3.1. Software Tools Used for Epitope and Ligand Binding Site Determination
In silico prediction of the linear hPSA epitope RHSLFHP was carried out using the consensus result of 10 servers presented in
Table 2: ExPASy–ProtScale (Kyte & Doolittle hydropathy scale, window size 9), BcePred (antigenic propensity method), BepiPred-2 (random forest regression algorithm), LBtope (support vector machine models), SCRATCH–COBEpro (support vector machine based propensity score;
Scheme S1), Sann and RVP-net (solvent accessibility algorithms), NetTurnP and LEPS (β-turn determination) and the informational spectrum method based on electron-ion interaction potential (ISM-EIIP) [
27,
28,
29,
30,
31,
32,
33,
34,
35,
36,
37,
38,
39,
40]. The hPSA sequence is given in
Scheme S1.
The 3D structures of the hPSA molecule and its binding sites were predicted by coupled Phyre2–3DLigandSite servers for protein modeling, prediction and analysis (
Figure 1a) [
41,
42]. 100% of hPSA residues were modeled at >90% confidence, the 3D structure template, obtained with X-ray diffraction, was protein data bank (PDB) file 2ZCH chain P (
Scheme S2) [
41]. The results obtained by Phyre2–3DLigandSite-based prediction were confirmed by RaptorX web portal Binding Prediction (
Figure 1b) [
44].
The antisense peptide, i.e., paratope AVRDKVG, was designed by the translation of the RHSLFHP epitope in a 3′ to 5′ direction (
Scheme 1) [
19,
20]. Potential paratopes to hPSA
53–59 epitope were selected using BLAST, with the blastp quadripeptide option (protein–protein BLAST) [
19,
20,
45].
The 3D structures of the sense epitope RHSLFHP (
Scheme S3) and its antisense paratope AVRDKVG
Scheme S4), presented in
Figure 2, were modeled using PEPstrMOD method [
46]. The PDB files were visualized using CCP4 software version 7.0 [
62].
Protein–peptide interactions between hPSA and its antisense peptide AVRDKVG were investigated using the pepATTRACT and CABS-dock web servers [
47,
48]. Both web services required the hPSA (protein) input file in PDB format (
Scheme S2) and docking peptide sequence (AVRDKVG).
The pepATTRACT server is a novel fully blind docking protocol that does not require any information about the binding site [
47]. In a very short time (~10 min) this web server returns an analysis of the most prevalent protein–peptide contacts among the top 50 generated models [
47]. The pepATTRACT protocol is well tuned to assist users to identify the binding site, performs large-scale in silico experiments, and represents a useful starting point to rationalize the design for further protein–peptide docking experiments [
47]. Model No. 24, presented in
Figure A1a, is a precise description of hPSA–AVRDKVG docking. Results are given in PDB format as above (
Scheme S5).
The CABS-dock server enables the modeling of protein–peptide interactions through an efficient method for the flexible docking of peptides to proteins without pre-defining the localization of the binding site [
48]. The CABS-dock protocol was tested over the largest dataset of non-redundant protein–peptide interactions available to date, which includes bound and unbound docking cases [
48]. Using a ligand RMSD cutoff of 5.5 Å, the best 10 models are selected with reference to the quality of docking models, which represent low-to-medium-accuracy models [
48]. Model No. 5, presented in
Figure A1b and
Table A1, gives a precise description of hPSA–AVRDKVG docking. This result was given in the PDB format as
Scheme S6.
3.3. Tryptophan Fluorescence (Binding of hPSA Epitope RHSLFHP and its Antisense Peptide)
The fluorescence spectra of the sense and antisense peptides, and their complexes, were measured at 25 °C by OLIS RSM 1000F spectrofluorimeter (Olis, Inc., Bogart, GA, USA) equipped with a thermostatted cell holder. The excitation wavelength was 280 nm (
Figure 3a) [
19,
20,
49]. The antisense peptide AVRDKVGW became bound to the hPSA peptide RHSLFHP, and their complex exhibited fluorescence, whereas the ligand RHSLFHP did not. The fluorescence units in
Figure 3a are given as a ratio of signals obtained from sample and reference photomultiplier tubes (PMTs). SPECFIT software was used to analyze data obtained from the titrations [
19,
20,
49]. The titration curve, in
Figure 3a―Insert: Fitting curve at 360 nm, shows a good fit to data (
r2 = 0.947). The dissociation constant was 2.6 ± 0.19 µM (
Kd = mean ± SEM).
3.4. Microscale Thermophoresis (Binding of hPSA Epitope RHSLFHP to its Antisense Peptide)
The binding between the biotinylated antisense peptide AVRDKVG and a titrant, i.e., hPSA peptide RHSLFHP (
Figure 3b), was observed by the method of microscale thermophoresis (MST) [
19,
20,
51,
52]. MST-analysis was performed using the Monolith.NT.115 instrument (NanoTemper Technologies GmbH, Munich, Germany). Biotin-AVRDKVG antisense directed to the hPSA region 53–59 was labeled with DY-495 (Red). The labeling procedure and the subsequent removal of unattached dye were performed within 45 min. A serial dilution of the non-labeled hPSA titrant was prepared in a 10 mM phosphate buffer. The concentration of biotin-hPSA antisense labelled with DY-495 was kept constant, while the concentration of the non-fluorescent binding partner (hPSA peptide RHSLFHP
53–59) was varied between 0.0854 µM and 2.8 mM. After a short incubation, the samples were loaded into MST NT.115 standard glass capillaries and the MST-analysis was performed [
19,
20,
51,
52]. The data were analyzed using GraphPad Prism software version 5. The titration curve shows a good fit to data (
r2 = 0.989),
Kd = 24.3 ± 0.74 µM (mean ± SEM).
3.5. Magnetic Particle Enzyme Immunoassay (Binding of hPSA Epitope RHSLFHP and its Antisense Peptide)
SPHERO™ Carboxyl Magnetic Particles (2.5%
w/
v, 1.14 μm) were coated with hPSA peptide (RHSLFHP) using a slightly modified one step 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) coupling method suggested by manufacturer [
20,
50]. Briefly, the reaction mixture was composed of 2 mL sodium acetate buffer (0.01M, pH 5.0), 2 mg of peptide and 2 mL of 2.5%
w/
v carboxyl magnetic particles―1.14 μm, 20 mg of EDC. Reactions proceeded in glass reaction tubes overnight at room temperature in a rotary mixer. Tubes were centrifuged at 3000×
g for 15 min, supernatant was carefully discarded, and the pellets were washed twice in 4 mL isotonic buffered saline (IBS), followed by centrifugation [
20,
50]. After washing, coated magnetic particles were resuspended in 4 mL of PBS containing 0.05% Tween 20.
Two folds serial dilutions (1.09–70 µM) of biotinylated antisense peptide AVRDKVG were made in 96-well microtiter plates to generate standard curves (
Figure 4b). Mismatched peptide Biotin-EHFRW was used as a negative control (
Figure 4b). A suspension (1.25%
w/
v) of carboxyl magnetic particles, coated with sense peptide RHSLFHP, was added to each well (40 µL/well) [
20,
50]. The plates were incubated at room temperature for 30 min and washed with PBS-Tween 20 five times. The washing solution was removed each time using the Spherotech UltraMag Separator (Sperotech, Inc., Lake Forest, IL, USA).
A blocking solution (PBS with 1% BSA) was added to the wells and incubated at room temperature for one hour. Wells were washed (PBS with 0.05% Tween 20) and ultrasensitive streptavidin-peroxidase polymer conjugate, diluted in the ratio 1:200 in PBS with 0.05% Tween 20, was added to the wells and incubated at room temperature for one hour (S 2438, SIGMA
®, Saint Louis, MO, USA). Wells were washed five times using PBS with 0.05% Tween 20. SIGMA
FAST™ OPD solution (o-phenylenediamine dihydrochloride) was added to the wells and incubated, in the dark, for 30 min at room temperature. The absorbances were read at 450 nm on a multiwell plate reader. The data were analyzed using GraphPad Prism software version 5. The titration curve in
Figure 4b shows a good fit to data with
r2 = 0.996, and
Kd = 24.6 ± 0.20 µM (mean ± SEM).
3.6. Immunohistochemical Staining of hPSA: Protocol 1 and Protocol 2
The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of the University Hospital Centre Zagreb, No. 01-20/32-1-2006 (approved 28 February 2006). The project code of the Croatian Ministry of Science and Education was 098-0982929-2524.
Immunohistochemistry was performed on deparafinized slides (xylen and a series of ethanol solutions) at room temperature (20–25 °C). The phosphate-buffered saline (PBS) was used, with a pH of 7.2 and containing disodium hydrogen phosphate, sodium chloride, potassium chloride and potassium dihydrogen phosphate was used. PBS stock solution: 4 g KH2PO4, 23 g Na2HPO4, 4 g KCl, 160 g NaCl and 810 mL distilled water. Before staining, 100 mL of stock solution was diluted with 1900 mL of distilled water.
All slides (from seven patients) were selected from the repository of prostate cancer tissue, with diagnosis confirmed by consensus of two independent pathologists. The two pathologists who examined the slides stained with antisense peptides were unconnected with the pathologists who established the initial diagnosis.
Each of the two immunohistochemistry protocols for PSA staining consisted of six steps. Steps 1, 4, 5 and 6 of protocol 1 and protocol 2 were identical, while steps 2 and 3 differed (
Figure 5 and
Figure 6). For this study, the slides were kept in the dark at room temperature (20–25 °C), to prevent fading as a result of exposure to strong light. They were analyzed using an Olympus BX41 microscope, with an Olympus DP71 camera set to three levels of magnification: less than 40×, 100×, and 500×.
3.6.1. Protocol 1 (Standard IHC Staining Technique)
Staining protocol 1 is a standard IHC staining technique using commercially available kit from Dako (Dako EnVision+System-HRP (AEC), Glostrup, Denmark) and primary monoclonal mouse anti-human PSA antibody. The kit and primary mouse antibody are intended for the quantitative identification of hPSA by light microscopy (
Figure 5). For this study the endogenous peroxidase activity was first quenched by incubating the specimen with Dako’s Peroxidase block (step 1). Then, the specimen was incubated with diluted mouse primary antibody, followed by incubation with the HRP labeled polymer conjugated to goat anti-mouse immunoglobulins―steps 2 and 3, respectively. The staining was completed by incubation with AEC + substrate-chromogen (step 4), followed by hematoxylin counterstain and mounting―step 5 and 6, respectively. The negative control was the primary monoclonal mouse anti-human CD20 (Clone L26; Dako, Glostrup, Denmark).
IHC staining―protocol 1 was performed in accordance with the manufacturer’s instruction, as follows:
The specimen was covered with peroxidase block, i.e., 100 μL of 0.03% hydrogen peroxide containing sodium azide (Dako, Glostrup, Denmark), and incubated for four minutes. Following this procedure, the specimen was gently rinsed with phosphate buffered saline (PBS), and placed in a fresh buffer bath.
Primary monoclonal mouse anti-human PSA antibody (Clone ER-PR8, Code M0750; Dako, Glostrup, Denmark) was diluted with standard diluent containing 0.05 mol/L TRIS-HCl buffer and a 1% bovine serum albumin (BSA). 100 μL of primary antibody diluted in the proportion 1:100 was used to cover the specimen. After 30 min of incubation at room temperature, the specimen was gently rinsed with PBS and placed in a fresh buffer bath.
100 μL of HRP-labelled polymer conjugated to goat anti-mouse immunoglobulins in Tris-HCl buffer containing stabilizing protein and an anti-microbial agent (Dako, Glostrup, Denmark) was applied to cover the specimen, followed by a 30 min incubation. After 30 min of incubation, the specimen was gently rinsed with PBS and placed in a fresh buffer bath.
The specimen was covered with 100 μL of AEC + substrate-chromogen solution for 10 min, i.e., 3-amino-9-ethylcarbazole containing hydrogen peroxide, stabilizers, enhancers and anti-microbial agent (Dako, Glostrup, Denmark). After that period the specimen was again rinsed with PBS.
The slides were immersed in a bath of aqueous hematoxylin (Mayerr’s hematoxylin), and rinsed gently in a distilled water bath. Slides were dipped 10 times into a bath of ammonia (0.037 mol/L), and rinsed in a bath of distilled water for four minutes.
The specimens were mounted and coverslipped with the non-aqueous permanent mounting medium Ultramount.
3.6.2. Protocol 2 (Modified IHC Staining Using Antisense Peptide Instead of Primary Antibody)
The staining protocol 2 was a modified IHC protocol 1, as follows: In step 2 we used antisense peptide Biotin-AVRDKVG, instead of the primary monoclonal mouse anti-human PSA antibody, in step 3 we used rabbit polyclonal antibody to biotin conjugated to HRP, instead of HRP labeled polymer conjugated to goat anti-mouse immunoglobulins (
Figure 5), with peptide Biotin-EHFRW as a negative control (
Figure 7). Otherwise, the procedures and chemicals were identical. Quality control for the PSA staining was investigated using NordiQC scoring criteria [
57]: 0—Poor; 1—Borderline; 2—Good; 3—Optimal. The scale is presented in
Table A2, and the final results given in
Table 4 are expressed as a sum of scores.
IHC staining―protocol 2:
The specimen was covered with peroxidase block, i.e., 100 μL of 0.03% hydrogen peroxide containing sodium azide (Dako, Glostrup, Denmark), and incubated for four minutes. Following this, the specimen was gently rinsed with phosphate buffered saline (PBS), and placed in a fresh buffer bath.
Biotinylated antisense peptide AVRDKVG (GenScript, Piscataway, NJ, USA) directed to PSA epitope 53–59 (RHSLFHP) was used to cover the specimen. Five milligrams of Biotin-AVRDKVG antisense was diluted in 2.5 mL PBS. Dilutions 1:10, 1:50, 1:100, 1:200 and 1:500 were applied to cover the specimen. A 100 μL volume was used per section. After 30 min of incubation, each specimen was gently rinsed with PBS and placed in a fresh buffer bath.
Anti-biotin antibody conjugated to HRP (ab34645, Abcam, Cambridge, UK) was applied to cover the specimen. Of the antibody 10 mg/2 mL was diluted 1:100. One hundred microliters of the diluted antibody was applied to cover each specimen, followed by a 30 min incubation. After the incubation, the specimen was gently rinsed with PBS and placed in a fresh buffer bath.
The specimen was covered with 100 μL of AEC + substrate-chromogen solution for 10 min, i.e., 3-amino-9-ethylcarbazole containing hydrogen peroxide, stabilizers, enhancers and an anti-microbial agent (Dako, Glostrup, Denmark). After that period the specimen was again rinsed with PBS.
The slides were immersed in a bath of aqueous hematoxylin (Mayerr’s hematoxylin), and rinsed gently in a distilled water bath. The slides were dipped 10 times into a bath of ammonia (0.037 mol/L), and then rinsed in a bath of distilled water for four minutes.
The specimens were mounted and coverslipped with non-aqueous permanent mounting medium Ultramount.