Biodegradation of composite resin with ester linkages: Identifying human salivary enzyme activity with a potential role in the esterolytic process

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Biodegradation of composite resin with ester linkages: Identifying human salivary enzyme activity with a potential role in the esterolytic process Kuihua Cai a , Yasaman Delaviz b , Michael Banh a , Yi Guo c , J. Paul Santerre a,b,c,∗ a b c

Faculty of Dentistry, University of Toronto, ON, Canada Institute of Biomedical and Biomaterials Engineering, University of Toronto, ON, Canada Materials Science Engineering, University of Toronto, ON, Canada

a r t i c l e

i n f o

a b s t r a c t

Article history:

Objectives. The ester linkages contained within dental resin monomers (such as Bisphenol

Received 5 March 2014

A-glycidylmethacrylate (BisGMA) and triethylene glycol dimethacrylate (TEGDMA)) are sus-

Received in revised form

ceptible to hydrolytic degradation by salivary esterases, however very little is known about

20 May 2014

the specific esterase activities implicated in this process. The objective of this work was to

Accepted 21 May 2014

isolate and identify the dominant proteins from saliva that are associated with the esterase activities shown to be involved in the degradation of BisGMA. Methods. Human whole saliva was collected and processed prior to separation in a HiPrep

Keywords:

16/60 Sephacryl S-200 HR column. The fraction with the highest esterase activity was fur-

Composite resin

ther separated by an anion exchange column (Mono-Q (10/100G)). Isolated fractions were

Saliva

then separated by gel electrophoresis, and compared to a common bench marker esterase,

Albumin

cholesterol esterase (CE), and commercial albumin which has been reported to express

Zinc-␣2-glycoprotein

esterase activity. Proteins suspected of containing esterase activity were analyzed by Mass

Esterase

Spectroscopy (MS). Commercially available proteins, similar to the salivary esterase pro-

Biodegradation

teins identified by MS, were used to replicate the enzymatic complexes and confirm their

Hydrolysis

degradation activity with respect to BisGMA.

BisGMA

Results. MS data suggested that the enzyme fraction with the highest esterase activity was

Methacrylic acid

contained among a group of proteins consisting of albumin, Zn-␣2-glycoprotein, ␣-amylase, TALDO1 protein, transferrin, lipocalin2, and prolactin-induced protein. Studies concluded that the main esterase bands on the gels in each fraction did not overlap with CE activity, and that albumin activity emerged as a lead candidate with significant esterase activity relative to BisGMA degradation, particularly when it formed a complex with Zn-␣2-glycoprotein, under slightly basic conditions. Significance. These enzyme complexes can be used as a physiologically relevant formulation to test the biostability of composite resins. © 2014 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

∗ Corresponding author at: Faculty of Dentistry and Institute of Biomaterials and Biomedical Engineering, University of Toronto, 124 Edward Street, Room 464D, Toronto, ON, Canada M5G 1G6. Tel.: +1 416 979 4903x4341; fax: +1 416 979 4760. E-mail address: [email protected] (J.P. Santerre). http://dx.doi.org/10.1016/j.dental.2014.05.031 0109-5641/© 2014 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

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1.

Introduction

An increasing demand for esthetic restorations, continued public concern over the release of mercury from amalgam fillings, the improved mechanical properties of composite resin materials, as well as more and improved adhesive resin systems have fueled the use of composite resins in dentistry [1,2]. The combination of BisGMA and the diluent monomer TEGDMA remain two of the most common composite resin monomers still in use today due to their many advantages with respect to rapid hardening within the oral environment and ease of handling and manipulation. Both monomers contain ester linkages linking Bis-phenol-A and triethylene glycol segments to polymerizable vinyl segments. However, it is now well recognized that these ester groups are highly susceptible to hydrolysis by salivary enzymes, and can produce toxic and/or pro-biotic products such as methacrylic acid (MA), triethyleneglycol (TEG) and bishydroxypropoxyphenylpropane (BisHPPP) [3,4]. Concern over these products and their influence on the function of host cells (for example cell morbidity, cellular adhesive function and inhibition of intracellular biomolecular synthesis) and microorganisms [3–8] has been raised over the past few decades, however, there has been very little work directed towards adapting the materials toward addressing these challenges. Rather the focus of material development has remained primarily on the mechanical function of the materials [4]. One study has shown that the residue from a bis-phenol diglycidyl ether used in dental composites has resulted in allergic reactions, and much controversy has arisen around the issue of the estrogenic cell response to bisphenol A derivatives [3,9,10]. The biodegradation of composite resins has also been reported to lead to a softening of the surface layers of resins and predispose the material to mechanical wear during mastication. Once the softened surface layers are removed, newly exposed material would be prone to further degradation and this can finally lead to the failure of interfacial structures at the margins of the restoration [3,5,8,11]. Human whole saliva is a complex mixture with salivary proteins and food residues. Salivary proteins have many functions such as digestive, protective, calcium and mineral homeostasis, antibacterial and antifungal function, protease inhibition, lubrication [12]. There are only a very small number of esterases reported to be found in saliva and these are typically involved in food hydrolysis and more recent years are suspected to be involved in the degradation of composite resins. Studies by the Santerre group have demonstrated that human saliva exhibits cholesterol esterase-like (CE) and pseudocholinesterase (PCE) hydrolase activities, which are able to degrade composite resin components [5,13]. However, there is still very little information published on the actual salivary enzyme proteins related to the CE-like components which are involved in the in vivo process of composite degradation. In order to mimic salivary esterases and to apply them in assessing new products being developed, leading to enhanced function with respect to the physical stability, ultimate chemical stability of composite resins, and biological character of the degradation products from composite resin interactions with cells and bacteria, the current study has sought to

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isolate and analyze the predominant components in saliva and to identify the specific source of esterase activity that could be involved in catalyzing the breakdown of polymer resins. In the current study, the commercial monomer BisGMA is used as a model substrate solely for the purpose of identifying sources of esterase activity in saliva relevant to the hydrolysis of esters contained within the resin monomer components of composites. Since the susceptible linkages to chemical hydrolysis in methacrylate based polymers are equivalent to that of the monomeric form in its formulations, using the monomer in solution allows for an accelerated and easy investigation of multiple salivary enzyme species, in addition to minimizing the number of parameters that must be controlled for in the study. For example, it removes considerations of surface area, filler loading, and degree of conversion and cross-linking, all of which can affect the rate of degradation and could hinder the focus of the current study.

2.

Materials and methods

2.1.

Preparation of human saliva

Healthy human whole saliva (20 mLs) was collected in centrifuge tube from 10 donors, 2 h after breakfast and prior to lunch [14]. All human subjects provided informed consent (protocol #8469, reviewed by the human ethics committee at the University of Toronto). The saliva from each donor was thoroughly homogenized with a Polytron PT2100 tissue homogenizer (Kinematica, Switzerland) for 1 min and centrifuged at 3700 rpm for 1.0 h at 4 ◦ C (Allegra 6R Centrifuge, Beckman Instruments, Mississauga, ON). The supernatant was filtered through 0.8/0.2 ␮m syringe filters (Acrodisc, NQ105018/1, Pall Corporation, Cornwall, UK) to remove undissolved particulate. The filtrate from each donor was pooled together; quickly frozen by liquid nitrogen and then lyophilized. All dry saliva samples were stored at −78 ◦ C until required.

2.2.

Esterase-like activity (CE) assay

The CE-like esterase activity from the human salivary fractions was measured at 401 nm on a Beckman Coulter DU800 spectrophotometer (Beckman Coulter Inc., Fullerton, CA, USA, St. Louis, MO, USA) using 4 mM of para-nitrophenylbutyrate (pNPB) (Sigma–Aldrich, N-9876) as the substrate [14]. One unit of esterase activity was defined as generating 1 nmol/min of p-nitrophenol from p-nitrophenylbutyrate.

2.3.

Isolation of proteins from saliva

Saliva samples were reconstituted to a volume ratio of 10–1 with 40 mM sodium phosphate buffer, pH 7.2. Reconstituted saliva (1.5 mL) was centrifuged at 14,000 rpm for 15 min. The supernatant was injected into a High Performance Liquid Chromatograph system (WatersTM 600 Controller, 996 Photodiode Array Detector) with a HiPrep 16/60 Sephacryl S-200 HR gel filtration column (Amersham Biosciences, 17-1166-01, Baie d’Urfé, PQ, Canada). Gel filtration was performed with 40 mM sodium phosphate buffer, pH 7.2, and a flow rate of

850

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0.5 mL/min. Product recovery was monitored at a wavelength of 230 nm. Fractions (3 mL) were collected and measured for esterase activity, and those fractions with the highest activity were pooled for a given peak area, concentrated by lyophilizing, and then dialyzed in a 3500 MWCO, SlideA-Lyzer® Dialysis Cassette (PIERCE, prod# 66110) to remove sodium phosphate using a dialysis buffer made up of 20 mM Tris–HCl, 2 mM CaCl2 , pH 7.0. The dialyzed fractions were further purified by ion exchange chromatography with a Mono-Q (10/100G) column (Amersham Biosciences, 17-5167-01, Baie d’Urfé, PQ, Canada). Buffer A (20 mM Tris–HCl, 2 mM CaCl2 , pH 7.0) and Buffer B (0.5 M NaCl, 20 mM Tris–HCl, 2 mM CaCl2 , pH 7.0) were used to perform a linear gradient elution, with a flow rate of 1.0 mL/min. Dialyzed fractions (1.0–5.0 mL) were injected into the HPLC system (WatersTM 600 Controller, 996 Photodiode Array Detector) using carrier Buffer A for the initial 10 min. From 10 to 12 min, Buffer B was introduced in a linear gradient, increasing up to 25%, followed by its continued introduction from 25 to 100% over 12–50 min. The system was kept at 100% Buffer B for a 10 min elution time, and then was equilibrated with Buffer A for 30 min prior to the next injection. Fractions were collected with a Pharmacia® Fraction Collector using a 1 min fraction interval, and the esterase activity was measured for each fraction. The fractions with elevated esterase activity levels were kept in −78 ◦ C until required for further analysis.

excess DTT solution was removed. 50 ␮L of 20 mM iodoacetamide in 100 mM NH4 HCO3 was added into the protein samples and incubated for 30 min at room temperature in the dark to alkylate the sulfhydryl group of the cysteine residue. The excess liquid was removed and the gel pieces were washed with 200 ␮L of 100 mM NH4 HCO3 and completely dried in a vacuum centrifuge. The gel pieces were swollen in a digestion buffer containing 40 ␮L of 50 mM NH4 HCO3 and 12.5 ng/␮L of trypsin (Promega, sequencing grade) in an ice bath for 30 min. The supernatant was removed and replaced with 20 ␮L 50 mM NH4 HCO3 . The samples were digested at 37 ◦ C overnight. Peptides were extracted from the gel pieces by shaking with 50 ␮L of 5% formic acid twice, then 50 ␮L of 5% formic acid in 30% acetonitrile once (20 min for each extraction) at room temperature. The volume of the peptide extract solution was reduced to about 20 ␮L by vacuum centrifuge. The peptide solution was injected into an LC–MS/MS system (Agilent 1100 HPLC-chip and 6340 ion trap MSD system, Agilent Technologies). Raw MS/MS data files were matched against an NCBInr human subset database, using the Spectrum Mill MS Proteomics Workbench (Agilent Technologies). Proteins with two or more peptides identified from the MS/MS search were reported on.

2.4.

In order to investigate the nature of esterase activity associated with albumin, salivary samples were processed to remove albumin and then the samples, with and without albumin, were characterized for composition and esterase character. The Fractions of interest were rapidly thawed and maintained on ice. The buffer was changed to a ProteoExtractTM binding buffer (Calbiochem Cat. No. 122641 ProteoExtract® Albumin Removal Kit, Maxi) using Ultra-4 10 K Centrifugal filter devices (Millipore Corporation catalog #UFC801024). Samples were centrifuged in a Beckman GPR centrifuge at 3000 rpm and 4 ◦ C, washed with 4 mL ProteoExtractTM binding buffer, and then concentrated to approximately 0.75 mL. The concentrated sample was loaded onto a ProteoExtractTM column (Calbiochem Cat. No. 122641 ProteoExtract® Albumin Removal Kit, Maxi) and the flow-through fraction was collected while the columns were eluted twice with 1 mL of binding buffer. All flow-through fractions were pooled together, concentrated with Amicon Ultra-15 5,000MWC centrifugal filter devices, and reconstituted with 20 mM Tris–HCl pH 7.2, 2 mM CaCl2 . This latter purification work was performed by staff at Lelial Proteiomics Inc. Toronto, ON.

Gel electrophoresis and mass spectroscopy

Fractions containing esterase activity from the gel filtration column preparation were concentrated using an Amicon Ultra-15 5,000MWC centrifugal filter device (Millipore Corporation catalog #UFC900524) and mixed with native PAGE sample loading buffer containing 0.03% (w/v) bromophenol blue, 0.5 M Tris/HCl (pH 6.8), 30% glycerol, and then directly loaded onto two pieces of a 12% polyacrylamide gel. Gels were run at 200 V in a Mini-PROTEAN 3 Electrophoresis system for 1 h. Esterase activity was identified by soaking the gel in 50 mM pH 6.0 sodium phosphate staining solution with 0.04% Fast Blue RR salt (Sigma Cat. No. F-0500) and 0.02% ß-naphthylbutyrate (Sigma Cat. No. N8125) for 1 h with gentle agitation in the dark. Gels were fixed in 12% (w/v) trichloroacetic acid (Sigma Cat No. T8657) and destained in 10% acetic acid [15,16]. A second gel was stained by Coomassie blue to stain for non-specific protein. The fractions containing esterase activity from the MonoQ column separation were also run on 12% native PAGE and 12% SDS-PAGE in order to isolate proteins prior to mass spectrometry (MS), define protein molecular weight and obtain information on the denatured protein state. Stained bands were excised and sent for mass spectroscopy (MS) analysis at the Advanced Protein Technology Centre at the Hospital for Sick Children (Toronto, ON, Canada) and the Proteomics Core Facility for Molecular and Cellular Biology Research, Sunnybrook & Women’s College Health Sciences Centre (Toronto, ON, Canada). The protein bands were cut into small pieces and dehydrated in acetonitrile, then dried in a vacuum centrifuge. The protein was reduced by incubating with 50 ␮L of 10 mM dithiotreitol (DTT) in 100 mM NH4 HCO3 for 30 min at 56 ◦ C. Samples were allowed to cool to room temperature and

2.5. Removal of albumin from salivary fractions of interest

2.6. Esterase activity from commercial human salivary ˛-amylase preparations Amylase was identified as an enzyme whose presence corresponded with gel fractions containing high esterase activity. Hence, it was desired to determine whether there was any potential esterase function specifically associated with this enzyme. The esterase activity of amylase (Sigma A-1031) was measured with p-NPB. Amylase activity was assayed by the dinitrosalicylic acid method [17]. 1% (w/v) starch in 50 mM pH 7.0 sodium phosphate buffer was added into amylase samples

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and incubated at 37 ◦ C for 5 min. A 3,5-dinitrosalicylic acid (DNSA) solution containing 1.0% DNSA, 0.4 M NaOH, 30% sodium potassium tartrate was added into equal volumes of the incubation mixture and boiled for 10 min at 100 ◦ C to stop the enzymatic reaction. The mixture was cooled down by tap water, and a 2.0 ml aliquot was taken to measure the absorbance at 540 nm. One unit of amylase activity was defined as the amount of enzyme releasing 1 ␮mol glucose per minute at 37 ◦ C pH 7.0. Commercial glucose solutions (0.1–2.0 mM) were used to produce a standard curve. Either the specific ␣-amylase inhibitor Triticum aestivum (Sigma A-3535) or esterase inhibitor PMSF (phenylmethyl sulfonyl fluoride, 2 mM) (Sigma P-7626) were added to amylase samples and incubated at 37 ◦ C for 30 min before measuring the amylase or esterase activity, respectively. Purification of the amylase from proteins containing esterase activity was carried out by dissolving approximately 109 mg of ␣-amylase (Sigma A-1031) from human saliva in 10 mL Milli-Q filtered water, desalted and concentrated to about 2.5 mL by Ultra-15 centrifugal filter (MW cut: 10,000). 250 ␮L samples were applied into a FPLC (Pharmacia) system with Mono-Q HR (10/10) (Pharmacia), anion-exchange column, eluted for 10 min at a flow rate of 1.0 mL/min. The mobile phase consisted of Buffer A (0.02 M Tris/HCl-0.002 M CaCl2 , pH 7.2), followed by a linear gradient of 0 to 100% of Buffer B (0.02 M Tris/HCl-0.002 M CaCl2 , 0.5 M NaCl, pH 7.2) over 40 min. Subsequently, the column was then eluted with 100% Buffer B for 10 min, followed by re-equilibration with Buffer A for 30 min between runs. The eluted solution was monitored at a wavelength of 230 nm and fractions were collected in 1 min intervals. Where chromatographic peaks were observed, fractions were pooled, and esterase activity with respect to p-NPB was measured. Two active fractions of interest were processed by the Advanced Protein Technology Center at the Hospital for Sick Children and analyzed by MS.

2.7. Identification of albumin complexes with esterase activity on native gel In order to investigate the contribution of albumin protein complexes to esterase activity in saliva, 20 ␮g of the commercially available single proteins: Zn-␣2-glycoprotein (Zn␣2G), ␣-amylase, transferin, or lactotransferrin were individually mixed with 20 ␮g albumin (CalBiochem Cat. 126654) in 20 mM, Tris–HCl pH 7.2 buffer. All mixtures and single protein controls were incubated at 4 ◦ C for overnight, speed vacuum dried, re-dissolved in sample loading buffer, loaded onto a 4–12% native gel, and run in a cold room for 45 min. All gels were then stained with 1% 2-naphthyl-butyrate for an hour under dark conditions. Single protein controls for, Zn␣2G, lactotransferrin, lipocalin, or albumin were assayed under the same conditions.

2.8.

Biodegradation of BisGMA

For the time course study, BisGMA monomer (Esschem, Linwood, PA, USA) was prepared in methanol and diluted with 0.02 M Tris/HCl-0.002 M CaCl2 , 0.005 M MgCl2 , pH 7.2 to a desired concentration. This preparation was then added to enzyme solution (see below) containing 100 ␮g of albumin and

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yielding a final BisGMA concentration 0.5 × 10−4 M and total reaction volume 25 ␮L. Samples were incubated at 37 ◦ C for 24, 48, and 72 h. 25 ␮L of acetonitrile was added to denature the enzymes and deactivate its esterase activity at the end of the incubation period. All samples were kept in −70 ◦ C until HPLC analysis. Incubation enzyme solution containing either 100 ␮g albumin, 20 ␮g Zn␣2G, or a combined mixture of 100 ␮g albumin with 20 ␮g Zn␣2G were individually prepared in 0.02 M Tris/HCl-0.002 M CaCl2 , 0.005 M MgCl2 , pH 7.2 buffer and allowed to stand at 4 ◦ C overnight prior to use. BisGMA was added into each protein solution to make up the final BisGMA concentration (0.5 × 10−4 M) and yield a total reaction volume 25 ␮L. At the end of the 48 h degradation period, 25 ␮Ls of acetonitrile were added to denature the enzymes and cease activity. All samples were stored at −70 ◦ C until required for analysis. To study the effect of pH on esterase activity, incubation enzyme solutions containing 100 ␮g albumin were individually prepared in 0.02 M Tris/HCl, 0.002 M CaCl2 , 0.005 M MgCl2 , pH buffers adjusted to 6.3, 7.2, 8.0 or 8.8 using HCl and NaOH. BisGMA monomer was prepared in methanol and added into each protein solution to make up the final BisGMA concentration 0.5 × 10−4 M ([MeOH] < 2 vol%) and a reaction volume 25 ␮L (note: the presence of MeOH at this concentration did not affect esterase activity). Samples were incubated at 37 ◦ C for 72 h and subsequently, 25 ␮Ls of acetonitrile were added to denature the protein solutions and cease esterase activity. All samples were stored at −70 ◦ C until required for analysis. Comparison between Cholesterol esterases (CE, Toyobo, COE-313) activity and optimized albumin esterase activity were also investigated in 0.02 M Tris/HCl-0.002 M CaCl2 , 0.005 M MgCl2 , pH 8.8 buffer solution at different concentration of the proteins. The enzymatic activity of the CE incubation solutions was adjusted to 10 units/mL with respect to p-NPB substrates. Albumin incubation solutions contained 2, 4, or 10 mg/mL of albumin. The control contained the buffer solution without proteins. Prior to incubating BisGMA with enzyme solutions, stability studies were conducted to assess the effect of the monomer on the enzymatic activity; solution volumes were kept identical to that of the biodegradation studies. Based on the CE stability experiment, the CE containing groups were replenished daily (24 h) in order to maintain the esterase activity near 10 units/mL. Buffer solution was added in equivalent volume to the control and albumin samples to maintain a consistent volume among all three conditions. All solutions were sterile filtered using a 0.22 ␮m filter (Millipore SLGP033RS). 50 ␮L of a BisGMA methanol solution (with BisGMA concentration of 2.5 mM) was added into each protein solution to make up the final BisGMA concentration (0.5 × 10−4 M) and yield a total reaction volume of 2.5 mL. The final methanol concentration in the incubated solutions was less than 2 vol%. The experiment was run with triplicate sample groups to allow for statistical analysis. At specific time points (0, 1, 2, 3 and 4 days), 500 ␮L samples of the incubation solution were removed and an equal volume of methanol was added to denature the esterase protein and stop the hydrolysis. All samples were stored at −70 ◦ C until required for analysis.

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2.9.

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HPLC analysis of degradation products

Incubation solutions from the BisGMA biodegradation studies (n = 3) were analyzed using high performance liquid chromatography (HPLC). Each sample was centrifuged at 14,000 rpm for 30 min, and the supernatant was injected into a Luna C18 (2) (phenomenex® 00G-4252-E0) column. The sample was eluted at a flow rate 1.0 mL/min for 20 min, using a linear gradient beginning from (70:30) methanol (solution A): pH 6.8, 20 mM ammonium acetate (solution B) to (100:0), respectively. This was followed by a 100% methanol elution for 5 min. The column was equilibrated in a 70:30 ratio of solution A: solution B for 30 min before the next injection. The retention time for BisHPPP (terminal degradation product from the hydrolysis of BisGMA) was 5.43 min. The peak fractions were collected and analyzed by mass spectroscopy at the Molecular and Cellular Biology Research Sunnybrook & Women’s College Health Sciences Center. The amount of BisHPPP was calculated from a standard curve equation, r2 = 0.9991, covering a mass range from 1.8 to 171.5 ng.

2.10.

Statistical analysis

Statistical analysis was performed using the SPSS program (version 20.0). One-way analysis of variance (ANOVA) and Turkey’s multiple comparison tests were performed to determine the effect of incubation time and condition on the amount of BisHPPP released. Data are all plotted with standard deviation of the mean (N = 3). Independent sample t-test was used to determine the statistical significance of any differences between the mean values of two groups. For such analysis, Levene’s test for homogeneity of the variances was conducted. The significance threshold for all analyses was set to ˛ = 0.05.

3.

Results

3.1. Separation of proteins from human saliva and CE-like activity The UV absorbance data (measure of protein) for the processed human saliva fraction are plotted as a function of retention time (Fig. 1a). When the UV data are compared to the CElike esterase activity curve, it is noted that there are three defined fractions of protein that contain CE-like activity in human saliva. The fraction from 90 to 110 min possesses the highest esterase activity which overlaps with protein peaks. Given the broad distribution of the UV peak, this region was concluded to be a mixture of multiple proteins. The retention time of the peak CE-like activity overlapped the elution time of an albumin standard (CALBIOCHEM Cat.126654) which eluted near 107.86 min (Fig. 1a). To further analyze the dominant esterase fraction (90–110 min), this isolate was dialyzed to change the buffer, and then was eluted using a Mono-Q (10/100G) with a linear gradient. The solid line in Fig. 1b shows the relative amounts of protein coming from the ion exchange chromatography, as monitored at 230 nm. Each individual peak fraction was collected and tested for CE-like activity. The bar plots in Fig. 1b

Fig. 1 – Protein separation for human saliva. Fig. 1a, Gel chromatography of the reconstituted lyophilized human saliva supernatant (1.5 mL) on HiPrep 16/60 Sephacryl S-200 HR column, eluant: 40 mM sodium phosphate buffer, pH 7.2. Flow rate 0.5 mL/min, wavelength 230 nm. Esterase activity was measured for every 5 mL fraction volume with p-nitrophenyl butyrate. Fig. 1b, Ion exchange chromatography of the fraction with the highest CE-like activity from gel filtration (Fig. 1a) was further separated on a Mono-Q (10/100G) column with a linear gradient and monitored at a wavelength of 230 nm. The bars show the total esterase activity in each peak fraction.

show that the CE-like activities reside in two main fractions included within retention times from 22 to 27 min and 34 to 39 min, with the latter having the highest esterase activity relative to protein content.

3.2.

Native gel electrophoresis

In order to further characterize the esterase containing fractions from gel filtration, three desalted and concentrated fractions (97–102 min, 103–108 min, and 109–114 min from Fig. 1a) were separated on a 12% native polyacrylamide gel. Fig. 2a shows that there are two main esterase activity components that exist within the three isolated fractions, reflecting the two isolated peaks shown in Fig. 1b, as well as differences in their relative amounts of protein. When comparing Gel 1 (esterase) with Gel 2 (protein) in Fig. 2a, the esterase active components in the protein mixture fractions (97–102 min, 103–108 min, and 109–114 min from Fig. 1a) are not all stained

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Fig. 2 – Gel electrophoresis of salivary esterase fractions. Fig. 2a, 12% Native PAGE, lanes 1, 2, 3, 4 and 5 are respectively, commercial CE, 97–103 min, 103–108 min, 109–114 min (from Fig. 1a), and the molecular weight marker, stained for esterase by naphthylbutyrate (Gel 1) and for protein by Coomassie blue (Gel 2). Gel 1 in Fig. 2b is 12% Native PAGE, stained for esterase. Lanes 1, 2, 3 and 4 are respectively fraction 34–36 min, 22–26 min (from Fig. 1b), albumin, and the molecular weight marker. Gel 2 is 12% SDS PAGE stained for protein. Lanes 1, 2, 3 and 4 are respectively fraction 34–36 min, 22–26 min (from Fig. 1b), albumin, and molecular weight marker. Fig. 2c, 12% Native PAGE, stained for esterase. Lanes 1, 2, 3 and 4 are respectively 10 ␮g fraction 34–36 min before albumin depleting; 34–36 min after albumin depleting; commercial albumin; and the molecular weight marker. Fig. 2d, 12% native gel run at 4–8 ◦ C for 45 min, and then stained for esterase activity with 1% 2-naphthyl-butyrate for an hour in the dark. Lanes 1 and 2 are single Zn␣2G, 3–5 are single albumin, 6–8 are complex albumin-Zn␣2G.

by Coommossie blue. Since a common bench marker esterase, used to model the human salivary derived esterase activity in past studies [18] was CE, the latter commercial enzyme (lane 1) was used here to compare with the isolated enzymes in the salivary fractions. It was noted that most of the esterase activity from the two bands in the salivary extractions did not overlap with CE activity where the esterase stained band of CE was around 103 kDa, and was very strong, while the dominant esterase activity bands in saliva show up between 60 and 90 kDa. Furthermore, the active CE itself did not stain with Coomassie. Hence, CE is ruled out as being a dominant esterase component in human saliva. Over the past few years there have been reports of albumin derived esterase activity [19–22]. Given the abundance of albumin in saliva [23] and its proximity to the esterase activity retention time in Fig. 1a, it was considered that albumin might be a candidate protein for the esterase activity of interest. Fractions 22–26 min and 34–36 min were further purified using a Mono-Q column (Fig. 1b) and were run on native PAGE gels and compared to commercial albumin in the crystal form, and analyzed for esterase activity with naphthylbutyrate (Fig. 2b, Gel 1). It is confirmed from the data that the fraction from 22–26 min (lane 2 in Fig. 2b) has esterase activity among its protein and shows low migration in the native gel. As well, it is noted that the 22–26 min peak does not overlap with the dense protein stain (Fig. 2b, Gel 2) that corresponded with pure albumin (Fig. 2b, Lane 3, Gel 2). The longer retention time

fraction (34–36 min), which had the higher levels of esterase activity components (Fig. 1b), contains a high amount of protein overlapping with the albumin stained band (Fig. 2b Gel 2). Interestingly, it is noted that the pure albumin has minimal esterase activity (Fig. 2b, Lane 3, Gel 1). Lane 1 data in Gel 2 of Fig. 2b for the SDS-PAGE stained with Coomassie blue shows that albumin is likely to be present in the salivary protein make-up. Another important observation was that there was a clear difference between lanes 1 and 2 for Gel 1 in Fig. 2b, indicating that there are multiple proteins, or possibly a complex of proteins, that are contributing to the esterase activity of interest. The next step was to investigate the composition of the proteins contained within the bands observed on the gel for the proteins with esterase activity in Fig. 2b (Gel 1). The one band from lane 1, two bands from lane 2, and the region where albumin was expected to be found in lane 3 Gel 1 (Fig. 2b), were excised and underwent on-gel digestion with Trypsin. Digested samples were run on an ABI/MDS Sciex QSTAR-XL LC/MS/MS system; and the collected mass spectra of proteins were searched using the Mascot Search Engine. Table 1 lists the main proteins found in the three dominant esterase bands and commercial albumin band. The dataset also includes the number of distinct peptide fragments found in the mass spectrum, total peptide spectral intensity and the percent sequence coverage for each protein that is identified. A high value in any of the latter parameters means that the identified protein had

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Table 1 – Salivary proteins identified by LC/MS/MS. Protein samples were derived from bands isolated from gels on Fig. 2b, Gel 1 (bands 1, 2 and 3); Fig. 2c (lane 2); and Fig. 2d (commercial albumin and Albumin/Zn␣2G complex bands). Fraction name

Protein name

Database accession number

Distinct peptides number

Lipocalin 2 Alpha 1A–amylase lactotransferrin Immunoglobulin heavy chain hemopexin

55961101 55587618 16198359 25987833

5 13 15 3

55635243

2

Serum albumin precursor Alpha 1 anti-trypsin Zn␣2G Trypsin precursor Keratin 1

52001697

32

11493443 38026 136429 181402

8 5 3 2

Serum albumin precursor Prolactin-induced protein Transferrin Variant Lipocalin 2 Zn␣2G Immunoglobulin kappa light chain VLJ region Triosephosphate isomerase Alpha 1A–amylase Hypothetical protein XP-518733 Keratin 1

6013427

MS/MS search score

Total peptide Spectral intensity

Protein MW (Da)

Protein pI

38 34 27 11

7.35E+07 1.02E+09 2.1E+08 6.21E+07

22,788.3 57,719.2 78,345.6 35,752.7

8.66 6.86 8.49 7.51

29.97

9

3.08E+07

51,689.7

6.55

512.42

52

4.03E+09

69,367.1

5.92

108.66 73.23 43.13 35.81

23 22 16 3

19.4E+08 1.01E+08 3.81E+08 1.94E+07

46,736.8 34,736.4 24,409.6 65,865.6

5.37 5.71 7 8.07

31

502.62

45

5.59E+09

69,226

5.92

4505821

6

99.6

43

1.67E+09

16,572.6

8.26

62897069 55961101 38026 21669299

25 6 9 4

410.21 93.29 140.45 58.03

39 38 31 23

5.68E+09 5.81E+08 1.40E+09 2.68E+08

77,080.4 22,788.3 34,736.4 29,099.7

6.68 8.66 5.71 8.45

41058276

4

66.26

21

2.03E+08

26,942.9

8.21

55587618 55665009

7 5

116.4 71.15

16 13

9.94E+08 1.55E+09

57,719.2 37,926.2

6.86 5.78

7331218

7

100.37

11

1.79E+08

66,018

8.16

L-plastin Zn␣2G Albumin precursor Alpha 1A–amylase TALDO1 protein Transferin Variant Prolactin-induced protein Enolase 1 variant hemopexin

4504965 38026 4502027 40254482 48257056 62897069 4505821

15 11 10 9 10 7 4

221.6 143.23 142.63 133.63 133.14 99.29 63.88

29 34 16 25 27 12 36

1.74E+09 2.48E+09 2.67E+09 1.94E+09 2.85E+09 6.38E+08 4.05E+09

70,289.7 34,736.4 69,367.1 57,768.1 37,409.1 77,080.4 16,572.6

5.2 5.71 5.92 6.47 6.35 6.68 8.26

62897945 11321561

4 4

59.61 58.54

13 10

3.75E+08 6.55E+08

47,197.2 51,676.7

7.01 6.55

Commercial Albumin lanes 3–5, Fig. 2d

Albumin precusor

4502027

32

527.62

49

9.77E+09

69,367.1

5.92

Alb-Zn␣2G complex lanes 6–8, Fig. 2d

Albumin precusor Zn␣2G

4502027 38026

33 15

527.46 224.6

48 50

1.23E+10 1.17E+09

69,367.1 34,736.4

5.92 5.71

Alb-Zn␣2G complex with BisGMA

Albumin isoform CRA-h Zn␣2G

119626071

35

572.34

56

2.30E+10

68,613.2

5.92

114614917

13

197.75

47

2.06E+09

34,328

5.86

22–26 min Sample from Mono-Q (band 1 on Fig. 2b, Gel 1)

22–26 min Sample from Mono-Q (band 2 on Fig. 2b, Gel 1)

34–36 min sample from Mono-Q (band 3 on Fig. 2b, Gel 1)

34–36 min sample from Mono-Q after depleted albumin (Lane 2 on Fig. 2c)

a high probability of being a good match. It can therefore be seen that serum albumin has a very strong signal for the band related to fraction 34–36 min (lane 1) and band 2 of fraction 22–26 min (lane 2). A ProteoExtractTM column was used to deplete albumin from the 34–36 min fraction in order to enable samples to provide a more clear detection for other proteins. The esterase activity relative to the total amount of the depleted albumin fraction was shown to increase dramatically (Fig. 2c), when

68.59 207.11 204.03 39.32

% AA Cover-age

comparing lane 2 (fraction 34–36 min after depleting albumin) to lane 1 (fraction 34–36 min before depleting albumin) in Fig. 2c, thereby confirming that proteins other than albumin were important contributors to the esterase activity. MS analysis of this preparation confirmed the presence of several other proteins (Table 1). In order to confirm that a complex of proteins was contributing to the esterase activity in saliva, each commercially available pure single protein for albumin, Zn␣2G, ␣-amylase,

d e n t a l m a t e r i a l s 3 0 ( 2 0 1 4 ) 848–860

855

Fig. 3 – Analysis of BisGMA degradation by-product BisHPPP with Albumin Derived Esterase Activity (37 ◦ C). All data are reported with standard deviation (±SD, N = 3); values were significantly different from each other: =/ p < 0.05, * p < 0.01,  p ≤ 0.001. Fig. 3a, typical HPLC Chromatogram of BisGMA biodegradation products. BisGMA has a retention time of 16.58 min and BisHPPP has a retention time of 5.52 min. Fig. 3b, the effect of different protein ratios for the albumin/Zn␣2G complex, on the degradation of BisGMA, pH = 7.2; n = 3; 48 h. Fig. 3c, the effect of incubation time on BisGMA degradation with albumin (100 ␮g) and albumin (100 ␮g) – Zn␣2G (20 ␮g), pH = 7.2; n = 3. Fig. 3d, effect of pH on BisGMA degradation with albumin (100 ␮g) and control buffer without albumin, n = 3; 72 h.

transferin, and lactotransferrin were individually measured for esterase activity by p-NPB method. The results indicate that only albumin and ␣-amylase derived from human saliva contained esterase activity (data not shown). It was subsequently shown that purified ␣-amylase contained no esterase activity on its own (data not shown) and that band 1 (Fig. 2b, Gel 1) which contained the high ␣-amylase fraction had the lowest intensity of esterase activity of the 3 bands studied by mass spectrometry. Attention was then focused on albumin and proteins in bands 2 and 3 (Fig. 2b, Gel 1). Given the knowledge that albumin has been reported to have esterase activity [19–22], and that it was shown to be found in the salivary fractions that had the highest esterase activity, this protein was a prime target of interest. However, when studied in purified form, it showed only traces of esterase activity (lane 3, Gel 1, Fig. 2 b). Hence, it was hypothesized that some of the proteins identified in Table 1 for bands labeled as 2 and 3 in Gel 1 (Fig. 2b) could possibly complex with albumin to enhance albumin’s esterase activity. Protein complexes were formed by incubating albumin

overnight with either Zn␣2G, lactotransferrin, or lipocalin at 4 ◦ C, and then assaying for esterase activity to compare each protein complex and its single protein controls. Lactotransferrin, lipocalin, and Zn␣2G on their own did not exhibit any detectible esterase activity on the gel. In addition, the combination of albumin-lactotransferrin, and albumin-lipocalin did not exhibit any dose response related intensity of the esterase activity stain relative to albumin alone (data not reported). However, the bands for the albumin-Zn␣2G complex (Fig. 2d), exhibited stronger esterase activity than the band for albumin alone. These results suggest that Zn␣2G must have complementary binding sites on the albumin chain that render its esterase function more available to the 2-naphthylbutyrate substrate. Further support that a protein complex was possibly forming was indicated by the MS analysis, which showed intense signals for both albumin with Zn␣2G (Table 1). Based on these findings, it was then hypothesized that the increased esterase activity could translate to activity that would catalyze the degradation of common dental composite resin monomer substrates such as BisGMA monomer. It was concluded that

856

d e n t a l m a t e r i a l s 3 0 ( 2 0 1 4 ) 848–860

CE-like activity with respect to p-NPB) at pH 8.8 (Fig. 4). After 24 h of incubation, a significantly higher amount of BisHPPP is produced by albumin (150 units/mL) vs. CE (10 units/mL), and no significant difference in BisHPPP production is observed at 48 and 72 h of incubation.

4.

Fig. 4 – Analysis of BisGMA degradation by-product BisHPPP with albumin and CE derived esterase activity (37 ◦ C). All data are reported with standard deviation (±SD, N = 3); values were significantly different from each other: = / p < 0.05, * p < 0.01,  p ≤ 0.001. Esterase activity is reported with respect to p-NPB substrate, rather than mass of protein.

the combination of albumin with Zn␣2G was a good candidate protein complex for further investigation.

3.3.

Biodegradation of BisGMA monomer

BisGMA was subsequently incubated with albumin vs albumin/Zn␣2G complexes. Incubation solutions were analyzed for potential degradation products by HPLC. Fig. 3a depicts the HPLC chromatograph of biodegradation products for BisGMA and proteins. A peak at a retention time of 5.43 ± 0.19 min was identified by mass spectroscopy as BisHPPP, a degradation product of BisGMA with the terminal methacrylate groups cleaved [13]). Fig. 3b demonstrates that following the incubation of BisGMA with a complex of albumin and Zn␣2G there is more BisHPPP generated than without the complex (p < 0.05). However, it was noted that the protein ratio of the complex was very important. A ratio of 5:1 albumin: Zn␣2G had a greater differentiating effect than did a 1:1 ratio of the proteins. Increasing the amount of albumin alone increased the amount of BisHPPP but the added effect of Zn␣2G at a select ratio was much more potent. Fig. 3c shows that the degradation of BisGMA in both albumin and albumin-Zn␣2G complex system increases as a function of time. The degradation of BisGMA by the albumin-Zn␣2G complex confirmed that the complex esterase activity remained more active with respect to BisGMA over time than with the albumin alone. The degradation rate for the complex was 0.4 ng/h vs 0.1 ng/h for albumin alone. The esterase activity of albumin, with respect to the BisGMA substrate over 72 h, on its own became more potent as pH increased from 6.3 to 8.8 (Fig. 3d). A trend of increasing BisHPPP release with time throughout the incubation period was observed with respect to albumin and CE at pH 8.8 (Fig. 4). The amount of BisHPPP released was elevated in the presence of protein vs. control, and greater in the presence of CE vs. albumin. In comparison to CE, albumin has an overall lower activity toward p-NPB per gram of protein. However, a comparable rate of degradation to 10 units/mL of CE activity can be achieved with 10 mg/mL of albumin (150 units/mL of

Discussion

Esterases are a family of enzymatic hydrolases (EC 3.1.1), that show differences in their substrate specificity, as some have higher activity toward long acyl chain substrates (C12–C18) while others prefer short chain substrates (C2–C6) because of providing easier accessibility to active sites [24]. There is a very small amount of non-defined esterases found in human saliva which shows an affinity for dental resin monomers, as found from previous studies in the field, using p-NPB as a substrate [14]. The data in Fig. 1a confirms that there are three fractions of esterase proteins specific to p-NPB. The proteins eluted out of the gel filtration column of Sephacryl S-200 HR between 90 and 110 min show a broad distribution in the UV peak, suggesting that there is a mixture of proteins. This chromatography region showed the highest esterase activity and contained a significant amount of protein with a molecular weight around 65 kDa or larger. Therefore, further identification of esteraseassociated proteins in the latter peak was focused on this dominant esterase fraction between 90 and 110 min. Three fractions (97–102, 103–108, and 109–114 min) were collected from the Sephacryl S-200 HR column and were run on two 12% native polyacrylamide gels. The gels were respectively stained by 2-naphathylbutyrate (Gel 1 Fig. 2a) or Coomassie blue (Gel 2 Fig. 2a) to analyze for esterase activity distribution and protein content. Since a common bench marker for these esterases, used to model the human salivary derived esterase activity in past studies [18], was CE, the latter commercial enzyme was used to compare to the isolated enzymes in the salivary fractions. Gel 1 (Fig. 2a) shows that there are two main esterase activities that are common to the three isolated fractions, reflecting the two isolated regions shown in the chromatography Fig. 1b (22–26 min and 34–36 min). The relative amounts of protein content differ among the three fractions from the visible bands in Gel 1 (Fig. 2a) since they do not overlap with the bands stained for Coomassie blue in Gel 2 in Fig. 2a, demonstrating that fraction 90–110 min from Fig. 1a contains multiple proteins and that the esterase related proteins in the fraction are possibly post-translationally modified. It was noted that most of the esterase activity from the two protein bands in the salivary extractions did not overlap with the prominent band for CE around 103 kDa. The dominant esterase activity bands in saliva show up between 60 and 90 kDa. Hence, CE was ruled out as being a dominant esterase in human saliva. An interesting observation was that the esterase activity band of CE could not be stained by Coomassie blue. This may due to the fact that CE possesses a glycosylated C-terminus (residues 536–714) in the form of an O-linked carbohydrate and an N-linked glycosylation site within the catalytic domain at Asn181 [24]. The Coomassie dye binds to proteins through ionic interactions between the dye’s sulfonic acid groups and the positive protein amine groups such

d e n t a l m a t e r i a l s 3 0 ( 2 0 1 4 ) 848–860

as lysine arginine and histidine, as well as binding to aromatic amino acids through Van der Waals attractions. Since the O-linked glycosylation usually occurs on the side chain of hydroxylysine, hydroproline, serine or threonine, and the N-linked glycosylation occurs on the side chain of the asparagines, the glycoprotein carries less charges on the protein’s exterior surface. In the native state, glycosylation yields relatively hydrophobic aromatic amino acids buried deeper inside of the protein core. It is a common observation that glycoproteins are poorly stained by Coomassie blue. Given this observation for CE, a similar finding may be relevant for the salivary esterase proteins which did not show overlapping esterase and Coomassie bands as they may also contain a high carbohydrate component. While multiple protein peaks were found in the anion exchange chromatograph (Fig. 1b), the esterase activity was primarily distributed between the regions of 22–27 min and 34–39 min (Fig. 1b), and the order of elution time from the anion exchange column suggests that proteins in the 22–27 min fraction have lower levels of negatively charged groups on the proteins, external domains at pH 7.2, versus the protein in the 34–36 min fraction. Over the past decade there have been several reports which have suggested that albumin exhibits esterase activity [19–22]. There is 39 ± 3 to 216 ± 174 mg/l of albumin in human whole saliva [23]; and hence it was considered that albumin might be an implicated protein for the source of esterase activity within the multi-protein complexes. Commercial crystal albumin was compared to fractions 22–26 min and 34–36 min (Fig. 1b). Fig. 2b Gel 1 showed that the 22–26 min fraction (lane 2) contains a very faint esterase activity band for protein, nearest to top of the gel, with an approximate molecular weight of a 100 kDa. The denatured protein information in Fig. 2b Gel 2 reveals that the apparent molecular weights of all visible proteins are lower than 62 kDa, and that most of the denatured proteins in fraction 22–26 min do not overlap with the dense protein stain that corresponded with pure albumin (Fig. 2, Gel 2, lane 3). Hence, the activity in the 22–26 min fraction contains proteins other than that of albumin. The above findings suggest that a protein complex, or glycosylated proteins may be involved. The protein fraction with the longer retention time (Fig. 1, 34–36 min fraction), and which contained higher levels of esterase activity (Fig. 1b), migrated faster on the native PAGE (lane 1, Gel 1 Fig. 2b). As well, the molecular weight of this protein overlapped with the pure albumin stain (Fig. 2b, Gel 2), showing a very strong protein band with a molecular weight of about 56 kDa. It was noted that the pure albumin sample showed only a faint intensity of esterase activity in Gel 1 Fig. 2b. Fig. 2b Gel 2 data for the SDS-PAGE suggests that albumin was present in the salivary protein sample, but it is unlikely that it is contributing to the observed esterase activity on its own. Mass spectroscopy data in Table 1 show that the serum albumin derived peptides always appear with a characteristic number of poly-peptides, MS/MS search score, percentage of amino acid sequence coverage and total peptide spectral intensity for band 2 associated with the 22–26 min fraction and band 3 from fraction 34–36 min in Gel 1 Fig. 2b). This suggests that serum albumin protein exists among these two esterase fractions at high levels.

857

In order to investigate the possibility that the strong albumin signal from saliva in the MS data may mask the signals from other important proteins present in smaller amounts; a ProteoExtractTM column was used to deplete large amounts of albumin from the 34–36 min fraction of the band 3 specimen. The esterase activity of the depleted albumin fraction showed a dramatic increase relative to the non-depleted sample (lane 1 vs lane 2 in Fig. 2c), thereby confirming that proteins other than albumin were important contributors to the esterase activity. High MS/MS search scores and elevated amino acid (AA) sequence coverage indicated that other proteins present with albumin included prolactin-induced protein, Zn␣2G, L-plastin, TALDO1 protein, alpha 1A-amylase, and others. (Table 1). An analysis of the 22–26 min fraction (Table 1) shows that the protein from band 1 is distinct from band 2 by the fact that band 1 exhibits ␣-amylase and albumin whereas band 2 only exhibits albumin. It should also be noted that a higher level of esterase activity corresponds with band 2 (Fig. 2b, Gel 1). When comparing the MS data for fraction 34–36 min with that of fraction 22–26 min, the identified proteins in both fractions look quite similar (Table 1). The proteins that show the highest intensity and % AA coverage from the native gels of these two fractions are serum albumin, Zn␣2G, ␣-amylase, TALDO1protein, transferrin, lipocalin2, and prolactin-induced protein. These findings further confirm that the esterase activity in fractions 34–36 min and 22–26 min is most likely derived from a complex of proteins with perhaps different degrees or different ratios of albumin and related binding proteins. In order to investigate which proteins contribute to the dominant esterase activity in saliva, each commercially available single protein: albumin, Zn␣2G, ␣-amylase, transferin, lipocalin 2 and lactotransferrin were individually measured for esterase activity by the p-NPB method. The results indicate that only albumin and ␣-amylase preparations derived from human saliva contained esterase activity on their own (data not shown). Since purified commercial ␣-amylase on its own does not exhibit esterase activity [25], it is suspected that the faint esterase activity in band1 of lane 2 (Gel 1 Fig. 2b) is likely attributed to a complex of other proteins with or without ␣–amylase. Lactoferrin contains a Fuc ␣ 1, 3 (Gal ␤ 1, 4) GlcNAc terminal structure in its oligosaccharides [26] and has been reported to be a polyfunctional and conformationally active protein [27]; its activity can be influenced not only by iron ions, but also by other metal ions such as K+ , Na+ , Mg2+ , Mn2+ , Ca2+ , Zn2+ , Cu2+ and ligands like DNA, RNA, polyanions, etc. The Fuc ␣ 1, 3 (Gal ␤ 1,4) GlcNAc terminal structure that is also present in one or both outer branches of the oligosaccharide in ␣-amylase could be a unique ligand to lactoferrin [26]. Since the fraction containing the ␣-amylase had the lowest intensity of esterase activity (Fig. 2b, Gel 1) of the three identified bands studied by mass spectrometry, the attention was focused on albumin and the proteins in bands 2 and 3 of Fig. 2b. After an analysis of the literature [19–22] and an examination of the findings of this current work, salivary albumin was considered to be a key protein contributing to salivary esterase activity. Its inherent low levels of esterase activity when present in pure form led to the hypothesis that albumin likely complexes, with other proteins identified in Table 1, in order to enhance its esterase activity. Albumin-associated

858

d e n t a l m a t e r i a l s 3 0 ( 2 0 1 4 ) 848–860

Fig. 5 – Simulated mechanisms for the hydrolysis of PNPP by human serum albumin. Adapted from ref. [21].

esterase activity is dependent on ligand binding sites Tyr-411 and Arg-410 (see Fig. 5 [3,21]). The substrate combines rapidly and reversibly to Tyr-411 and Arg-410, and the hydroxyl group of Ser-489 and the aromatic residue of Phe-488 help to stabilize the albumin-substrate intermediate. Modification of those sites or changes within the microenvironments of those sites will change the esterase activity of albumin [19–22]. In addition, seven fatty acid binding sites are distributed asymmetrically across all of the above three domains of human serum albumin, and three of them overlap with the two primary drug-binding sites, IIA and IIIA. This forms the active site of the albumin-associated esterase activity, which favors binding to fatty acids shorter than C18 in which the carboxylate moiety salt-bridges to Arg 410, Try 411 and Ser489. Within these binding sites, the fatty acids appear to be capable of rapid internal motions. The change in environment initiated by the fatty acid transport channel will affect the rate for deacetylation of acetyl-albumin, which is related to the esterase activity of serum albumin. This hypothesis was proven in subsequent experiments where the albumin esterase activity increased as the pH value was increased from 7.2 to 8.8 in Fig. 3d). In considering the previous discussion where salivary esterase proteins were shown to contain high carbohydrate content (groups that are influenced by pH) related to the glycoproteins Zn␣2G, lactotransferrin, lipocalin2, the latter proteins remain promising candidates to form the albumin complex of interest. The cavity formed by residues within lipocalin shows an affinity for some R-OH groups [28]. Lipocalin is able to perform side-chain conformational changes in order to optimize its cavity to accommodate the naturally occurring ligands. The affinity for R-OH could generate an irreversible albuminsubstrate intermediate and enhance the esterase activity of albumin. On the other hand, lactotransferrin is a Fe3+ binding glycoprotein, also referred to as lactoferrin. Its molecular weight is between 76 and 80 kDa. When bacteria in saliva bind to lactoferrin, they will be deprived of Fe3+ and die [27]. There

are no reports of this protein being a co-factor for esterase activity. Zn␣2G is a member of the major histocompatibility complex (MHC) class I family of proteins, and is present in different body fluids such as sweat, saliva, cerebrospinal, milk and urine. X-ray crystallography has demonstrated an open groove between the helices of Zn␣2G ‘s ␣1 and ␣2 domains with additional electron density which is useful for guiding the binding to natural ligands like fatty acid. This makes Zn␣2G able to bind to a variety of ligands [29,30]. The biological function of Zn␣2G varies with the nature of the bound compound. If Zn␣2G complexes with albumin and forms a fatty acid relay transporter, thereby releasing the esterase active site Arg 410 albumin, the albumin esterase activity will be enhanced. Hence, there is the possibility to attribute higher esterase activity from saliva, when some of the glycoproteins form a complex with albumin. In the current study, albumin complexes were formed by incubating albumin with either Zn␣2G (recombinant protein), lactotransferrin, or lipocalin at 4 ◦ C overnight, and the level of esterase activity were evaluated. Single protein lactotransferrin, lipocalin, and Zn␣2G did not exhibit any detectible esterase activity on their own. In addition, the combination of albumin-lactotransferrin, and albumin-lipocalin did not exhibit any increase in the intensity of the esterase activity stain relative to albumin (data not reported). However, the bands for the albumin-Zn␣2G complex (Fig. 2d), exhibited stronger esterase activity than the band for albumin alone. These results suggested that Zn␣2G must have complementary binding sites on the albumin chain that render its esterase function more available to the 2naphthylbutyrate substrate or Zn␣2G complex with albumin to form a fatty acid relay transporter. Further support that a protein complex was likely forming was indicated from the MS analysis which showed an intense band formed for the complex of albumin with Zn␣2G. These findings supported the hypothesis that this increased activity could translate to activity that would catalyze the degradation of common dental composite resin monomer substrates such as the BisGMA monomer. It was very interesting to note that albumin (Mr 69367 Da) and Zn␣2G (Mr 34736 Da), in complex form, both changed their conformation state in the presence of BisGMA monomer when MS data were compared (Table 1). Albumin changed to be albumin isoform CRA-h (Mr 68613) and Zn␣2G (pI 5.71) changed to the isoform pI 5.86 (Table 1). The latter suggests a specific interaction with the BisGMA monomer. The degradation products of Bis-GMA with albumin or its Zn␣2G complex were measured by HPLC. The HPLC results proved that a complex, which could enhance esterase activity with respect to BisGMA, was likely forming. Fig. 3b demonstrates that the incubation of BisGMA with a complex of albumin and Zn␣2G produced more BisHPPP, a degradation product from BisGMA [4], than what was generated without the complex (p < 0.05). It was also noted that the ratio of the complexed protein was very important. A ratio of 5:1 albumin: Zn␣2G had a greater hydrolytic effect than did a 1:1 ratio of the proteins. It should be noted that it may therefore not just be a coincidence that the total peptide spectral intensity/MW for albumin is approximately 5 times the total peptide spectral intensity/MW of Zn␣2G, as shown in the MS data (Table 1). Increasing the amount of albumin

d e n t a l m a t e r i a l s 3 0 ( 2 0 1 4 ) 848–860

alone increased the amount of BisHPPP but the added effect of Zn␣2G at a select ratio was much more potent (Fig. 3b). Fig. 3c shows that the degradation of BisGMA in both the albumin and the albumin-Zn␣2G complex system increases as a function of time. The time course study for the degradation of BisGMA by the albumin-Zn␣2G complex confirmed that the esterase activity complex remained more active with respect to BisGMA over time than with the albumin alone, and that the hydrolysis rate for the complex was approximately 0.4 ng/h vs 0.1 ng/h for albumin alone. As can be seen in Fig. 3d, the esterase activity of albumin increased as the pH value increased from 7.2 to 8.8. An increase in pH value will contribute to generating more negative electrostatic potential for Ser 489 and Tyr 411. After cleavage of the ester bond from BisGMA, the deprotonated free fatty acid will be easily ejected from the active site due to electrostatic repulsion [31]. The faster the fatty acid is removed from active site, the higher the albumin esterase activity appears. This result suggests that Zn␣2G could have enhanced albumin esterase activity by enhancing the fatty acid depleting function. At pH of 8.8, 10 mg/mL (150 units/mL) of albumin degraded BisGMA and released terminal degradation by-product BisHPPP at levels comparable to 10 units/mL of CE. Although a greater quantity of protein is required with albumin to achieve the same amount of degradation by-product as 10 units/mL of CE, it has the benefit of a stable CE-like activity even after 16 days of incubation at 37 ◦ C while CE solutions are required to be replenished on a daily basis where sample volume is affected and must be taken into consideration. Despite experimental data supporting the albumin-Zn␣2G as being the dominant contributor to the esterase activity of human saliva with respect to BisGMA, the sample related to the protein in fraction 34–36 min contains 5 times more esterase activity with respect to BisGMA when compared to the albumin-Zn␣2G complex alone. One possible explanation for this could be related to the differences between the natural proteins and those of the commercial Zn␣2G, which is a recombinatant protein with twelve extra amino acids fused in the N-terminus. Alternatively, natural salivary derived Zn␣2G has been reported to be different from the Zn␣2G derived from serum [32]. As well, real salivary esterase activity may be enhanced by other factors such as specific metal ion binding or surfactant-type molecules existing in the natural extract from saliva. Surfactant-like molecules have the ability to modulate the biodegradation kinetics of composites [3]. It is also possible that additional proteins are involved in the esterase complexes and this could be further explored in future work.

5.

Conclusion

The findings of the study have further clarified the nature of the esterase activity associated with human saliva that is potentially involved in the biochemical breakdown of composite resins used in the production of dental composite fillings. The formation of albumin and Zn␣2G into a complex with a mass ratio of 5–1 can effectively degrade the resin through a mechanism that appears to enhance the elimination of cleaved ester substrates from the reactive site of albumin’s esterase activity. This action can be further stimulated by

859

changing the pH at which the single protein (albumin)/BisGMA reaction occurs. These enzyme complexes can now be used as a physiologically relevant formulation to test the biostability of new composite resins undergoing development for commercial use.

Acknowledgment This study was supported by a Natural Sciences and Engineering Research Council (NSERC) Discovery grant (360520).

references

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