Glutamine-Binding Protein from Escherichia Coli Specifically Binds a Wheat Gliadin Peptide. 2. Resonance Energy Transfer Studies Suggest a New Sensing Approach for an Easy Detection of Wheat Gliadin Maria Staiano,† Viviana Scognamiglio,† Gianfranco Mamone,‡ Mauro Rossi,‡ Antonietta Parracino,† Mose’ Rossi,† and Sabato D’Auria*,† Institute of Protein Biochemistry, C.N.R., Via Pietro Castellino, 111 80131, Naples, Italy, and Institute of Food Sciences, C.N.R., Avellino, Italy Received May 27, 2006
Abstract: In this work is presented the first attempt to develop a fluorescence assay for detection of traces of gluten in food by utilizing the recombinant glutaminebinding protein (GlnBP) from E. coli. We found that GlnBP specifically binds the sequence of amino acids present both in gliadin and other prolamines classified as toxic for celiac patients. Affinity chromatography experiments together with mass spectrometry experiments demonstrated that GlnBP can bind the following amino acid sequence XXQPQPQQQQQQQQQQQQL. Sequence alignment experiments pointed out that this sequence is exclusively representative of the gliadin and the other prolamines considered toxic for celiac patients. These findings suggest the development of a competitive resonance energy transfer (RET) assay for an easy and rapid detection of this sequence in raw and cooked food. Keywords: Gliadin • optical biosensors • fluorescence • celiac disease
In recent years, fluorescent sensors have been developed based upon the natural affinity and specificity between a protein and its substrate.1,2 Site-directed mutagenesis has allowed alterations in amino acid sequences, resulting in changes in protein binding constants and insertion of new positions for reporter group labeling. Such amino acid changes have allowed the signal transduction of the binding event to be evaluated by using a variety of physical and chemical techniques.3,4 In particular, optical methods of detection using fluorescence energy transfer, polarization, and solvent sensitivity have been shown to offer high signal- to-noise ratios and the potential to construct simple and robust devices.5,6 As a result, the use of such methods has allowed for the development of highly sensitive optical protein biosensors for a variety of analytes, including amino acids, sugars, and metabolic byproducts. * To whom correspondence should be addressed. E-mail: s.dauria@ ibp.cnr.it. Fax: +39-0816132277. Address: Institute of Protein Biochemistry, Consiglio Nazionale delle Ricerche, Via Pietro Castellino, 111, 80131 Naples, Italy. † Institute of Protein Biochemistry. ‡ Institute of Food Sciences. 10.1021/pr060258+ CCC: $33.50
2006 American Chemical Society
The Escherichia coli periplasmic space contains a diverse group of binding proteins whose main function is to present various biomolecules such as sugars, amino acids, peptides, and inorganic ions for transport into the cell.7 Although these proteins range in size and primary sequence, they have remarkably similar structures.8,9 The three-dimensional structures are characterized by two globular domains connected by a hinge region which forms a cleft for substrate binding. X-ray crystallographic data consistently show that the binding of substrate induces large conformational changes in the tertiary structure of the protein. The changes in global structure of these proteins make them compelling targets to serve as the basis for new biosensor design. Celiac disease is an inflammatory disease of the small intestine affecting genetically susceptible people.10,11 This pathogenesis is related to inappropriate intestinal T-cell activation in HLA-DQ2 and -DQ8 individuals triggered by peptides from wheat gliadin and related prolamins from barley and rye. Changes in intestinal permeability, secondary to alterations in intercellular tight junctions or in the processing of the food antigen, have also been implicated in the loss of tolerance to gliadin.12 At present, a strict gluten-free lifelong diet is mandatory for celiacs for both intestinal mucosal recovery and prevention of complicating conditions such as lymphoma and refractory sprue. However, dietary compliance has been shown to be poor in most patients mainly because of inadvertent gluten consumption. From this point of view, a very sensitive assay for detection of toxic components of prolamins in food represents a perspective worth pursuing. In the present report, we introduce an optical assay for the detection of gliadin, a very well characterized toxic prolamin, based upon the E. coli glutamine binding protein (GlnBP).
Materials and Methods Materials. All the chemicals used were commercial samples of the purest quality. Glutamine-binding protein from E. coli (GlnBP) was prepared and purified according to D’Auria.13 The protein concentration was determined by the method of Bradford14 with bovine serum albumin as standard on a double beam Cary 1E spectrophotometer (Varian, Mulgrade, Victoria, Australia). Purification of Prolamines. Cereal flours from wheat cultivar Sagittario, barley cultivar Giacinto, rye cultivar Picasso, and oat Journal of Proteome Research 2006, 5, 2083-2086
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communications Table 1. Alignment of Gliadin Peptides Homologous to QPQPQQQQQQQQQQQQ(LI)
a Boxed residues indicate substituted amino acid residues along the peptide chain found under the indicated accession number in the NCBI Data Bank archive.
cultivar Nave were generous gifts from Dr. Stanca (Istituto Sperimentale per la Cerealicoltura, SOP di Fiorenzuola D’Arda, Italy). Commercial supplies of rice and corn kernels were milled to finely ground flours by using a laboratory mill. An amount of 100 mg of each flour was extracted with 60% (v/v) aqueous ethanol by magnetic stirring for 30 min at 50 °C. The suspensions were centrifuged for 20 min at 15000g at room temperature. The supernatants were freeze-dried and stored at -20 °C until use. Purified gliadin and zein were purchased from Sigma (Sigma Chemical Co., St. Louis, MO). Peptic-Tryptic (PT) Digestion of Prolamines. An amount of 10 mg of purified prolamines was dissolved in 1.0 mL of 0.2 N HCl and incubated in a water bath with pepsin (1:100 enzyme/substrate ratio) for 2 h at 37 °C. Then the pH was adjusted to 8.0 with 2 N NaOH, and trypsin was added to an enzyme/substrate ratio of 1:100. After 2 h incubation at 37 °C, the reaction was stopped by heating for 5 min. The samples were freeze-dried and stored at -20 °C. Steady-State Fluorescence Spectroscopy. Steady-state emission experiments were done on the ISS-K2 fluorometer (ISS, Urbana-Champaign) with excitation wavelength of 480 nm and the emission slit width of 1 nm. Samples were placed in the thermostatic holder and kept at 25 °C for 5 min before measurements. The presented data do not require a statistical examination since the fluorescence measurements (each of which has been performed at least three times) gave the emission spectra almost superimposable. In addition, our RET measurements for the gliadin determination showed a high degree of accuracy (data not shown). Labeling of GlnBP. Solutions of homogeneous GlnBP (2.0 mg/mL in 1.0 mL of 0.1 M bicarbonate buffer, pH 9.0) and PT were mixed with 10 µL of fluorescein isothiocyanate (FITC) (Molecular Probes) solution in N,N-dimethylformamide (DMF) (1.0 µg FITC/100, µl DMF) and 10 µL of rhodamine isothiocyanate (FITC) (Molecular Probes) solution in DMF (1.0 µg RITC/ 100 µl DMF), respectively. The reaction mixtures were incubated for 1 h at 30 °C and the labeled molecules were separated from unreacted probes by passing them over a Sephadex G-25 column equilibrated in 50 mM phosphate buffer, 100 mM NaCl, pH 7.0. For fluorescence measurements we used FITC-GlnBP samples with absorbance 0.05 OD at the excitation wavelength. Mass Spectrometry Analysis. MALDI-TOF experiments were carried out on a PerSeptive Biosystems (Framingham, MA) Voyager DE-PRO instrument equipped with a N2 laser (337 nm, 3 ns pulse width). Each spectrum was taken with the following procedure: A 1 µL aliquot of sample was loaded on a stainless steel plate together with 1 µL of matrix R-cyano-4-hydroxycinnamic acid (10 mg in 1 mL aqueous 50% acetonitrile). Mass 2084
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spectrum acquisition was performed in both positive linear and reflectron mode by accumulating 200 laser pulses. The accelerating voltage was 20 kV. External mass calibration was performed with mass peptide standards (SIGMA). Tandem mass spectrometry (MS/MS) data for the extracted peptides were obtained using a Q-STAR mass spectrometer (Applied Biosystems) equipped with nanospray interface (Protana, Odense, Denmark). Dried samples were resuspended in 0.1% TFA, desalted using Zip-Tip C18 microcolumns (Millipore), and sprayed from gold-coated “medium length” borosilicate capillaries (Protana). The capillary voltage used was 800 V. Doubly charged ion isotopic clusters was selected by the quadrupole mass filter (MS1) and then induced to fragment by collision. The collision energy was 30-40 eV, depending on the size of the peptide. The collision-induced dissociation was processed using Analyst 5 software (Applied Biosystems). The deconvolution MS/MS was manually interpreted with the help of the Analyst 5 software.
Results and Discussion GlnBP is a monomeric protein that binds with high affinity glutamine (gln) and poly-gln residues.13 Since gluten proteins are rich of gln residues, we questioned if GlnBP was able to bind amino acid sequences present in gluten proteins such as gliadin, a protein toxic for celiac patients. To check it, GlnBP was covalently bound to a CNBr-activated Sepharose 4B resin according to the manufacturer’s instructions (Amersham Biosciences Europe GmbH, Cologno Monzese, Italy) and PTgliadin was passed through the column. The column was washed with three volumes of phosphate buffer saline and the peptide(s) bound to GlnBP was eluted with 0.2 M glycine/HCl, pH 3.0. The eluted fractions were utilized for mass spectrometric experiments. Mass spectrometric investigation of the isolated gliadin sequences showed that GlnBP binds the following amino acid sequence: XXQPQPQQQQQQQQQQQQL. The two amino acid residues located at the peptide N-terminus could be either MetThr or Thr-Met. For this reason, we have indicated them as XX.15 Alignment analysis of the prolamine amino acid sequences retrieved from Swiss-Prot, showed that this sequence was present only into the toxic prolamines (Table 1). This result prompts us to develop a resonance energy transfer (RET) assay for sensing toxic sequences for celiac patients. We labeled GlnBP and PT-gliadin with fluoresceine isothiocyanate and rhodamine isothiocyanate, respectively. Figure 1 shows the SDS-PAGE of the labeled fluoresceine-GlnBP and rhodamine PT-gliadin upon exposure to UV light. The emission spectra of fluoresceine-labeled GlnBP alone and upon addition of
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Figure 1. SDS-PAGE of fluorescein-labeled GlnBP (lane 1) and rhodamine-labeled PT-gliadin (lane 2). The protein bands were visualized by UV illumination.
Figure 2. (a) Normalized fluorescence emission spectra at different concentration of rhodamine-labeled PT-gliadin; (b) effect of rhodamine-labeled PT-gliadin on the emission of fluoresceinlabeled GlnBP in the absence (closed squares) and in the presence (closed circles) of 250 nmol unlabeled PT-gliadin.
rhodamine-labeled PT-gliadin showed the presence of RET between fluorescein and rhodamine (data not shown). The resonance energy transfer process observed upon the addition of rhodamine-labeled PT-gliadin indicates a close interaction between GlnBP and PT-gliadin. Figure 2a shows the effect of rhodamine-labeled PT-gliadin addition on fluoresceine-GlnBP. To better visualize the RET process, we have normalized the
Figure 3. Effect of rhodamine-labeled zein on the emission of fluorescein-labeled GlnBP.
emission spectra at the maximum fluorescence intensity. Figure 2b shows the variation of the fluorescence intensity ratio (I520 nm/I572 nm) at different concentrations of rhodamine-PTgliadin. The obtained results indicate that the sensitivity of this assay is up to 1.0 µg of PT-gliadin addition; that means 33 nM if we consider the average molecular weight of gliadin before enzyme digestion (30000 Da) and the volume of the RET assay (1 mL). Figure 2b also shows the effect of the unlabeled PT-gliadin on the fluorescein-GlnBP-rhodamine-PT-gliadin complex fluorescence emission. In the presence of 250 nmol unlabeled PT-gliadin, a marked reduction of the RET efficiency is observed as a consequence of the competition between unlabeled PTgliadin and rhodamine-labeled PT-gliadin. This result suggests the use of this assay for determining the presence of gliadin in food. In addition, the specificity of the RET assay for PT-gliadin based on the utilization of GlnBP is confirmed by the fact that GlnBP does not bind a peptic-tryptic digest of zein, the corresponding prolamin from corn that together with rice, is a safe cereal for celiac patients (Figure 3). This result also indicates that the increased intensity of the acceptor molecule is due to energy transfer and it is not a side effect of the excitation of the increased concentration of the acceptor. The rationale of the proposed strategy is that gluten proteins (gliadins and glutenins) are characterized by a high content of glutamine (gln) residues and, consequently, they represent a substrate for the GlnBP. As a consequence, the relevance of these results for the topic of gluten determination has been addressed. The detection of residual gluten in food is fundamental for celiac patients. Among toxic prolamins, gliadin has been very well characterized. Gliadin resulted to be a mixture of many proteins; biochemical analysis revealed the existence of a relationship among the various constituents, so gliadin fractions have been grouped in three classes named R, γ, and ω gliadins. To date several immuno-active gliadin peptides in the celiac population have been identified.16,17 Some of these epitopes are encompassed in a 33 mer gliadin peptide found to be resistant to digestion by gastric and pancreatic enzymes.18 It is noteworthy that most of these T-cell epitopes were recognized following deamidation catalyzed by tissue transglutaminase (tTG), in which some specific glutamine residues were converted to glutamic acids.19 Moreover, T-cell epitopes cluster in regions that are rich in proline residues. Taken together, these observations highlighted the difficulties in Journal of Proteome Research • Vol. 5, No. 9, 2006 2085
communications identifying sequences useful for developing immunoassays addressed toward the toxic portions of gliadin. Ideally, a method to determine gliadin should be applicable in a wide range of food, irrespective of processing, and should be directly related with toxicity. Until now, none of the produced methods are considered to be fully satisfactory. On the other hand, the use of reducing agents that are present in the proposed solvent can improve the extraction of prolamines but affect their immunochemical quantification. From this point of view our method can potentially work also in reducing conditions, so overcoming the problem related to prolamin extraction. In conclusion, in this work we have presented useful data for the design of a competitive RET assay for an easy and rapid detection of gliadin. Genetic manipulation experiments for improving the protein affinity to PT-gliadin are in progress.
Acknowledgment. This project was realized in the frame of the CRdC-ATIBB and CRdc-NTP POR UE-Campania Mis 3.16 activities and the CNR Commessa “Diagnostica Avanzata ed Alimentazione”. References (1) Coulet, P. R.; Bardeletti, G. Biochem. Soc. Trans. 1991, 19, 1-4. (2) Hellinga, H. W.; Marvin, J. S. Trends Biotechnol. 1998, 16, 83189. (3) Ramsden, J. J. J. Mol. Recognit. 1997, 10, 109-120. (4) Sorochinskii, V. V.; Kurganov, B. I. Appl. Biochem. Microbiol. 1997, 33, 515-529.
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(5) Giuliano, K. A.; Post, P. L.; Hahn, K.; Taylor, D. L. Annu. Rev. Biophys. Biomol. Struct. 1995, 24, 405-434. (6) Giuliano K. A.; Taylor, D. L. Trends Biotechnol. 1998, 16, 135140. (7) Higgins C. F. Annu. Rev. Cell Biol. 1992, 8, 67-113. (8) Adams, M. D.; Oxender, D. L. J. Biol. Chem. 1989, 264, 1573915742. (9) Hsiao, C. D.; Sun, U. J.; Rose, J.; Wang, B.-C. J. Mol. Biol. 1996, 262, 225-242. (10) Maki, M.; Collin, P. Lancet 1997, 349, 1755-1759. (11) ]. Tursi, A.; Giorgetti, G.; Brandimarte, G.; Rubino, E.; Lombardi, D.; Gasbarrini, G. Hepatogastroenterology 2001, 48, 462-464. (12) Fasano, A.; Not, T.; Wang, W.; Uzzau, S.; Berti, I.; Tommasini, A.; Goldblum, S. E. Lancet 2000, 355, 518-1519. (13) D’Auria, S.; Scire`, A.; Varriale, A.; Scognamiglio, V.; Staiano, M.; Ausili, A.; Rossi, M.; Tanfani, F. Proteins 2005, 58, 80-87. (14) Bradford, M. M. Anal. Biochem 1976, 72, 248-254. (15) De Stefano, L.; Rossi, M.; Staiano, M.; Mamone, G.; Parracino, A.; Rossi, M.; D’Auria, S. J. Protein Res. 2006, 5, 1241-1245 (16) Vader, L. W.; de Ru, A.; van der Wal, Y.; Kooy, Y. M. C.; Benckhuijsen, W.; Merin, M. L.; Wouter Drijfhout, J. W.; van Veelen, P.; Koning, F. J. Exp. Med. 2002, 195, 643-649. (17) Arentz-Hansen, H.; McAdam, S. N.; Molberg, O.; Fleckenstein, B.; Lundin, K. E.; Jorgensen, T. J.; Jung, G.; Roepstorff, P.; Sollid, L. M., Gastroenterology 2002, 123, 803-809. (18) Shan, L.; Molberg, O.; Parrot, I.; Hausch, F.; Filiz, F.; Gray, G. M.; Sollid, L. M.; Khosla, C. Science 2002, 297, 2275-2279. (19) Molberg, O.; McAdam, S. N.; Korner, R.; Quarsten, H.; Kristiansen, C.; Madsen, L.; Fugger, L.; Scott H.; Noren, O.; Roepstorff, P.; Lundin, K. E.; Sjostrom, H.; Sollid, L. M. Nat. Med. 1998, 4, 713-717.
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