1

Introduction

Dentin, less mineralized than enamel, is composed of 50 vol% mineral, 30 vol% organic materials and 20 vol% fluids , therefore the dentin caries process undergoes demineralization and degradation of the organic matrix. Carious lesion in dentin is described as two differential lesions, the outer lesion (infected dentin) and the inner lesion . The outer lesion is infected and irreversibly destroyed in terms of chemical structure, which is incapable of remineralization. The inner lesion is also destroyed, but not infected, which has potential for remineralization. The remineralization of inner carious dentin is clinically important for retaining tooth tissues to restore its function. Within a minimally invasive and tissue-preserving approach, the caries-infected dentin is removed, and the partially demineralized caries-affected (but not infected) dentin is preserved to undergo remineralization .

In light of remineralization of dentin lesion, the collagen matrix in dentin serves as a scaffold for mineral deposition and further crystallites . The preservation of collagen matrix dentin could facilitate mineral precipitation and consequently remineralization . Collagen is the main organic component in dentin, which is subject to degradation by endogenous enzymes such as matrix metalloproteinases (MMPs) and cysteine cathepsins . To date, there are two pharmacological approaches to protect collagen against enzymatic degradation, the use of collagenase inhibitors such as chlorhexidine and the use of collagen cross-linkers. However, the collagenase inhibitors should be effective only when being applied, because their pharmacological actions diminish over time after application .

In contrast, the use of collagen cross-linker agents is thought to be a stable approach to modify collagen matrix to increase resistance to collagenase degradation . Collagen cross-linker has been proposed to modify collagen networks to a highly cross-linked structure. The effect of cross-linker agents such as glutaraldehyde, carbodiimide and natural cross-linkers, such as proanthocyanidin has been reported using demineralized dentin collagen . In our previous study, we examined the effect of natural-derived reagents, proanthocyanidin, epigallocatechin gallate, genipin, and hesperidin (HPN) using bacterial collagenolytic digestion on demineralized collagen . The use of HPN, proanthocyanidin and epigallocatechin gallate, was effective in stabilizing dentin collagen by increasing its mechanical properties and resistance to collagenase challenges. Especially, HPN demonstrated its effect on collagen modification even at lower concentrations than 0.5%; meanwhile, proanthocyanidin and epigallocatechin gallate did not show such an effect. This finding implied a possible interaction between HPN and collagen, which might be more prominent than proanthocyanidin and epigallocatechin gallate.

Although there have been various experiments to investigate the interaction between dentin collagen and these reagents, there is not enough information at molecular level. From the aspect of biomechanical properties and biostability of collagen, the aforementioned studies were carried out on dentin matrices, which were generally in solid phases as assembles of collagen microfibrils or collagen fibres . Even in the case where soluble collagen was used, in almost of those experiments, the interactions caused insoluble or poorly soluble collagen complexes which precipitated and consequently complicated the characterization of the interaction. Thus it was rather hard to obtain the molecular level information about the interaction. Exceptionally, using not collagen but easily soluble gelatin, the molecular mechanism of interaction with polyphenolic flavonoid has been investigated by isothermal titration calorimetry to determine the thermodynamic binding parameters . As a result, these investigations made it possible to discuss the fact that the conformational flexibility and molecular weight were important complementary factors leading to strong interactions.

HPN, a glycoside flavonoid, is not categorized as a polyphenolic flavonoid. Since HPN does not contain multiple phenol structural units, the amphiphilic property of HPN may be different from that of proanthocyanidin and epigallocatechin gallate. In our previous experiments using human and bovine dentin, the application of HPN demonstrated a potential to preserve dentin collagen, resist caries progression and promote dentin remineralization . Therefore, it is important to investigate the mechanism of the direct interaction of HPN to collagen molecules.

Using soluble collagen, we attempted to analyze in detail the interaction between HPN and collagen in solution, avoiding the aggregation of collagen , by the physico-chemical techniques, circular dichroism (CD) spectroscopic analysis, sedimentation velocity measurement by ultracentrifuge and saturation transfer difference measurement (STD) by NMR.

The CD spectroscopy is used to analyze the secondary structure or conformation of macromolecules, particularly proteins, where secondary structure changes with environmental conditions or on interaction with other molecules. Structural, kinetic and thermodynamic information about macromolecules can be obtained from circular dichroism spectroscopy. The sedimentation velocity method has been developed to determine size and shape of macromolecule and to analyze simple association–dissociation between macromolecules. We used the technological-innovated methods to enable association–dissociation analysis on samples with differences in molecular weight and/or concentration . For further molecular analysis, we investigated HPN-collagen interaction using STD- ¹ H NMR techniques. STD-NMR spectroscopy is a powerful method to study protein–ligand interactions in solution. This method is capable of identifying the binding epitope of ligands when bound to proteins. Ligand protons that are proximally situated to the receptor protein receive a higher degree of saturation and produce stronger STD NMR signals. As a result, epitope mapping was used to identify the site where ligand binds to protein. Previously we introduced this NMR method to analyze the interaction between a collagen model and dental adhesive monomers, and suggested the possibility of hydrophobic interaction between collagen and specific monomers .

The present approach to investigate the interactions of HPN and collagen by these methods provides biophysical and molecular-level information on their interaction, thus offering evidence of potential use of HPN to stabilize collagens.

2

Materials and methods

As the Type I collagen model, atelocollagen powder CLP-01 was purchased from Koken (Tokyo, Japan). This atelocollagen (ATCL) was an aqueous soluble collagen obtained by extraction from bovine dermis and treated enzymatically to remove the non-helical telopeptides at both N and C terminal ends. Chemicals used in this study, including HPN were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

For CD analysis, ATCL powder was dissolved in a 10% aqueous acetic acid to be 10 mg/mL. After being kept at room temperature for 12 h, the solution was centrifuged by 800 × g for 10 min. The supernatant was diluted five-fold to be the ATCL sample solution with ca 2 mg/mL concentration. The HPN solution was prepared by dissolving HPN in methanol to be 0.5 mg/mL and was diluted by aqueous acetic acid to make a 0.014 mg/mL HPN solution in 2% aqueous acetic acid with 3% methanol. To investigate the effect of HPN, aliquots of HPN solution were added to ATCL solution to obtain various mixtures in the ratio of up to 40:1 (HPN:ATCL). The CD spectra at various temperatures were obtained using a 0.1 mm path length quartz cell on a Jasco spectrometer J-720W (Tokyo, Japan). Thermally induced unfolding of the ACTL was monitored at 220 nm with an ascending temperature gradient of 1 °C/min.

For sedimentation analysis, the ATCL powder was dissolved to be about 2 mg/mL in 10 mM acetate buffer pH 4 containing 100 mM NaCl and then the solution was diluted twice to make a 1 mg/mL ATCL solution with the buffer. HPN was dissolved in methyl alcohol to be 0.5 mg/mL. The mixed solution of ATCL and HPN was prepared by diluting each stock solution. The final concentration of ATCL is 1 mg/mL and that of HPN is 0.03 mg/mL (0.05 mM).

Sedimentation analysis was carried out with a Beckman Optima XL-A or XL-I analytical ultracentrifuge (Beckman Coulter, Inc., Brea, CA) equipped with a 4-hole An60 Ti rotor using double-sector charcoal-filled 12-mm Epon centerpieces and quartz windows. Sedimentation equilibrium studies were performed at rotor speeds of 8000 rpm and at 20 °C. Absorbance scans at 230 nm were measured in the radial step mode at 0.001 cm intervals and data were collected taking the average of 16 measurements at each radial distance. Approach to equilibrium was considered to be complete when replicate scans separated by ≥6 h were indistinguishable. The partial specific volume of the protein was assumed to be 0.73 mL/g and the density of the solvent was assumed to be 1.0025 g/mL. Analysis of the data was carried out utilizing the program Origin 6.0 (Origin Lab., Northampton, MA). Sedimentation velocity runs were conducted at 30,000 rpm and 20 °C. Radial absorbances scanned stepwise with a radial increment of 0.003 cm were collected at four minute intervals. The wavelengths used were 280 nm for the solutions of HPN, 230 nm for the solution of ATCL and 280 nm for the mixtures of collagen and HPN. The distribution of sedimentation coefficients was analyzed using the Program SEDFIT which involves direct fitting of the boundaries using numeric solutions to the Lamm equation .

For NMR analysis, ATCL powder was dissolved to saturation in a deuterated aqueous buffer solution containing a 50 mM d ₄ -acetic acid, 150 mM NaCl, 50 mM CaCl ₂ and 0.02% NaN ₃ and the pH was adjusted to 4.0 with NaOD. The saturated solution was then diluted four-fold by the buffer to be the ATCL solution. HPN was dissolved in deuterated d ₆ -DMSO to a concentration of 20 mM. The DMSO solution was diluted five-fold with the above buffer solution, so that the final HPN concentration was 4 mM, where the concentration of d ₆ -DMSO was 20%. For the STD experiment, the mixed solution of ATCL and HPN was prepared by adding the above ACTL solution in the d ₆ -DMSO solution of HPN to be diluted five-fold. All NMR experiments were carried out at 25 °C on a Bruker 900, 800 or 600 MHz spectrometer equipped with a shielded gradient cryogenic probe (Bruker BioSpin Corporation, Billerica MA). The ¹ H COSY, ¹ H- ¹³ C HMQC, and ¹ H- ¹³ C HMBC spectra were acquired conventionally for resonance assignments of HPN. The ¹ H NOESY spectra were acquired with mixing time of 400 m s. In the STD NMR experiments, ATCL signals were saturated by irradiation at 1.36 ppm or at 0.86 ppm in the methyl region of the spectrum and off-resonance at 40 ppm, where no signals of ATCL or HPN occur, with a cascade of 40 selective Gaussian-shaped pulses of 50 m s duration (50 dB), which correlates to a strength of 190 Hz. A 100 μ s delay between each soft pulse was applied, resulting in a total saturation time of 2 s. On- and off-resonance spectra were processed separately, and the final STD NMR spectrum was obtained by subtracting the individual on- and off-resonance spectra, resulting in less subtraction artifacts. All NMR data were processed and analyzed with Topspin (Bruker BioSin) or NMR Pipe . The WATERGATE pulse train was used to suppress HDO signals.

2

Materials and methods

As the Type I collagen model, atelocollagen powder CLP-01 was purchased from Koken (Tokyo, Japan). This atelocollagen (ATCL) was an aqueous soluble collagen obtained by extraction from bovine dermis and treated enzymatically to remove the non-helical telopeptides at both N and C terminal ends. Chemicals used in this study, including HPN were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

For CD analysis, ATCL powder was dissolved in a 10% aqueous acetic acid to be 10 mg/mL. After being kept at room temperature for 12 h, the solution was centrifuged by 800 × g for 10 min. The supernatant was diluted five-fold to be the ATCL sample solution with ca 2 mg/mL concentration. The HPN solution was prepared by dissolving HPN in methanol to be 0.5 mg/mL and was diluted by aqueous acetic acid to make a 0.014 mg/mL HPN solution in 2% aqueous acetic acid with 3% methanol. To investigate the effect of HPN, aliquots of HPN solution were added to ATCL solution to obtain various mixtures in the ratio of up to 40:1 (HPN:ATCL). The CD spectra at various temperatures were obtained using a 0.1 mm path length quartz cell on a Jasco spectrometer J-720W (Tokyo, Japan). Thermally induced unfolding of the ACTL was monitored at 220 nm with an ascending temperature gradient of 1 °C/min.

For sedimentation analysis, the ATCL powder was dissolved to be about 2 mg/mL in 10 mM acetate buffer pH 4 containing 100 mM NaCl and then the solution was diluted twice to make a 1 mg/mL ATCL solution with the buffer. HPN was dissolved in methyl alcohol to be 0.5 mg/mL. The mixed solution of ATCL and HPN was prepared by diluting each stock solution. The final concentration of ATCL is 1 mg/mL and that of HPN is 0.03 mg/mL (0.05 mM).

Sedimentation analysis was carried out with a Beckman Optima XL-A or XL-I analytical ultracentrifuge (Beckman Coulter, Inc., Brea, CA) equipped with a 4-hole An60 Ti rotor using double-sector charcoal-filled 12-mm Epon centerpieces and quartz windows. Sedimentation equilibrium studies were performed at rotor speeds of 8000 rpm and at 20 °C. Absorbance scans at 230 nm were measured in the radial step mode at 0.001 cm intervals and data were collected taking the average of 16 measurements at each radial distance. Approach to equilibrium was considered to be complete when replicate scans separated by ≥6 h were indistinguishable. The partial specific volume of the protein was assumed to be 0.73 mL/g and the density of the solvent was assumed to be 1.0025 g/mL. Analysis of the data was carried out utilizing the program Origin 6.0 (Origin Lab., Northampton, MA). Sedimentation velocity runs were conducted at 30,000 rpm and 20 °C. Radial absorbances scanned stepwise with a radial increment of 0.003 cm were collected at four minute intervals. The wavelengths used were 280 nm for the solutions of HPN, 230 nm for the solution of ATCL and 280 nm for the mixtures of collagen and HPN. The distribution of sedimentation coefficients was analyzed using the Program SEDFIT which involves direct fitting of the boundaries using numeric solutions to the Lamm equation .

For NMR analysis, ATCL powder was dissolved to saturation in a deuterated aqueous buffer solution containing a 50 mM d ₄ -acetic acid, 150 mM NaCl, 50 mM CaCl ₂ and 0.02% NaN ₃ and the pH was adjusted to 4.0 with NaOD. The saturated solution was then diluted four-fold by the buffer to be the ATCL solution. HPN was dissolved in deuterated d ₆ -DMSO to a concentration of 20 mM. The DMSO solution was diluted five-fold with the above buffer solution, so that the final HPN concentration was 4 mM, where the concentration of d ₆ -DMSO was 20%. For the STD experiment, the mixed solution of ATCL and HPN was prepared by adding the above ACTL solution in the d ₆ -DMSO solution of HPN to be diluted five-fold. All NMR experiments were carried out at 25 °C on a Bruker 900, 800 or 600 MHz spectrometer equipped with a shielded gradient cryogenic probe (Bruker BioSpin Corporation, Billerica MA). The ¹ H COSY, ¹ H- ¹³ C HMQC, and ¹ H- ¹³ C HMBC spectra were acquired conventionally for resonance assignments of HPN. The ¹ H NOESY spectra were acquired with mixing time of 400 m s. In the STD NMR experiments, ATCL signals were saturated by irradiation at 1.36 ppm or at 0.86 ppm in the methyl region of the spectrum and off-resonance at 40 ppm, where no signals of ATCL or HPN occur, with a cascade of 40 selective Gaussian-shaped pulses of 50 m s duration (50 dB), which correlates to a strength of 190 Hz. A 100 μ s delay between each soft pulse was applied, resulting in a total saturation time of 2 s. On- and off-resonance spectra were processed separately, and the final STD NMR spectrum was obtained by subtracting the individual on- and off-resonance spectra, resulting in less subtraction artifacts. All NMR data were processed and analyzed with Topspin (Bruker BioSin) or NMR Pipe . The WATERGATE pulse train was used to suppress HDO signals.

3

Results

The CD spectra of the mixture of ATCL and HPN with various molecular ratios at 4 °C are shown in Fig. 1 . The spectrum of solution of ACTL alone displayed the typical CD pattern for native collagen with a strong negative peak at 197 nm and a weak positive peak at 222 nm. As shown in Fig. 1 , the spectra of the mixture with three different molar ratios are overlaid to show practically the same spectra even though there are small differences around 197 nm. These spectra showed that addition of various amounts of HPN in the ACTL solution did not induce any clear difference in the spectra of the mixture up to the ratio of 40:1 (HPN:ATCL). Thus it was concluded that no large change occurred in the structure of collagen in this range of HPN concentration, which corresponds to ca 0.34 mM.

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