Acquired enamel pellicle protects gastroesophageal reflux disease patients against erosive tooth wear

Abstract The objective of this study was to compare the protein profile of the acquired enamel pellicle (AEP) formed in vivo in patients with or without gastroesophageal reflux disease (GERD), and with or without erosive tooth wear (ETW). Twenty-four volunteers were divided into 3 groups: 1) GERD and ETW; 2) GERD without ETW; and 3) control (without GERD). The AEP formed 120 min after prophylaxis was collected from the lingual/palatal surfaces. The samples were subjected to mass spectrometry (nLC-ESI-MS/MS) and label-free quantification by Protein Lynx Global Service software. A total of 213 proteins were identified, or 119, 92 and 106 from each group, respectively. Group 2 showed a high number of phosphorylated and calcium-binding proteins. Twenty-three proteins were found in all the groups, including 14-3-3 protein zeta/delta and 1-phosphatidylinositol. Several intracellular proteins that join saliva after the exfoliation of oral mucosa cells might have the potential to bind hydroxyapatite, or participate in forming supramolecular aggregates that bind to precursor proteins in the AEP. Proteins might play a central role in protecting the dental surface against acid dissolution.


Introduction
Erosive ooth wear (ETW) is characterized by the cumulative loss of mineralized tooth substances, resulting from exposure to nonbacterial acids of intrinsic and/or extrinsic origin, where erosion is the primary causative factor. 1 In recent years, several studies have sought to investigate the biochemical process involving ETW, prompted by an increase in the average global prevalence from 30% to 50%. 2,3To this end, it is important to identify what individuals are likely to develop ETW, so that early diagnosis and preventive measures can be established. 2 n i mpor t a nt cl i n ica l condit ion associated w it h ETW is gastroesophageal reflux disease (GERD), typical manifestations being regurgitation, dysphagia, and vomiting.It affects 15% to 25% of the high-income and 10% of the low-income population. 4However, the prevalence of ETW in patients with GERD is 5-47%, 3 caused by direct contact of regurgitated gastric contents (pH between 1 and 2) with the tooth surface. 2,5The low pH of the gastric content seems to suggest that GERD patients would have a higher expected prevalence of ETW.This means that GERD patients without ETW might have some protective factor.The acquired enamel pellicle (AEP) is considered one of the most important factors guarding against ETW. 6Previously, our team compared the proteomic profile of AEP of GERD patients with and without ETW, in an effort to find what proteins in the AEP would resist removal by intrinsic acids.Among the acidresistant proteins, hemoglobin was found to have increased more than threefold in the AEP of GERD volunteers without ETW, compared with those presenting ETW. 7n our previous study, 7 AEP was collected from the buccal surface of the upper and lower teeth.However, AEP composition varies depending on its location in the dental arches. 8Notably, the severity of the reflux above the upper esophageal sphincter is correlated with the severity of tooth erosion, especially on the palatal/lingual surfaces, 9 which are directly exposed to gastric acids.
Therefore, the aim of this in vivo study was to compare the proteomic profile of the AEP of GERD patients with and without ETW, in order to investigate what proteins in the palatal/lingual AEP would resist removal by intrinsic acids, for the purpose of their use in future AEP engineering procedures.

Methodology Ethical aspects and subjects
T h is st udy was approved by t he Et h ics Committees of the Bauru and Ribeirão Preto School of Dentistry, (#CAAE 44.007.415.1.0000.5417and 44.007.415.1.3001.5419,respectively).Twenty-four volunteers signed a consent form to take part in the study (n = 8 per group).These patients were the same as those who participated in our previous study, in which AEP was collected from the buccal surface of the teeth. 7They were of both genders, and between 20 and 60 years of age.The criteria for inclusion and exclusion, as well as the clinical examination, have been cited elsewhere. 7The patients were divided into 3 groups, 10 as follows:

In vivo experiment
The AEP collection procedures were conducted exactly as described in our previous study. 7The patients were submitted to prophylaxis, and the AEP was collected from the lingual/palatal surfaces of the upper and lower teeth after 120 min, using a 5 X 10 mm electrode filter paper (Bio-Rad, Hercules, USA) pre-dipped in 3% citric acid. 11One filter paper was used for each quadrant.The batches of paper were stored at −80 °C until the analysis was performed.

Proteomic analysis
Proteomic analysis of the AEP was conducted as previously reported. 7The protein was extracted by cutting strips of paper collected from all the patients in the same group into small pieces, and then grouping these pieces to form a pool in a single microtube.The processes for AEP sample preparation and shotgun proteomic analysis were performed as previously described. 8The equipment used was a nanoACQUITY UPLC-Xevo QT-MS system (Waters, Manchester, UK).ProteinLynx Global Server (PLGS) version 3.0 (Waters, Manchester, UK) was used to process and search the continuum LC-MSE data.The proteins were identified using the ion accounting algorithm embedded in the software and the Homo Sapiens database (only reviewed, UniProtKB/Swiss-Prot) downloaded in February 2020 from UniProtKB (http://www.uniprot.org/).The label-free quantitative proteomic analysis was performed by analyzing three MS raw files from each pool using PLGS software.All the identified proteins with a confidence score > 95% were included in the quantitative analysis.Identical peptides from each triplicate by sample were grouped based on mass accuracy (< 10 ppm), and on a retention time tolerance of <0.25 min, using the clustering software embedded in the PLGS.Search results were filtered for a false discovery rate (FDR) of 1%.The difference in expression between the groups was analyzed by the t-test (p < 0.05), using PLGS software.The comparisons made were GE vs. C, GNE vs. C, and GNE vs. GE.

Results
The characterization of the patients as to age, gender, BEWE score, and % time esophageal pH < 4 was reported elsewhere (Martini et al., 2019), and the proteins were identified (Table 1).
In all, 213 proteins were identified (Table 2), or 106, 119, and 92 from groups C, GE, and GNE, respectively.Figure 1 shows the number of proteins common to the groups, as well as the number of proteins found in just one of the groups.Twentythree proteins were found in all groups, including 14-3-3 protein zeta/delta, 1-phosphatidylinositol 4_5-bisphosphate phosphodiesterase beta-4, actin isoforms, alpha-internexin, ankyrin-3, annexin A1, apolipoprotein A-II, Ig lambda isoforms, myeloblastin, and myosin light polypeptide 6.Some proteins typically described as existing in the AEP were also common to all groups, such as protein S100-A8, serotransferrin, and serum albumin.The number of proteins found exclusively in groups C, GE and GNE groups was 51, 41 and 40, respectively (Figure , Table 2).Regarding the proteins found exclusively in one or two of the groups, some factors should be highlighted: a) histone H3 isoforms were found only in group C, while histone H2B isoforms were found only in the reflux groups (GE and GNE); b) group GNE had a high number of phosphorylated and calciumbinding proteins; c) isoforms of the spectrin betachain were found exclusively in GNE.
As for the quantitative analyses (Table 3), a comparison of GE vs. C showed that the number of GE proteins increased significantly by 4, and that of C decreased significantly by 14.The proteins that increased included the zinc finger and BTB domain-containing protein 21, negative elongation factor E, serotransferrin, and protein S100-A8.On the other hand, the proteins that decreased in the GE vs. C group included myeloblastin, RNA-binding protein 25, 1-phosphatidylinositol 4_5-bisphosphate phosphodiesterase beta-4, basic salivary proline-rich protein 1, breast cancer type 1 susceptibility protein, centrosomal protein of 170 kDa, neurofilament medium polypeptide, neutrophil defensin 1, alphainternexin, and actin isoforms.As for GNE vs. C, actin_cytoplasmic 1 increased, while protein S100-A8, alpha-internexin, and annexin A1 decreased.The most important comparison (GNE vs. GE) showed that two isoforms of actin as well as myeloblastin were higher, while protein PRR14L, annexin A1, and protein S100-A8 decreased.

Discussion
In the previous study by our team, we compared the proteomic profile of the AEP of GERD patients with ETW vs. without ETW for the first time, in an effort to find what proteins in the AEP would help protect against ETW.However, the AEP was collected from the vestibular surface of the teeth. 7][12] That is why we decided to collected AEP from the palatal and lingual surfaces of the patients in the present study.Notably, the site of AEP collection is an important factor that should be taken into account in proteomic studies of this integument, because deep changes in the proteomic profile of AEPs occur according to its location in the dental arches. 8The profile of the proteins found herein was very different from that observed in our previous

P68133
Actin_ alpha skeletal muscle (b, d, m, n, q, u, w) Yes Yes Yes P62736 Actin_ aortic smooth muscle (b, d, m, n, q, u) Yes Yes Yes

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Voltage-dependent calcium channel subunit alpha-2/delta-1 (b, f, m, t, w) -Yes -Continue study, in which the AEP was collected from the vestibular surfaces. 7ne interesting finding of the present study was the number of different types of histones.These proteins are very rich in lysine and arginine residues.This makes it easier to identify these proteins in proteomic studies, since tryptic peptides are generated upon the polypeptide chain cleavage of these residues.However, one finding was quite notable: H3 histones were found solely in the C group, while histone H2B isoforms were found only in the reflux groups (GE and GNE).The reason is not clear, but we should consider that H2B histones have a serine residue that can be phosphorylated in the N-terminus region.On the other hand, histone H3 has 2 serine residues that can be phosphorylated at the N-terminus.However, this region also contains at least 4 lysine residues that can be methylated. 13,14This can make access of the phosphate ion to the tooth surface difficult, thus reducing the binding of this type of histone to the calcium in the hydroxyapatite.The increase in after-radiotherapy histones is related to the DNA methylation process, which acts concomitantly with histone acetyltransferases.Acetylation is a type of histone alteration that plays an important role in modulating gene expression and cell cycles, and in neoplasm diffusion. 16The acetylation process starts with the action of acetyltransferases, prompted by the binding of acetyl radicals and lysine residues of histone proteins, and results in the decompression of chromatin and transcriptional activity.Histones H2A and H2B are examples of proteins whose function is not only to act in DNA replication and repair, but also to constitute octamers with histones H3 and H4.They are also involved in the packaging of DNA in nucleosomes. 17Interestingly, a study by Ventura et al. 18 showed that histone isoforms can be considered a strong prognostic biomarker in patients with head and neck cancer.This shows how an analysis of the AEP can benefit the patient by providing previous diagnosis and adequate treatment.AEP proteins have long been known for their protective effect in the homeostasis of the oral cavity. 11It is interesting to highlight that the focus on these proteins a few years ago was placed on those that were secreted in saliva. 11However, other important sources of AEP proteins are exfoliated oral mucosa cells, and gingival cells that deliver their content to saliva.In this context, many proteins with an affinity for hydroxyapatite might be immobilized in the AEP, and play an important protective role against acid challenges.This is the case of histones, as well as other proteins recently identified in the AEP as acid-resistant, such as hemoglobin (HB). 7,19- 22Nevertheless, HB was evaluated in the present study because it was found to be higher in the AEP 7 and saliva 22 of GERD patients without ETW, compared with GERD patients with ETW.Moreover, bear in mind that HB has a strong affinity for hydroxyapatite, and that hydroxyapatite columns are used to purify this protein. 23Interestingly, the adsorption rate of HB to hydroxyapatite increases as pH decreases. 24GERD patients have an oral pH typically lower than that of healthy people. 10This might increase the chance of HB adsorption onto dental surfaces.

This process is based on the epigenetic mechanisms associated with cellular memory and identity, which influence the cell environment and control
Furthermore, a high number of phosphorylated and calcium-binding proteins were found among the proteins identified exclusively in the GNE group.Nearly 50% of a total of 40 distinct proteins of the GNE group (the vast majority being intracellular proteins) are phosphorylated or Ca-binding proteins, thus suggesting that they might interact intensively with hydroxyapatite.This finding was also observed in our previous publication 7 , and might also be implicated in acid resistant mechanisms.
Another important finding was that several isoforms of the spectrin beta-chain were found only in the AEP collected from the GNE group.These proteins interact with actin.Curiously, there were two actin isoforms that increased more than twofold in the GNE group, in comparison with the GE group.The spectrin beta-chain can also form protein complexes.Because it binds to actin, it could be involved in the formation of supramolecular aggregates in the second stage of AEP formation. 25s mentioned above, several intracellular proteins delivered to saliva after exfoliation of oral mucosa cells have the potential to bind to hydroxyapatite, or participate in supramolecular aggregates that bind to the precursor proteins in the AEP.This has also been observed in other recent studies, [26][27][28][29][30][31] and could have a central role in protecting the tooth surface against acid dissolution.Hence, it is worthwhile noting that not only do secreted salivary proteins participate in AEP formation, but oral mucosa proteins, gingival crevicular fluid and even bacteria also act strongly on the enamel pellicle.This indicates that the AEP protein could play a protective role against dental erosion caused by intrinsic acids.This topic should be investigated further in future studies.

a.
Patients with GERD-related symptoms and ETW (GE; n = 8): The inclusion criteria for ETW were BEWE (basic erosive wear examination) ≥ 9, or grade 3 in the upper anterior sextant (with all incisors affected); b.Patients with GERD-related symptoms without ETW (GNE; n = 8): Patients without ETW were included in this group (BEWE = 0); c.Control group (C; n = 8): Patients in this group did not have GERD-related symptoms or ETW (BEWE = 0).

Figure .
Figure.Venn diagram showing the number of proteins identified in the control (no GERD, no dental erosion), GE (GERD and dental erosion), and GNE (GERD but no dental erosion) groups.

Table 1 .
Characterization of the volunteers according to age, gender and BEWE score.

Table 2 .
Classification of proteins identified in the acquired enamel pellicle collected from volunteers with gastro-esophageal reflux disease (GERD) and erosive tooth wear (GE), GERD but no erosive tooth wear (GNE), or controls (no GERD, no erosive tooth wear; C).

Table 3 .
Proteins expressed differentially in the acquired enamel pellicle collected from patients with gastroesophageal reflux disease (GERD) and erosive tooth wear (GE), GERD but no erosive tooth wear (GNE), or controls (no GERD, no erosive tooth wear; C).
Identification is based on ID proteins from Uniprot protein database, only reviewed (http://www.uniprot.org/);b Proteins with significantly altered expression are organized according to the ratio; c Indicates proteins found exclusively in the acquired enamel pellicle (AEP) of C patients (in alphabetical order); d Indicates proteins found exclusively in the acquired enamel pellicle (AEP) of GE patients (in alphabetical order); e Indicates proteins found exclusively in the acquired enamel pellicle (AEP) of GNE patients (in alphabetical order).Ratios highlighted in bold indicate proteins with higher increase or decrease in GNE compared with GE patients. a