Comparison of N-linked Glycoproteins in Human Whole Saliva, Parotid, Submandibular, and Sublingual Glandular Secretions Identified using Hydrazide Chemistry and Mass Spectrometry

INTRODUCTION: Saliva is a body fluid that holds promise for use as a diagnostic fluid for detecting diseases. Salivary proteins are known to be heavily glycosylated and are known to play functional roles in the oral cavity. We identified N-linked glycoproteins in human whole saliva, as well as the N-glycoproteins in parotid, submandibular, and sublingual glandular fluids. MATERIALS AND METHODS: We employed hydrazide chemistry to affinity enrich for N-linked glycoproteins and glycopeptides. PNGase F releases the N-peptides/proteins from the agarose-hydrazide resin, and liquid chromatography-tandem mass spectrometry was used to identify the salivary N-glycoproteins. RESULTS: A total of 156 formerly N-glycosylated peptides representing 77 unique N-glycoproteins were identified in salivary fluids. The total number of N-glycoproteins identified in the individual fluids was: 62, 34, 44, and 53 in whole saliva, parotid fluid, submandibular fluid, and sublingual fluid, respectively. The majority of the N-glycoproteins were annotated as extracellular proteins (40%), and several of the N-glycoproteins were annotated as membrane proteins (14%). A number of glycoproteins were differentially found in submandibular and sublingual glandular secretions. CONCLUSIONS: Mapping the N-glycoproteome of parotid, submandibular, and sublingual saliva is important for a thorough understanding of biological processes occurring in the oral cavity and to realize the role of saliva in the overall health of human individuals. Moreover, identifying glycoproteins in saliva may also be valuable for future disease biomarker studies.     

A functional and chemical study of radiation effects on rat parotid and submandibular/sublingual glands.

The aim of this study was to monitor composition and rate of secretion of rat parotid and submandibular/sublingual saliva following local single doses of X-rays ranging from 5 to 20 Gy. Pilocarpine-stimulated samples of parotid and submandibular/sublingual saliva were simultaneously collected with miniaturized Lashley cups before and 1-30 days after irradiation. The lag phase (period between injection of pilocarpine and start of the secretion) and flow rate were recorded and the concentrations of sodium, potassium, calcium, phosphate, and amylase were measured. With increasing dose and time, the salivary flow rate as well as sodium concentration decreased, while potassium concentrations increased throughout the follow-up period. The lag phase and the concentration of amylase reached their maximum at 3 and 10 days after irradiation, respectively. The changes in lag phase and flow rate were most obvious after doses of 15 or 20 Gy and showed a great similarity for parotid and submandibular/sublingual saliva. No dose-response relationship was observed for the changes in concentrations of calcium and phosphate. It is concluded that for radiation doses of 10 Gy and above, irreversible changes (lag phase, flow rate, potassium, sodium) were observed. A saturation of the irradiation effects (lag phase, flow rate) seems to exist at doses larger than 15 Gy. No significant differences were observed between the radiation-induced functional changes in parotid and submandibular/sublingual salivary gland tissue.     

Mapping Relative Differences in Human Salivary Gland Secretions by Dried Saliva Spot Sampling and nanoLC–MS/MS

Dried saliva spot sampling is a minimally invasive technique for the spatial mapping of salivary protein distribution in the oral cavity. In conjunction with untargeted nano‐flow liquid chromatography tandem mass spectrometry (nanoLC–MS/MS) analysis, DSS is used to compare the proteomes secreted by unstimulated parotid and submandibular/sublingual salivary glands. Two hundred and twenty proteins show a statistically significant association with parotid gland secretion, while 30 proteins are at least tenfold more abundant in the submandibular/sublingual glands. Protein identifications and label‐free quantifications are highly reproducible across the paired glands on three consecutive days, enabling to establish the core proteome of glandular secretions categorized into eight salivary protein groups according to their biological functions. The data suggest that the relative contributions of the salivary glands fine‐tune the biological activity of human saliva via medium‐abundant proteins. A number of biomarker candidates for Sjögren's syndrome are observed among the gland‐specifically expressed proteins, which indicates that glandular origin is an important factor to consider in salivary biomarker discovery.     

The proteomes of human parotid and submandibular/sublingual gland salivas collected as the ductal secretions

Saliva is a body fluid with important functions in oral and general health. A consortium of three research groups catalogued the proteins in human saliva collected as the ductal secretions: 1166 identifications--914 in parotid and 917 in submandibular/sublingual saliva--were made. The results showed that a high proportion of proteins that are found in plasma and/or tears are also present in saliva along with unique components. The proteins identified are involved in numerous molecular processes ranging from structural functions to enzymatic/catalytic activities. As expected, the majority mapped to the extracellular and secretory compartments. An immunoblot approach was used to validate the presence in saliva of a subset of the proteins identified by mass spectrometric approaches. These experiments focused on novel constituents and proteins for which the peptide evidence was relatively weak. Ultimately, information derived from the work reported here and related published studies can be used to translate blood-based clinical laboratory tests into a format that utilizes saliva. Additionally, a catalogue of the salivary proteome of healthy individuals allows future analyses of salivary samples from individuals with oral and systemic diseases, with the goal of identifying biomarkers with diagnostic and/or prognostic value for these conditions; another possibility is the discovery of therapeutic targets.     

Major salivary gland output differs between users and non-users of specific medication categories

Background: The intake of medications is a major aetiologic factor of xerostomia. The purpose of this study was to investigate the selective influence of medication categories on flow rates of individual major salivary glands. Methods: The effect of each medication category on salivary flow rates was determined by dichotomy comparisons between users and non‐users. A total of 246 patients were included, 79 males and 167 females aged 13–92 years (mean 63 years). Of these, 200 used medications, which were grouped according to their category. A comprehensive medical and oral examination was performed. Both unstimulated and stimulated saliva was collected separately from the parotid and submandibular/sublingual glands. Results: Parotid flow rate was decreased among users of tranquillisers and sedatives (unstimulated flow), cardiovascular drugs and gastrointestinal drugs (stimulated flow). Submandibular/sublingual unstimulated output was lower in patients taking cardiovascular drugs, antihistamines, tranquillisers/sedatives and antidepressants, while the stimulated flow, in those taking cardiovascular drugs, antihistamines, tranquillisers/sedatives and gastrointestinal drugs. Conclusions: Users of many common medication categories display significantly reduced unstimulated and/or stimulated salivary flow rate from the major salivary glands compared with non‐users. A larger number of medication categories are associated with reductions in salivary flow rate from submandibular/sublingual glands than parotid glands.     

Factors That Influence the Extensional Rheological Property of Saliva

The spinnbarkeit of saliva reflects the ability of saliva to adhere to surfaces within the mouth, thereby serving as a protective role and aiding in lubrication. Therefore, alterations in the extensional rheology of saliva may result in the loss in adhesiveness or the ability to bind onto surfaces. Mucin glycoproteins and their structures are known to be important factors for the extensional rheological properties of saliva. The conformation of mucin depends on factors such as pH and ionic strength. Chewing is one of the main stimuli for salivary secretion but creates significant sheer stress on the salivary film which could influence mouthfeel perceptions. The current study investigates the possible factors which affect the extensional rheological properties of saliva by comparing submandibular/sublingual saliva with different oral stimuli within the same group of subjects. Unstimulated and stimulated saliva (chew, smell and taste) salivas were collected primarily from submandibular/sublingual glands. The saliva samples were measured for Spinnbarkeit followed by the measuring mucin, total protein, total calcium and bicarbonate concentrations. The results indicated correlations between rheological properties and mucin/ion concentrations. However, chewing stimulated submandibular/sublingual saliva is shown to have significantly lower Spinnbarkeit, but factors such as mucin, protein and calcium concentrations did not account for this variation. Analysis of the concentration of bicarbonate and pH appears to suggest that it has a prominent effect on extensional rheology of saliva.     

Differences in saliva collection location and disparities in baseline and diurnal rhythms of alpha-amylase: A preliminary note of caution

Identified in the early 1980s as a surrogate marker of the sympathetic nervous system component of the stress response, there has been renewed interest in measuring salivary alpha-amylase (sAA) to test biosocial models of stress vulnerability. This brief report presents studies that document that oral fluids from the parotid and submandibular gland areas had higher sAA values than did whole saliva specimens, and sAA values in whole saliva were higher than levels measured in oral fluids from the sublingual gland area. sAA in oral fluids from the parotid and submandibular gland areas showed the highest and more pronounced diurnal variation than levels in whole saliva, and sAA in sublingual saliva showed the lowest and shallowest diurnal variation. When this source of inherent variability in sAA activity levels is not controlled for by collecting oral fluids consistently from specific gland areas, the detection of individual differences, associations between sAA and “behavioral” variables, and intra-individual change in sAA levels may be compromised. Awareness, and management, of this ubiquitous source of measurement error in sAA are essential to ensure the success of future research on the correlates and concomitants of sAA levels, stress-related reactivity and recovery, and diurnal variation.     

Differences in saliva collection location and disparities in baseline and diurnal rhythms of alpha-amylase: a preliminary note of caution

Identified in the early 1980s as a surrogate marker of the sympathetic nervous system component of the stress response, there has been renewed interest in measuring salivary alpha-amylase (sAA) to test biosocial models of stress vulnerability. This brief report presents studies that document that oral fluids from the parotid and submandibular gland areas had higher sAA values than did whole saliva specimens, and sAA values in whole saliva were higher than levels measured in oral fluids from the sublingual gland area. sAA in oral fluids from the parotid and submandibular gland areas showed the highest and more pronounced diurnal variation than levels in whole saliva, and sAA in sublingual saliva showed the lowest and shallowest diurnal variation. When this source of inherent variability in sAA activity levels is not controlled for by collecting oral fluids consistently from specific gland areas, the detection of individual differences, associations between sAA and “behavioral” variables, and intra-individual change in sAA levels may be compromised. Awareness, and management, of this ubiquitous source of measurement error in sAA are essential to ensure the success of future research on the correlates and concomitants of sAA levels, stress-related reactivity and recovery, and diurnal variation.     

Adherence ofStreptococcus gordonii HG 222 in the presence of saliva

The influence of the presence of saliva from different salivary glands on the adherence ofStreptococcus gordonii strain HG 222 to saliva-coated polystyrene surfaces was tested. In the presence of undiluted parotid saliva or diluted whole, submandibular and sublingual saliva the adherence of HG 222 was enhanced by the formation of small aggregates on the attachment surface. In the presence of undiluted whole, submandibular and sublingual saliva large aggregates were formed and the adherence to saliva-coated polystyrene surfaces was inhibited.Adherence in the presence of whole saliva compared to adherence in buffer was decreased when lower densities of bacterial suspension were used, although in this case in the presence of whole saliva smaller bacterial aggregates were formed.In conclusion, these results suggest that the presence of saliva in solution may both enhance and decrease the adherence ofS. gordonii HG 222 to saliva-coated polystyrene surfaces, partly depending on the size of bacterial aggregates that are formed in the presence of saliva.Abbreviations CHW saliva clarified human whole saliva - PAR saliva parotid saliva - SM saliva submandibular saliva - SL saliva sublingual saliva     

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.     

Characterization of mouse salivary glands by water content and type

The thermoanalytical analysis was applied to samples of sublingual, submandibular and parotid glands from sexually mature mice of both sexes. Findings indicated that the three salivary glands show a behaviour of water release characteristic for each type of gland. Derivative thermogravimetry curves concerned with the sublingual and parotid glands belonging to male and female subjects exhibited overlapped results. As regards submandibular gland, instead, some differences emerged between subjects of different sex. Water content and types in sublingual, submandibular and parotid glands were discussed and related to the different morphological expression, histochemical reactivity and chemical composition of these organ tissues.     

Interaction of the salivary low-molecular-weight mucin (MG2) with Actinobacillus actinomycetemcomitans

Periodontitis is associated with the presence of certain Gram-negative bacteria in the oral cavity, among these Actinobacillus actinomycetemcomitans. In order to determine which types of salivary components interact with A. actinomycetemcomitans two strains (HG 1175 and FDC Y4) were incubated with whole saliva and individual glandular secretions, viz. parotid, submandibular, and sublingual saliva. Immunochemical analysis by immunoblotting of bacteria-bound salivary proteins showed that IgA, the low-molecular mucin MG2, parotid agglutinin, and a 300 kDa sublingual and submandibular glycoprotein, were bound to the bacterial strains tested. In addition, adherence of A. actinomycetemcomitans to salivary proteins in a solid-phase was studied. After electrophoresis and transfer of salivary proteins to nitrocellulose membranes A. actinomycetemcomitans adhered only to MG2. In this assay periodate treatment, mild acid hydrolysis or neuraminidase digestion of the saliva glycoproteins abolished binding of two clinical isolates (HG 1175 and NY 664), suggesting that sialic acid residues on MG2 are involved in the binding. In contrast, adherence of the smooth laboratory strain Y4 was not affected by removal of sialic acid residues or even periodate treatment of MG2.Abbreviations S-IgA Secretory IgA - MG1 high-molecular-weight mucin - MG2 low-molecular-weight mucin - EP-GP extra parotid-glycoprotein - PRPs proline-rich proteins - SNA Sambucus nigra agglutinin - MAA Maackia amurensis agglutinin - PNA peanut agglutinin - UEA Ulex europaeus agglutinin     

Effects of chronic clonidine administration on parasympathetic-evoked rat saliva.

Effects of chronic administration of clonidine on parasympathetic-evoked saliva from both parotid and submandibular glands were investigated. Clonidine at 1 mg/kg/day for 5 or 7 days caused a significant reduction in the salivary secretion (flow rate and total volume) evoked by parasympathetic nerve stimulation of parotid but not submandibular glands. Ion concentrations (Na, K and Ca) of parasympathetically nerve-evoked parotid saliva were not altered. However, the total protein concentration as well as output, amylase activity, and output of such saliva were markedly increased. Possible mechanisms for clonidine-induced increase in nerve-elicited salivary protein concentration include release of neuropeptides, and changes in adrenergic receptor binding which need further study.     

Expression and distribution of osteopontin and matrix metalloproteinase (MMP)-3 and -7 in mouse salivary glands

We investigated the expression and distribution of osteopontin in mouse salivary glands. Western blot analysis showed intense positive bands at the predicted molecular mass (about 60 kDa) in mouse parotid and sublingual glands. However, a cross-reacted band around 30 kDa was strongly detected in submandibular glands. Indirect immunofluorescent analysis showed that osteopontin was localized at the luminal (apical) membranes of the acinar cells in parotid and sublingual glands. However, it was not detected in acinar cells of submandibular glands. No expression was found in ductal cells of any glands. We also examined the expression of matrix metalloproteinase (MMP)-3 and -7. In parotid gland, MMP-3 was observed at 57 kDa, indicating a latent form, but MMP-7 was not detected. In contrast, MMP-7 definitely was observed at 28 kDa area in submandibular gland, whereas MMP-3 was not detected. These results suggest that osteopontin localizes at luminal sites of acinar cells and may be associated with saliva secretion in mouse salivary gland. It is also suggested that osteopontin may be cleaved by MMP-7 in mouse submandibular gland.     

Effect of parotid submandibular and sublingual saliva on wound healing in rats.

1. The effectiveness of wound licking with parotid, submandibular or sublingual saliva on wound healing was evaluated in selectively sialadenectomized rats. 2. The rate of healing of experimentally induced cutaneous wounds was evaluated macroscopically by photography at 0, 2, 4 and 6 days after surgery. 3. Sialadenectomy of all major glands significantly slowed down wound healing compared to sham-operated controls. 4. Parotid licking had no effect compared to controls; submandibular licking and sublingual licking appeared to be very effective. 5. The results suggest that saliva promotes wound healing in experimentally induced cutaneous wounds by communal licking; this is a result of the submandibular and sublingual saliva and not the parotid saliva.     

Localization of amylase and mucins in the major salivary glands of the mouse

Summary Antibodies against murine submandibular and sublingual mucins have been raised in rabbits. Both antisera appeared to be specific. Using these antibodies, the mucins were localized in the acinar cells of the submandibular and sublingual glands respectively.The dyed amylopectin method was used to estimate the activity of amylase in the salivary glands. The enzyme was localized either by a starch-substrate film method or with antibodies against purified parotid amylase. The activity of amylase in parotid homogenates is about 1000-fold higher than that in homogenates of either submandibular or sublingual glands, in which the activity was comparable. Amylase was localized in the acinar cells of the parotid gland with both localization techniques. In the sublingual gland, amylase was found predominantly in the stroma around the acini, and there was some evidence that amylase was present in the demilune cells as well. In the submandibular gland, contradictory results were obtained with both techniques. With the starch-substrate film method, amylase activity was found in the granular convoluted tubular cells, whereas immuno-reactive amylase could only be demonstrated in the acinar cells of this gland. It is concluded that in the submandibular gland amylase and mucin are present in the same cell type.     

Induction of a specific (LM) protein in the submandibular gland of the rat by repeated amputation of the lower incisor teeth

Chronic administration of the beta-adrenergic agonist isoproterenol (IPR) leads to marked hyperplastic/hypertrophic enlargements of the parotid and submandibular glands in rats and mice with concomitant changes in the composition of both the glands and the saliva. Conspicuous among the alterations of the submandibular saliva is the appearance of a 13,000 Mr protein, termed LM (large mobile) protein. Repeated amputation of the lower incisor teeth also causes enlargements of the major salivary glands in rats. In this study, we have compared the enlargements of submandibular glands of rats produced by IPR administration or teeth amputation with respect to the relative levels of the LM protein in gland extracts and saliva. Administration of IPR-HCl (40 mg/kg) twice daily for 5 days or amputation of the lower incisor teeth 3 times a week for 3 weeks resulted in a 2.2-fold increase in the weight of the submandibular gland. Amputation for one week led to a 1.4-fold increase in gland weight. Double immunodiffusion in agar antibodies against the purified LM protein gave a single precipitin line with gland extracts and saliva of IPR-treated and teeth-amputated rats, indicating immunological identity of the reacting antigens. No precipitin lines were seen with gland extracts or saliva of untreated rats. Immunoblots of pooled saliva obtained from IPR-treated or teeth-amputated rats revealed a single protein band of the same electrophoretic mobility in SDS-polyacrylamide gels when stained using anti-LM antibodies. The relative concentrations of LM protein in gland extracts and saliva were measured by a solid-phase enzyme-linked immunoabsorption assay using antibodies against the purified LM protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Developmental changes in the activities of prolinase and prolidase in rat salivary glands,and the effect of thyroxine administration

Summary As the salivary glands are interesting tissues to study proliferation, we studied the activities of prolinase and prolidase using Pro-Ala and Pro-Hyp as substrates, respectively, in developing rat salivary glands between day 1 and week 10 after birth. Developmental changes of prolinase activity in the submandibular and sublingual glands were similar to those in the parotid gland, which steadily increased and reached the adult level by 20–25 days after birth. However, the changes in the activity of prolidase in the submandibular and sublingual glands were different from those in the parotid gland: the activity in the parotid gland slowly increased with maturation and reached a maximum level on day 30, but the activity in the submandibular and sublingual glands continuously increased with maturation. When thyroxine was injected every two days from day 1 to day 19, both enzyme activities were induced precociously in the parotid gland but not in the submandibular and sublingual glands. On the study of regional distribution in rat tissues, the correlation coefficient between prolinase and prolidase activities was high in the peripheral but not high in the brain regions.These results indicate that the physiological roles of prolinase and prolidase are very similar but not the same.