N- and O-linked glycosylation site profiling of the human basic salivary proline-rich protein 3M.

In the present study, we show that the heterogeneous mixture of glycoforms of the basic salivary proline-rich protein 3M, encoded by PRB3-M locus, is a major component of the acidic soluble fraction of human whole saliva in the first years of life. Reversed-phase high-performance liquid chromatography with high-resolution electrospray ionization mass spectrometry analysis of the intact proteoforms before and after N-deglycosylation with Peptide-N-Glycosidase F and tandem mass spectrometry sequencing of peptides obtained after Endoproteinase GluC digestion allowed the structural characterization of the peptide backbone and identification of N- and O-glycosylation sites. The heterogeneous mixture of the proteoforms derives from the combination of 8 different neutral and sialylated glycans O-linked to Threonine 50, and 33 different glycans N-linked to Asparagine residues at positions 66, 87, 108, 129, 150, 171, 192, and 213.

Chrono-proteomics of human saliva: variations of the salivary proteome during human development.

An important contribution to the variability of any proteome is given by the time dimension that should be carefully considered to define physiological modifications. To this purpose, whole saliva proteome was investigated in a wide age range. Whole saliva was collected from 17 preterm newborns with a postconceptional age at birth of 178-217 days. In these subjects sample collection was performed serially starting immediately after birth and within about 1 year follow-up, gathering a total of 111 specimens. Furthermore, whole saliva was collected from 182 subjects aged between 0 and 17 years and from 23 adults aged between 27 and 57 years. The naturally occurring intact salivary proteome of the 316 samples was analyzed by low- and high-resolution HPLC-ESI-MS platforms. Proteins peculiar of the adults appeared in saliva with different time courses during human development. Acidic proline-rich proteins encoded by PRH2 locus and glycosylated basic proline-rich proteins encoded by PRB3 locus appeared following 180 days of postconceptional age, followed at 7 months (±2 weeks) by histatin 1, statherin, and P-B peptide. The other histatins and acidic proline-rich proteins encoded by PRH1 locus appeared in whole saliva of babies from 1 to 3 weeks after the normal term of delivery, S-type cystatins appeared at 1 year (±3 months), and basic proline-rich proteins appeared at 4 years (±1 year) of age. All of the proteinases involved in the maturation of salivary proteins were more active in preterm than in at-term newborns, on the basis of the truncated forms detected. The activity of the Fam20C kinase, involved in the phosphorylation of various proteins, started around 180 days of postconceptional age, slowly increased reaching values comparable to adults at about 2 years (±6 months) of age. Instead, MAPK14 involved in the phosphorylation of S100A9 was fully active since birth also in preterm newborns.

Significant modifications of the salivary proteome potentially associated with complications of Down syndrome revealed by top-down proteomics.

People with Down syndrome, a frequent genetic disorder in humans, have increased risk of health problems associated with this condition. One clinical feature of Down syndrome is the increased prevalence and severity of periodontal disease in comparison with the general population. Because saliva plays an important role in maintaining oral health, in the present study the salivary proteome of Down syndrome subjects was investigated to explore modifications with respect to healthy subjects. Whole saliva of 36 Down syndrome subjects, divided in the age groups 10-17 yr and 18-50 yr, was analyzed by a top-down proteomic approach, based on the high performance liquid chromatography-electrospray ionization-MS analysis of the intact proteins and peptides, and the qualitative and quantitative profiles were compared with sex- and age-matched control groups. The results showed the following interesting features: 1) as opposed to controls, in Down syndrome subjects the concentration of the major salivary proteins of gland origin did not increase with age; as a consequence concentration of acidic proline rich proteins and S cystatins were found significantly reduced in older Down syndrome subjects with respect to matched controls; 2) levels of the antimicrobial α-defensins 1 and 2 and histatins 3 and 5 were significantly increased in whole saliva of older Down syndrome subjects with respect to controls; 3) S100A7, S100A8, and S100A12 levels were significantly increased in whole saliva of Down syndrome subjects in comparison with controls. The increased level of S100A7 and S100A12 may be of particular interest as a biomarker of early onset Alzheimer's disease, which is frequently associated with Down syndrome.

Top-down HPLC-ESI-MS detection of S-glutathionylated and S-cysteinylated derivatives of cystatin B and its 1-53 and 54-98 fragments in whole saliva of human preterm newborns.

Analysis by a HPLC-ESI-MS top-down proteomic platform of specimens of human preterm newborn whole saliva evidenced high relative amounts of cystatin B and its S-glutathionylated,S-cysteinylated, and S-S 2-mer (on Cys(3)) derivatives, decreasing as a function of postconceptional age (PCA). The percentage of S-unmodified cystatin B was higher than the S-modified isoforms in the early PCA period, differently from adults where cystatin B was detectable only as S-modified derivatives. The percentage of S-modified derivatives increased as a function of PCA, reaching at the normal term of delivery values similar to those determined in at-term newborns, babies, and adults. Moreover, in the early PCA period, high relative amounts of the 1-53 and 54-98 cystatin B fragments were detected, decreasing as a function of PCA and disappearing at the normal term of delivery. In agreement with intact cystatin B, fragment 1-53 was detectable as S-unmodified and S-modified derivatives, and their percentages changed accordingly with the percentages of intact proteins, suggesting that the fragmentation process could be subsequent to and independent from the S-modification of the protein. This study highlights specific enzymatic activity in the oral cavity of preterm newborns not present in at-term newborns and adults, which can be a clue to specialized pathways occurring during fetal oral development.

The surprising composition of the salivary proteome of preterm human newborn.

Saliva is a body fluid of a unique composition devoted to protect the mouth cavity and the digestive tract. Our high performance liquid chromatography (HPLC)-electrospray ionization-MS analysis of the acidic soluble fraction of saliva from preterm human newborn surprisingly revealed more than 40 protein masses often undetected in adult saliva. We were able to identify the following proteins: stefin A and stefin B, S100A7 (two isoforms), S100A8, S100A9 (four isoforms), S100A11, S100A12, small proline-rich protein 3 (two isoforms), lysozyme C, thymosins ß(4) and ß(10), antileukoproteinase, histone H1c, and α and γ globins. The average mass value reported in international data banks was often incongruent with our experimental results mostly because of post-translational modifications of the proteins, e.g. acetylation of the N-terminal residue. A quantitative label-free MS analysis showed protein levels altered in relation to the postconceptional age and suggested coordinate and hierarchical functions for these proteins during development. In summary, this study shows for the first time that analysis of these proteins in saliva of preterm newborns might represent a noninvasive way to obtain precious information of the molecular mechanisms of development of human fetal oral structures.

HPLC-ESI-MS and MS/MS structural characterization of multifucosylated N-glycoforms of the basic proline-rich protein IB-8a CON1+ in human saliva.

This study describes 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. 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 showing from zero to four additional fucoses in the antennal region. The sixth glycoform carries a monoantennary monofucosylated oligosaccharide. The glycoform cluster was detected on 28 of 71 adult saliva specimens. Level of fucosylation showed interindividual variability with the major relative abundance for the trifucosylated glycoform. Nonglycosylated IB-8a CON1(+) and the variant IB-8a CON1(-), lacking of the glycosylation site, have been also detected in human saliva.

Alterations of the salivary secretory peptidome profile in children affected by type 1 diabetes.

The acidic soluble fraction of whole saliva of type 1 diabetic children was analyzed by reversed phase (RP)(1)-HPLC-ESI-MS and compared with that of sex- and age-matched control subjects. Salivary acidic proline-rich phosphoproteins (aPRP), histatins, α-defensins, salivary cystatins, statherin, proline-rich peptide P-B (P-B), beta-thymosins, S100A8 and S100A9*(S100A9* corresponds to S100A9 vairant lacking the first four amino acids), as well some naturally occurring peptides derived from salivary acidic proline-rich phosphoproteins, histatins, statherin, and P-B peptide, were detected and quantified on the basis of the extracted ion current peak area. The level of phosphorylation of salivary acidic proline-rich phosphoproteins, histatin-1 (Hst-1), statherin and S100A9* and the percentage of truncated forms of salivary acidic proline-rich phosphoproteins was also determined in the two groups. The study revealed that statherin, proline-rich peptide P-B, P-C peptide, and histatins, were significantly less concentrated in saliva of diabetic subjects than in controls, while concentration of α-defensins 1, 2 and 4 and S100A9* was higher. The low concentration of P-C peptide was paralleled by high levels of some of its fragments. On the whole, the study highlighted the severe impairment of the repertoire of peptides involved in the safeguard of the oral cavity in children who have diabetes, as well as an higher concentration of the proinflammatory mediator S100A9* with respect to healthy children.

Characterization of two isoforms of human SPRR3 from saliva of preterm human newborn and autoptic fetal oral mucosa, parotid and submandibular gland samples.

RP-HPLC-ESI-MS profile of saliva samples from human preterm newborn showed a protein peak in the elution range 26.6-27.6min. Deconvolution of ESI-MS spectra revealed the presence of two proteins with average molecular mass (Mav) values of 17,239+/-3Da and 18,065+/-3Da in 9 samples, with Mav value of 17,239+/-3Da in 4 samples and Mav value of 18,065+/-3Da in 2 samples. MALDI-TOF-MS analysis of tryptic digest allowed identifying the proteins as two isoforms of small proline-rich protein 3 and cDNA amplification of RNA extracts from oral mucosa, parotid and submandibular gland samples, obtained at fetal autopsy, provided two nucleotide sequences in agreement with those reported in the literature. The two proteins differ for an octapeptide repeat (GCTKVPEP) and the substitution Leu-->Val, at position 148 and 140 of the mature form of the 18,065 and 17,239Da protein, respectively. During maturation the two proteins undergo two post-translational modifications, corresponding to N-terminal acetylation and removal of the initiator methionine. cDNA amplification did not allow to clarify if the proteins found in saliva originated from cellular shedding of the epithelium and/or secretion.

Trafficking and postsecretory events responsible for the formation of secreted human salivary peptides: a proteomics approach.

To elucidate the localization of post-translational modifications of different classes of human salivary proteins and peptides (acidic and basic proline-rich proteins (PRPs), Histatins, Statherin, P-B peptide, and "S type" Cystatins) a comparative reversed phase HPLC-ESI-MS analysis on intact proteins of enriched granule preparations from parotid and submandibular glands as well as parotid, submandibular/sublingual (Sm/Sl), and whole saliva was performed. The main results of this study indicate the following. (i) Phosphorylation of all salivary peptides, sulfation of Histatin 1, proteolytic cleavages of acidic and precursor basic PRPs occur before granule storage. (ii) In agreement with previous studies, basic PRPs are secreted by the parotid gland only, whereas all isoforms of acidic PRPs (aPRPs) are secreted by both parotid and Sm/Sl glands. (iii) Phosphorylation levels of aPRPs, Histatin 1, and Statherin are higher in the parotid gland, whereas the extent of cleavage of aPRP is higher in Sm/Sl glands. (iv) O-Sulfation of tyrosines of Histatin 1 is a post-translational modification specific for the submandibular gland. (v) The concentration of Histatin 3, Histatin 5, and Histatin 6, but not Histatin 1, is higher in parotid saliva. (vi) Histatin 3 is submitted to the first proteolytic cleavage (generating Histatins 6 and 5) during granule maturation, and it occurs to the same relative extent in both glands. (vii) The proteolytic cleavages of Histatin 5 and 6, generating a cascade of Histatin 3 fragments, take place after granule secretion and are more extensive in parotid secretion. (viii) Basic PRPs are cleaved in the oral cavity by unknown peptidases, generating various small proline-rich peptides. (ix) C-terminal removal from Statherin is more extensive in parotid saliva. (x) P-B peptide is secreted by both glands, and its relative quantity is higher in submandibular/sublingual secretion. (xi) In agreement with previous studies, S type Cystatins are mainly the product of Sm/Sl glands.

HPLC-ESI-MS analysis of oral human fluids reveals that gingival crevicular fluid is the main source of oral thymosins beta(4) and beta(10).

Thymosin beta(4) (Tbeta(4)), its sulfoxide, and thymosin beta(10 )(Tbeta(10)) were detected in human saliva and identified by different strategies based on RP HPLC coupled to electrospray multidimensional IT MS. Tbeta(4 )was almost always detected in whole saliva, its sulfoxide sporadically, Tbeta(10) rarely. Tbeta(4) was undetectable in parotid saliva and less concentrated in submandibular/sublingual saliva than in whole saliva. Analysis of gingival crevicular fluid revealed high relative amounts of Tbeta(4), Tbeta(4) sulfoxide, and Tbeta(10) in all the samples. Tbeta(4) mean concentration was 200 times higher in crevicular fluid (20 micromol/L, N = 9) than in whole saliva (0.1 micromol/L, N = 9). Crevicular fluid concentration of Tbeta(4 )(ca. 5% represented by its sulfoxide) and beta(10 )significantly correlated (r = 0.856; N = 9), and their ratio was about 5. A significant correlation was also observed between Tbeta(4 )concentrations in whole saliva and gingival crevicular fluid (r = 0.738; N = 9). Immunohistochemical analysis of the major salivary glands showed that immunoreactivity for Tbeta(4) is restricted to ductal cells, with minor degree of focal positivity in some acinar cells. On the whole, results indicate that gingival sulcus is a main, although not the sole, source for oral Tbeta(4 )and Tbeta(10).

Usefulness of real time PCR for the differentiation and quantification of 652 and JP2 Actinobacillus actinomycetemcomitans genotypes in dental plaque and saliva.

BACKGROUND: The aim of our study is to describe a fast molecular method, able to distinguish and quantize the two different genotypes (652 and JP2) of an important periodontal pathogen: Actinobacillus actinomycetemcomitans. The two genotypes show differences in the expression of an important pathogenic factor: the leukotoxin (ltx). In order to evidence this, we performed a real time PCR procedure on the ltx operon, able to recognize Aa clinical isolates with different leukotoxic potentials. METHODS: The specificity of the method was confirmed in subgingival plaque and saliva specimens collected from eighty-one Italian (Sardinian) subjects with a mean age of 43.9, fifty five (68 %) of whom had various clinical forms of periodontal disease. RESULTS: This procedure showed a good sensitivity and a high linear dynamic range of quantization (10(7)-10(2) cells/ml) for all genotypes and a good correlation factor (R2 = 0.97-0.98). Compared with traditional cultural methods, this real time PCR procedure is more sensitive; in fact in two subgingival plaque and two positive saliva specimens Aa was only detected with the molecular method. CONCLUSION: A low number of Sardinian patients was found positive for Aa infections in the oral cavity, (just 10 positive periodontal cases out of 81 and two of these were also saliva positive). The highly leukotoxic JP2 strain was the most representative (60 % of the positive specimens); the samples from periodontal pockets and from saliva showed some ltx genotype for the same patient. Our experience suggests that this approach is suitable for a rapid and complete laboratory diagnosis for Aa infection.

Top-down platform for deciphering the human salivary proteome.

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 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 is presented in this study. Of them, 233 have been identified, while 23 are still pending for the definitive characterization. The present review reports average and mono-isotopic masses of the peptides and proteins detected, RP-HPLC elution times, PTMs, origin and quali-quantitative variations observed in several physiological and pathological conditions. The information reported can be a reference for users of top-down RP-HPLC-ESI-MS proteomic platforms applied to the study of the human salivary proteome as well as of other human bodily fluids.

Distribution of human papillomavirus genotypes in sardinian patients with oral squamous cell carcinoma.

Human papillomaviruses (HPVs) seem to play an important role in the pathogenesis of gynecological carcinomas and in head and neck carcinomas. The aim of this study was to detect and genotype HPVs in fresh oral squamous cell carcinoma (OSCC) from a Sardinian population, and to determine whether HPV presence was significantly associated with the development of OSCC.The oral mucosa tissues were obtained from 120 samples (68 OSCC and 52 control samples) taken from a Sardinian population seen at the Dental Clinic of the Department of Surgery and Odontostomatological Sciences, University of Cagliari (Italy) and the " Ospedale SS Trinità", Cagliari (A.S.L. 8) between 2007 and 2008. PCR was used for the detection of HPV DNA and the genotype was determined by DNA sequencing. The frequency of HPV infection was evaluated in relation to age, sex, smoking and alcohol use. Statistical analysis was performed using the SPSS 11.5 software.The results showed the presence of HPV-DNA in 60.3% of OSCC with HPV-16 (51.2%) being the most frequent genotype. In these Sardinian OSCC patients, HPV-DNA was detected more in males (65.8%) than in females (34.1%) while controls show a 0% of HPV presence. HPV positive was highly associated with OSCC among subjects with a history of heavy tobacco and alcohol use and among those with no such history.A greater frequency of high risk HPV presence was observed in patients with OSCC compared to health control patients. In addition these results suggested that oral HPV presence could be associated in OSCC subjects. Our results need more analyses to detect the HPV-DNA integration into tumoral cells.

Age-dependent modifications of the human salivary secretory protein complex.

Physiological variability of the naturally occurring, human salivary secretory peptidome was studied as a function of age. The qualitative and quantitative changes occurring in the secretion of proteins/peptides specific to the oral cavity (i.e., basic salivary proline-rich proteins, salivary acidic proline-rich phosphoproteins, statherin, proline-rich peptide P-B, salivary cystatins, and histatins) were investigated by high-performance liquid chromatography-electrospray ionization-mass spectrometry in 67 subjects aged between 3 and 44 years. Subjects were divided into five age groups: group A, 8 donors, 3-5 years; group B, 11 donors, 6-9 years; group C, 20 donors, 10-12 years; group D, 15 donors, 13-17 years; group E, 13 donors, 24-44 years. Basic salivary proline-rich proteins, almost undetectable in the 3-5 and 6-9 years groups, reached salivary levels comparable to that of adults (24-44 years) around puberty. Levels of peptide P-D, basic peptide P-F, peptide P-H, peptide P-J (a new basic salivary proline-rich protein characterized in this study), and basic proline-rich peptide IB-1 were significantly higher in the 10-12-year-old group than in the 3-5-year-old group, whereas the increase of proline-rich peptide II-2 was significant only after the age of 12 years. The concentration of salivary acidic proline-rich phosphoproteins, histatin-3 1/24, histatin-3 1/25, and monophosphorylated and diphosphorylated cystatin S showed a minimum in the 6-9-year-old group. Finally, the histatin-1 concentration was significantly higher in the youngest subjects (3-5 years) than in the other groups.

Detection and genotyping of human papillomavirus DNA in samples from healthy Sardinian patients: a preliminary study.

The human papillomavirus (HPV) is involved in the development of different benign and malignant lesions that include in particular squamous tumours of the cervix, skin and the respiratory tracts. In particular, the 'high risk' HPV type 16 (HPV 16) causes genito-rectal epithelial cancers and is suspected of causing epithelial cancers of the head and neck. To determine the presence and genotypes of HPV was determined in saliva samples from 164 subjects recruited from the Department of Surgery and Odontostomatological Sciences (University of Cagliari). For this study a sensitive seminested polymerase chain reaction (PCR) method was used to detect HPV-DNA; moreover in all positive samples, HPV genotyping was based on sequencing of the HPV genome L1 region. The results obtained with these patients (who were ethnically homogeneous), showed an interesting percentage of positive samples for HPV-DNA (30 samples out of 164-18.3%). Only two HPV genotypes have been identified in these patients, HPV 16 and HPV 31 with 76.7% and 23.3% of the positive specimens, respectively, both correlating with high carcinogenic risk. This preliminary result leads us to reflect on the presence of HPV in saliva, in particular in young asymptomatic subjects (15.38%), and its prognostic value for the possible incidence in Sardinia of oral carcinoma.

HPLC-MS characterization of cyclo-statherin Q-37, a specific cyclization product of human salivary statherin generated by transglutaminase 2.

In the present study the analytical potential of HPLC-MS/MS was utilized for the structural characterization of a post-translational modification of statherin. Human salivary statherin (M(av)5380.0 +/- 0.3 Da) is transformed by the action of transglutaminase 2 into a cyclic derivative with an average molecular mass of 5363.0 +/- 0.3 Da. The intra-molecular bridge is generated by the loss of an ammonia molecule between the unique Ione-pair donating nucleophile Lys-6 and one acceptor among the seven glutamine residues of statherin. Digestion of the cyclic derivative with chymotrypsin, proteinase K, and carboxypeptidase Y, monitored by HPLC-electrospray ionization-ion trap-mass spectrometric analysis, demonstrated that cyclization involved almost specifically Gln-37 (> 95%), with the percentage of Gln-39 implicated in the cross-linkiing being less than 5%. The main derivative was named cyclostatherin Q37. Guineapig transglutaminase 2 showed high affinity for statherin in vitro (Km = 0.65 +/- 0.06 microM). Cyclo-statherin was detected in vivo by HPLC-electrospray ionization ion trap-mass spectrometry analysis of whole human saliva and it accounted for about 1% of total statherin. Detection of cyclo-statherin in whole saliva is suggestive of a putative role of this molecule in the formation of the "oral protein pellicle".

Peptides of human gingival crevicular fluid determined by HPLC-ESI-MS.

The acidic-soluble protein content of human gingival crevicular fluid was analyzed by reverse-phase high-performance liquid chromatography (RP-HPLC), and the eluent deriving from the chromatography separation was directly introduced into an ion-trap mass spectrometer through electrospray ionization (ESI-IT MS). By this technique the molecular weight of peptides/proteins was determined with a precision of approximately 1/10,000 amu. On the basis of the chromatographic behavior and the knowledge of the molecular mass value, some peptides and proteins soluble in acidic solution were unambiguously recognized. Besides high quantities of human serum albumin, alpha-defensins 1-4 and minor amounts of cystatin A, statherin, basic PB salivary peptide and other unidentified components were detected. The presence of alpha-defensins in gingival crevicular fluid is in agreement with their relevant contribution to protein composition deriving from granulocyte secretions. Other peptides and proteins abundant in human saliva, such as proline-rich proteins (PRPs) and histatins, were not detected in gingival crevicular fluid. Further investigations will be necessary to establish the origin of statherin and PB salivary peptide in gingival crevicular fluid.