with peroxidase-conjugated streptavidin for biotin labels on MS analysis and Ms. Li-Wen Lo for technical support on confocal microscopic on blots. analysis. We also thank Academia Sinica for financial support.

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