DOI:
10.1039/C6RA00105J
(Paper)
RSC Adv., 2016,
6, 28612-28618
A fluorescence-enhanced inorganic probe to detect the peptide and capsid protein of human papillomavirus in vitro†
Received
3rd January 2016
, Accepted 7th March 2016
First published on 8th March 2016
Abstract
Cervical cancer is the second-largest killer of women worldwide. Development of biomarkers that can be used to efficiently screen cervical cancer would be extremely useful for clinical management. The presence of human papillomavirus (HPV) capsid proteins, L1 and L2, is extremely important clinically and warrants further examination of cervical cancer. The present study supplied an easy, cost-effective and efficient fluorescence-enhanced method to detect the cationic peptides of HPV capsid proteins by using an Eu-containing polyoxometalate. The binding-induced luminescence enhancement of EuW10 was further successfully used to detect HPV L1 pentamers expressed from Escherichia coli, which could be extended to detect other proteins involving a large amount of polybasic segments. The present study showed an excellent application of a type of inorganic material, polyoxometalate, to viral and biological science.
1. Introduction
Cervical cancer of the uterus is one of the most common cancers in women, with an estimated 500
000 new cases and half the number of deaths every year worldwide, and approximately 99% of them harbour at least one type of human papillomavirus (HPV).1–3 Currently in the clinical setting the screening of cervical cancer relies mainly on cytology and high-risk HPV detection, while histologic diagnosis is the main parameter that drives clinical management of screen-positive women.4 Cytology-based cervical screening identifies dyskaryosis cervical cells and treats cervical intraepithelial neoplasia (CIN) to prevent progression to invasive disease. However, as a consequence, this invasive procedure once being performed too frequently is of high cost and potentially has a negative impact on reproductive outcomes. In addition, although histologically diagnosed CIN regresses spontaneously in more than half of cases, morphology alone is an insufficient predictor of lesion behavior.5–7 Therefore, development of biomarkers that can be used to efficiently screen cervical cancer would be extremely useful for clinical management.
Various possible prognostic markers have been suggested, including HPV genotype and L1 capsid protein. A number of commercial DNA-based approaches to detect HPV have been developed.8 However, these methods are too complicated, expensive, time consuming and/or difficult to produce on a large scale for extensive use. Molecular testing of HPV capsid proteins has higher sensitivity for high-grade disease in comparison to cytology, facilitating identification of patients in need of more aggressive treatment while avoiding interventions in patients who may require only close surveillance.9 HPV L1 capsid protein is the major protein comprising the viral capsid and can assemble into virus-like particles (VLPs) that are associated with immune responses; while L2 is a minor component of the capsid, thought to aid in the assembly and the packaging of viral DNA within the virions.10 The detection of HPV L1 used as a prognostic marker for management of HPV high-risk positive abnormal pap smears was reported by Hilfrich several years ago.11 The particular methodical advantage of L1 capsid protein detection is that the protein is synthesized in the cells of the superficial layer of the epithelium that are easy to obtain by taking a smear or to image using fluorescent microscopy. In addition, HPV vaccine protein L1 was used to predict disease outcome of high-risk HPV+ early squamous dysplastic lesions.12 Recently, a study from a prospective international multicenter indicated that HPV L1 detection could be used to discriminate efficiently cervical precancer from transient HPV infection.13 Therefore, the presence of L1 and L2 could be used as a reliable biomarker and would be extremely useful clinically and warrants further examination.4 However, currently, making available easy-to-perform, cost-effective and efficient methods to detect HPV capsid proteins is still a challenge.
Therefore, in the present study we aim to exploit the fluorescence detection method of HPV capsid proteins through the high binding affinity between the Arg/Lys-rich basic peptides of HPV capsid proteins (L1 or L2) and a suitable polyoxometalate (POM). Previously, a bi-Lindqvist type europium-containing POM, Na9[EuW10O36]·32H2O (EuW10) (Fig. S1†),14 has been used to monitor the assembly of well-defined nanospheres with the cationic peptides of HPV L1.15 And especially the binding mechanisms between them were revealed in detail at a molecular level. In addition, the largely enhanced luminescence response of EuW10 to the cationic peptide of L1 was suggested as possibly being a good bio-inorganic probe and useful to detect HPV capsid proteins, at least in vitro.15 In addition, studies of interactions of EuW10 with several proteins16–18 have suggested EuW10 might be used as a novel biological labeling agent for protein detection. Therefore, the present study used EuW10 as a fluorescence probe. Firstly, by using a cationic peptide from HPV16 L1 protein (HPV16L1Ctb) as a model we will show the intrinsic mechanism of it for the detection of HPV capsid protein. Furthermore, it will be extended to another cationic peptide from L1 as well as to two peptides from the minor capsid protein L2 (Table 1) to validate the proposed method. Finally, the successful detection of the recombinant L1 from E. coli is demonstrated. The present study is vital in finding a novel fluorescence-enhanced method to detect the capsid proteins of HPV, which is easy, cost-effective and efficient and being also possible to be extended to other viruses and/or proteins with identical properties.
Table 1 Sequences and originations of the peptides from HPV capsid protein
Peptide |
Sequence of peptide |
Position in protein |
HPV16L1Cta |
LKAKPKFTLGKRKAT |
474–488 of HPV16 L1 |
HPV16L1Ctb |
SSTSTTAKRKKRKL |
492–505 of HPV16 L1 |
HPV16L2Nt |
MRHKRSAKRTKRA |
1–13 of HPV16 L2 |
HPV16L2Ct |
MLRKRRKRL |
454–462 of HPV16 L2 |
2. Materials and methods
2.1 Materials
The peptides (HPV16L1Cta, L1Ctb, L2Nt and L2Ct, Table 1) were purchased from Shanghai Apeptide Co. Ltd (Shanghai, China), the purity of them being 99.11% as confirmed by HPLC. As illustrated in Table 1, HPV16L1Cta and L1Ctb are the peptides corresponding to the amino acids (AAs) in C-terminal from residues 474 to 488 and 492 to 505 of the HPV16 L1 protein, respectively; while HPV16L2Nt and L2Ct are the similar positively charged sequences from residues 1 to 13 and 454 to 462 present at the N- and C-terminus of the HPV16 L2 protein, respectively. EuW10 was synthesized and characterized according to a published procedure.19 Pure water (ρ = 18.2 MΩ cm, 25 °C) was obtained from a Millipore Milli-Q instrument. The stock solution of EuW10 was prepared at 2.0 mM in aqueous solution and stored in a refrigerator at 4 °C. Then it was diluted to exact concentrations according to the fluorescence and isothermal titration calorimetry (ITC) experiment requirements.
2.2 Fluorescence methods
A Shimadzu (Japan) RF-5301 spectrophotometer was used for the fluorescence spectral measurements. To get reliable spectra, the excitation lamp was kept on for 0.5 h and the samples aged for 1 h before the spectral recording. A quartz cuvette of 1.0 × 1.0 cm was used to contain the sample. To detect the luminescence of EuW10, the excitation wavelength was selected at 265 nm. The measurements were performed at 25 °C either in buffer A (10 mM MES-NaOH, pH 6.0) or buffer B (50.0 mM Tri–HCl, 200 mM NaCl, pH 7.5) solution as indicated.
Time-resolved fluorescence spectra were obtained by using an FLS980-Edinburgh instrument. A quartz cuvette of 1.0 × 1.0 cm was used to contain the sample. The excitation wavelength was selected at 265 nm for EuW10, while the intensity at 591 nm was collected in buffer A solution. The lifetimes of EuW10 were calculated by fitting the decay curves performed by the software of the instrument.
2.3 Isothermal titration calorimetry (ITC)
ITC experiments were conducted using a MicroCal ITC200 (GE) instrument. Peptide and EuW10 were prepared in buffer A solution, while protein solutions were prepared either in buffer A or buffer B as indicated. The concentrations of peptide and EuW10 used for titration were 700 and 50 μM, respectively, while that for protein was 10.0 μM. 0.7 μL peptide solution was injected into the calorimeter cell (Vcell = 200 μL) containing EuW10 solution, and measurement performed under stirring of 1000 rpm with a delay of 180 s every time. The method to calculate the binding constant (Kb) was based on the literature.20 Thermodynamic parameters were obtained by fitting the data using a one-set binding model, performed by the Origin 7.0 program with which the instrument was equipped.
2.4 Expression and purification of the recombinant HPV L1 protein
The HPV16 L1 coding sequences, which were lacking 4 AAs at the N-terminus and 30 AAs at the C-terminus for better expression, were cloned into pGEX-6p-1 based vectors. The protein was expressed with glutathione-S-transferase (GST) fusion in Escherichia coli under IPTG induction. The protein from the cell lysate was purified by using a protocol previously reported.21,22
3. Results and discussion
3.1 The fluorescence-enhanced response of EuW10 to HPV16L1Ctb
In buffer A solution, a slightly acidic condition, EuW10 shows two weak fluorescence emissions at 591 and 618 nm that are assigned to the transitions of 5D0 → 7F1 and 5D0 → 7F2 of Eu3+, respectively.15,23 The luminescence intensity of EuW10 increases dramatically upon the addition of HPV16L1Ctb, as shown in Fig. 1. The limit of detection (LOD) for HPV16L1Ctb is obtained at 1.9 μM when using 30.0 μM EuW10 (Fig. S2†); once the concentration of EuW10 is reduced to 10.0 μM, the LOD for HPV16L1Ctb is increased to 0.5 μM (Fig. S3†). This means that a decrease in the concentration of EuW10 can increase the sensitivity of it to the peptide. However, to reduce the fluctuation in detection we chose 30.0 μM EuW10 as the final concentration.
 |
| Fig. 1 The fluorescence spectra of EuW10 (30.0 μM) in buffer A solution upon gradual addition of HPV16L1Ctb; the inset shows the intensity at 591 nm induced by different concentrations of HPV16L1Ctb. | |
The enhanced luminescence intensity reaches its maximum when the concentration of peptides increases up to ∼120 μM, and then it is constant (inset in Fig. 1). That is, a linear luminescence response to HPV16L1Ctb can be obtained from 1.9 to 114 μM by using 30.0 μM EuW10. The induced luminescence enhancement was attributed to the strong binding between EuW10 and HPV16L1Ctb, which changes the microenvironment of Eu3+ largely by expelling the surrounding water.15,18 Such results illustrate that the enhanced luminescence response of EuW10 to basic peptide might be used to detect the capsid proteins of HPV L1 and/or L2, as several typical cationic peptides are involved there.
3.2 Optimization of L1 peptide segments for HPV detection
It was reported that the C-terminal polybasic patch plays a role in the encapsidation of the viral genomic DNA, at least for some papillomavirus species.24 As the polybasic patch is proposed to be situated within the lumen of the mature virion, it might be less possible that it could be involved in interactions of real virions with POMs, as suggested in a study of HPV11 VLPs in binding with heparin.25 Therefore, more suitable peptides for use as a model for the detection of HPV capsid protein should be explored. As described in Table 1, the basic segment of HPV16 L1 includes two regions at the C-termini, which were generally investigated separately and named as Cta and Ctb, respectively, according to their sequences.26,27 HPV16L1Cta (Table 1) will be the first candidate beyond HPV16L1Ctb to be considered for such purpose. The fluorescence-enhanced responses of EuW10 to HPV16L1Cta are illustrated in Fig. 2, and the intensity at each peptide concentration is illustrated and compared with those induced by HPV16L1Ctb. At low concentration of POM the induced intensities of HPV16L1Cta are relatively strong, but are much less efficient at higher concentrations than those of HPV16L1Ctb. Therefore, the more sensitive response of EuW10 to HPV16L1Cta at lower peptide concentration (<40.0 μM) in Fig. 2 illustrates it might be a better model for the sensitive detection of HPV capsid protein, as in general the population of HPV is extremely low. Actually, a higher LOD at 1.0 μM was obtained for HPV16L1Cta than HPV16L1Ctb when using 30.0 μM EuW10 as fluorescence probe (Fig. S4†); however, a less sensitive luminescence response of EuW10 to HPV16L1Cta at higher peptide concentration (≥40.0 μM) proved that it might not be a good model for the detection of HPV L1 when the concentration of HPV capsid proteins is high using EuW10 as an enhanced fluorescence probe.
 |
| Fig. 2 (A) The fluorescence spectra of EuW10 (30.0 μM) in buffer A solution upon gradual addition of HPV16L1Cta. (B) The intensity comparison of EuW10 at 591 nm upon the titration of each of HPV16L1Cta and HPV16L1Ctb. | |
The above results demonstrate the strong interactions between EuW10 and two peptides from HPV16 L1. And the different level of luminescence enhancement of POM by them indicates that EuW10 might bind to HPV16L1Ctb more compactly. As revealed by ITC between EuW10 and HPV16L1Ctb, the driving forces between them are essentially electrostatic interactions, as well as some contribution from hydrogen bonds.15 Therefore, the number of basic residues involved in peptides should be considered firstly. While each has six basic residues in HPV16L1Cta and HPV16L1Ctb, there is a large difference of induced luminescence enhancement between them. Such a difference may be attributed to the polybasic residues (KRKKRK) in HPV16L1Ctb, which has been suggested to have higher compatibility and binding affinity to another POM, EuSiWMo, in a previous study.28 Being similar here, the dispersive positive charge in HPV16L1Cta may not match precisely with EuW10 either in size and/or charge distance. Further evidence will be supplied by ITC plots between them in the following.
3.3 Optimization of L2 peptidic segments for HPV detection
In addition, two Arg/Lys-rich cationic peptides from the C- and N-terminus of HPV16 minor capsid protein L2, HPV16L2Ct and HPV16L2Nt, should have strong binding affinity with EuW10, both of which contain a large amount of basic AAs.27 It was reported that when the HPV capsid interacts with a proposed cellular receptor, heparan sulfate proteoglycan, it could result in a subtle conformational change that is caused following the exposure of an N-terminal portion of the minor capsid protein L2.29 This surface-exposed region of L2 would be able to further facilitate the uptake of virions to host cells.30 Moreover, it has been suggested that in the L2 of bovine papillomavirus, some parts of the sequence of 61–123 are exposed on the surface of the virion and can be recognized by monoclonal antibodies although the majority of them appear to be buried inside the surface.31 Therefore, it would be interesting to perform an investigation focusing on L2 peptide as a molecular biomarker to detect HPV. Thus we illustrate the binding of two L2 peptides from the C- and N-terminal respectively, HPV16L2Ct and HPV16L2Nt (Table 1), in binding with EuW10 by using fluorescence spectra (Fig. 3). The interactions of them with EuW10 indeed induce more extensive luminescence enhancement of EuW10 than those of HPV16L1Ctb and HPV16L1Cta (Fig. 1 and 2), suggesting stronger binding affinities between them and EuW10 and potentially more sensitive detection of L2 protein. Actually, a higher LOD obtained at 0.6 μM for HPV16L2Ct (Fig. S5†) and at 0.4 μM for HPV16L2Nt (Fig. S6†) when using 30.0 μM EuW10 as fluorescence probe. Therefore, it is possible that the two peptides from L2 of HPV16 could also be a very good target for the detection of HPV capsid.
 |
| Fig. 3 (A) The fluorescence spectra of EuW10 (30.0 μM) in buffer A solution upon the gradual addition of HPV16L2Ct. (B) The intensity comparison of EuW10 at 591 nm upon the addition of each of HPV16L2Ct and HPV16L2Nt. | |
The above results demonstrate the existence of strong interactions between EuW10 and the two L2 peptides. The more extensive luminescence enhancement of EuW10 than those of HPV16 L1 peptides suggests the L2 peptides may bind to POM more strongly. The number of basic residues involved in the peptides may be the first key point to be considered as contributing to the strong interaction. Possessing seven basic residues, HPV16L2Nt induced larger luminescence enhancement than HPV16L2Ct and HPV16L1Ctb, as each has an equal number of six basic residues. In addition, albeit both involve polybasic residues for HPV16L2Ct (MLRKRRKRL) and HPV16L1Ctb (SSTSTTAKRKKRKL), the former induced luminescence enhancement of POM (29.9-fold) is ∼2-fold greater than that of the latter (16.2-fold), when they are at the same concentration of 86 μM. Therefore, factors other than the electrostatic interaction, which induce higher compatibility and binding affinity to EuW10, should also be considered. For example, the distinction of K and R proportions in the two peptides should be paid attention as both possess opposite fractions of K and R. That is, HPV16L2Ct possesses two K and four R, while HPV16L1Ctb has four K and two R. Despite the apparent electrostatic nature, Arg and Lys have different numbers of hydrophobic methylene moieties linking charged functionality to the backbone. The effects of side chain length on lateral ion pairing interactions between carboxylate and ammonium groups have been explored in detail.32–34 These reports indicated the two extra methylenes donate more electron density to and withdraw less electron density from the paired negative groups; especially the longer side chain would provide more flexibility and increase the entropic penalty upon forming electrostatic interaction.32–34 In fact, this shows that an increase in arginine content also results in better capacity to stimulate the binding of peptide with POM, where it has shown that the matching of number and/or distance between the charged residues of peptide and EuW10 is crucially important for the specific interaction between POM and peptide.
In summary, the results show a sufficiently strong binding affinity between EuW10 and the four HPV16 peptides, which induced a large enhancement of the luminescence of EuW10. The large difference of EuW10 luminescence enhancement induced by the four peptides should be extended to peptides from other subtypes of HPV capsid protein, and hopefully can be used to distinguish the different subtypes of HPV after further optimization of either POMs or peptides. Finally, we hope to find a more appropriate, efficient target on which to focus to detect the HPV capsid, once we choose a suitable POM as fluorescence probe model.
3.4 Lifetime changes of EuW10 upon binding with peptides
To get a deeper insight into the origin of luminescence enhancement of EuW10 in binding with peptides, the time-resolved fluorescence of EuW10 in the absence and presence of four peptides was measured. The decay curves of fluorescence intensity at 591 nm for EuW10 before and after binding with the peptides are illustrated in Fig. 4, which shows directly the lifetime changes upon peptide binding. The lifetime is measured to be 250 μs for 30.0 μM EuW10, proving the dispersive state of it in solution. Being different, the full complex of EuW10 and HPV16L1Cta (1
:
3 molar ratio) shows a bi-exponential decay curve, supplying two different lifetimes and proportions (Table S1†) and being consistent with previous reports.15,18 The observed large enhancement of luminescence lifetimes is understandable in the context of the interaction of EuW10 with peptides. It is known that in the presence of water molecules, the OH oscillators surrounding EuW10 can strongly quench the luminescence of the europium ion (Eu3+) and shorten its luminescence lifetime.16,18 However, displacing water molecules by other ligands such as peptide can reduce this quenching effect.16,18 In the current case, the peptide residues serve as ligands and displace the water molecules which surround EuW10, inducing a significant enhancement of the luminescence lifetimes. Therefore, the luminescence enhancements are positively correlated with the lifetime increases in the present case.
 |
| Fig. 4 Time-resolved decay curves of EuW10 (30.0 μM) luminescence in buffer A, at room temperature (25 °C), before and after binding with the four peptides (90 μM). The sample was excited at 265 nm and monitored at 591 nm. | |
Similarly, the complex of EuW10 and HPV16L1Ctb exhibits a bi-exponential decay curve as well, giving two lifetimes of τ1 = 419 μs (8.79%) and τ2 = 2419 μs (91.21%) (Table S1†). However, the increase in magnitude of fluorescence lifetime induced by HPV16L1Ctb is obviously higher than that induced by HPV16L1Cta (9.0- vs. 6.0-fold), and that by HPV16L2Nt is also higher than that by HPV16L2Ct (14.8- vs. 13.9-fold). Such results are well consistent with the trend of the EuW10 fluorescence quantum yield induced by binding with them. The differences of τ2 (Table S1†) between them should be attributed to the larger binding constant of HPV16L1Ctb than HPV16L1Cta, and that of HPV16L2Nt than HPV16L2Ct when interacting with EuW10. The stronger combination with peptide and better drainage capacity result in a greater lifetime increase for EuW10, as well as more fluorescence enhancement, which will be revealed by ITC in the following.
3.5 ITC of EuW10 with peptides
We then investigated the interaction of HPV16L1Cta with EuW10 in buffer A solution by using ITC, which could supply the thermodynamic parameters for the binding properties of EuW10 to the peptide. Fig. 5A shows a calorimetric heat-flow trace and corresponding titration curve obtained by adding HPV16L1Cta into the EuW10 solution. The fitting of the integrated data by using a one-set binding model supplies standard thermodynamic parameters (Table 2). The results illustrate that the binding reaction should be driven by enthalpy, whereas the entropy is unfavorable (ΔH < 0, ΔS < 0). The Gibbs free energy change (ΔG) is summarized in Table 2 calculated by using the thermodynamic equation ΔG = ΔH − TΔS.
 |
| Fig. 5 The titration isotherms for the interaction of EuW10 and the peptides. EuW10 (50 μM) in the cell was titrated by (A) HPV16L1Cta (1200 μM, in the syringe), (B) HPV16L2Ct and (C) HPV16L2Nt (700 μM, in the syringe); the measurement was performed in buffer A solution at 25 °C. The integrated heat and fitted lines of the reactions are shown in the bottom panes. | |
Table 2 The thermodynamic parameters for the binding of EuW10 with the four peptides in buffer A solution at 25 °C
Peptide |
Temp./°C |
n |
Kb/M−1 |
ΔH/kcal mol−1 |
ΔG/kcal mol−1 |
TΔS/kcal mol−1 |
HPV16L1Cta |
25 |
0.43 ± 0.06 |
(3.01 ± 0.26) × 104 |
−49.26 ± 5.73 |
−6.62 ± 5.73 |
−42.64 |
HPV16L1Ctb15 |
25 |
0.79 ± 0.01 |
(5.06 ± 0.36) × 105 |
−12.80 ± 0.14 |
−7.79 ± 0.14 |
−5.01 |
HPV16L2Ct |
25 |
1.59 ± 0.01 |
(2.71 ± 0.27) × 106 |
−20.04 ± 0.13 |
−8.77 ± 0.13 |
−11.27 |
HPV16L2Nt |
25 |
1.29 ± 0.01 |
(4.88 ± 0.85) × 106 |
−27.45 ± 0.23 |
−9.11 ± 0.23 |
−18.34 |
The ITC results of HPV16L2Ct and HPV16L2Nt with EuW10 (Fig. 5B and C) are shown to be different from those of HPV16L1Cta. Therefore, two different titration curves are obtained and the thermodynamic parameters are shown in Table 2 for comparison. The calculated binding constants (Kb) of EuW10 with HPV16L2Ct and HPV16L2Nt are about 2 orders of magnitude higher than that with HPV16L1Cta, and 1 order higher than that with HPV16L1Ctb, being consistent with the fluorescence enhancement of EuW10 after interaction with them. In summary, the results together show strong interaction between EuW10 and peptides, which supplies a possible platform to detect the HPV capsid protein in biological applications.
3.6 Binding of EuW10 with the recombinant HPV L1 protein from E. coli
The most ideal way to validate the developed protocol in detection of HPV capsid protein is to use the recombinant HPV L1 proteins expressed in E. coli as a demonstration. The HPV16 L1 coding sequences, lacking 4 AAs at the N-terminus and 30 AAs at the C-terminus, HPV16L1ΔN4ΔC30, for better soluble expression purpose were cloned into pGEX-6p-1 based vectors.35 It was expressed in E. coli under IPTG induction with a glutathione-S-transferase (GST) fusion, and was purified by using the same protocols as reported previously.21,22 The interaction of EuW10 with HPV16GST-L1 (L1 monomer) is firstly assayed by using fluorescence titration spectra (Fig. S7†). Fig. S7† illustrates that with the addition of HPV16GST-L1 to EuW10 solution, the luminescence intensity of EuW10 increases and reaches saturation at a concentration of 4 μM proteins. Albeit that the titration of GST with EuW10 solution induces an increase of the luminescence intensity of EuW10 as well (Fig. S7B†), the increase is much less than that induced by GST-L1 which is saturated at a concentration of 6.7 μM. That is, either HPV16GST-L1 or GST can interact with EuW10, both leading to an enhancement of POM luminescence. The large difference of the EuW10 fluorescence enhancement between GST-L1 and GST indicates the interaction between EuW10 and L1 protein occurs; however, the interference of GST here cannot be ignored. Therefore, the interaction of EuW10 with the recombinant HPV16 L1 pentamers will be tested by using fluorescence spectra.
After removing the GST tag by PPase cleavage in a GST-affinity column, the liberated L1 can assemble into pentamers with structural features that resemble “donuts”.35,36 The formed L1 pentamers generally were stored in buffer B to keep their structural stability. However, the titration of HPV16L1ΔN4ΔC30 pentamers in buffer B into EuW10 induces only a luminescence quenching other than the enhancement of EuW10 (data not shown), which is mainly attributed to the quenching effects of high concentration of ions in buffer B solution. Once we changed the solution of HPV16 L1 protein from buffer B to A by dialysis, the additions of it into the POM solution induce indeed luminescence enhancement and the LOD is 0.5 μM when using 30.0 μM EuW10 (Fig. 6B). That means that at the optimized condition the addition of L1 pentamers to EuW10 solution could induce a luminescence enhancement, which could be used to detect the L1 pentamers. However, the drawback is that for the present condition the luminescence enhancement of EuW10 was not as great as those induced by the specific peptides. Such a result may be attributed to the truncation of coding sequences in HPV16 L1, especially those at the C-terminus where most of the basic arginines and lysines are situated. Therefore, albeit that the present method is not sensitive enough in real application at the moment, it opens a way to develop such an easy-to-perform, cost-effective and efficient method, and will be improved by using more appropriate and/or sensitive protein as well as POM as probe.
 |
| Fig. 6 (A) The fluorescence spectra of EuW10 (30.0 μM) in buffer A solution upon the gradual addition of HPV58 L1 pentamer. (B) The intensity comparison of EuW10 at 591 nm upon the titration of each of HPV16 L1 pentamer and HPV58 L1 pentamer. | |
In addition, the luminescence intensity changes of EuW10 induced by addition of capsid protein HPV58 L1, another high-risk subtype of HPV, show similar but stronger enhancement than HPV16 L1 (Fig. 6). Although the extensive intensity of the double wavelength from protein (340 nm) changed somewhat the band shape, it finally results in a LOD of 0.3 μM when using 30.0 μM EuW10 (Fig. 6B). This illustrates the universality for the binding of EuW10 with the recombinant HPV L1 protein from E. coli, and which should be extended to the detection of other HPV subtypes and/or even other kinds of proteins with identical properties. As we do not have the recombined L1 of low-risk HPV subtype to hand, this cannot be checked at this moment. However, one peptide from a subtype of low-risk HPV44, HPV44L1Ctb, was used as control to test and the results show its response is similar to that of HPV16L1Ctb (data not shown). Therefore, we speculate that the low-risk subtypes of HPV L1, at least some of them, may have a response to EuW10 similar to the high-risk subtypes of HPV16 and HPV58, which may interfere with the detection of high-risk subtypes and should be improved in a following study.
In addition, we have investigated the luminescence response of EuW10 in binding with large abundant proteins such as BSA and HSA to test the selectivity of the method and/or interference of any other proteins in the detection. The results in Fig. S8† show that when more HSA was added into the EuW10 solution, the luminescence of it at 591 nm increased slightly, while the addition of BSA induced a decrease at 591 nm firstly and then it remained stable. That is, neither of them is sensitive enough to disturb the L1 detection strongly.
Furthermore, the interaction of EuW10 with HPV16L1ΔN4ΔC30 pentamer is directly confirmed by using ITC (Fig. S9†). The fitting of the isotherm plot reveals a binding constant of Kb = (1.09 ± 0.45) × 105 M−1 (Table S2†), illustrating a moderate interaction between EuW10 and the recombinant HPV16 L1 pentamer. Taking all these results together reveals strong interactions between HPV L1 proteins and POMs, and especially the interaction-induced large luminescence enhancements of POMs lead to an easy-to-perform, cost-effective, and efficient fluorescence enhanced method to detect the capsid protein of HPV in vitro.
4. Conclusions
The Arg/Lys-rich cationic peptides from HPV capsid protein, L1 and L2, induced large luminescence enhancement of polyoxometalates, which were further used as fluorescence-enhanced probes to detect HPV capsid protein in vitro. The strong dependence of luminescence enhancement on both the number and, especially, the sequences of basic residues in peptide was well revealed, and several strategies to monitor the POM–peptide interactions and finally to improve the detection of recombinant HPV capsid protein have been proposed. The present study supplied an easy, cost-effective and efficient fluorescence-enhanced method to detect HPV capsid proteins. Especially, it supplied an excellent application of POM as a type of inorganic material in viral and biological science, and would be expected to be used as an efficient agent to explore the mechanism of virus infection as well in the future.
Conflict of interest
The authors declare no competing financial interest.
Acknowledgements
We greatly appreciate the financial support from the projects of NSFC (91027027, 21373101 and 91227110), the National 973 Program (2013CB834503), the China Scholarship Council (CSC) and the Innovation Program of State Key Laboratory of Supramolecular Structure and Materials, Jilin University.
References
- J. M. Walboomers, M. V. Jacobs, M. M. Manos, F. X. Bosch, J. A. Kummer, K. V. Shah, P. J. Snijders, J. Peto, C. J. Meijer and N. Muñoz, J. Pathol., 1999, 189, 12–19 CrossRef CAS PubMed.
- J. Haedicke and T. Iftner, Radiother. Oncol., 2013, 108, 397–402 CrossRef CAS PubMed.
- M. Arbyn, X. Castellsagué, S. Sanjosé, L. Bruni, M. Saraiya, F. Bray and J. Ferlay, Ann. Oncol., 2011, 22, 2675–2686 CrossRef CAS PubMed.
- A. Yemelyanova, P. E. Gravitt, B. M. Ronnett, A. F. Rositch, A. Ogurtsova, J. Seidman and R. B. Roden, Mod. Pathol., 2013, 26, 268–274 CrossRef CAS PubMed.
- P. E. Castle, M. Schiffman, C. M. Wheeler and D. Solomon, Obstet. Gynecol., 2009, 113, 18–25 CrossRef PubMed.
- G. Y. Ho, R. Bierman, L. Beardsley, C. J. Chang and R. D. Burk, N. Engl. J. Med., 1998, 338, 423–428 CrossRef CAS PubMed.
- A. B. Moscicki, S. Shiboski, N. K. Hills, K. J. Powell, N. Jay, E. N. Hanson, S. Miller, L. K. Canjura-Clayton, S. Farhat, J. M. Broering and T. M. Darragh, Lancet, 2004, 364, 1678–1683 CrossRef.
- A. Szarewski, L. Ambroisine, L. Cadman, J. Austin, L. Ho, G. Terry, S. Liddle, R. Dina, J. McCarthy, H. Buckley, C. Bergeron, P. Soutter, D. Lyons and J. Cuzick, Cancer Epidemiol., Biomarkers Prev., 2008, 17, 3033–3042 CrossRef CAS PubMed.
- J. Hernandez, A. Elahi, E. Siegel, D. Coppola, B. Riggs and D. Shibata, Am. J. Clin. Pathol., 2011, 135, 436–441 CrossRef CAS PubMed.
- C. B. Buck, N. Cheng, C. D. Thompson, D. R. Lowy, A. C. Steven, J. T. Schiller and B. L. Trus, J. Virol., 2008, 82, 5190–5197 CrossRef CAS PubMed.
- R. Hilfrich and J. Hariri, Anal. Quant. Cytol. Histol., 2008, 30, 78–82 Search PubMed.
- H. Griesser, H. Sander, C. Walczak and R. A. Hilfrich, Am. J. Clin. Pathol., 2009, 132, 840–845 CrossRef CAS PubMed.
- G. Mehlhorn, E. Obermann, G. Negri, L. Bubendorf, C. Mian, M. Koch, H. Sander, B. Simm, M. Lütge, Z. Bánrévi, A. Weiss, G. Cathomas, R. Hilfrich, M. W. Beckmann and H. Griesser, Mod. Pathol., 2013, 26, 967–974 CrossRef PubMed.
- W. Bu, H. Li, W. Li, L. Wu, C. Zhai and Y. Wu, J. Phys. Chem. B, 2004, 108, 12776–12782 CrossRef CAS.
- T. Zhang, H. W. Li, Y. Wu, Y. Wang and L. Wu, J. Phys. Chem. C, 2015, 119, 8321–8328 CAS.
- L. Zheng, Y. Ma, G. Zhang, J. Yao, B. Keita and L. Nadjo, Phys. Chem. Chem. Phys., 2010, 12, 1299–1304 RSC.
- G. Hungerford, F. Hussain, G. R. Patzke and M. Green, Phys. Chem. Chem. Phys., 2010, 12, 7266–7275 RSC.
- L. Zheng, Z. Gu, Y. Ma, G. Zhang, J. Yao, B. Keita and L. Nadjo, J. Biol. Inorg. Chem., 2010, 15, 1079–1085 CrossRef CAS PubMed.
- R. D. Peacock and T. J. R. Weakley, J. Chem. Soc. A, 1971, 1836–1839 RSC.
- Y. S. N. Day, C. L. Baird, R. L. Rich and D. G. Myszka, Protein Sci., 2002, 11, 1017–1025 CrossRef CAS PubMed.
- X. S. Chen, G. Casini, S. C. Harrison and R. L. Garcea, J. Mol. Biol., 2001, 307, 173–182 CrossRef CAS PubMed.
- S. Jin, D. Pan, X. Zha, X. Yu, Y. Wu, Y. Liu, F. Yin and X. S. Chen, Mol. BioSyst., 2014, 10, 724–727 RSC.
- T. R. Zhang, R. Lu, H. Y. Zhang, P. C. Xue, W. Feng, X. L. Liu, B. Zhao, Y. Y. Zhao, T. J. Li and J. N. Yao, J. Mater. Chem., 2003, 13, 580–584 RSC.
- F. Schäfer, L. Florin and M. Sapp, Virology, 2002, 295, 172–181 CrossRef PubMed.
- J. G. Joyce, J. S. Tung, C. T. Przysiecki, J. C. Cook, E. D. Lehman, J. A. Sands, K. U. Jansen and P. M. Keller, J. Biol. Chem., 1999, 274, 5810–5822 CrossRef CAS PubMed.
- J. Sun, J. S. Yu, S. Jin, X. Zha, Y. Wu and Z. Yu, J. Phys. Chem. B, 2010, 114, 9854–9861 CrossRef CAS PubMed.
- L. Bousarghin, A. Touzé, A. Lucia, C. Rojas and P. Coursaget, J. Gen. Virol., 2003, 84, 157–164 CrossRef CAS PubMed.
- T. Zhang, H. W. Li, Y. Wu, Y. Wang and L. Wu, Chem.–Eur. J., 2015, 21, 9028–9033 CrossRef CAS PubMed.
- R. M. Richards, D. R. Lowy, J. T. Schiller and P. M. Day, Proc. Natl. Acad. Sci. U. S. A., 2006, 103, 1522–1527 CrossRef CAS PubMed.
- Y. Kawana, K. Kawana, H. Yoshikawa, Y. Taketani, K. Yoshiike and T. Kanda, J. Virol., 2001, 75, 2331–2336 CrossRef CAS PubMed.
- W. J. Liu, L. Gissmann, X. Y. Sun, A. Kanjanahaluethai, M. Müller, J. Doorbar and J. Zhou, Virology, 1997, 227, 474–483 CrossRef CAS PubMed.
- H. T. Kuo, C. J. Fang, H. Y. Tsai, M. F. Yang, H. C. Chang, S. L. Liu, L. H. Kuo, W. R. Wang, P. A. Yang, S. J. Huang, S. L. Huang and R. P. Cheng, Biochemistry, 2013, 52, 9212–9222 CrossRef CAS PubMed.
- R. P. Cheng, P. Girinath and R. Ahmad, Biochemistry, 2007, 46, 10528–10537 CrossRef CAS PubMed.
- S. Liu, Q. S. Fu, J. Zhao and H. Y. Hu, Biochim. Biophys. Acta, 2013, 1834, 2672–2678 CrossRef CAS PubMed.
- B. Bishop, J. Dasgupta, M. Klein, R. L. Garcea, N. D. Christensen, R. Zhao and X. S. Chen, J. Biol. Chem., 2007, 282, 31803–31811 CrossRef CAS PubMed.
- D. D. Zheng, D. Pan, X. Zha, Y. Wu, C. Jiang and X. Yu, Chem. Commun., 2013, 49, 8546–8548 RSC.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra00105j |
|
This journal is © The Royal Society of Chemistry 2016 |
Click here to see how this site uses Cookies. View our privacy policy here.