Medical Research Project on Human Saliva as a Diagnostic And Prognostic Body Fluid
Project location: ITALY, Milan, Rome, Cagliari
Project start date: June 2011 - Project end date: October 2013
Project number: 2011-28
Beneficiary: Università Cattolica del Sacro Cuore Facoltà di Medicina e Chirurgia - Istituto di Biochimica e Biochimica Clinica
According to the project, in the period November 2011- May 2012 the following studies and researches have been carried out.
1) Investigation of the function of some salivary peptides.
A study devoted to establish the function of basic proline-rich proteins (bPRPs) and other salivary peptides is in progress. In particular interesting correlation was evidenced between the level of specific bPRPs and bitter taste (PROP) perception in the adult. Taste perception intensity evoked by PROP and NaCl solutions was measured in sixty-three volunteers (21 males, 42 females, age 2563 y) to establish their PROP taster status, and 24 PROP super-tasters and 21 nontasters were selected to participate in the study. TAS2R38 and gustin gene molecular analysis were performed using PCR techniques. Qualitative and quantitative determination of salivary proteins was performed by HPLC-ESI-MS before and after PROP taste stimulation. PROP super-tastings was strongly associated with the ‘taster' variant (PAV haplotype) of TAS2R38 and the A allele of rs2274333 polymorphism in the gustin gene and non tasting was associated with the minor alleles at both loci. ANOVA revealed that basal levels of II-2 and Ps-1 proteins, belonging to the basic proline-rich protein (bPRPs) family, were significantly higher in PROP super-taster than in non taster un-stimulated saliva (Figure 1), and that PROP stimulation elicited a rapid increase in the levels of the same proteins only in PROP super-taster saliva (Figure 2). These data show for the first time that responsiveness to PROP is associated with salivary levels of II-2 peptide and Ps-1 protein, which are products of the PRB1 gene. These findings suggest that PRB1, in addition to TAS2R38 and gustin, could contribute to individual differences in thiourea sensitivity, and the expression of the PROP phenotype as a complex genetic trait. This study was published .
These studies are suggesting that a significant correlation is present between the saliva composition, particularly in the highly polymorphic class of basic proline-rich proteins, and taste perception.
2) Characterization of protein and derivatives detectable in human saliva.
Saliva from healthy subjects has been investigated to determine inter-individual qualitative and quantitative variability of several proteins/peptides involved in the safety of the oral cavity. This research could have relevant implication in the determination of the control group for further studies on the use of saliva for diagnostic and prognostic purposes.
a) Cystatin B.
Researches performed on cystatin B have evidenced that this protein is present in human saliva mainly as S-glutathionyl, S-cysteinyl and S-S-dimer derivatives (Figure 3). The identification was confirmed by high resolution HPLC-ESI-MS-MS experiments on the intact naturally occurring proteins and their tryptic digests. S-unmodified cystatin B is rarely detectable in whole saliva of healthy adults (5 subjects out of 65) and its percentage does not overcome approximately 20% of total cystatin B (11±9%). In the majority of subjects (60 out of 65) the mean percentages of the S-modified derivatives were the following: S-glutathionyl 53±13%, S-cysteinyl 15±5%, S-S 2-mer 32±13%. Variations of the percentages of these S-modified derivatives of cystatin B could be indicative of oral oxidative stress. This is the first time that S-glutathionylation and S-cysteinylation were described as extensive PTM of a salivary protein and the first time that these PTMs were detected in naturally occurring cystatin B. The results of the study have been published 
c) Proline-rich protein Ib8a
The characterization of the glycan moieties and the peptide backbone of six glycoforms of IB-8a CON1+, a basic proline-rich protein present in human saliva has been carried out. MS analyses on the intact glycoproteins before and after N-deglycosylation with PNGase F and high-resolution MS/MS sequencing by LTQ Orbitrap XL of peptides and glycopeptides from tryptic digests allowed the structural characterization of the glycan moieties and the polypeptide backbone, as well as to establish the glycosylation site at the asparagine residue at 98th position. Five of the glycoforms carry a biantennary N-linked glycan fucosylated in the innermost N-acetylglucosamine of the core and show from zero to four additional fucoses in the antennal region (Figure 4). The sixth glycoform carries a monoantennary monofucosylated oligosaccharide (Figure 4). The glycoform cluster was detected on 28 of 71 adult saliva specimens. Level of fucosylation showed inter-individual variability with the major relative abundance for the trifucosylated glycoform. Non glycosylated IB-8a CON1+ and the variant IB-8a CON1-, lacking of the glycosylation site, have been also detected in human saliva. The results of the study have been published .
S100A9 has been detected in human saliva in ten different isoforms. They are:
1) long isoform, missing the N-terminal Met residue, and N-terminally acetylated
2) long isoform, S-glutathionylated on Cys2, missing the N-terminal Met residue, and N-terminally acetylated.
3) long isoform, S-cysteinylated on Cys2, missing the N-terminal Met residue, and N-terminally acetylated.
4) short isoform, missing the N-terminal pentapeptide (MTCKM), and N-terminally acetylated
5) short isoform, missing the N-terminal pentapeptide (MTCKM), N-terminally acetylated, and oxidized at Met89 residue.
All the five isoforms have a counterpart phosphorylated on their penultimate Thr residue.
As observed for cystatin B the long isoform has been detected in adult saliva only as S-glutathionylated and S-cysteinylated derivatives at Cys2.
In 22 subjects (out of 60) none isoform of S100A9 was detected. 17 subjects showed only the short isoform, 4 subjects only the long isoform, and 17 subjects both the long and the short isoforms. In these last subjects the two isoforms contributed about for the same amount (50%) to the total S100A9.
21 out of the 34 subjects (62 %) displaying the short isoform evidenced also the presence of the corresponding phosphorylated isoform. 14 out of the 21 subjects (66%) displaying the long isoform evidenced also the presence of the corresponding phosphorylated isoform too. Interestingly, 12 out of 17 subjects with the short and the long isoforms displayed simultaneously the phosphorylated counterparts, while the other 5 did not. In the samples showing the phosphorylated derivatives the percentage of the phosphorylated isoform corresponded roughly to 40%. On the whole, these results suggest that the expression of the different isoforms of S100A9 is characterized by a complex genetic polymorphism.
Preliminary results have been presented at the VII ItPA Congress (Viterbo 12-15 June 2012).
All the researches described in Section 1)a-c could have relevant implications for the use of saliva as a diagnostic bodily fluid. The variations of the percentages of the different isoforms in some systemic diseases should be utilized as early diagnostic markers. Moreover, these studies can provide relevant information on the molecular mechanism modulating the functions and the roles of these proteins in the oral cavity and in other organs and tissues.
3) Study of the age related composition variability, exploring saliva in the pediatric age.
Different studies on the variation of saliva composition as a function of the age are in progress, with a particular concern to pre-term newborns and children in pediatric age. Interesting differences with respect to the adults are emerging, particularly in the different percentages of the isoforms of cystatin B and S100A9. The evaluation of the level of phosphorylation of histatin 1 and statherin confirms that the activity of the Golgi casein kinase responsible for this PTM is not fully active in the last period of fetal life. These researches can be useful in order to investigate by non invasive methods the molecular mechanisms at the basis of human development and the sequels of their impairment. For instance, they can provide some clues for the mechanism at the basis of the outcome of some autism spectrum disorders.
4) Investigation of the variations of human salivary proteome in different pathologies.
The analysis of the variations detectable in whole saliva of Down Syndrome (DS) patients is almost concluded. The profile of salivary peptides of DS patients was characterized by RP-HPLC-ESI-MS and compared to that of a sex and age-matched healthy control group in order to evidence eventual significant qualitative and quantitative differences. According to the age, patients and controls were divided in two groups: 10-17 and 18-40 years. Intriguingly, the age-dependent modification of the salivary aPRPs, and cystatins, demonstrated for normal subjects was not evidenced in DS patients. As a consequence their levels were found significantly lower in DS patients with respect to controls only in the 18-40-year-old group. Conversely, levels of antimicrobial alpha-defensins 1-3 and histatins were higher in DS subjects. Levels of S100A7 (D27 isoform) and S100A12 proteins, practically absent in saliva of healthy subjects, were found significantly higher in saliva of DS patients independently from the age. In this respect, it is of particular interest the recent finding of an association between high levels of S100A7 in CSF of Alzheimer's disease patients and AD clinical severity.
The studies on the salivary proteome on different tumor pathologies in the pediatric age, before and after drug or radiation treatment, that ones on different patients affected by schizophrenia and other bipolar disorders, as well as patients with the Wilson disease are in progress.
All these researches can provide interesting clues on the molecular mechanisms at the basis of these diseases and can provide suggestions for targeted therapies.
5) General studies
Several reviews describing the relevance of the study of the salivary proteome for diagnostic and prognostic purposes, as well a review critically describing the different results obtainable by different proteomic platforms have been published [4,5].
 Cabras T., Melis M., Castagnola M., Padiglia A., Tepper B.J., Messana I. Tomassini Barbarossa I. Responsiveness to 6-n-propylthiouracil (PROP) is associated with salivary levels of two specific basic proline-rich proteins in humans. PLoS ONE 7(2) (2012) e30962.
 Cabras T., Manconi B., Iavarone F., Fanali C., Nemolato S., Fiorita A., Scarano E., Passali G.C., Manni A., Cordaro M., Paludetti G., Faa G., Messana I., Castagnola M. RP-HPLC-ESI-MS evidenced that salivary cystatin B is detectable in adult human saliva mostly as S-modified derivatives: S-glutathionyl, S-cysteinyl and S-S 2-mer. J. Proteomics 75 (2012) 908-913.
 Cabras T., Boi R., Pisano E., Iavarone F., Fanali C., Nemolato S., Faa G., Castagnola M, Messana I. HPLC-ESI-MS and MS/MS structural characterization of multifucosylated N-glycoforms of the basic proline-rich protein IB-8a CON1(+) in human saliva. J Sep. Sci. 35(9) (2012) 1079-1086.
 Castagnola M., Picciotti P.M., Messana I., Fanali C., Fiorita A., Cabras T., Calò L., Pisano E., Passali G.C., Iavarone F., Paludetti G., Scarano E. Potential application of human saliva as diagnostic fluid. Acta Otorhinolaryngol. Ital., 31 (2011) 347-357.
 Castagnola M., Cabras T., Iavarone F., Fanali C., Nemolato S. Peluso G., Bosello S.L., Faa G., Ferraccioli G., Messana I. The human salivary proteome: a critical overview of the results obtained by different proteomic platforms. Expert Rev. Proteomics 9(1) (2012) 33-46.
According to the project in the period June 2012- December 2012 the following studies and researches have been carried out.
1) Characterization of protein and derivatives detectable in saliva.
a) Determination in adult human saliva of fragments of bigger proteins
A top-down discovery proteomic platforms centered on high-resolution micro-HPLC-ESI-MS allowed the identification the characterization in human saliva of two fragments of PIgR (Polymeric immunoglobulin receptor, Swiss Prot accession number P01833), the fragment 609-648 and the fragment 623-648, until now not detected. PIgR binds polymeric IgA and IgM at the basolateral surface of epithelial cells. The complex is then transported across the cell to be secreted at the apical surface. During this process a cleavage occurs that separates the extracellular (known as the secretory component) from the transmembrane segment. The secretory component corresponds to the fragment 1-585. Therefore, fragments 609-648 and 623-648 derive from the transmembrane segment and suggest unknown molecular events occurring during the secretion of the complex. Moreover, the fragment 1-45 of Zymogen granule protein 16 homolog B was identified in whole saliva in paediatric age. Interestingly, the function of this protein is not well established until now and the terminal sequence of the fragment identified does not correspond to the sequence reported in international data banks (Swiss Prot accession number Q96DA0). Further studies are in progress on the meaning of these findings.
Currently, by the top-down proteomic platforms a repertoire of 256 masses belonging to naturally occurring proteins and peptides consistently detected by RP-HPLC-ESI-MS analysis of the acidic soluble fraction of human whole saliva has been characterized. Of them, 233 have been identified, while 23 are still pending for the definitive characterization. 
Proteomic platforms can be classified in bottom-up strategies, which analyze the sample after proteolytic digestion, and top-down strategies, which analyze the intact naturally occurring proteome. Bottom-up platforms are high-throughput because they can investigate a large number of proteins, regardless of their dimension. Nonetheless, information on post-translational modifications (PTMs) can be lost, especially those regarding naturally occurring cleavages and alternative splicing. Top-down platforms cannot cover vast proteomes, however, they can disclose subtle structural variations occurring during protein maturation and allow label-free relative quantifications in an unlimited number of samples. A review describing the meaning of different proteomic platforms has been recently published .
3) Investigation of the age related composition variability, exploring saliva in the pediatric age.
The detection of high amount of thymosin β4 (Tβ4) in pre-term newborn saliva but not in the adult induced to further investigate the properties of this interesting peptide by integrated proteomic and immunohistochemical platforms. The evaluation of the level of thymosin β4 evidenced that this peptide is highly expressed in saliva of human newborns but not in adults. Immunoreactivity for Tβ4 in human salivary glands showed high quantities before birth, followed by down-regulation of expression in adulthood. In contrast, Tβ4 is detected in tumors of salivary glands, suggesting that tumor cells might utilize fetal programs, including Tβ4 synthesis. Immunohistochemical analyses in the gastrointestinal tract showed strong reactivity for Tβ4 in enterocytes during development, but weak immunostaining in mature enterocytes. In colorectal cancer, the association of a high expression of Tβ4 with epithelial-mesenchymal transition was observed. On the basis of these data, the process ofepithelial-mesenchymal transition could represent the unifying process explaining the role of Tβ4 during fetal development and in cancer progression .
4) Investigation of the variations of human salivary proteome in different pathologies.
The analysis of the variations detectable in whole saliva of Down Syndrome (DS) patients is almost concluded and the study has been recently submitted to Molecolar and Cellular Proteomics. The studies on the salivary proteome on different tumor pathologies in the pediatric age, before and after drug or radiation treatment, that ones on different patients affected by schizofrenia and other bipolar disorders, celiac patients, as well as patients with the Wilson disease are in progress. Two chapters on books have been published [4,5].
The first, inserted on a book on autism, describes the results obtained in studies previously carried out evidencing that phosphorylation level of specific salivary phospho-peptides, namely statherin, histatin 1 and acidic proline-rich proteins (both entire and truncated isoforms) were significantly lower in autistic patients, being hypo-phosphorylation of at least one peptide observed in 18 ASD subjects (66%). Developmental scale assessment (Griffith or WISC-R), possible only on 14 ASD, highlighted a normal to border-line cognitive development on 10 subjects, all included in the hypo-phosphorylated group. Phosphorylation of salivary peptides is under the control of Golgi casein kinase common to many organs and tissues, CNS included. The data obtained on saliva of preterm newborns suggest that activity of the kinase involved in aPRP phosphorylation increases with PCA, synchronizing with that of at term newborns and reaching the adult levels at about 500-600 days of PCA, in concomitance with the beginning of deciduous dentition. Thus, hypo-phosphorylation of salivary peptide in the subgroup of ASD subjects suggests potential asynchronies in the phosphorylation of other secretory proteins, which could be relevant in the CNS development either during foetal life or in early infancy. Further studies are necessary to increase the statistics, to structurally characterize the Golgi casein kinase and to establish the proteins phosphorylated by this kinase during the human CNS development.
The second, inserted in a book on dysphagia, describes the different function of saliva, salivary reflexes and the effect of different hormones and drugs on salivary secretion. Moreover highlight the principal families of salivary secretory proteins and recent results obtained in the study of the human salivary proteome.
5) Further investigations
Research on the antimicrobial, antiviral activity of some proline-rich peptide of human and pig origin are in progress, as well as the study of some proline-rich protein on the bitter taste perception. Moreover, some studies are in progress for the top-down characterization of the rat salivary proteome. The study of the animal kingdom offer always interesting examples of how evolution has generated different structures in order to fulfill similar purposes. Comparative studies of the proteins present in whole saliva of some mammals allow establishing molecular similarities and differences with respect to human saliva and to gather precious information to better understand the function of different families of human salivary proteins. Finally, the development of a CE-ESI-MS targeted platform for the absolute quantitative determination of some salivary peptides is in progress.
 Castagnola M, Cabras T, Iavarone F, Vincenzoni F, Vitali A, Pisano E, Nemolato S, Scarano E, Fiorita A, Vento G, Tirone C, Romagnoli C, Cordaro M, Paludetti G, Faa G, Messana I. Top-down platform for deciphering the human salivary proteome. J. Matern. Fetal Neonatal Med. 25 (Suppl 5) (2012) 27-43.
 Messana I, Cabras T, Iavarone F, Vincenzoni F, Urbani A, Castagnola M. Unraveling the different proteomic platforms. J Sep Sci. 2012 in press PMID: 23212829
 Faa G, Nemolato S, Cabras T, Fanni D, Gerosa C, Fanari M, Locci A, Fanos V, Messana I, Castagnola M.
Thymosin β4 expression reveals intriguing similarities between fetal and cancer cells. Ann. N. Y. Acad. Sci. 1269(1) (2012) 53-60.
 Castagnola M, Messana I, Cabras T, Iavarone F, Fanali C, Morelli A, Pecoraro AM, Neri G, Torrioli MG, Gurrieri F. Hypo-phosphorylation of salivary peptidome as a clue of the molecular pathogenesis of autism spectrum disorders. In The Comprehensive Guide to Autism · V.B.Patel, V.R.Preedy, C.R. Martin Eds. pringer, London 2012 - Article ID: 331196 · Chapter ID: 87
 Ekström, J., Khosravani, N., Castagnola M., Messana I. Saliva and the control of its secretion. In "Dysphagia. Diagnosis and treatment." Olle Ekberg Ed., In Medical Radiology & Diagnostic Imaging Series. Reiser M.F., Hricak, H., Knauth, M. Eds., Springer-Verlag, Berlin-Heidelberg - 2012. Part I, Chapter 2, pp. 19-48.