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Salivary proteomic profile of dogs with and without dental calculus

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Background: Dogs' saliva is a complex mixture of inorganic and organic constituents, rich in proteins. Therefore, knowing the saliva composition of these animals is extremely important to identify the presence of proteins that may be involved in physiological and pathological mechanisms of their oral cavity. The present study aimed to characterize the proteomic profile of saliva from dogs with and without dental calculus. Results: Saliva samples were collected from 20 dogs. Before the collection, a visual clinical examination was performed and 8 subjects (40%) did not present any signs of dental calculus, while 12 (60%) presented dental calculus. After saliva collection, the samples were submitted to protein quantification (mBCA), and then they were prepared for analysis by nLC-ESI-MS/MS. A total of 658 unique proteins were identified, of which 225 were specific to dogs without dental calculus, 300 were specific to dogs with dental calculus, and 133 were common to all subjects. These proteins presented functions including transportation, immune response, structural, enzymatic regulation, signal transduction, transcription, metabolism, and some proteins perform functions as yet unknown. Several salivary proteins in dogs with dental calculus differed from those found in the group without dental calculus. Among the abundant proteins detected in periodontal affected cases, can be highlighting calcium-sensing receptor and transforming growth factor beta. Enrichment analysis reveled the presence of Rho GTPases signaling pathway. Conclusions: This research identified salivary proteins, that should be further investigated as potencial biomarkers of chronic periodontits with dental calculus formation in dogs.
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R E S E A R C H A R T I C L E Open Access
Salivary proteomic profile of dogs with and
without dental calculus
Mayara Bringel
1,2
, Paula Karine Jorge
1,2
, Priscila Amanda Francisco
1
, Cadance Lowe
3
, Robinson Sabino-Silva
1,4
,
Bella Luna Colombini-Ishikiriama
2
, Maria Aparecida de Andrade Moreira Machado
2
and Walter Luiz Siqueira
1*
Abstract
Background: Dogssaliva is a complex mixture of inorganic and organic constituents, rich in proteins. Therefore,
knowing the saliva composition of these animals is extremely important to identify the presence of proteins that
may be involved in physiological and pathological mechanisms of their oral cavity. The present study aimed to
characterize the proteomic profile of saliva from dogs with and without dental calculus.
Results: Saliva samples were collected from 20 dogs. Before the collection, a visual clinical examination was performed
and 8 subjects (40%) did not present any signs of dental calculus, while 12 (60%) presented dental calculus. After saliva
collection, the samples were submitted to protein quantification (mBCA), and then they were prepared for analysis by
nLC-ESI-MS/MS. A total of 658 unique proteins were identified, of which 225 were specific to dogs without dental
calculus, 300 were specific to dogs with dental calculus, and 133 were common to all subjects. These proteins
presented functions including transportation, immune response, structural, enzymatic regulation, signal transduction,
transcription, metabolism, and some proteins perform functions as yet unknown. Several salivary proteins in dogs with
dental calculus differed from those found in the group without dental calculus. Among the abundant proteins
detected in periodontal affected cases, can be highlighting calcium-sensing receptor and transforming growth factor
beta. Enrichment analysis reveled the presence of Rho GTPases signaling pathway.
Conclusions: This research identified salivary proteins, that should be further investigated as potencial biomarkers of
chronic periodontits with dental calculus formation in dogs.
Keywords: Mass spectrometry, Saliva, Dogs, Proteome, Dental Calculus
Background
Saliva has a complex mixture of organic and inorganic con-
stituents. The organic constituents are predominantly saliv-
ary proteins [1]. Its proteome is represented especially by
glycoprotein, enzymes, immunoglobulins, and several pep-
tides [2].Moreover,thewholesalivaiscomposedofgingival
crevicular fluid (containing plasma proteins), food debris
and substances produced by oral microorganisms [3,4].
In mammals, the main saliva functions are lubrication
and protection of the buccal tissue, buffer capacity,
maintenance of teeth integrity, and antimicrobial effect
[3]. In some species, some saliva components represent an
essential part on the enzymatic digestion assisting masti-
cation and deglutition [5]. Besides these beneficiais func-
tions, dogssaliva has an alkaline pH that varies between
7.2 to 8.5 [6,7], a range that facilitates dental calculus for-
mation, through calcification of dental biofilm present on
teeth cervical region [810]. Dental calculus formation is
always preceded by the development of a saliva protein
layer on the tooth enamels surface, called acquired en-
amel pellicle (AEP), which serves both to protect dental
enamel, reduce friction between teeth and oral mucosa
[11], and as basis to formation and maturation of dental
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data made available in this article, unless otherwise stated in a credit line to the data.
* Correspondence: walter.siqueira@usask.ca
1
College of Dentistry, University of Saskatchewan, Saskatoon, SK, Canada
Full list of author information is available at the end of the article
Bringel et al. BMC Veterinary Research (2020) 16:298
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biofilm, which constitutes the organic matrix to its pos-
sible calcification [12].
In dogs, the formation of dental calculus is typical since
the first year of life and it appears as granular, yellow
brown masses on the buccal surfaces of molar teeth of the
upper jaw near salivary duct orifices [7,13]. Dental calcu-
lus consists in a mixture of calcium carbonate and calcium
phosphate, presenting a rough surface that increases the
occurrence of periodontal disease [8], due to the calcifica-
tion of more dental biofilm, bringing it closer to the soft
tissues [14]. Similar to humans, there is a link in dogs
between the calculus presence with the increasing rate of
periodontal diseases, which in some cases may lead to the
animal starvation as a result of feeding difficulties [13,15,
16]. Furthermore, in dogs, dental calculus is considered
one of the main conditions involved in the development of
periodontal disease resulting in teeth loss. Studies report
prevalence of periodontal disease varying from 80 to 85%
[10,1719].
Other than constituents involved in the formation of
dental calculus, saliva possesses chemical and physical
properties that may serve to detect evidence, through
proteomic analysis, of systemic diseases [2]. Mass spec-
trometry is an indicated method for studies focused on
protein identification, characterization and quantification
[20]. This analysis is available both for humans [21,22]
and animals [2325]. Some proteins are involved in the
formation and in the inhibition of minerals precipitation
over human dental surface, such as cystatins, statherin
and acidic proline-rich proteins (PRPs), which have been
already identified in saliva protein composition of some
mammal species like monkeys, rats, mices and pigs [23].
However, literature is still limited concerning identifica-
tion of these proteins in dogssaliva. Studies with a
Table 1 Distribution of the participating dogs by breed, gender, age, weight, diet and volume of saliva collected
Shih
tzu
Lhasa
apso
Gender Age
(years)
Weight
(Kg)
Diet
a
Volume of saliva
(μl)
Degree of dental
calculus
Total Protein concentration (ug/
ml)
Dog 1 X Female 6 4 Mixed 450 0 2453.49
Dog 2 X Female 9 8 Dry 400 3 742.24
Dog 3 X Male 5 6.5 Dry 500 2 520.76
Dog 4 X Male 3 6 Dry 500 2 441.77
Dog 5 X Female 0.42 4 Dry 500 0 616.30
Dog 6 X Female 2 5.5 Dry 400 1 546.82
Dog 7 X Female 6 8 Dry 550 0 485.16
Dog 8 X Female 4 8 Dry 550 1 968.05
Dog 9 X Female 1 3 Dry 400 0 1151.45
Dog
10
X Female 4 11 Dry 800 0 1212.73
Dog
11
X Male 0.25 4 Dry 400 0 3171.34
Dog
12
X Male 9 10 Dry 550 3 1972.65
Dog
13
X Male 2 3 Dry 500 1 2168.71
Dog
14
X Female 3 4.5 Dry 500 1 1155.82
Dog
15
X Female 7 4 Dry 100 1 1995.13
Dog
16
X Male 4 6.7 Mixed 400 3 1820.04
Dog
17
X Male 0.66 7 Dry 1000 0 1633.00
Dog
18
X Female 7 8 Mixed 600 1 2302.20
Dog
19
X Female 0.25 2.4 Dry 700 0 1020.11
Dog
20
X Female 7 5 Food 500 3 2360.05
N/A (Not available)
a
A mixed diet refers to dry dog food and human food
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proteomic approach may be used to develop biological
strategies for controlling both bacterial dental biofilm and
dental calculus formation. Thus, the purpose of this study
was to characterize the salivary proteomic profile of dogs
with and without dental calculus.
Results
Characterization of the study participants
Of the 20 dogs that participated in the study, the visual
clinical examination showed that 8 (40%) had no signs
of dental calculus and 12 (60%) presented with some de-
gree of dental calculus (Table 1). The distribution of
these dogs according to breed, gender, age, weight, diet
and volume of saliva collected is also shown in Table 1.
Total protein concentration
The concentrations of total proteins present by the Micro
Bicinchoninic Acid (Micro BCA) assay in each dog studied
are shown in Table 1. Students t-test was used to com-
pare total protein concentrations between the groups with
and without dental calculus and between genders. There
were no statistically significant differences (p=0.892) be-
tween groups and gender (p= 0.3822).
Correlations between variables
Pearsons correlation test (p< 0.05) was used to investi-
gate the associations between animal age, saliva volume
and total protein in the saliva. As expected, there was no
statistically significant correlation between the variables.
Proteome identification and quantification
The proteome from all different samples showed a con-
sistent elution of protein/peptides range from 20 to 45
min. Representative base-peak chromatograms of saliva
from dogs with and without dental calculus are repre-
sented in Fig. 1.
In the present study, 1662 proteins were identified, of
which 658 (39.6%) were unique proteins. Of these 658
unique proteins found, 623 (9.7%) were characterized
and 35 (15.3%) were uncharacterized proteins, therefore
identified for the first time.
Regarding the oral clinical condition of each animal,
133 (20.2%) specific proteins were common to all par-
ticipating dogs (Supplementary Table S1), 225 (34.2%)
specific proteins were identified in dogs without clinical
signs of dental calculus (Supplementary Table S2), 300
(45.6%) specific proteins in dogs with dental calculus
(Supplementary Table S3). This distribution is described
in Venn diagram (Fig. 2). Of these 225 specific proteins
of dogs without dental calculus, 220 (97.8%) were char-
acterized and 5 (2.2%) were uncharacterized proteins.
From the 300 specific proteins of dogs with dental calcu-
lus, 287 (95.7%) present characterization and 13 (4.3%)
were uncharacterized proteins.
Biological functions of proteins
Among the 658 unique proteins identified, the most
abundant based on spectral count and ion abundance
[26] had their biological functions analyzed by UniProt
software (www.uniprot.org). These proteins exhibited
functions of substance transport, immune response, en-
zymatic regulation and metabolism (Table 2and Supple-
mentary Table S4with peptide sequences).
Regarding the 225 specific proteins identified in dogs
without dental calculus, the most abundant were: Can f 4
variant allergen, Keratin, type II cytoskeletal 1 and others.
Fig. 1 Examples of base-peak chromatograms from samples of dogs without dental calculus (upper graph) and dogs with dental calculus (lower
graph) performed by nano-flow RP-HPLC column, and elution gradient ranging from 0 to 80%
Bringel et al. BMC Veterinary Research (2020) 16:298 Page 3 of 12
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These proteins also exhibited functions of transport, im-
mune response, transcription, structural, enzymatic regu-
lation, metabolism and signal transduction (Table 3and
Supplementary Table S4with peptide sequences).
And finally, from the 300 specific proteins identified in
dogs with dental calculus, the most abundant analyzed
were: Calcium-sensing receptor, Voltage-dependent T-type
calcium channel subunit alpha, Multidrug and toxin extru-
sion protein (Fragment), Phosphatase and actin regulator,
Poly [ADP-ribose] polymerase, Zinc alpha-2-glycoprotein 1
(Fragment), Transforming growth factor beta (TGFB1) and
others. These proteins exhibited functions of transport, im-
mune response, enzymatic regulation, signal transduction,
transcription and metabolism (Table 4and Supplementary
Table S4with peptide sequences).
Fig. 3shows and compares the functions of the most
abundant proteins for dogs with and without dental calcu-
lus. The group with dental calculus presented with more
proteins related to immune response, enzymatic regula-
tion and uncharacterized proteins, as compared to the
group without dental calculus.
Analysis by the STRING database
Two protein-protein networks were created with the 225
unique proteins identified in the group of dogs without
dental calculus, and 300 unique proteins in the group of
dogs with dental calculus. Canis lupus was selected as
the studied organism and the highest score of confidence
(0.900) was set. Supplementary Figures 1and 2shows
the constituents of both networks, dogs without dental
calculus and dogs with dental calculus, respectively. Al-
though it is possible to see an increase of connections
from the group without dental calculus (42 edges) to the
group with dental calculus (90 edges), only Reactome
pathways such as cell cycle, mitosis, signaling by Rho
GTPases, M phase and mitotic prometaphase were
found in the second group (Supplementary Table S5).
No pathways (from Reactome Pathway Datasabe or
Kyoto Encyclopedia of Genes and Genomes) were en-
countered in the first group.
Discussion
The current study characterized the salivary proteome
profile of dogs with and without dental calculus. In total,
we identified 1662 proteins through the SEQUEST filter
criteria applied to MS/MS spectra. Among these salivary
proteins, there were 658 (39.6%) described for the first
time in saliva of dogs. Besides, it is important enphazises
that 225 specific proteins were identified in dogs without
clinical signs of dental calculus and 300 specific proteins
Fig. 2 Venn diagram showing salivary proteins found in animals
with and without dental calculus and their interrelationship
Table 2 Functions of the most abundant proteins identified in all participants dogs
Function Accession number Protein name (Gene name)
Transport F2Z4Q6 Serum albumin (AFP ALB)
P49822 Albumin (allergen Can f 3) (ALB)
P60524 Hemoglobin subunit beta (HBB)
J9P430 Transferrin (TF)
P02648 Apolipoprotein A-I (APOA1)
P60529 Hemoglobin subunit alpha (HBA)
Immune response J9P732 EF-hand domain-containing protein (S100A9)
E2RCC8 Uncharacterized protein
C0LQL0 Protein S100 (S100A8 or S100A6)
F1PCH3 Enolase 1 (ENO1)
P19006 Haptoglobin (HP)
F1PR54 Uncharacterized protein
Enzymatic regulation E2R0H6 Prolactin induced protein (PIP)
F1PGM1 Complement C3 (C3)
F6USN4 Uncharacterized protein
Metabolism F1PE28 Transketolase (TKT)
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Table 3 Functions of the most abundant specific proteins identified in dogs without dental calculus
Functions Accession number Protein name (Gene name)
Transport J9P950 Can f 4 variant allergen (OBP)
E2QX44 Solute carrier family 29 member 4 (SLC29A4)
E2RSV0 Importin 4 (IPO4)
Immune response E2RF74 Sphingosine-1-phosphate receptor 3 (S1PR3)
F1PIL9 Interleukin 1 receptor accessory protein (IL1RAP)
Transcription F1PII4 Uncharacterized protein
E2RR25 BTB domain containing 8 (BTBD8)
Structural F1PKA4 MTSS I-BAR domain containing 1 (MTSS1)
F1PTY1 Keratin 3 (KRT3)
Enzymatic regulation E2RKG0 Dermokine (DMKN)
Metabolism E2QW50 Zinc finger protein 532 (ZNF532)
E2RTI2 Chromodomain helicase DNA binding protein 3 (CHD3)
F6Y4F1 Dachsous cadherin-related 1 (DCHS1)
F1PBU5 SMG1 nonsense mediated mRNA decay associated PI3K related kinase (SMG1)
F1Q0K5 Triokinase and FMN cyclase (TKFC)
Signal transduction F1PSR2 Dedicator of cytokinesis 5 (DOCK5)
Table 4 Functions of the most abundant specific proteins identified in dogs with dental calculus
Functions Accession number Protein name (Gene name)
Transport A2SXS6 Calcium-sensing receptor (CASR)
E2R9S8 Voltage-dependent T-type calcium channel subunit alpha (CACNA1I)
E2RAB8 Multidrug and toxin extrusion protein (SLC47A2)
Immune response F1PI70 Transforming growth factor beta (TGFB1)
E2R141 Complement C8 beta chain (C8B)
F1PSJ1 Uncharacterized protein
Enzymatic regulation E2RGH9 HECT domain E3 ubiquitin protein ligase 1(HECTD1)
F1PVE1 PH domain and leucine rich repeat protein phosphatase 1 (PHLPP1)
F1P6R1 Phosphatase and actin regulator (PHACTR4)
Metabolism E2R0T6 Heat shock protein family A (Hsp70) ember 8 (HSPA8)
E2RF62 Unc-51 like autophagy activating kinase 2 (ULK2)
E2RD14 Pleckstrin homology domain containing A5
(PLEKHA5)
J9P7C9 Poly [ADP-ribose] polymerase (PARP14)
Signal transduction E2RSB9 Misato mitochondrial distribution and morphology regulator 1 (MSTO1)
Transcription F1PQQ7 Nuclear receptor corepressor 2 (NCOR2)
Uncharacterized F1PPE5 Biorientation of chromosomes in cell division 1 like 1 (BOD1L1)
Q4GX49 Zinc alpha-2-glycoprotein 1(AZGP1)
F1PI98 NCK associated protein 5 (NCKAP5)
F1P9Y0 NHS like 1 (NHSL1)
J9P0U6 NHS actin remodeling regulator (NHS)
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of dogs with dental calculus, which demonstrated that
saliva could be a valuable medium to get biomarkers of
the dental calculus formation on dogs.
The whole saliva collection was performed by mechan-
ical stimulation with aid of a device formulated for such
purpose. The device used was MicroSALsaliva collec-
tion device (Oasis Diagnostics® Corporation - Vancouver,
WA, USA). The saliva collection device has no cellulose
in its composition [27] and allows immediate visual con-
firmation of the volume of saliva obtained; the plunger
compresses the absorbent pad and the saliva is collected
in the Eppendorf tube portion of the device. Foremost,
differently from previous studies where the collection of
saliva in dogs was performed under anesthesia with
stimulation with acid [28,29], we have opted for a non-
invasive collection without any other stimulation mech-
anism or general anesthesia. However, there were diffi-
culties regarding the sample collection. The sample
volumes obrained varied significantly due to the dogs
defense movements, such as head shaking or trying to
move away from the owners containment position.
These reports were also described by dogs owners in
the study of Wenger-Riggenbach et al. [30]. Moreover,
some dogs refused to open their mouths and became ag-
gressive, consequently resulting in them being excluded
from the study.
The visual clinical examination of the 20 analyzed
dogs identified that 8 (40%) did not present any signs of
dental calculus, and 12 (60%) presented some degree of
dental calculus. The mean dental calculus scores of dogs
with dental calculus was 1.8, indicating a moderate cal-
culus scoring in these dogs. Since our main goal is to
study dental calculus, we selected the breeds Shih Tzu
and Lhasa Apso that possesses brachycephalic skulls and
tend to show malocclusion, dental crowding and rota-
tion [31,32], which facilitates accumulation of dental
calculus. It is known that dental calculus is a predispos-
ing factor to periodontal disease, which is more frequent
in middle-aged (above 7 years old) and small size animals
(below 10 kg) [33]. In addition, the breeds studied here are
similar in appearance and the genetic investigation pre-
sented a close relationship between them [34]. As our
study only sampled these genetically similar breeds, this
may have influenced the number of proteins identified.
Sousa-Pereira et al. [25] identified 249 proteins on a mixed
breed group and Torres et al. [35] identified 2.491 proteins
in healthy dogs among 19 breeds. Notwithstanding, there
is no description of oral health on the animals evaluated
in the study of Sousa-Pereira et al. [25] nor the proteins
functions. In this present study were identified 1.662 pro-
teins, of which 658 (39.6%) were unique. The biological
functions of the most abundant proteins of each group
Fig. 3 Comparison of functional characteristic of the most abundant proteins found in dogs with and without dental calculus
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identified were: transportation, immune response, struc-
tural, enzymatic regulation and metabolism, and some
proteins perform still unknown functions. Besides that, in
our study, there was no difference in total protein concen-
tration between males and females. The same result was
found in the study by Lucena et al. [29], in which there
was no major differences between genders.
In the last years, several studies have been carried out
on proteomic analysis in different mammals. Sousa-
Pereira et al. [25], analyzed the salivary proteome of dis-
tinct species, including dogs, and identified, the alpha
amylase (among other) was not found in the present
study. There are, differences between that study and the
present one that make a direct comparison of results dif-
ficult. In Sousa Pereira study [23], the samples were col-
lected in mixed breed while in our study two specific
breeds were selected. Sanguansermsri et al. compared
the salivary proteome between dogs and humans, and
observed that alpha amylase was present in dogs saliva,
but in lower levels than in humans saliva; however, the
presence of this protein in dogs in that study may be
due the fact that in Thailand these animals are often fed
with rice and starch [35]. Most likely due to a diet adap-
tion, the dogs used in this study do not have this pro-
tein. This lack of amylase in dogs saliva was also
observed by other studies [4,36,37].
To the best of our knowledge, there are no proteomic
studies regarding dental calculusinthesalivaofdogs,sothis
is a pioneering study. Among the unique proteins identified,
the existence of serum albumin was noted on every dogs
sample due to, as described in humans, a small volume of
gingival crevicular fluid that enters the oral cavity even in
healthy subjects, free of gingivitis, resulting the measurement
of levels of this component of serum in saliva [38]. Other
components of serum have also been identified, among
these elements, are: Hemoglobin subunit beta, Apolipopro-
tein A-I, Hemoglobin subunit alpha and Haptoglobin.
Further, mucin and lysozyme C proteins were identified.
Mucins are glycoproteins of high molecular weight with
elongated structure that contribute significantly to salivas
viscoelastic behavior [39]. They also play an antibacterial
function of modulating selectively the coherence of micro-
organisms to the oral tissue surface, which assist with the
control of bacterial and fungal colonization [3]. Lyso-
zymes, on the other hand, are enzymes with hydrolytic ac-
tivities. In other words, they promote cell lysis in bacteria
[40], since its biochemically function hydrolyze beta-1,4
bond between N-acetyl glucosamine and N-acetyl mura-
mic acid residues of bacterial peptidoglycan, which is an
essential part of bacterial cell wall and also promotes
structural stiffness and neutralize osmotic pressure of the
cytoplasm [41]. Therefore, the lysozyme is considered a
natural antibiotic and an important part of the innate im-
mune system [4143]. In humans, C-type lysozyme is
found in all biofluids, including saliva. The C-type lyso-
zymes, as the ones identified, are the main lysozymes pro-
duced by vertebrates [44].
Concerning the analyses in the two different groups,
the most abundant proteins in the group of dogs without
dental calculus participated in metabolism functions,
whereas in the group with dental calculus there were
major proteins related to immune response, enzymatic
regulation and uncharacterized proteins, potentially this
may be due the presence of dental calculus near the gin-
giva, which has tendency of more pronounced inflamma-
tory response [45].
Among the specific proteins of dogs without dental
calculus, the presence of Sphingosine-1-Phosphate Re-
ceptor Protein 1 (S1P1) was observed. This protein is
highly expressed in humans in endothelial cells, brain,
heart and immune system cells [46,47]; it is coupled to
G protein and binds to sphingosine-1-phosphate, which
is a bioactive sphingolipid that behaves as an intracellu-
lar messenger of some cytokines and also as an auto-
crine and paracrine extracellular mediator [48]. In
addition, sphingosine-1-phosphate stimulates events of
intracellular signaling, such as activation of phospholip-
ase C, increased cytoplasmic calcium concentration,
regulation of adenylate cyclase, activation of the MAP
kinase pathway and the Rho cascade [49]. When bound,
they participate in several cellular processes such as pro-
liferation, differentiation, adhesion, motility, angiogen-
esis, apoptosis, migration, morphogenesis and changes in
the cytoskeleton [50]. S1P1 proteins were also described
in memory T cells and a cell immunophenotyping re-
vealed that humans secrete CD4(+) T cells in saliva [51].
Besides, the Voltage-dependent T-type calcium channel
subunit alpha protein encoded by CACNA1G gene was
equally identified as a specific protein of dogs without
dental calculus. Interestingly, a similar protein encoded
by CACNA1I gene was only identified in the saliva of
dogs with dental calculus. It is deemed that proteins T-
type calcium channel play important roles in neuronal
activity and have been described in studies with rats [52]
and humans [53]; but there are no reports in the litera-
ture relating these proteins to the dogssaliva.
According to Zhang and collaborators [54]upto40
proteins can be named as protein biomarkers for peri-
odontal diseases in humans, these biological mediators
are released from host defense cells due to the presence
of periodontopathic bacteria in the oral enviromment.
They include numerous cytokines, such as prostaglandin
E2; tumor necrosis factor (TNF); interleukins IL-1 and
IL-6, proteinases as matrixmetalloproteinases (MMPs);
elastase-like enzymes; trypsin-like proteases; aminopepti-
dases and dipeptidylpeptidases, epidermal; platelet-
derived and vascular growth factors, pyridino-line cross-
linked carboxyterminal telopeptide, osteocalcin, among
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others. Still, there is a lack of proteomic researches re-
garding dogs with chronic periodontitis, which compro-
mises comparisons within this species.
Howsoever, amidst the specific proteins identified in
dogs with dental calculus in this study, some were found
to function as immune response to the current peridodon-
titis. Between then it can be pointed out the presence of
fragments of the Polymeric immunoglobulin receptor pro-
tein, which has been described in human saliva having the
function of transporter of IgA, the main salivary antibody
[39]. Transforming growth factor beta (TGFB1) and com-
plement C8 beta chain (C8B), were equally present in high
abundance. TGFB1 was previously correlated to chronic
periodontitis [55], its importance lies in the fact that this
growth factor stimulate tissue remodeling and wound
healing through increasing fibroblast proliferation, angio-
genesis, and extracellular matrix production [56], and by
inhibiting MMPs [57]. Paradoxically, TGFB1 was also
found to promote inflammation-associated tissue degrad-
ation by enhanced production of mediators that raise re-
cruitment of eosinophils, lympocytes and monocytes, cells
known to participate in chronic inflammation and tissue
destruction [58]. These facts could explain why TGFB1 is
upregulated in cases of chronic periodontal pathologie.
Furthermore, another abundant protein detected by
this research was the Calcium-sensing receptor (CaSR),
whose function is calcium (Ca
2+
) transportation and
regulation [54]. In humans, CaSR is a G protein-coupled
receptor that detects extracellular levels of Ca
2+
, which
is expressed on plasma membranes of a broad variety of
epithelial tissues including parathyroid, kidney, gastro-
intestinal tract and salivary glands [59,60]. In salivary
glands, it was showed that functional CaSR proteins can
be stimulated by Ca
2+
concentration, that is, CaSR can
serve as a Ca
2+
sensor in the luminal membrane of saliv-
ary gland ducts and regulate reabsorption of Ca
2+
from
the saliva via transient receptor potential canonical 3
(TRPC3), thus contributing to maintenance of salivary
Ca
2+
levels and representing a possible important pro-
tective mechanism against formation of salivary gland
stones [60]. Hence, as the CaSR stimulation in salivary
glands can be related to an imbalance in the salivary
Ca
2+
concentration, it could as well contribute to the
dental calculus accumulation that consist essentially of
calcium phosphate. It is also important to note that in
the kidney, the formation of stones is associated with
changes in calcium reabsorption, causing hypercalcemia
[59]. Remarkably in the current investigation, CaSR was
identified only in the saliva of dogs with dental calculus,
which could suggest, together with its high abundance,
an association with dental calculi presence. Further re-
search is needed to better understand the relevance of
CaSR protein in saliva and dental calculi formation, as it
was demonstrated mainly in other human tissues [59].
An additional layer of information on possible pathways
and processes involved in periodontal disease was granted
by enrichment analysis. Overall, four of the five biological
process that were encountered in dental calculus cases re-
lated to mitotic pathways that is the most common
eukaryotic cell cycle. However, one critical finding merit
being highlighted, which is the presence of the Rho
GTPases signaling pathway. Rho GTPases can modulate
the effects on human periodontal ligament cells of TNFB1,
an important cytokine already known by its role in peri-
odontal pathologies [61]. Rho is a notable coordinator of
the cytoskeleton [62], it was suggested in a previous study
that the small Rho GTPase and its downstream effector
Rho kinase (ROCK) regulate TGFB1-induced remodelling
of mammary epithelial cell-to-cell contact [63]. In accord-
ance to the stated, Wang et al. showed that TGFB1 can in-
duce proliferation and cytoskeletal rearrangement in
periodontal ligament cells via Rho GTPase-dependent
pathways [64]. The high abundance of TGFB1 in dogs
with dental calculus associated with the presence of Rho
GTPase pathway is a relevant finding that may suggest, for
futher investigations, TGFB1 as a biomarker candidate of
periodontal disease in this species.
Future studies are needed for the evaluation of param-
eters such as pH values and determination of the buffer
activity of each saliva sample collected. Quantification of
electrolytes such as calcium and phosphate, which par-
ticipate in the formation of the dental calculus and pro-
cesses of demineralization and remineralization of dental
enamel, should also be performed, not forgetting to
mention sodium, potassium, zinc and magnesium, that
are important in the metabolism of the salivary glands
[65,66]. The final task would be to combine the gen-
omic, proteomic and other omic profiles together in an
attempt to obtain a broader vision of how dental calcu-
lus accumulation impact dog salivary proteomic profile.
Conclusion
In this study, the proteome salivary profile of dogs
showed a large number of unique proteins, 225 belong-
ing to dogs without dental calculus, 300 exclusives to
dogs with dental calculus and 133 belonging to both
groups. Therefore, this research identified salivary pro-
teins, that should be further investigated as potencial
biomarkers of chronic periodontits with formation of
dental calculus in dogs. Besides, it could open opportun-
ities to study and potentially develop new substances,
that would aid in preventing or delaying the formation
of dental calculus in dogs.
Methods
Sample selection
This study was approved by the Ethics Committee in the
Teaching and Research on Animals of the Dental School
Bringel et al. BMC Veterinary Research (2020) 16:298 Page 8 of 12
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
of Bauru, University of São Paulo (number 002/2016);
moreover, all the dogsowners signed consent forms.
The study was composed by 20 dogs (Canis lupus
familiaris) ranging in age from three months to nine
years with and without dental calculus. General inclu-
sion criteria of the groups with and without dental cal-
culus included: dog breed of either Shih Tzu or Lhasa
Apso, of both sex, that were, dewormed, vaccinated,
antibiotic-free at least 6 months prior to data collection,
free from any other concurrent oral disorders, and for
females, out of the estrus period. Specific inclusion cri-
teria were in the control group (without dental calculus)
the dogs should not present any signs of dental calculus
and; in the experimental group (with dental calculus) the
dogs should necessarily present signs of dental calculus.
Dental evaluation
Previously to collection of saliva samples, a visual clinical
examination of the oral cavity was carried out in order
to evaluate the dental conditions, concerning the pres-
ence or absence of dental calculus.
A visual oral inspection was carried out by a veterin-
ary, where each participant of the study was categorized
based on the criteria adopted to evaluate the dental cal-
culus: 0 for absence of dental calculus; 1 for supragingi-
val calculus covering not more than one-third of the
exposed surface of the examined tooth; 2 for supragingi-
val calculus covering more than one third but not more
than two thirds of the exposed tooth surface and 3 for
supragingival calculus covering more than two third of
the exposed tooth surface [67,68].
Saliva collection and preparation
A non-invasive collection, without general anesthesia, and
after 2 h of the animal fasting was performed. Whole sal-
iva samples were collected under stimulation using
MicroSALsaliva collection device (Oasis Diagnostics®
Corporation - Vancouver, WA, USA) for approximately 5
to 10 min. The tip end of the white absorbent collection
pad of the device was inserted into the mouth of the dog
where saliva pools and collect until the pad was saturated.
Saliva was totally transferred from the absorbent part to
the collection tube using the compression tube.
The samples were centrifuged at 14,000 g for 20 min
at 4 °C and whole saliva supernatants (WSS) separated
from the pellet. This was followed by the lyophilization
of samples for further analyses. The total protein
concentration of WSS was measured by Micro BCA
Protein Assay Kit (Thermo Scientific Pierce, Rockford,
IL, USA) with bovine serum albumin used as the stand-
ard and it was stored at 80 °C until further analysis
[69,70].
Insolution digestion
The equivalent of 20 μg of each WSS sample was dried
by a rotary evaporator, denatured and reduced for 1 h at
room temperatute by the addition of 50 μl of solution 1
(4 M urea, 10 mM dithiothreitol), and 50 mM
NH4HCO3, pH 7.8. After, four-fold dilution with 50 mM
NH4HCO3, pH 7.8, tryptic was carried out for 16 h at
37 °C, after the addition of 4% (w/w) sequencing-grade
trypsin (Promega, USA). Finally, aliquots from each sam-
ple were dried again in a rotary evaporator, de-salted by
C18 Pipette Tips (Millipore, USA) and finally subjected
to mass spectrometry analyses [71].
Mass spectrometry analyses
Mass spectrometry analyses were carried in triplicates for
each sample with a nano-HPLC Proxeon (Thermo Scien-
tific, San Jose, CA, USA) which allows in-line liquid chro-
matography with the capillary column, 60 μm × 100 mm
(Pico TipEMITTER, New Objective, Woburn, MA) filled
with C
18
resin of 5 mm diameter and 200 pores sizes
(Michrom BioResources, Auburn, CA) linked to the mass
spectrometer (LTQ-Velos, Thermo Scientific, San Jose, CA,
USA) using an electrospray ionization in a survey scan in
therangeofm/zvalues3902000 tandem MS/MS.
The equivalent of 20 μg of each sample already dried by
rotary evaporator was re-suspended in 20 μgof0.1%for-
mic acid and then subjected to reversed-phase LC-ESI-
MS/MS. The nano-flow reversed-phase HPLC was devel-
oped with linear gradient of 85 min ranging from 0 to
100% of solvent B (97.5% acetonitrile, 0.1% formic acid) at
a flow rate of 200 nl/min with a maximum pressure of
280 bar. Electrospray voltage and the temperature of the
ion transfer capillary were 1.8 kV and 250 °C respectively.
Database searches
The acquired MS/MS spectra were compared to canus
lupus familiaris protein database (UniPROT and
TREMBL, Swiss Institute of Bioinformatics, Geneva,
Switzerland, http://ca.expasy.org) using SEQUEST and
Proteome Discoverer 1.3 software (Thermo, USA). In
order to infer protein with high confidence, the SEQUEST
filter criteria applied to MS/MS spectra were: 1.5; 2.5; 3.1;
3.1; 4.5 for the XCorr applied in addition to the Percolator
filter. Search results were filtered at a false discovery rate
of 1% using a reverse database search strategy [26,27].
As described previously [26], after identification of the
proteome profile, the most abundant proteins had their
biological functions verified through the accession num-
ber using the database www.uniprot.org. In addition, the
STRING database (http://string-db.org/) was searched
for protein-proteins networks in the group of dogs with-
out dental calculus and the group with dental calculus
separately.
Bringel et al. BMC Veterinary Research (2020) 16:298 Page 9 of 12
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Statistical analyses
The results obtained were analysed by SPSS for WIN-
DOWS, version 19.0 (SPSS Inc., Chicago, IL, USA), using
descriptive statistics. Additionally, Studentst-testwas
used to compare total protein concentrations between the
groups with and without dental calculus and between gen-
ders. And, Pearsons chi-square test was chosen to exam-
ine the null hypothesis that there is no relationship
between animal age, saliva volume and total protein in the
saliva. Significance levels were set at 5% (p<0.05).
Supplementary information
Supplementary information accompanies this paper at https://doi.org/10.
1186/s12917-020-02514-0.
Additional file 1: Supplementary Table S1. Proteins common to all
participating dogs. Supplementary Table S2. Specific proteins
identified in dogs without dental calculus. Supplementary Table S3.
Specific proteins identified in dogs with dental calculus. Supplementary
Table S4. Peptide sequence of the most abundant proteins from all
groups and; Supplementary Table S5. Proteins-protein relationship in-
volved in Reactome pathways in the group of dogs with dental calculus.
Additional file 2: Supplementary Figure 1. Protein-protein interaction
network of the specific proteins identified in dogs without dental calcu-
lus, based on STRING database and showing only connected proteins.
Most abundant proteins in the network (Table 3) are marked with a rect-
angular outline. Legend: Amyloid-beta A4 protein (APP); Rho GDP dissoci-
ation inhibitor (GDI) alpha (ARHGDIA); Additional sex combs like
transcriptional regulator 1 (ASXL1); BUB1 mitotic checkpoint serine/threo-
nine kinase (BUB1); Calmodulin 3 (phosphorylase kinase, delta) (CALM3);
Chemokine (C-C motif) receptor 7, belongs to the G-protein coupled re-
ceptor 1 family (CCR7); Centrosomal protein 152 kDa (CEP152); Centroso-
mal protein 162 kDa (CEP162); Chromodomain helicase DNA binding
protein 3 (CHD3); Choroideremia (Rab escort protein 1) (CHM); Cytoplas-
mic linker associated protein 1 (CLASP1); CAP-GLY domain containing
linker protein 1 (CLIP1); Component of oligomeric golgi complex 2
(COG2); Deltex 3 like, E3 ubiquitin ligase (DTX3L); Histone H2B (ENSC
AFG00000031879); GTP binding protein (GTPBP2); Host cell factor C1
(HCFC1); HECT and RLD domain containing E3 ubiquitin protein ligase
family member 1 (HERC1); Histidine-rich glycoprotein (HRG); Integrin beta
(ITGB2); Uncharacterized protein, Kalirin, RhoGEF kinase (KALRN); Keratin,
type I cytoskeletal 10 (KRT10); Keratin 24, belongs to the intermediate fila-
ment family (KRT24); Keratin 5, belongs to the intermediate filament fam-
ily (KRT5); Keratin, type I cytoskeletal (KRT9); Histone H3 (LOC488263);
Mitogen-activated protein kinase 6 (MAPK6); Neural precursor cell
expressed, developmentally down-regulated 4-like, E3 ubiquitin protein
ligase (NEDD4L); Neuromedin U receptor 1, belongs to the G-protein
coupled receptor 1 family (NMUR1); Papillary renal cell carcinoma (trans-
location-associated) (PRCC); RAN binding protein 2 (RANBP2); Ras protein-
specific guanine nucleotide-releasing factor 2 (RASGRF2); RAB6A GEF
complex partner 1 (RIC1); RNA binding protein S1, serine-rich domain
(RNPS1); Reticulon 4 (RTN4); Sphingosine-1-phosphate receptor 3, belongs
to the G-protein coupled receptor 1 family (S1PR3); Serine/arginine repeti-
tive matrix 2 (SRRM2); Serine/threonine kinase 10 (STK10); Transferrin (TF);
Talin 2 (TLN2); Ubiquitin protein ligase E3 component n-recognin 1
(UBR1).Representation of protein-protein network inside the group of
dogs without dental calculus with confidence score adopted at highest
confidence-0.900
Additional file 3: Supplementary Figure 2. Protein-protein interaction
network of the specific proteins identified in dogs with dental calculus,
based on STRING database and showing only connected proteins. Most
abundant proteins present in the network (Table 4) are marked with a
rectangular outline. Legend: ATP-binding cassette, sub-family A, member
13 (ABCA13); Actinin, alpha 4 (ACTN4); AT hook containing transcription
factor 1 (AHCTF1); Fructose-bisphosphate aldolase (ALDOA); Annexin
(ANXA1); ATR serine/threonine kinase (ATR); B9 protein domain 2; Canis
lupus familiaris transforming growth factor, beta 1 (TGFB1), mRNA (B9D2);
Canis lupus familiaris calcium-sensing receptor (CASR); Centriolar coiled
coil protein 110 kDa (CCP110); Cyclin-dependent kinase 12 (CDK12); Canis
lupus familiaris carcinoembryonic antigen-related cell adhesion molecule
1 (CEACAM1); Uncharacterized protein (CENPF), Centrosomal protein 250
kDa (CEP250); Cytoplasmic linker associated protein 1 (CLASP1); Cytoplas-
mic linker associated protein 2 (CLASP2); Ceruloplasmin (ferroxidase); Be-
longs to the multicopper oxidase family (CP); Dedicator of cytokinesis 2;
Belongs to the DOCK family (DOCK2); Cyclin N-terminal domain-
containing protein (ENSCAFG00000016600); Fas (TNFRSF6) binding factor
1 (FBXO43); Fibronectin (FN1); Uncharacterized protein (GOLGA2); General
transcription factor IIF, polypeptide 1, 74 kDa (GTF2F1); HECT domain con-
taining E3 ubiquitin protein ligase 1 (HECTD1); Uncharacterized protein;
Belongs to the heat shock protein 70 family (HSPA8); Heat shock protein
beta-1 (HSPB1); HECT, UBA and WWE domain containing 1, E3 ubiquitin
protein ligase (HUWE1); Histone-lysine N-methyltransferase; Lysine (K)-spe-
cific methyltransferase 2D (KMT2D); Keratin 13 (KRT13); Keratin 3 (KRT3); L-
lactate dehydrogenase (LDHA); Glyceraldehyde-3-phosphate dehydrogen-
ase (LOC477441); Histone H3 (LOC483167); Matrix metalloproteinase-9
(MMP9); Nibrin (NBN); non-SMC condensin II complex, subunit D3 (NCAP
D3); Condensin complex subunit 2 (NCAPH); Nuclear receptor corepressor
1 (NCOR1); Nuclear receptor corepressor 2 (NOL10); Nucleolar protein 6
(NOL6); Profilin 1 (PFN1); Phosphatidylinositol binding clathrin assembly
protein (PICALM); Polycystin 2 (PKD2); Serine/threonine-protein kinase
PLK; Polo-like kinase 1 (PLK1); Polymerase (DNA directed), epsilon, cata-
lytic subunit (POLE); Protein phosphatase 1, regulatory subunit 12A
(PPP1R12A); Protein tyrosine kinase 2 (PTK2); Uncharacterized protein
(RAB3IP); RB1-inducible coiled-coil 1 (RB1CC1); RB binding protein 6, ubi-
quitin ligase; Retinoblastoma binding protein 6 (RBBP6); Regulatory asso-
ciated protein of MTOR, complex 1 (RPTOR); U4/U6.U5 tri-snRNP-
associated protein 1; Squamous cell carcinoma antigen recognized by T
cells (SART1); SEC31 homolog A, COPII coat complex component; SEC31
homolog A (SEC31A); SET domain containing 1A (SETD1A); SH2 domain
containing adaptor protein B (SHB); Spectrin, beta, non-erythrocytic 1
(SPTBN1); Uncharacterized protein (SRGAP2); Transaldolase (TALDO1);
Transcobalamin I (TCN1); Thrombospondin 1 (THBS1); Tenascin R (TNR);
Tumor protein p53 binding protein 1 (TP53BP1); Trafficking protein par-
ticle complex 6A (TRAPPC6A); Thyroid hormone receptor interactor 12
(TRIP12); U-box domain containing 5 (UBOX5); Unc-51 like autophagy ac-
tivating kinase 2 (ULK2); Versican (VCAN). Representation of protein-
protein network inside the group of dogs with dental calculus with confi-
dence score adopted at highest confidence-0.900.
Abbreviations
AEP: Acquired enamel pellicle; PRPs: Proline-rich proteins; WSS: Whole saliva
supernatants; Micro BCA: Micro Bicinchoninic Acid; S1P1: Sphingosine-1-
Phosphate Receptor Protein 1; TNF: Tumor necrosis factor;
MMPs: Matrixmetalloproteinases; TGFB1: Transforming growth factor beta;
C8B: Complement C8 beta chain; CaSR: Calcium-sensing receptor;
Ca
2+
: Calcium; TRPC3: Transient receptor potential canonical 3; ROCK: Rho
kinase
Acknowledgements
Not applicable.
Authorscontributions
MAAMM, WLS, MB, PKJ and BLCI designed the experiments. PKJ performed
the experiments. MB, PKJ, PAF and WLS, analyzed and interpreted the data.
MB, managed study participant recruitment and sample collection. MAAMM,
WLS, MB, PAF, RSS, CL, BLCI and PKJ, wrote and reviewed the manuscript. All
authors have read and approved the final version of the manuscript.
Funding
This research was funded by the Brazilian Research Council (CNPq grant
#401390/20144), Canadian Institutes of Health Research (CIHR grants
#106657 and 97577) and, Natural Sciences and Engineering Research Council
of Canada (NSERC grant #06119). The funding body had no role in the
design of the study, collection, analysis, and interpretation of data or in the
writing of this manuscript.
Bringel et al. BMC Veterinary Research (2020) 16:298 Page 10 of 12
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Availability of data and materials
The datasets supporting the conclusion of this article are included within the
article and Additional files.
Ethics approval and consent to participate
This study was approved by the Ethics Committee in the Teaching and
Research on Animals of the Dental School of Bauru, University of São Paulo
(number 002/2016); moreover, all the dogsowners signed consent forms.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1
College of Dentistry, University of Saskatchewan, Saskatoon, SK, Canada.
2
Department of Pediatric Dentistry, Bauru School of Dentistry - University of
São Paulo, Bauru, SP, Brazil.
3
College of Veterinary Medicine, University of
Saskatchewan, Saskatoon, SK, Canada.
4
Department of Physiology, Institute of
Biomedical Sciences, Federal University of Uberlandia, Uberlandia, Minas
Gerais, Brazil.
Received: 23 October 2019 Accepted: 6 August 2020
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