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From the salivary proteome to the OralOme: Comprehensivemolecular oral biology

Nuno Rosa a,*, Maria Jose Correia a, Joel P. Arrais c, Pedro Lopes c,Jose Melo c, Jose Luıs Oliveira c, Marlene Barros a,b

aHealth Sciences Institute, Portuguese Catholic University – Viseu, PortugalbCentre for Neurosciences and Cell Biology, University of Coimbra, PortugalcDepartment of Electronics, Telecommunications and Informatics (DETI), Institute of Electronics and Telematics Engineering of Aveiro (IEETA),

University of Aveiro, Portugal

a r c h i v e s o f o r a l b i o l o g y x x x ( 2 0 1 2 ) x x x – x x x

a r t i c l e i n f o

Article history:

Accepted 28 December 2011

Keywords:

Saliva

Salivary proteome

Oralome

Bioinformatics

Oral biology

Oral pathology

Systems biology

a b s t r a c t

Objectives: There have been several efforts to identify the protein components of saliva.

Some of these studies were conducted in healthy individuals and other in individuals with

different oral and systemic disorders. However, a resource compiling and reviewing all of the

proteins identified in proteomic studies is still lacking. The aim of this project is to develop

such a resource.

Design: The proteins identified by proteomic studies were compiled and all information

concerning them was manually curated according to ‘‘IPI History search’’ and UniProt.

Proteins were classified according to gene ontology using PANTHER. The involvement of

each protein in disease was scrutinized using DAVID and a classification into protein disease

classes was performed.

Results: This survey of proteins in the oral cavity lead to the identification of 3397 non-

redundant proteins, 605 altered in pathological conditions and 51 present only in disease.

These proteins originate from different sources: 3115 from saliva, 990 from oral mucosa and

1929 from plasma. All protein sources contribute with different numbers and types of proteins

to identical functions. However, each source produces specific proteins. Examples of the use of

this proteomics database of saliva included the analysis of the changes in the proteome

associated with periodontitis and a survey of systemic disease potential biomarkers in saliva.

Conclusion: The database generated with this work and the information therein stands as a

resource for investigators/clinicians studying the oral biology, searching for molecular

disease markers, developing diagnostic and prognostic tests, and contributing to the dis-

covery of new therapeutic agents.

# 2012 Elsevier Ltd. All rights reserved.

Available online at www.sciencedirect.com

journal homepage: http://www.elsevier.com/locate/aob

1. Introduction

The human oral cavity is a complex ecosystem where host,

microbial and external factors combine in a dynamic

equilibrium which is reflected in saliva. The understanding

* Corresponding author at: Health Sciences Institute, Catholic PortugueTel.: +351 232430200; fax: +351 232428344.

E-mail address: nrosa@crb.ucp.pt (N. Rosa).

Please cite this article in press as: Rosa N, et al. From the salivary proteomeBiology (2012), doi:10.1016/j.archoralbio.2011.12.010

0003–9969/$ – see front matter # 2012 Elsevier Ltd. All rights reservedoi:10.1016/j.archoralbio.2011.12.010

of oral biology hinges upon the compilation and integration of

all information generated by high-throughput techniques,

particularly proteomic studies of saliva.

Saliva is an aqueous biological fluid, with many and diverse

functions, essential for the maintenance of healthy oral

tissues.1 It consists predominantly of water (99%) but includes

se University, Estrada da Circunvalacao, 3504-505 Viseu, Portugal.

to the OralOme: Comprehensive molecular oral biology. Archives of Oral

d.

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a complex mixture of electrolytes (sodium, potassium,

calcium, chloride, magnesium, bicarbonate and phosphate),

proteins (enzymes, immunoglobulins and other antimicrobial

factors, mucosal glycoproteins, albumin and other polypep-

tides and oligopeptides) and, in even smaller amounts, glucose

and nitrogenous products (urea and ammonia).2

The proteins present in whole saliva are secreted mainly

from 3 pairs of major salivary glands: the parotid, the

submandibular, and the sublingual, making up approximately

90% of total salivary secretion. The remaining proteins result

from the minor salivary glands (located at various oral mucosal

sites),3 the gingival crevicular fluid (GCF) and the oral mucosa

(including tongue and other oral mucosae). Saliva also has

plasmatic proteins. There are several ways by which plasma

proteins can reach saliva. The most common include passive

diffusion, ultrafiltration (which occurs through the tight

junctions between the cells) and as a result of GCF outflow.4

The complete role of salivary proteins in oral physiology

and as indicators of disease states is still poorly understood.

The determination of the salivary proteome enables not only

the functional characterisation of saliva, and thus the

clarification of its role in oral biology, but also the identifica-

tion of disease biomarkers. The fact that whole saliva includes

plasma derived proteins and exfoliated epithelial cells, has led

to the suggestion that it may in the future provide a means for

diagnosis of conditions that currently require blood or tissue

samples.

In the last decade, the development of powerful and

discriminating proteomics techniques, allowed an exponen-

tial increase in the identification of protein components of

saliva. Some studies include exclusively samples from healthy

individuals5–23 and others use samples from individuals with

several oral24–39 and systemic27,40–50 disorders. Despite the

vast amount of data generated, these are still scattered and

there is, no database publicly available with a compilation and

characterisation of the proteins identified by proteomic

studies. Bioinformatics tools have a key role in exploring

these data by modelling the inter-relationships between the

sequences, structures and functions of proteins, extracting

biological meaning from the data generated by these studies.51

The aim of this work was to compile and curate the

proteins reported in proteomic studies of saliva and charac-

terize the saliva proteome (OralOme). An integrated database

was created in a local repository and will be publically

available to the scientific community shortly.

We expect this study and the associated database to be a

valuable resource for investigators aiming to clarify the oral

biology, identify molecular disease markers, develop diagnos-

tic tests and improve prognostic, as well as providing

information for the design of biological pathways setting

the ground for the discovery of new therapeutic agents.

2. Materials and methods

2.1. Compilation and curation of human OralOme

To create the database the first step was to compile the

proteins identified in published proteomic studies of saliva

samples. We analysed several bibliographic references of

Please cite this article in press as: Rosa N, et al. From the salivary proteomeBiology (2012), doi:10.1016/j.archoralbio.2011.12.010

proteomic studies in which a complete lists of proteins was

provided.5–50 These studies used saliva from different sources

and are classified in Table 1 regarding sample type.

Because proteins present in whole saliva could originate

from blood transudates, we checked plasma proteomic

studies52,53 to verify which of the proteins present in salivary

proteomic results could have originated from the plasma. To

this subset of proteins we called the salivary plasma.

In this paper, ‘‘saliva’’ is the set of proteins identified by

proteomics techniques, obtained from salivary glands, crevi-

cular fluid and whole saliva samples. ‘‘Whole saliva’’ is

understood as saliva whose samples were not collected

directly from the gland ducts or the gingival crevice. The

proteins identified in the different studies were compared and

repeated entries eliminated. All proteins showing expression

changes in certain pathologies were stored with an indication

of up-regulation or down-regulation under these conditions.

Since there are differences in expression of proteins depen-

dent on donor age, whenever possible we also recorded the age

group for the sample donors.

We registered the existence and type of post translational

modifications of salivary proteins due to the crucial role these

may have in protein function and consequently in oral

physiology.

From the first publication of saliva proteomes, many of the

original identified proteins, catalogued as different entries in

biological databases, have been merged with others and some

were deleted due to misidentification. Therefore, all informa-

tion concerning the identified proteins was manually curated

and updated as of February 2011. The update of the IPI

(International Protein Index) entries was carried out with ‘‘IPI

History search’’ tool (http://www.ebi.ac.uk/IPI/IPIhelp.html).54

All other updates have been made according to UniProt

database (http://www.uniprot.org/).55,56 The curated list of

proteins identified in this work was stored in the database

OralCard—Web Information System for Oral Health developed

for this purpose.

2.2. Human saliva proteome cataloguing

2.2.1. Gene ontology (GO) analysisThe proteins in the database were classified according to

molecular function, biological process and cellular component

by using the PANTHER database (http://www.pantherdb.org/

).57,58 PANTHER is a unique resource that classifies genes and

proteins by their functions, using published scientific experi-

mental evidence and evolutionary relationships inferred by

curators with the goal of predicting function even in the

absence of direct experimental evidence. PANTHER applies

both software tools and manual curation to perform these

inferences as accurately as possible, and to keep them up-to-

date as new experimental results accumulate. To accomplish

this task we conducted a Batch ID Search with the PANTHER

Classification Tool for each group of proteins, using the AC

UniProtKB identifiers (without protein isoforms). We also used

the PANTHER gene expression data analysis tool to compare

classifications of multiple clusters of lists with a reference list

(total number of human proteins in PANTHER database) to

statistically determine over- or under-representation of

PANTHER classification categories. Each list is compared to

to the OralOme: Comprehensive molecular oral biology. Archives of Oral

Table 1 – List of oral cavity proteomic studies analysed by sample origin.

Saliva source References N8 of identified proteins Condition

Health Disease

Salivary glands

Parotid Gonzalez-Begne et al., 20097 431

Preza et al. 200936 69 69 Dental caries

Siqueira et al. 200815 46

Denny et al. 20085 989

Ramachandran et al. 200821 33

Ryu et al. 200649 10 10 Sjogren’s syndrome

Walz et al. 200617 13

Hardt et al. 20059 6 Diurnal variation

Hardt et al. 20058 16

SM/SL Siqueira et al. 200815 46

Denny et al. 20085 963

Ramachandran et al. 200821 56

Walz et al. 200617 14

Minor Hjelmervik et al. 200945 365 431 Sjogren’s syndrome

Siqueira et al. 200815 56

Gingival crevicular fluid

Bostanci et al. 201024 66 76 Chronic periodontitis

Aggressive periodontitis

Oral mucosae

Tongue Negishi et al. 200935 5 5 HNSCC

He et al. 200431 12 12 HNSCC

Other mucosae Chi et al. 200926 962 80 HNSCC

Lo et al. 200734 11 HNSCC

Turhani et al. 200637 20 20 HNSCC

Whole saliva

Brinkmann et al. 201125 7 7 HNSCC

Haigh et al. 201030 10 10 Chronic periodontitis

Cabras et al. 201040 15 15 Diabetes type 1

Loo et al. 201012 2290

Goncalves et al. 201029 25 25 Chronic periodontitis

Jou et al. 201033 8 8 HNSCC

Costa et al. 201027 3 3 Chronic periodontitis diabetes type 2

Castagnola et al. 201041 15 Preterm newborn

Streckfus et al. 200950 158 158 Mammary carcinoma

Fleissig et al. 200942 16 16 Sjogren’s syndrome

Wu et al. 200938 11 11 Aggressive periodontitis

Quintana et al. 200914 12 Inter-individual variability

Ito et al. 200947 1 1 Sjogren’s syndrome

Rao et al. 200948 491 61 Diabetes type 2

Yan et al. 200920 140

Dowling et al. 200828 6 6 HNSCC

Hu et al. 200832 39 60 HNSCC

Ramachandran et al. 200821 60

Fang et al. 20076 1479

Giusti et al. 200743 18 9 Sjogren’s syndrome

Hu et al. 200746 26 28 Sjogren’s syndrome

Giusti et al. 200744 9 Diffuse systemic sclerosis

Larsen et al. 200723 45

Ramachandran et al. 200622 45

Yang et al. 200639 2 2 Lichen Planus

Walz et al. 200617 29

Xie et al. 200519 423

Hu et al. 200510 266

Vitorino et al. 200416 34

Messana et al. 200413 7

Huang 200411 25 10 Oral cavity bleeding

Wilmarth et al. 200418 90

Abbreviations: HNSCC, head and neck squamous cell carcinoma.

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the reference list using the binomial test59 for each GO cellular

component, molecular function or biological process term in

PANTHER.

2.2.2. Disease associationHuman saliva and plasma proteins were scrutinized for their

involvement in diseases and grouped into protein disease

classes by the use of DAVIDBioinformatics Resources 6.7 (http://

david.abcc.ncifcrf.gov/home.jsp).60,61 DAVID bioinformatics

resources consist of an integrated biological knowledgebase

and analytic tools aimed at systematically extracting biological

meaning from large gene/protein lists. For this task, the lists of

proteins derived from the saliva, salivary plasma (plasma

proteins found in saliva) and total plasma (all plasma proteins)

were subjected to Functional Annotation Tool. The proteins

were annotated according to ‘‘genetic association database

disease class’’ and presented as a ‘‘chart report’’. This is an

annotation-term-focused view which lists annotation terms

and their associated genes under study. To avoid over counting

duplicated genes, the Fisher exact statistics is calculated based

on corresponding DAVID gene IDs by which all redundancies in

original IDs are removed. All results of the Chart Report have to

pass the thresholds (by default, Max.Prob. � 0.1 and Min.-

Count � 2) in the Chart Option section to ensure only statisti-

cally significant ones are displayed.

3. Results

3.1. Compilation and curation of human oral proteome

This work leads to the documentation of 3397 non-

redundant proteins that may be found in the oral cavity.

Fig. 1 – Venn diagram illustrating human OralOme constitution

sources; (c) saliva protein sources. Numbers in parentheses rep

Underlined numbers represent proteins unique to a particular s

intersections account for proteins common to intersected group

from patients with oral or systemic diseases.

Please cite this article in press as: Rosa N, et al. From the salivary proteomeBiology (2012), doi:10.1016/j.archoralbio.2011.12.010

Of these, 3115 were found in saliva, 990 in oral mucosa (17 in

tongue mucosa31,35 and 984 in other oral mucosae26,34,37)

and 1929 were plasmatic proteins found in the oral

cavity52,53 (Fig. 1a). The numbers with asterisks in Fig. 1

represent 707 proteins identified in patients with several

oral and systemic conditions. Of these proteins, 637 were

identified in samples of saliva (7336,49 in parotid glands,

43145 in minor glands, 7424 in crevicular fluid and 22825,27–

30,32,33,38–44,46–48,50 in whole saliva samples) and 118 in oral

mucosa (17 in tongue31,35 and 106 in other oral muco-

sa26,34,37). The complete list of the conditions and respective

proteomic studies included in the analysis is available in

Table 1. Fifty-one of the proteins were identified exclusively

in pathological situations.

The human parotid5,7–9,15,17,21,36,49 present 1193 proteins

(including 431 from parotid exosome7). Since submandibular/

sublingual (SM/SL)5,15,17,21 glands present 999 proteins, they

contribute to total protein number almost in the same

proportion, with 701 proteins being shared between them.

Salivary minor glands5,15,45 with 554 proteins and GCF24 with

100 proteins contribute to salivary proteome to a much lesser

extent (Fig. 1c).

All sources contribute with unique proteins (Fig. 1—

underlined numbers). Note that 1283 proteins (40%) appeared

only in saliva, and were absent in blood plasma or oral

mucosae samples. Of all the proteins obtained from saliva

samples, 470 are not found in the salivary glands, GCF or oral

mucosae. The parotid glands secrete a higher percentage

(15.3%) of unique proteins compared to SM/SL (8.5%) and

minor (5.4%) glands as well as GCF (8%). Of the proteins

present in the oral cavity 96 have been identified as

glycosylated and 9 of these were only found in studies of

post translational modification.21–23

: (a) OralOme main protein sources; (b) oral mucosa protein

resent the total proteins obtained from a particular source.

ource relatively to total OralOme. Numbers inside

s. Numbers with an asterisk represent proteins obtained

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3.2. Characterisation of human oral proteome

The human proteins present in the oral cavity have several

origins including the salivary glands, oral mucosa and even

blood plasma. The proteins associated to each of these

compartments were characterised according to diverse

aspects including cellular component, molecular function

and biological process in order to understand their functional

organization.

3.2.1. Saliva, oral mucosae and salivary plasma proteinscomparisonWhen the different sources of oral cavity proteins are

compared according to cellular component (GO: 0005575 –

‘‘The part of a cell or its extracellular environment in which a gene

product is located’’) (Fig. 2a) we found that most of the proteins

in the OralOme are intracellular. Most of these intracellular

proteins are derived from the oral mucosa (60.2%), which also

contributes to the oral proteome with a higher percentage of

ribonucleoprotein complexes (12.4%) than saliva (5.9%) and

salivary plasma (7.7%). Furthermore, we can see that the oral

cavity has a large percentage of protein complexes (almost

twice what would be expected relative to total human

proteins).

A comparison of the different sources of oral cavity

proteins regarding molecular function (GO: 0003674 – ‘‘Elemen-

tal activities, such as catalysis or binding, describing the actions of a

gene product at the molecular level’’), using PANTHER Classifica-

tion System is presented in Fig. 2b. In saliva, oral mucosa and

salivary plasma, proteins participate in the same sort of

functions. However, each source contributes with a different

number and type of proteins that participate in common

functions, which reflects the specific role of each of these

sources to salivary functions.

Fig. 2b shows that a large percentage of the proteins present

in the oral cavity are involved in the molecular functions

‘‘catalytic activity’’ and ‘‘binding’’. Because some proteins can

have more than one function, we tried to find which of the

proteins with catalytic and binding activities are also involved

in other molecular functions (Fig. 2c). We confirmed that the

molecular functions ‘‘catalytic activity’’ and ‘‘binding’’ are

quite broad and redundant since the proteins involved in them

also have other activities. Note that many proteins with

catalytic activity also have binding activity. Likewise, many of

the proteins with binding activity are also structural mole-

cules, enzyme regulators or have catalytic activity, amongst

other functions.

Looking at the antioxidant function we see that, despite the

small percentage of proteins involved in it, all sources

contribute with proteins to carry out this important function

of saliva. Furthermore, the oral cavity has a higher percentage

(almost four times, p � 0.05) of proteins with antioxidant

activity than would be expected relative to total human proteins

(results not shown). The contribution of each source of OralOme

to the antioxidant activity function is almost the same (salivary

plasma 35%, saliva 30% and oral mucosae 30%). More interesting

is the fact that amongst the plasma proteins with antioxidant

function, almost all appear in the oral cavity (salivary plasma).

Only 5% of all plasma proteins with antioxidant function are not

present in the oral cavity (Fig. 2b—pie chart). We can also see

Please cite this article in press as: Rosa N, et al. From the salivary proteomeBiology (2012), doi:10.1016/j.archoralbio.2011.12.010

that some of these proteins with antioxidant function (e.g.

glutathione peroxidase and extracellular superoxide dismutase

[Cu–Zn]), are produced in all sources of saliva, except in the

crevicular fluid (Fig. 3b).

3.2.2. Human saliva protein sourcesMost of the proteins present in OralOme come directly from

saliva sources (major and minor salivary glands and GCF).

Thus, these proteins have a prominent role in saliva functions

and consequently in oral biology. In order to evaluate the

specific contribution of different saliva sources to the saliva

functions, we investigated the molecular functions of the

proteins produced from each source and the biological

processes (GO: 0008150 – ‘‘Any process specifically pertinent to

the functioning of integrated living units: cells, tissues, organs, and

organisms. A process is a collection of molecular events with a defined

beginning and end’’) in which they are involved, using PANTHER

Classification System (Fig. 3). The proteins produced by the

major and minor salivary glands contribute to the same

molecular functions. However, the number of proteins

dedicated to each molecular function varies slightly amongst

the major salivary gland proteins and at a greater degree,

between the proteins of all the major salivary glands and the

proteins of the minor ones. This variation consists mainly of

an increase in the number of proteins involved in catalytic and

structural activities and a decrease in the number of proteins

involved in enzyme regulatory and receptor activities (Fig. 3a).

The gingival crevicular fluid does not contain proteins

involved in ion channel and receptor activities neither

translation regulator activity. Furthermore, the number of

proteins from GCF with structural molecular activity is

markedly increased in relation to the proteins produced by

the salivary glands. We can also observe a sharp increase in

the number of unique proteins in GCF with transcription

regulatory activity (Fig. 3b).

Regarding biological processes involving proteins obtained

from each saliva source, it appears that they are very similar

and only the gingival crevicular fluid differs significantly

compared to other sources (Fig. 3c). Note that the GCF has an

increase of proteins involved in response to stimuli (8.4%

versus 5.9% of whole saliva) but has almost the same

percentage of proteins engaged in the ‘‘immune system

process’’ as other saliva sources. Moreover, we observed that

most proteins involved in immune response in saliva, were

also present in plasma (results not shown). Another difference

is that minor salivary glands produce a higher percentage

(25.1% versus 18% in parotid, 19.6% in SM/SL and 14.8% in GCF)

of proteins involved in metabolic processes.

3.3. Strategies for the analysis of molecular changes indisease

One of the most interesting uses of a database compiling all

the proteomic data available for the oral cavity is to enable the

analysis of proteomics data. This database is fundamental to

detect the possible changes in the quantity and type of

proteins present in saliva samples from diseased individuals.

This kind of approach allows the identification of functions/

biological processes compromised in the pathologies ana-

lysed. In Fig. 4 we show the classification of the proteins

to the OralOme: Comprehensive molecular oral biology. Archives of Oral

Fig. 2 – Classification of saliva, oral mucosae, salivary plasma proteins and total human proteins in PANTHER database

according to (a) cellular component (percentage of gene hit against total # component hits); (b) molecular function

(percentage of gene hit against total # function hits). Pie chart represents ‘‘antioxidant activity’’ function in detail.

(c) Number of proteins with catalytic and binding activities that participate in other molecular functions. Analysis

performed with PANTHER classification tool.

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present in saliva samples from donors with various oral

diseases, according to molecular functions and biological

processes, and compare it to the normal oral proteome (all

proteins of the oral cavity except those identified exclusively

in diseases). In each of these oral diseases, we can identify

molecular functions and biological processes markedly chan-

ged from the normal. For instance, in chronic periodontitis

there is a significant change in proteins with structural

molecular activity (Fig. 4a) and in proteins involved in cellular

Please cite this article in press as: Rosa N, et al. From the salivary proteomeBiology (2012), doi:10.1016/j.archoralbio.2011.12.010

component organization and immune system biological

processes (Fig. 4b).

3.4. Molecular evidence of potential salivary biomarkers

In order to understand how the plasma and salivary

proteins reflect the state of human health, and therefore

their relative value as a diagnostic medium, the proteins

obtained from plasma and saliva were classified according to

to the OralOme: Comprehensive molecular oral biology. Archives of Oral

Fig. 3 – Classification of the proteins obtained from different saliva sources according to: (a) molecular function of total

number of proteins obtained from each source (percentage of gene hit against total # function hits); (b) molecular function of

proteins obtained exclusively from each source relatively to the total number of proteins obtained from saliva samples

(percentage of gene hit against total # function hits); (c) biological process (percentage of gene hit against total # process

hits). Analysis performed with PANTHER classification tool.

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their involvement in diseases and grouped into classes using

the DAVID web tool (Fig. 5).

Salivary proteins are associated with almost the same

classes of diseases as plasma proteins. Moreover, the

Please cite this article in press as: Rosa N, et al. From the salivary proteomeBiology (2012), doi:10.1016/j.archoralbio.2011.12.010

percentage of proteins associated with each disease class is

very similar in saliva and salivary plasma. As expected, we find

a higher percentage of proteins associated with cardiovascular

diseases in plasma (17%) than in saliva (11%). We can also see

to the OralOme: Comprehensive molecular oral biology. Archives of Oral

Fig. 4 – Classification of the proteins obtained from saliva samples from donors suffering various oral diseases according to:

(a) molecular function (percentage of gene hit against total # function hits); (b) biological process (percentage of gene hit

against total # process hits). Analysis performed with PANTHER classification tool. ‘‘Normal OralOme’’ is understood as the

totality of human proteins identified in the oral cavity, except those identified exclusively in pathological conditions.

Fig. 5 – Human saliva, blood plasma and salivary plasma protein disease class association, determined by DAVID Functional

Annotation Tool.

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that there is a higher percentage of proteins associated with

immune system diseases in saliva (13%) than in plasma (7%).

More unusual is the presence of a higher percentage of proteins

related to neurological diseases in saliva (9.3%) than in plasma

(5.8%). In fact, we find, in saliva, an extensive range of proteins

involved in Alzheimer’s, Parkinson’s and Huntington’s disease.

Our analysis also identified salivary proteins involved in

diseases related to vision, which did not appear in plasma, as

is the case of ornithine aminotransferase (P04181), which, when

defective, is responsible for hyperornithinemia with gyrate

atrophy of choroid and retina (HOGA) [MIM:258870].

4. Discussion

4.1. Compilation and curation of human OralOme

Most of the attempts to elucidate the oral proteome, have

consisted of very specific studies focused on particular

situations24–50 or more general studies where saliva is

collected in well-defined conditions.7–20,24,25 Some attempts

were made at a broader approach. The first of these attempts

was by Denny et al. in 2008.5 They identified 1166 proteins

from saliva samples collected directly from major salivary

glands, developing the Salivary Proteome Knowledge Base.

Loo et al. compiled 2290 proteins in saliva12 and compared

it with proteins identified in plasma. Another workgroup, led

by Li, published a body fluid database62 that includes salivary

proteins (Sys-BodyFluid). Despite the innovative attempt to

integrate information from the application of proteomics to

different body fluids, the data pertaining to saliva was limited

to eight studies conducted from 2004 to 2008, identifying 2161

proteins. In the Sys-BodyFluid database the proteins were

classified with the IPI code, which is now discontinued and

hinders the match of these proteins with those stored in the

major protein databases such as UniProtKB. It is known that

saliva is more complex than what is secreted by the salivary

glands and may have proteins from the oral mucosa and even

blood plasma. Our database includes proteins from all of these

sources both under physiological and pathological conditions.

This database offers more than just a list of proteins including

further information on each protein.

4.2. Saliva, oral mucosae and salivary plasma proteinscomparison

The amount and type of proteins obtained from each of the

oral protein sources, elucidates their contribution to the

physiology of the oral cavity leading to the interpretation of

the OralOme. Saliva, oral mucosa and plasma proteins

probably participate in the same sort of functions. However,

each source contributes with a different set of proteins. All

sources have unique proteins that may indicate specific

functions carried out by them and therefore, a particular role

in the oral cavity physiology. Since some proteins found in the

oral cavity are not found in the salivary glands, GCF or oral

mucosae, we set forth the hypothesis that, because the

salivary gland acinar complexes are ‘‘leaky’’ epithelia, many of

these proteins must come from plasma entering in saliva by

diffusion across tight junction complexes.

Please cite this article in press as: Rosa N, et al. From the salivary proteomeBiology (2012), doi:10.1016/j.archoralbio.2011.12.010

We noted that a large percentage of the proteins that

appear in the oral cavity are intracellular and are derived from

the oral mucosa, which is not surprising considering that

saliva collects a large number of proteins resulting from the

constant scaling that occurs in the oral cavity both from oral

mucosa and salivary gland ducts. Other evidence that

supports this is the fact that our results indicate that the

mucosa contributes to the oral proteome with a higher

percentage of ribonucleoprotein complexes than saliva and

salivary plasma, presumably due to cell lysis. It is well

established that ribosomes are abundant in the cell and it is

estimated that the majority of eukaryotic cellular RNA is

ribosomal RNA. So, there is a great contribution, in terms of

the mass, of these proteins to the whole cell content.

Moreover, we saw that the oral proteome has a percentage

of protein complexes higher than what would be expected

relative to total human proteins. We propose that this reflects

the supramolecular complexes existing in the complex

membrane organelle system and in the rough endoplasmic

reticulum and therefore structures which are intracellular.

Although most of the proteins present in the oral cavity are

involved in ‘‘catalytic activity’’ and ‘‘binding’’ these functions

are not necessarily the most representative. These functions

are quite broad and redundant since the proteins involved in

them also have other activities. This is a good example of the

difficulties that still exist in the cataloguing of protein

functions, as well as, the definition of the different ontologies,

that can lead to the misinterpretation of results.

The higher percentage of proteins with antioxidant activity

observed in the oral cavity in relation to total human proteins

is probably explained by the fact that the oral cavity is

particularly exposed to oxidative stress.63

4.3. Human saliva protein sources

The absence of proteins in gingival crevicular fluid involved in

ion channel and receptor activities can be explained by the

fact they are essentially integral membrane proteins, and

therefore cannot be released to the extracellular fluid which

composes the GCF. Moreover, the absence of proteins with

translation regulator activities from the GCF may be related to

the lower percentage of proteins with this function, found in

blood plasma, since the GCF is formed when fluid exudes from

the vessels of the microcirculation into the sulcus or dental

pocket.64 The GCF exhibits a marked increase in the number of

proteins with structural molecular activity when compared to

the proteins produced by the salivary glands. This fact is

probably due to the remodelling of extracellular matrix

occurring in healthy periodontium and to the increased

degradation of this matrix observed in oral diseases such as

periodontitis.65 Proteins resulting from this degradation

accumulate in the GCF.

The observed higher number of unique proteins in GCF

with transcription regulatory activity reflects the need for an

increased protein synthesis in order to offset the degradation

taking place in gingival crevice which is consistent with the

increase in cellular processes and cellular component organi-

zation processes observed in GCF.

The increase of proteins identified in GCF involved in the

response to stimuli could be explained by the fact that the

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AOB-2738; No. of Pages 12

gingival sulcus is a ‘‘war zone’’ where the response of the host

tissue to microbial challenges is present.66 On the other hand, it

is strange that the GCF has almost the same percentage of

proteins engaged in the ‘‘immune system process’’ as other

saliva sources. These results suggest that, in the GCF the result

of ‘‘war’’ is more noticeable than the ‘‘warriors’’ themselves. In

other words, the GCF may be a good medium for studying the

degradome resulting from the host’s immune response to

microbial aggression. This is corroborated by the fact that there

is an increased amount of GCF produced in periodontal

disease24 and, in this instance, periodontal pockets comprise

not only the microorganisms growing in the sulcus, but also the

result of an overly aggressive immune response against these

microorganisms. Due to the equal participation of different

sources of saliva in the immune response, we can also speculate

that the participation in the ‘‘immune system process’’ is

shared by the whole saliva (because of its importance) and the

proteins involved can be recruited from plasma by infiltration in

the epithelium of glands and oral mucosa. The presence in

plasma of most of the proteins involved in immune response

carried out in saliva supports that idea.

With this work, we found that all sources of saliva,

especially the minor salivary glands, have a high percentage

of proteins involved in metabolism. This reveals the interest in

saliva as an object in metabolomics studies and in the

understanding of the molecular mechanisms of diseases

related to defects in metabolism, such as diabetes. Most of the

glycosylated proteins found in oral cavity, are involved in

metabolic diseases. This influence is through their role in the

complement and coagulation cascades pathways (results not

shown). Alterations in these pathways are known to be related

with poor wound healing.67 The characteristic diabetic

hyperglycaemic state may favour the glycosylation68 of the

proteins present in saliva and therefore be responsible for the

difficulty in healing observe in the oral cavity of diabetic

patients.

4.4. Strategies for the analysis of molecular changes indisease

The analysis of proteomics data revealing changes in the

quantity and type of proteins present in the saliva of diseased

patients can lead to the identification of functions/biological

processes compromised in these conditions. This analysis is

substantially easier and more reliable if there is a methodology

that can be reproduced in different situations, eliminating most

of the analyser’s subjectivity. We presented an example of how

proteomics data can be analysed and interpreted. This approach

allowed us to identify molecular support for the importance of

proteins involved in cellular component organization and

immune system biological processes as well as structural

molecular functions in the molecular mechanisms of periodon-

titis. Further analyses may lead to the identification of possible

molecular markers and even potential therapeutic targets.

4.5. Molecular evidence of potential salivary biomarkers

Salivary proteins are associated with nearly the same classes

of diseases as plasma proteins, indicating that molecular

markers for all these diseases can be found in saliva,

Please cite this article in press as: Rosa N, et al. From the salivary proteomeBiology (2012), doi:10.1016/j.archoralbio.2011.12.010

enhancing the possibility of its use for diagnosis of both oral

and systemic diseases.

The presence, in saliva, of a higher percentage of proteins

related to neurological, immune system and ophthalmic

diseases may be indicative of a strong potential of saliva as

a source of molecular markers essential for designing

strategies for non-invasive diagnosis of these diseases.

In spite of remarkable advances in bioinformatics techni-

ques used in systems biology, there are still clear gaps in the

path between proteomics results and the elucidation of

molecular mechanisms involving the identified proteins. This

void can only be overcome with studies, making use of

bioinformatics techniques, always based on human interpre-

tation in the light of existing literature.

Information about the oral cavity is dispersed through

different databases focused on more general systems. In

addition to being dispersed, the data are not always

standardized, which makes their integration and comprehen-

sive study a difficult task. The present work is the largest and

most comprehensive survey of proteins of the oral cavity,

covering proteins from all salivary sources both under

physiological and pathological conditions, organized in an

integrated database.

Funding

None.

Competing interests

None declared.

Ethical approval

Not required.

Appendix A. Supplementary data

Supplementary data associated with this article can be

found, in the online version, at doi:10.1016/j.archoralbio.

2011.12.010.

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