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Saliva: a Dynamic Proteome

Boston University Goldman School of Dental Medicine, Department of Periodontology and Oral Biology, 700 Albany Street CABR W-201, Boston, MA 02118, USA

Correspondence: ^* corresponding author, helmer{at}bu.edu

	ABSTRACT

TOP ABSTRACT (1) INTRODUCTION (2) SALIVARY PROTEIN GENE... (3) POST-TRANSLATIONAL... (4) SALIVARY PROTEIN... (5) CONCLUSION REFERENCES

 
The proteome of whole saliva, in contrast to that of serum, is highly susceptible to a variety of physiological and biochemical processes. First, salivary protein secretion is under neurologic control, with protein output being dependent on the stimulus. Second, extensive salivary protein modifications occur in the oral environment, where a plethora of host- and bacteria-derived enzymes act on proteins emanating from the glandular ducts. Salivary protein biosynthesis starts with the transcription and translation of salivary protein genes in the glands, followed by post-translational processing involving protein glycosylation, phosphorylation, and proteolysis. This gives rise to salivary proteins occurring in families, consisting of structurally closely related family members. Once glandular secretions enter the non-sterile oral environment, proteins are subjected to additional and continuous protein modifications, leading to extensive proteolytic cleavage, partial deglycosylation, and protein-protein complex formation. All these protein modifications occur in a dynamic environment dictated by the continuous supply of newly synthesized proteins and removal by swallowing. Understanding the proteome of whole saliva in an environment of continuous turnover will be a prerequisite to gain insight into the physiological and pathological processes relevant to oral health, and be crucial for the identification of meaningful biomarkers for oral disease.

Key Words: saliva • proteomics • polymorphic • proteolysis • glycosylation • phosphorylation • degradation • complexation • diagnostics • oral

	(1) INTRODUCTION

TOP ABSTRACT (1) INTRODUCTION (2) SALIVARY PROTEIN GENE... (3) POST-TRANSLATIONAL... (4) SALIVARY PROTEIN... (5) CONCLUSION REFERENCES

 
The promise of understanding the functional significance of whole saliva, as well as its value for serving as a diagnostic fluid, is highly dependent on our ability to establish its composition. Most proteins and peptides present in whole saliva have undergone a complex series of molecular processes, which ultimately define their structures. The onset of these processes occurs at the biosynthetic level within the gland, while terminal processing of the protein/peptides takes place after secretion into the oral cavity. In the lifetime of a salivary protein, four principal phases can be distinguished. Phase 1 consists of the basic cellular process of protein biosynthesis, based on its genetic blueprint. Phase 2 is characterized by intracellular post-translational modifications prior to secretion of the protein into the ductal system. Phase 3 consists of modifications incurred during secretion and transit through the ductal tree. Phase 4 represents extensive modifications to salivary proteins after their release into the non-sterile environment of the oral cavity. This last phase has profound consequences for the proteome of whole saliva, since the population of proteins and peptides undergoes continued modifications in the time span between entry into and clearance from the oral cavity. The definition of the whole-saliva proteome is therefore highly variable, dependent on time and the nature and amounts of agents capable of protein/peptide modifications.

The purpose of this review is to summarize current knowledge on salivary protein alterations and modifications, from the moment the gene has been expressed in the gland until the moment the protein will be removed from the oral cavity by being swallowed. Recognizing the dynamic composition of the salivary proteome is important not only vis-à-vis salivary protein function, but also with regard to the growing interest in saliva-based diagnostics.

	(2) SALIVARY PROTEIN GENE TRANSLATION

TOP ABSTRACT (1) INTRODUCTION (2) SALIVARY PROTEIN GENE... (3) POST-TRANSLATIONAL... (4) SALIVARY PROTEIN... (5) CONCLUSION REFERENCES

 
The major salivary protein families together constitute > 95% of the salivary protein content. The genes encoding for the acidic proline-rich proteins are PRH1 and PRH2 (Maeda, 1985), for basic proline-rich proteins PRB1 to PRB4 (Maeda, 1985) and PBII(SMR3B) (Isemura, 2000), for amylase AMY1 (Nishide et al., 1986), for high-molecular-weight mucous glycoprotein MUC5B (Desseyn et al., 1997), for low-molecular-weight mucous glycoprotein MUC7 (Bobek et al., 1993), for agglutinin DMBT1 (Prakobphol et al., 2000), for cystatins CST1 to CST5 (Saitoh and Isemura, 1993), for histatins HIS1 and HIS2 (Sabatini and Azen, 1989), and for statherin STATH (Dickinson et al., 1987; Sabatini et al., 1993). The genes and their translation products are summarized in Table 1.

	(3) POST-TRANSLATIONAL MODIFICATIONS

TOP ABSTRACT (1) INTRODUCTION (2) SALIVARY PROTEIN GENE... (3) POST-TRANSLATIONAL... (4) SALIVARY PROTEIN... (5) CONCLUSION REFERENCES

(3.A) Glycosylation
By weight, the majority of the salivary proteins are glycosylated. The heavily glycosylated proteins are mucous glycoprotein 1 (MG1), mucous glycoprotein 2 (MG2), agglutinin, glycosylated proline-rich proteins (PRPs), and secretory immunoglobulin A (sIgA). In general, the non-glycosylated salivary proteins are smaller and consist of acidic and basic PRPs, cystatins, statherins, and histatins. The glycosylated proteins undergo N-linked glycosylation (reviewed in Kornfeld and Kornfeld, 1985; Weerapana and Imperiali, 2006) and/or O-linked glycosylation (Ten Hagen et al., 2003; reviewed in Hang and Bertozzi, 2005). With the combined use of nuclear magnetic resonance (NMR) and mass spectrometry (MS), it has become feasible for investigators to structurally unravel complex sugar moieties and to gain insight into the tremendous diversity in the glycosylation pattern of the major salivary glycoproteins.

(3.A.1) Glycosylation of Amylase
Amylase is secreted mainly by the parotid gland in both glycosylated and non-glycosylated isoforms (Kauffman et al., 1973). In pancreatic amylase, which is highly homologous to salivary amylase, it has been established that only the Asn461 residue is glycosylated (Rydberg et al., 1999). Salivary amylase contains neutral and sialic acid-containing sugar moieties, all expressing Lewis x (Le^x) epitopes (Yamashita et al., 1980) (Table 2). Originally, the various amylase isoforms in saliva were believed to arise solely from deamidation of the glycosylated and non-glycosylated isoforms (Keller et al., 1971; Karn et al., 1973, 1974), but revised models have been reported (Bank et al., 1991). Mass spectrometric analyses of the various isoforms of amylase in saliva have revealed that the amylase protein family contains more species than previously recognized (Hardt et al., 2005; Hirtz et al., 2005). The functional role of the sugar moiety in amylase has not yet been established. Amylase catalyzes the hydrolysis of 14 glycosidic linkages in starch, and both glycosylated and non-glycosylated amylases exhibit activity (Srivastava, 1991; Koyama et al., 2000). Apart from functioning as a digestive enzyme, an additional role for amylase in the oral cavity may relate to its capacity to bind to some oral micro-organisms, including S. gordonii (formerly called S. sanguis genotype I), S. mitis genotype II, and S. oralis (Scannapieco et al., 1989; Murray et al., 1992; Scannapieco, 1994). Both the glycosylated and non-glycosylated amylase preparations bind to S. gordonii, and it thus appears that glycosylation is a prerequisite neither for glycolytic activity nor for binding to bacteria. Salivary amylase is also a prominent constituent of the in vivo-formed acquired enamel pellicle and the mucosal pellicle, in which the glycosylated and non-glycosylated forms, respectively, predominate (Al-Hashimi and Levine, 1989; Bradway et al., 1992).

(3.A.3) Glycosylation of Mucous Glycoprotein 2 (MG2)
MG1 and MG2 constitute the bulk of the salivary proteins secreted by the submandibular/sublingual glands. These glycoproteins are frequently referred to by their gene names, MUC5B and MUC7, respectively. MG2 contains multiple threonine-, serine-, proline-, and alanine-rich repeats, which display extensive O-glycosylation (Bobek et al., 1993). About 68% of the weight of the protein is carbohydrate. Initial characterizations of the O-linked carbohydrate units of MG2 showed that approximately 80% of the oligosaccharides consist of Galβ13GalNAc that either are unsubstituted, or are substituted with a terminal fucose or sialic acid residue (Reddy et al., 1985). With further structural characterizations by mass spectrometry and nuclear magnetic resonance, 41 different oligosaccharide structures containing up to 12 sugar residues have been structurally characterized (Prakobphol et al., 1998). The most prominent structures expressed sialyl-T antigen, Le^x, and sialyl-Le^x (sLe^x) determinants. Le^x and sLe^x were identified in the latter study as the most prominent motifs on MG2 (Table 2). As described above for the glycosylated PRPs, the heavy glycosylation of MG2 explains its viscoelastic and rheological properties, important for masticatory lubrication. Each of the expressed lectin epitopes can serve as a specific ligand for oral microbial species. Indeed, a large array of different Gram-positive and Gram-negative oral microbial species bind to MG2 (Murray et al., 1992; Amerongen et al., 1995). The association between MG2 and bacteria in solution leads to the formation of aggregates and bacterial clearance from the oral cavity. As such, mucins can be considered part of the innate oral defense system. Surprisingly, the prominent Le^x and sLe^x epitopes in MG2 do not represent binding ligands for many oral bacterial strains, but rather seem to be involved in governing lymphocyte tracking, suggesting diverse functions for MG2 in the oral cavity (Prakobphol et al., 1999).

(3.A.4) Glycosylation of Mucous Glycoprotein 1 (MG1)
MG1 is the MUC5B gene product and represents the largest and the most heavily glycosylated protein fraction in human saliva. It has recently been suggested that MG1 may also contain an even larger product encoded by the MUC19 gene (Chen et al., 2004; Culp et al., 2004), but this product has not been identified in any of the saliva proteome analyses (e.g., Wilmarth et al., 2004; Hu et al., 2005). MG1 isolated from human submandibular/sublingual saliva contains about 78% carbohydrate (Loomis et al., 1987; Offner and Troxler, 2000). Over half of the oligosaccharides are neutral (56%), over one-quarter (26%) are sialylated, and 19% are sulfated. By mass spectrometry, at least 62 different types of neutral and 25 types of sialylated structures have been characterized, indicating that the level of heterogeneity in the glycosylation of MG1 exceeds that of PRG and MG2 (Thomsson et al., 2002). Fucose is present in the form of blood group epitopes, including H types 1 and 2, Le^a, Le^b, Le^x, sLe^x, and Le^y. In fact, MG1 is the primary carrier of the ABH, Le^a, and Le^b blood group antigens in saliva (Prakobphol et al., 1993). The heterogenous glycosylation of MG1 has several biological implications. The numerous O-glycosylations in MG1 result in a large, extended, soluble gel-forming mucin. MG1 is abundantly present in the early enamel pellicle and, as such, serves as an interface between the tooth surface and the oral environment (Tabak et al., 1985; Al-Hashimi and Levine, 1989). It is also part of mucosal pellicles, where it provides a permeability barrier for the protection of the oral mucosal epithelium against environmental insult and desiccation (Tabak et al., 1982; Bradway et al., 1992; Amerongen et al., 1995). Despite the expression of a wide variety of oligosaccharide structures on MG1, it appears to bind fewer bacteria than MG2 (Prakobphol et al., 2005). Helicobacter pylori strains bind to MG1, depending on the strain-dependent specificity for Lewis blood group epitopes (Mahdavi et al., 2002; Prakobphol et al., 2005). Such specificity may point toward the predisposition of certain individuals for colonization with certain microbial species, which would of great interest from a diagnostic point of view.

(3.A.5) Glycosylation of Agglutinin (gp-340)
Agglutinin is the second largest glycoprotein in salivary secretions. It is identical to the lung scavenger protein gp-340 and contains 14 potential N-glycosylation sites. Agglutinin contains about 25% carbohydrate (Prakobphol et al., 2005) and is N-glycosylated (Ramachandran et al., 2006). Purified agglutinin from either parotid saliva or submandibular/sublingual saliva expressed Le^a and Le^y as well as sLe^x epitopes, and some glycoforms contained ABH epitopes (Ligtenberg et al., 2000; Prakobphol et al., 2000; Eriksson et al., 2007) (Table 2). The functions of agglutinin have been mainly attributed to its capacity to aggregate bacteria. Microbial clearance following adhesion and aggregation is particularly beneficial if the protein binds pathogens. Agglutinin shows a high binding affinity for S. mutans, and this association is so specific that it has been used to isolate agglutinin from parotid secretions (Ericson and Rundegren, 1983; Ligtenberg et al., 2000; Prakobphol et al., 2000). Interestingly, the carbohydrate residues on agglutinin do not appear to be involved in the binding to S. mutans, and a peptidic recognition site for this micro-organism in agglutinin has been identified (Bikker et al., 2002, 2004). Agglutinin also binds to several other oral streptococci and to H. pylori (Prakobphol et al., 2000). It furthermore exhibits human immunodeficiency virus (HIV) and influenza A-neutralizing activities. While the non-glycosylated N-terminus of agglutinin is responsible for the interaction with the HIV virus (Wu et al., 2006), the types of sialic acid linkages on agglutinin are an important determinant for anti-influenza A virus activity (Hartshorn et al., 2006).

(3.A.6) Glycosylation of Secretory Immunoglobulin A (sIgA)
The most abundant immunoglubulin in saliva, sIgA, is a complex comprised of multiple polypeptides. It consists of a secretory component (SC), which is covalently attached to two IgA molecules containing a joining (J) chain. The SC component is produced by acinar cells, whereas the IgA and J molecules are synthesized by the plasma cells close to the glandular epithelium, indicating that two types of cells are involved in the production and post-translational modification of the sIgA complex (Norderhaug et al., 1999). The heavy chains, SC, and J chain are N-glycosylated, and the heavy chains of some sIgA isotypic variants are furthermore O-glycosylated. Notably, the Fab regions, involved in specific antigen recognition, are not glycosylated (Royle et al., 2003). The largest variation in N-linked oligosaccharide structures is found on SC, expressing all of the possible sialylated and non-sialylated Lewis isotopes (Table 2). The O-linked glycans on the heavy chain of sIgA1 are equally complex and contain many different sugar epitopes (Royle et al., 2003). The glycosylation of sIgA and other salivary glycoproteins may function to protect these proteins from proteolytic activity (Crottet and Corthesy, 1998). Furthermore, the N-linked glycosylation sites in the heavy chain are important for the secretion and dimerization of the antibody (Taylor and Wall, 1988; Atkin et al., 1996). The large variety of expressed Lewis-type structures on SC endows sIgA with the capacity to bind to a large number of bacteria via domains that do not involve the Fab region. Indeed, SC binds to H. pylori (Falk et al., 1993), S. pneumonia (Hammerschmidt et al., 1997), and E. coli (Wold et al., 1990). With respect to its lectin-binding properties and bacteria binding, sIgA shows striking similarities to some other salivary glycoproteins. It has been suggested that sIgA may participate both in adaptive immunity, via its Fab parts, as well as in innate immune systems, via its glycosylated regions (Royle et al., 2003).

(3.B) Phosphorylation
The phosphorylated salivary proteins are comprised of the acidic proline-rich proteins, statherin, histatin 1, and some cystatin family members (cystatins S and SA-III). Most salivary phosphoproteins are fully and faithfully phosphorylated at the serine residues dedicated to be phosphorylated. Recently, with sensitive mass spectrometric approaches, minor, unusually phosphorylated species of the known salivary phosphoproteins have been detected (Inzitari et al., 2005, 2006, 2007). It is widely recognized that the state of salivary supersaturation with respect to calcium phosphate salts is due to the biological roles of phosphorylated salivary proteins. Protein phosphorylation adds a negative charge to the molecule, which appears to dictate, in large part, the affinity of the protein for the enamel surface. Hence, an important biological role of the phosphorylation of salivary proteins is related to mineral homeostasis.

(3.B.1) Phosphorylation of Acidic PRPs
All acidic PRPs are phosphorylated at residues 8 and 22 (Wong et al., 1979; Wong and Bennick, 1980). Mono- and non-phosphorylated species are rarely observed. Interestingly, a recent report showed that, in newborns, the percentages of non-phosphorylated and monophosphorylated acidic PRPs are much higher than in adults, suggesting that phosphokinase expression or activity is not yet fully developed at a young age (Inzitari et al., 2007). The specific phosphorylation of the Ser8 and the Ser22 residues in acidic PRPs was demonstrated upon expression of PRP constructs into a human submandibular gland cell line (Drzymala et al., 2000). The Ser8 phosphorylation site in acidic PRPs is confined to the sequence Ser-Xaa-Glu/pSer, which is the recognition site for Golgi casein kinase (G-CK; Lasa et al., 1997). Interestingly, Ser22 in acidic PRP does not comply with this sequence requirement, but it is consistent with an alternative consensus sequence for G-CK, namely, Ser-Xaa-Gln-Xaa-Xaa-Asp/Glu (Brunati et al., 2000). Acidic PRPs are effective inhibitors of calcium phosphate crystal growth, but not of primary calcium phosphate precipitation at physiological concentrations (Hay et al., 1979). Their calcium-binding affinity and inhibitory activities reside in the highly charged N-terminal 30 amino acids containing the two phosphoserine residues (Bennick et al., 1981; Hay et al., 1987). Data have provided ample evidence for the importance of protein phosphorylation and the simultaneous presence of both phosphoserines for the biological functions of acidic PRPs pertaining to mineral homeostasis (Hay et al., 1979, 1987).

(3.B.2) Phosphorylation of Statherin
Statherin was the first fully sequenced salivary phosphoprotein (Schlesinger and Hay, 1977). It contains two vicinal phosphoserine residues (Ser2-Ser3), followed by two glutamic acid residues (Glu4-Glu5). This tetrapeptide domain (Ser2-Ser3-Glu4-Glu5) contains two tripeptide sequences of the Ser-Xaa-Glu type that, in principle, could be recognized by G-CK phosphokinase. Statherin exhibits a high affinity for hydoxyapatite, and inhibits both crystal growth of calcium phosphate salts and spontaneous precipitation of calcium phosphate from a supersaturated solution. Statherin is the most effective inhibitor of calcium phosphate precipitation among other salivary phosphoproteins. The capacity to prevent crystal growth is localized in the N-terminal 6 amino acid residues containing both phosphoserines. Overall, the negative charges responsible for these functions are not restricted to phosphoserines, but also include contributions made by aspartic and glutamic residues (Hay et al., 1979; Raj et al., 1992; Tamaki et al., 2002).

(3.B.3) Phosphorylation of Histatin 1
Histatins 1 and 3 are the two primary histatin gene products. Histatin 1 is phosphorylated at Ser2 in the Ser2-Asp3-Glu4 context. In histatin 3, Glu4 is substituted by Ala4, abolishing the kinase recognition site and preventing the phosphorylation of Ser2 (Oppenheim et al., 1986, 1988). Functional comparisons between native histatin 1 and recombinantly expressed histatin 1, lacking phosphate, have shown that the phosphate group has no significant impact on the antifungal properties of histatin 1 (Driscoll et al., 1995). This observation is consistent with the fact that the middle region, rather than the N-terminus, of histatins contains the fungicidal domain (Lamkin and Oppenheim, 1993). In contrast, non-phosphorylated recombinant histatin 1 exhibits reduced affinity for hydroxyapatite, as compared with native histatin 1, thus indicating that the phosphate group specifically, or its negative charge, is a determining factor in governing the functional interaction of histatin 1 with tooth enamel mineral (Driscoll et al., 1995).

(3.B.4) Phosphorylation of Cystatin S and SA-III
Among the family of salivary cystatins, only cystatin S is phosphorylated. In contrast to other salivary phosphoproteins, cystatin S shows an interesting extent of heterogeneous phosphorylation not observed in other salivary phosphoproteins. It is either non-phosphorylated, or phosphorylated at Ser3, or at Ser1 and Ser3 (Isemura et al., 1991; Ramasubbu et al., 1991). In view of the Ser-Xaa-Glu/Pser recognition site of the G-CK phosphokinase, Ser1 would become a recognition site for this enzyme after phosphorylation of Ser3. Analysis of a variant of cystatin S, called cystatin SA-III, provided evidence for four phosphate groups associated with Ser3, Ser99, Ser112, and Ser116, making this the only salivary protein to date with phosphorylated residues in both the N- and the C-termini (Lamkin et al., 1991). Removal of the phosphate groups by alkaline phosphatase treatment reduces the affinity of cystatins (called cysteine-containing phosphoproteins at the time) for hydroxyapatite (Shomers et al., 1982). The main function of cystatins in the oral cavity is related to their strong inhibitory activity toward various cysteine proteases (Minakata and Asano, 1984; Barrett, 1986; Isemura et al., 1986). Both non-phosphorylated and phosphorylated cystatin members display this activity, and the activity is retained upon dephosphorylation of the phosphorylated isoforms (Ramasubbu et al., 1991; Saitoh et al., 1991; Lamkin and Oppenheim, 1993). The main function of cystatin phosphorylation therefore remains elusive.

(3.C) Proteolytic Processing
Most salivary proteins with molecular weights < 40 kDa are post-translationally cleaved into smaller fragments before their release into the oral cavity. Proteolysis can be either complete, leaving no trace of the parent protein, or partial, yielding a mixture of intact and cleavage products (Cai and Bennick, 2004). In the latter case, the ratio of uncleaved to cleaved product is oftentimes strikingly constant and flow-rate-independent. The constancy of this ratio suggests that post-translational proteolysis is a tightly controlled process. The mechanism of control has not been elucidated, but could be explained by sequestration and selective fusion of protein- and protease-containing vesicles within the acinar cell. This possibility is schematically depicted in Fig. 1. The biological significance of proteolytic protein processing could be to expand the salivary protein repertoire without the need for additional genes encoding for such proteins. In most cases, the fragments generated retain their biological activity (Lu and Bennick, 1998), or may even be more active than the primary gene product (Madapallimattam and Bennick, 1990; Xu et al., 1991). When a protein is cleaved into two active fragments, degradation can be considered a means to increase the concentration of active components on a molar basis. The generation of different peptides from a single translation product could also be beneficial with regard to cooperativity and synergism of the products affecting various biological functions.

View larger version (38K): [in this window] [in a new window]   Figure 1. Hypothetical model explaining the constant ratio of histatin 3 to histatin 5 in glandular secretions.

(3.C.3) Proteolytic Processing of Histatins
The major proteolytic histatin fragment formed during biosynthesis is histatin 5, which arises from a chymotryptic-like cleavage in histatin 3 after Tyr24 (Oppenheim et al., 1986, 1988). The almost constant ratio of histatin 3 to histatin 5 in parotid secretions poses interesting questions regarding the biological control of the proteolytic processing of histatin 3. A possible mechanism consistent with this observation is shown in Fig. 1. Parotid secretions contain eight minor, chromatographically detectable, histatin fragments varying in length from 12 to 25 residues. In addition, smaller histatin fragments containing from 4 to 10 residues have been identified and characterized (Troxler et al., 1990; Perinpanayagam et al., 1995). More recent and sensitive mass spectrometric analysis of proteins present in parotid secretions has expanded the histatin peptide repertoire to at least 50, pointing toward extensive proteolysis of histatins in the glands (Hardt et al., 2005). Most of these peptides, however, are likely present in low concentrations, and the biological significance of the minor species remains to be explored. The enzymes responsible for histatin processing have not yet been identified. Furin, which has been implicated in the processing of PRPs (Chan and Bennick, 2001), does not seem a likely candidate for histatin processing, since histatins are inhibitors of this enzyme (Basak et al., 1997). Histatins exhibit multiple antifungal and antibacterial activities that may or may not be affected by proteolysis. The antifungal activity of histatin 5 exceeds that of histatin 3, pointing toward a potential biological advantage for the generation of this fragment (Xu et al., 1991; Helmerhorst et al., 2005). The fungicidal domain, consisting of residues 12–25 in histatin 3, is present in most of the longer naturally occurring histatin fragments (Troxler et al., 1990; Lamkin and Oppenheim, 1993; Xu et al., 1993), suggesting that post-translational proteolysis would not necessarily reduce the antimicrobial properties associated with the unprocessed parent protein.

(3.C.4) Proteolytic Processing of Statherin
The proteolytic processing of statherin and its splice variant SV-2 consists of the cleavage of the C-terminal Phe residue (Jensen et al., 1991). This modification renders the statherin degradation products somewhat less hydrophobic than the original protein, but overall, the protein remains quite hydrophobic, due to the presence of multiple Tyr residues comprising almost of the statherin sequence. It is not known if and how the removal of the C-terminal Phe residues affects the known functions of statherin.

	(4) SALIVARY PROTEIN MODIFICATIONS IN WHOLE SALIVA

TOP ABSTRACT (1) INTRODUCTION (2) SALIVARY PROTEIN GENE... (3) POST-TRANSLATIONAL... (4) SALIVARY PROTEIN... (5) CONCLUSION REFERENCES

 
The in vivo structure-function relationship of salivary proteins in the oral cavity is dictated not only by post-translational protein processing in the gland, but also by protein modifications that occur after glandular secretions are mixed with whole saliva. Whole saliva is non-sterile and contains various non-exocrine contributors, including gingival crevicular fluid, oral bacteria, desquamated epithelial cells, and neutrophils and their products. Not surprisingly, oral fluid contains a variety of host- and bacteria-derived enzymes, such as proteases, glycosidases, and transferases (Chauncey, 1961; Makinen, 1966). Exposure of glandular proteins to these enzymes results in multiple structural modifications. Indeed, the proteomes of parotid and submandibular secretions differ in many respects from that of whole saliva, as visualized by two-dimensional PAGE (Yao et al., 2003; Walz et al., 2006). Protein-protein interactions in the whole salivary environment, furthermore, lead to the formation of heterotypic complexes, which may aggregate to form globular super complexes (Young et al., 1999; Soares et al., 2004). In addition, novel and hybrid salivary molecules are generated through the formation of intra- and intermolecular covalent linkages (Iontcheva et al., 1997; Yao et al., 2000; Cabras et al., 2006). Our understanding of salivary protein modifications in whole saliva and its impact on function is far from complete and lags behind existing knowledge on protein processing occurring during biosynthesis in the gland. Knowledge of whole saliva protein modifications appears to be critically important in view of the fact that whole saliva directly surrounds oral soft and hard tissues and should contain the elements required for host protection. Furthermore, it is expected that whole saliva, rather than glandular secretions, will harbor the biomarkers useful for oral diagnostics.

(4.A) Protein Degradation in Whole Saliva
The concentrations of histatin and acidic PRPs in whole saliva are drastically lower than those in parotid secretions (Fig. 2) or in submandibular/sublingual secretions (data not shown), despite the fact that these glands produce the bulk of the whole saliva fluid volume. Their low levels in whole saliva coincide with the presence of multiple degradation fragments with a high electrophoretic mobility, indicative of proteolytic breakdown. Not all salivary proteins are equally susceptible to proteolytic breakdown, and those that are most susceptible are histatins, statherin, acidic PRPs, and basic non-glycosylated PRPs (Baum et al., 1976; Kousvelari et al., 1980; Minaguchi et al., 1988; Castagnola et al., 2004; Vitorino et al., 2004; Inzitari et al., 2005, 2006; Helmerhorst et al., 2006). The extensive proteolysis of these proteins in the oral environment emphasizes that whole saliva is not a suitable source for the study of their biosynthesis and post-translational modifications, as has been occasionally attempted (Castagnola et al., 2004; Inzitari et al., 2005). The structural and functional consequences of the degradation of histatins and acidic PRPs occurring in the oral cavity have been investigated in more detail.

View larger version (102K): [in this window] [in a new window]   Figure 2. Analysis of the composition of parotid and whole saliva from four individuals by anionic and cationic PAGE. Stimulated parotid secretion was collected on ice with the aid of a Lashley cup placed over the Stensen’s duct, with sour candies used for stimulation. Stimulated whole saliva was collected into tubes containing PMSF and EDTA which were placed on ice. All samples were boiled immediately after collection. Aliquots (100 µL) were analyzed by cationic PAGE (upper panel), or by anionic PAGE (lower panel), for visualization of histatins and acidic acidic PRPs, respectively (Davis, 1964; Ornstein, 1964; Baum et al., 1977). Lanes 1 to 4 contain samples from participants 1 to 4, respectively.

View larger version (17K): [in this window] [in a new window]   Figure 3. Early histatin 5 degradation fragments formed upon exposure of synthetic histatin 5 (residues 1-24) to whole saliva supernatant (solid lines) and naturally occurring histatin 5 peptides in whole saliva (dotted lines). Data from Helmerhorst et al.(2006) and Castagnola et al.(2004), respectively.

(4.A.3) Degradation of Cystatins in Whole Saliva
Whole saliva contains cystatin variants that are 113 residues in length (Isemura et al., 1984a,b, 1986, 1987) and arise from post-secretory degradation of the full-length cystatins, which are 121 residues in length (Hawke et al., 1987; Al-Hashimi et al., 1988; Saitoh et al., 1988). Both the full-length and the N-terminally truncated forms of cystatins are present in whole saliva, and both forms exhibit cystatin protease inhibitory activities (Bobek et al., 1994). This is an important observation, since it indicates that the biological activity of cystatins is retained in the proteolytic environment of the oral cavity.

(4.B) Protein Deglycosylation in Whole Saliva
Partial deglycosylation of salivary glycoproteins in whole saliva is caused by enzymes produced by oral bacteria which subsequently utilize the released carbohydrate moieties for growth. Streptococci produce a large variety of glycosidases, and many oral streptococci are able to grow in batch culture with mucin as the sole source of carbohydrates (van der Hoeven et al., 1990). One of the glycosidic enzymes produced by streptococci is neuraminidase, which cleaves terminal sialic acid residues from carbohydrate moieties (Perlitsh and Glickman, 1966; Briscoe et al., 1972). The structural alteration of terminal sugar epitopes creates new, and abolishes existing, binding ligands for oral bacteria (Leach, 1963). Neuraminidase treatment of mucous glycoproteins completely abolishes its ability to aggregate S. sanguis, whereas the binding of S. mutans is unaffected (McBride and Gisslow, 1977; Levine et al., 1978). Since many oral bacteria recognize sugar epitopes, modification of these ligands by salivary neuraminidase and other oral glycosidases may have profound effects on the binding epitopes of these organisms and their capacity to colonize the oral cavity.

(4.C) Protein-Protein Interactions in Whole Saliva
Most proteins in cellular biological systems function as part of intricate molecular networks that involve multiple protein-protein interactions. Protein-protein interactions are numerous in the oral environment, which contains a large variety of proteins originating from many different sources. The ensuing interactions reflect a whole spectrum of affinities, resulting in dissociable and non-dissociable protein-protein complexes (Iontcheva et al., 1997; Bruno et al., 2005). The salivary protein complexes formed in the oral cavity represent entirely new biological entities, and differ not only in structure, but likely also in function from isolated salivary proteins in solution.

(4.C.1) Protein Complex Formation in Whole Saliva
Salivary mucins and other glycoproteins have a high propensity to form homotypic and heterotypic complexes with other proteins. MG2 (MUC7) forms complexes with sIgA, amylase, acidic PRP2, basic PRP3, statherin, histatin 1, and lactoferrin (Loomis et al., 1987; Biesbrock et al., 1991; Soares et al., 2003; Bruno et al., 2005). The large-molecular-weight glycoprotein MG1 also interacts with multiple salivary constituents, but not with sIgA (Iontcheva et al., 1997, 2000). Investigators have speculated on the functional benefits of protein complex formation. Many of the protein-protein complexes bind to bacteria, and such associations, in some cases, have shown to enhance bacterial agglutination and clearance from the oral cavity (Rundegren and Arnold, 1987a,b; Biesbrock et al., 1991; Oho et al., 1998). Mucin binding could prolong the residence time of the binding partners in the oral cavity by retarding proteolytic degradation. Complexing of mucins with protective molecules, such as lysozyme, may concentrate these components at various tissue-environmental interfaces (Levine et al., 1987a). Protein complexes may also act as a vehicle for the slow release of proteins to sites in the oral cavity where their activity is required (Iontcheva et al., 1997).

Some of the protein-protein complexes in whole saliva further aggregate into globular, micelle-like structures. These structures are virtually undetectable in glandular secretions and are primarily present in whole saliva (Young et al., 1999). A calculated 4.7–20% of total whole salivary proteins are present in the form of micellar aggregates. Interestingly, only a selected number of salivary proteins can be immunologically identified in micelles (Soares et al., 2004). It appears that only protein-protein complexes containing MG2 would further organize to form the particulate, micelle-like globular structures (Soares et al., 2004). Since the amino acid composition of micelles is virtually identical to that of the early acquired enamel pellicle, salivary micelles have been considered the early participants of protein films formed on the tooth surface (Young et al., 1999).

(4.C.2) Covalent Cross-linking of Salivary Proteins
Strong evidence points to the formation of covalent protein-protein interactions at the mucosal pellicle (Bradway et al., 1989, 1992; Bradway and Levine, 1993). Buccal epithelial cells express the enzyme transglutaminase, which catalyzes the formation of a -glutamyl--lysine isopeptide bond between a glutamine and a lysine residue (Abe et al., 1977; Williams-Ashman and Canellakis, 1980). Incubation of parotid secretions with pure acidic PRPs with buccal epithelial cells leads to the formation of new, non-dissociable, high-molecular-weight macromolecules. In in vitro experiments, histatins and statherin have been found to be suitable lysyl substrates for transglutaminase-mediated cross-linking to a glutamyl donor. Statherin is also able to serve as a glutamyl donor for binding to a lysyl donor (Yao et al., 1999). Recently, a cyclic form of statherin has been identified in whole saliva resulting from intra-molecular cross-linking between Lys6 and Gln37 (Cabras et al., 2006). Evidence for the in vitro formation of PRP-statherin hybrid molecules has also been obtained (Yao et al., 2000). Transglutaminase-mediated intra- and intermolecular modifications leading to circularized and hybrid salivary proteins add an additional level of structural diversity to the whole salivary proteome.

Because transglutaminase levels are low in saliva, the functional role of transglutamination is likely restricted to processes occurring at the mucosal surface. The formation of non-dissociable complexes of salivary proteins to the mucosal pellicle can create new bacterial binding sites or mask existing binding sites. An example of the latter is the binding of germinated Candida albicans cells to buccal epithelium (Bradway and Levine, 1993; Staab et al., 1999). Since acidic PRPs as well as C. albicans cell wall proteins serve as substrates for epithelium-associated transglutaminase, it has been speculated that PRPs may compete with C. albicans for binding to the oral mucosal cell surface and interfere with Candida colonization (Bradway and Levine, 1993).

	(5) CONCLUSION

TOP ABSTRACT (1) INTRODUCTION (2) SALIVARY PROTEIN GENE... (3) POST-TRANSLATIONAL... (4) SALIVARY PROTEIN... (5) CONCLUSION REFERENCES

 
It is evident that a large body of knowledge exists on the structure of individual salivary proteins at the biosynthetic level. Considerable information has been obtained on the function of pure salivary proteins and derived peptides from experiments performed in vitro. More recent efforts in salivary research have begun to elucidate the complex interplay between salivary proteins and enzymes resident in whole saliva. This interplay leads to a whole host of novel components, and the interactions of such components with oral structures is pivotal for our understanding of oral health mechanisms. Whole saliva represents a very dynamic fluid. The flow-rate-dependent influx of glandular secretion and the intermittent removal of whole saliva by swallowing create an influx and efflux dynamic difficult to approach experimentally. Superimposed on those conditions are variables that relate to the enzymology associated with whole saliva dictated by micro-organisms and host cells. To aid our understanding of the physiological and pathological processes relevant to oral health, it is imperative that the dynamics of whole saliva be addressed at the molecular level. While this represents considerable challenges, the results of such studies will provide insights into salivary function and will be critical for the development of saliva-based diagnostic applications.

	ACKNOWLEDGMENTS

 
The authors pay tribute to the pioneering work of Patricia J. Keller, the first woman professor in the University of Washington’s School of Dentistry, who died on April 1, 2007. Her work in the field of salivary protein biochemistry, particularly her efforts on the characterization of salivary amylases and basic proline-rich proteins, represent benchmark contributions toward understanding of the polymorphisms dominating the salivary exocrine systems. The authors also acknowledge their NIH/NIDCR support from grants DE05672, DE07652, DE16699, and DE14950.

Received for publication April 18, 2007. Revision received May 21, 2007. Accepted for publication May 21, 2007.

	REFERENCES

TOP ABSTRACT (1) INTRODUCTION (2) SALIVARY PROTEIN GENE... (3) POST-TRANSLATIONAL... (4) SALIVARY PROTEIN... (5) CONCLUSION REFERENCES

 

• Abe T, Chung SI, DiAugustine RP, Folk JE (1977). Rabbit liver transglutaminase: physical, chemical, and catalytic properties. Biochemistry 16:5495–5501.
• Al-Hashimi I, Levine MJ (1989). Characterization of in vivo salivary-derived enamel pellicle. Arch Oral Biol 34:289–295.[CrossRef][Medline] [Order article via Infotrieve]
• Al-Hashimi I, Dickinson DP, Levine MJ (1988). Purification, molecular cloning, and sequencing of salivary cystatin SA-1. J Biol Chem 263:9381–9387.[Abstract/Free Full Text]
• Amerongen AV, Bolscher JG, Veerman EC (1995). Salivary mucins: protective functions in relation to their diversity. Glycobiology 5:733–740.[Free Full Text]
• Anderson LC, Kauffman DL, Keller PJ (1982). Identification of the Pm and PmS human parotid salivary proteins as basic proline-rich proteins. Biochem Genet 20:1131–1137.[Medline] [Order article via Infotrieve]
• Atkin JD, Pleass RJ, Owens RJ, Woof JM (1996). Mutagenesis of the human IgA1 heavy chain tailpiece that prevents dimer assembly. J Immunol 157:156–159.[Abstract]
• Ayad M, Van Wuyckhuyse BC, Minaguchi K, Raubertas RF, Bedi GS, Billings RJ, et al. (2000). The association of basic proline-rich peptides from human parotid gland secretions with caries experience. J Dent Res 79:976–982.
• Azen EA (1993). Genetics of salivary protein polymorphisms. Crit Rev Oral Biol Med 4:479–485.[Abstract/Free Full Text]
• Azen EA, Denniston C (1980). Polymorphism of Ps (parotid size variant) and detection of a protein (PmS) related to the Pm (parotid middle band) system with genetic linkage of Ps and Pm to Gl, Db, and Pr genetic determinants. Biochem Genet 18:483–501.[Medline] [Order article via Infotrieve]
• Azen EA, Maeda N (1988). Molecular genetics of human salivary proteins and their polymorphisms. Adv Hum Genet 17:141–199.[Medline] [Order article via Infotrieve]
• Azen EA, Yu PL (1984a). Genetic polymorphism of CON 1 and CON 2 salivary proteins detected by immunologic and concanavalin A reactions on nitrocellulose with linkage of CON 1 and CON 2 genes to the SPC (salivary protein gene complex). Biochem Genet 22:1–19.[CrossRef][Medline] [Order article via Infotrieve]
• Azen EA, Yu PL (1984b). Genetic polymorphisms of Pe and Po salivary proteins with probable linkage of their genes to the salivary protein gene complex (SPC). Biochem Genet 22:1065–1080.[CrossRef][Medline] [Order article via Infotrieve]
• Azen EA, Hurley CK, Denniston C (1979). Genetic polymorphism of the major parotid salivary glycoprotein (Gl) with linkage to the genes for Pr, Db, and Pa. Biochem Genet 17:257–279.[Medline] [Order article via Infotrieve]
• Azen EA, Latreille P, Niece RL (1993a). PRB1 gene variants coding for length and null polymorphisms among human salivary Ps, PmF, PmS, and Pe proline-rich proteins. Am J Hum Genet 53:264–278.[Medline] [Order article via Infotrieve]
• Azen EA, Prakobphol A, Fisher SJ (1993b). PRB3 null mutations result in absence of the proline-rich glycoprotein Gl and abolish Fusobacterium nucleatum interactions with saliva in vitro. Infect Immun 61:4434–4439.[Abstract/Free Full Text]
• Azen EA, Amberger E, Fisher S, Prakobphol A, Niece RL (1996). PRB1, PRB2, and PRB4 coded polymorphisms among human salivary concanavalin-A binding, IL-1, and Po proline-rich proteins. Am J Hum Genet 58:143–153.[Medline] [Order article via Infotrieve]
• Bank RA, Hettema EH, Arwert F, Amerongen AV, Pronk JC (1991). Electrophoretic characterization of posttranslational modifications of human parotid salivary alpha-amylase. Electrophoresis 12:74–79.[CrossRef][Medline] [Order article via Infotrieve]
• Barrett AJ (1986). The cystatins: a diverse superfamily of cysteine peptidase inhibitors. Biomed Biochim Acta 45:1363–1374.[Medline] [Order article via Infotrieve]
• Basak A, Ernst B, Brewer D, Seidah NG, Munzer JS, Lazure C, et al. (1997). Histidine-rich human salivary peptides are inhibitors of proprotein convertases furin and PC7 but act as substrates for PC1. J Pept Res 49:596–603.[Medline] [Order article via Infotrieve]
• Baum BJ, Bird JL, Millar DB, Longton RW (1976). Studies on histidine-rich polypeptides from human parotid saliva. Arch Biochem Biophys 177:427–436.[CrossRef][Medline] [Order article via Infotrieve]
• Baum BJ, Bird JL, Longton RW (1977). Polyacrylamide gel electrophoresis of human salivary histidine-rich-polypeptides. J Dent Res 56:1115–1118.
• Bennick A (1982). Salivary proline-rich proteins. Mol Cell Biochem 45:83–99.[Medline] [Order article via Infotrieve]
• Bennick A, Cannon M, Madapallimattam G (1981). Factors affecting the adsorption of salivary acidic proline-rich proteins to hydroxyapatite. Caries Res 15:9–20.[Medline] [Order article via Infotrieve]
• Bergey EJ, Levine MJ, Reddy MS, Bradway SD, Al-Hashimi I (1986). Use of the photoaffinity cross-linking agent N-hydroxysuccinimidyl-4-azidosalicylic acid to characterize salivary-glycoprotein-bacterial interactions. Biochem J 234:43–48.[Medline] [Order article via Infotrieve]
• Biesbrock AR, Reddy MS, Levine MJ (1991). Interaction of a salivary mucin-secretory immunoglobulin A complex with mucosal pathogens. Infect Immun 59:3492–3497.[Abstract/Free Full Text]
• Bikker FJ, Ligtenberg AJ, Nazmi K, Veerman EC, van’t Hof W, Bolscher JG, et al. (2002). Identification of the bacteria-binding peptide domain on salivary agglutinin (gp-340/DMBT1), a member of the scavenger receptor cysteine-rich superfamily. J Biol Chem 277:32109–32115.[Abstract/Free Full Text]
• Bikker FJ, Ligtenberg AJ, End C, Renner M, Blaich S, Lyer S, et al. (2004). Bacteria binding by DMBT1/SAG/gp-340 is confined to the VEVLXXXXW motif in its scavenger receptor cysteine-rich domains. J Biol Chem 279:47699–47703.[Abstract/Free Full Text]
• Bobek LA, Tsai H, Biesbrock AR, Levine MJ (1993). Molecular cloning, sequence, and specificity of expression of the gene encoding the low molecular weight human salivary mucin (MUC7). J Biol Chem 268:20563–20569.[Abstract/Free Full Text]
• Bobek LA, Ramasubbu N, Wang X, Weaver TR, Levine MJ (1994). Biological activities and secondary structures of variant forms of human salivary cystatin SN produced in Escherichia coli. Gene 151:303–308.[Medline] [Order article via Infotrieve]
• Boisgard R, Charpigny G, Chanat E (1999). Polymeric IgA are sulfated proteins. FEBS Lett 463:250–254.[Medline] [Order article via Infotrieve]
• Bolstad AI, Jensen HB, Bakken V (1996). Taxonomy, biology, and periodontal aspects of Fusobacterium nucleatum. Clin Microbiol Rev 9:55–71.[Abstract]
• Bradway SD, Levine MJ (1993). Do proline-rich proteins modulate a transglutaminase catalyzed mechanism of candidal adhesion? Crit Rev Oral Biol Med 4:293–299.[Abstract/Free Full Text]
• Bradway SD, Bergey EJ, Jones PC, Levine MJ (1989). Oral mucosal pellicle. Adsorption and transpeptidation of salivary components to buccal epithelial cells. Biochem J 261:887–896.[Medline] [Order article via Infotrieve]
• Bradway SD, Bergey EJ, Scannapieco FA, Ramasubbu N, Zawacki S, Levine MJ (1992). Formation of salivary-mucosal pellicle: the role of transglutaminase. Biochem J 284(Pt 2):557–564.[Medline] [Order article via Infotrieve]
• Bresnahan PA, Leduc R, Thomas L, Thorner J, Gibson HL, Brake AJ, et al. (1990). Human fur gene encodes a yeast KEX2-like endoprotease that cleaves pro-beta-NGF in vivo. J Cell Biol 111:2851–2859.[Abstract/Free Full Text]
• Briscoe JM Jr, Pruitt KM, Caldwell RC (1972). The effect of neuraminidase on the properties of salivary proteins. J Dent Res 51:819–824.
• Brunati AM, Marin O, Bisinella A, Salviati A, Pinna LA (2000). Novel consensus sequence for the Golgi apparatus casein kinase, revealed using proline-rich protein-1 (PRP1)-derived peptide substrates. Biochem J 351:765–768.[CrossRef][Medline] [Order article via Infotrieve]
• Bruno LS, Li X, Wang L, Soares RV, Siqueira CC, Oppenheim FG, et al (2005). Two-hybrid analysis of human salivary mucin MUC7 interactions. Biochim Biophys Acta 1746:65–72.[Medline] [Order article via Infotrieve]
• Cabras T, Inzitari R, Fanali C, Scarano E, Patamia M, Sanna MT, et al. (2006). HPLC-MS characterization of cyclo-statherin Q-37, a specific cyclization product of human salivary statherin generated by transglutaminase 2. J Sep Sci 29:2600–2608.[CrossRef][Medline] [Order article via Infotrieve]
• Cai K, Bennick A (2004). Processing of acidic proline-rich proprotein by human salivary gland convertase. Arch Oral Biol 49:871–879.[Medline] [Order article via Infotrieve]
• Castagnola M, Inzitari R, Rossetti DV, Olmi C, Cabras T, Piras V, et al. (2004). A cascade of 24 histatins (histatin 3 fragments) in human saliva. Suggestions for a pre-secretory sequential cleavage pathway. J Biol Chem 279:41436–41443.[Abstract/Free Full Text]
• Chan M, Bennick A (2001). Proteolytic processing of a human salivary proline-rich protein precursor by proprotein convertases. Eur J Biochem 268:3423–3431.[Medline] [Order article via Infotrieve]
• Chen Y, Zhao YH, Kalaslavadi TB, Hamati E, Nehrke K, Le AD, et al. (2004). Genome-wide search and identification of a novel gel-forming mucin MUC19/Muc19 in glandular tissues. Am J Respir Cell Mol Biol 30:155–165.[Abstract/Free Full Text]
• Chauncey HH (1961). Salivary enzymes. J Am Dent Assoc 63:360–368.[Medline] [Order article via Infotrieve]
• Crottet P, Corthesy B (1998). Secretory component delays the conversion of secretory IgA into antigen-binding competent F(ab')2: a possible implication for mucosal defense. J Immunol 161:5445–5453.[Abstract/Free Full Text]
• Culp DJ, Latchney LR, Fallon MA, Denny PA, Denny PC, Couwenhoven RI, et al. (2004). The gene encoding mouse Muc19: cDNA, genomic organization and relationship to Smgc. Physiol Genomics 19:303–318.[Abstract/Free Full Text]
• Davis BJ (1964). Disc electrophoresis. II. Method and application to human serum proteins. Ann NY Acad Sci 121:404–427.[Medline] [Order article via Infotrieve]
• Desseyn JL, Guyonnet-Duperat V, Porchet N, Aubert JP, Laine A (1997). Human mucin gene MUC5B, the 10.7-kb large central exon, encodes various alternate subdomains resulting in a super-repeat. Structural evidence for a 11p15.5 gene family. J Biol Chem 272:3168–3178.[Abstract/Free Full Text]
• Dickinson DP, Ridall AL, Levine MJ (1987). Human submandibular gland statherin and basic histidine-rich peptide are encoded by highly abundant mRNA’s derived from a common ancestral sequence. Biochem Biophys Res Commun 149:784–790.[Medline] [Order article via Infotrieve]
• Driscoll J, Zuo Y, Xu T, Choi JR, Troxler RF, Oppenheim FG (1995). Functional comparison of native and recombinant human salivary histatin 1. J Dent Res 74:1837–1844.
• Drzymala L, Castle A, Cheung JC, Bennick A (2000). Cellular phosphorylation of an acidic proline-rich protein, PRP1, a secreted salivary phosphoprotein. Biochemistry 39:2023–2031.[CrossRef][Medline] [Order article via Infotrieve]
• Ericson T, Rundegren J (1983). Characterization of a salivary agglutinin reacting with a serotype c strain of Streptococcus mutans. Eur J Biochem 133:255–261.[Medline] [Order article via Infotrieve]
• Eriksson C, Frangsmyr L, Danielsson Niemi L, Loimaranta V, Holmskov U, Bergman T, et al. (2007). Variant size- and glycoforms of the scavenger receptor cysteine-rich protein gp-340 with differential bacterial aggregation. Glycoconj J 24:131–142.[Medline] [Order article via Infotrieve]
• Falk P, Roth KA, Boren T, Westblom TU, Gordon JI, Normark S (1993). An in vitro adherence assay reveals that Helicobacter pylori exhibits cell lineage-specific tropism in the human gastric epithelium. Proc Natl Acad Sci USA 90:2035–2039.[Abstract/Free Full Text]
• Gibbons RJ, Hay DI, Schlesinger DH (1991). Delineation of a segment of adsorbed salivary acidic proline-rich proteins which promotes adhesion of Streptococcus gordonii to apatitic surfaces. Infect Immun 59:2948–2954.[Abstract/Free Full Text]
• Gillece-Castro BL, Prakobphol A, Burlingame AL, Leffler H, Fisher SJ (1991). Structure and bacterial receptor activity of a human salivary proline-rich glycoprotein. J Biol Chem 266:17358–17368.[Abstract/Free Full Text]
• Hagerman AE, Butler LG (1981). The specificity of proanthocyanidin-protein interactions. J Biol Chem 256:4494–4497.[Abstract/Free Full Text]
• Hammerschmidt S, Talay SR, Brandtzaeg P, Chhatwal GS (1997). SpsA, a novel pneumococcal surface protein with specific binding to secretory immunoglobulin A and secretory component. Mol Microbiol 25:1113–1124.[CrossRef][Medline] [Order article via Infotrieve]
• Hang HC, Bertozzi CR (2005). The chemistry and biology of mucin-type O-linked glycosylation. Bioorg Med Chem 13:5021–50234.[CrossRef][Medline] [Order article via Infotrieve]
• Hardt M, Thomas LR, Dixon SE, Newport G, Agabian N, Prakobphol A, et al. (2005). Toward defining the human parotid gland salivary proteome and peptidome: identification and characterization using 2D SDS-PAGE, ultrafiltration, HPLC, and mass spectrometry. Biochemistry 44:2885–2899.[CrossRef][Medline] [Order article via Infotrieve]
• Hartshorn KL, Ligtenberg A, White MR, Van Eijk M, Hartshorn M, Pemberton L, et al. (2006). Salivary agglutinin and lung scavenger receptor cysteine-rich glycoprotein 340 have broad anti-influenza activities and interactions with surfactant protein D that vary according to donor source and sialylation. Biochem J 393:545–553.[Medline] [Order article via Infotrieve]
• Hatton MN, Loomis RE, Levine MJ, Tabak LA (1985). Masticatory lubrication. The role of carbohydrate in the lubricating property of a salivary glycoprotein-albumin complex. Biochem J 230:817–820.[Medline] [Order article via Infotrieve]
• Hawke DH, Yuan PM, Wilson KJ, Hunkapiller MW (1987). Identification of a long form of cystatin from human saliva by rapid microbore HPLC mapping. Biochem Biophys Res Commun 145:1248–1253.[Medline] [Order article via Infotrieve]
• Hay DI, Moreno EC, Schlesinger DH (1979). Phosphoprotein inhibitors of calcium phosphate precipitation from human salivary secretions. Inorg Persp Biol Med 2:271–285.
• Hay DI, Carlson ER, Schluckebier SK, Moreno EC, Schlesinger DH (1987). Inhibition of calcium phosphate precipitation by human salivary acidic proline-rich proteins: structure-activity relationships. Calcif Tissue Int 40:126–132.[CrossRef][Medline] [Order article via Infotrieve]
• Hay DI, Bennick A, Schlesinger DH, Minaguchi K, Madapallimattam G, Schluckebier SK (1988). The primary structures of six human salivary acidic proline-rich proteins (PRP-1, PRP-2, PRP-3, PRP-4, PIF-s and PIF-f). Biochem J 255:15–21.[Medline] [Order article via Infotrieve]
• Hay DI, Ahern JM, Schluckebier SK, Schlesinger DH (1994). Human salivary acidic proline-rich protein polymorphisms and biosynthesis studied by high-performance liquid chromatography. J Dent Res 73:1717–1726.
• Helmerhorst EJ, Venuleo C, Beri A, Oppenheim FG (2005). Candida glabrata is unusual with respect to its resistance to cationic antifungal proteins. Yeast 22:705–714.[CrossRef][Medline] [Order article via Infotrieve]
• Helmerhorst EJ, Alagl AS, Siqueira WL, Oppenheim FG (2006). Oral fluid proteolytic effects on histatin 5 structure and function. Arch Oral Biol 51:1061–1070.[CrossRef][Medline] [Order article via Infotrieve]
• Hirtz C, Chevalier F, Centeno D, Rofidal V, Egea JC, Rossignol M, et al. (2005). MS characterization of multiple forms of alpha-amylase in human saliva. Proteomics 5:4597–4607.[CrossRef][Medline] [Order article via Infotrieve]
• Hu S, Xie Y, Ramachandran P, Ogorzalek Loo RR, Li Y, Loo JA, et al. (2005). Large-scale identification of proteins in human salivary proteome by liquid chromatography/mass spectrometry and two-dimensional gel electrophoresis-mass spectrometry. Proteomics 5:1714–1728.[CrossRef][Medline] [Order article via Infotrieve]
• Inzitari R, Cabras T, Onnis G, Olmi C, Mastinu A, Sanna MT, et al. (2005). Different isoforms and post-translational modifications of human salivary acidic proline-rich proteins. Proteomics 5:805–815.[CrossRef][Medline] [Order article via Infotrieve]
• Inzitari R, Cabras T, Rossetti DV, Fanali C, Vitali A, Pellegrini M, et al. (2006). Detection in human saliva of different statherin and P-B fragments and derivatives. Proteomics 6:6370–6379.[CrossRef][Medline] [Order article via Infotrieve]
• Inzitari R, Vento G, Capoluongo E, Boccacci S, Fanali C, Cabras T, et al. (2007). Proteomic analysis of salivary acidic proline-rich proteins in human preterm and at-term newborns. J Proteome Res 6:1371–1377.[Medline] [Order article via Infotrieve]
• Iontcheva I, Oppenheim FG, Troxler RF (1997). Human salivary mucin MG1 selectively forms heterotypic complexes with amylase, proline-rich proteins, statherin, and histatins. J Dent Res 76:734–743.
• Iontcheva I, Oppenheim FG, Offner GD, Troxler RF (2000). Molecular mapping of statherin- and histatin-binding domains in human salivary mucin MG1 (MUC5B) by the yeast two-hybrid system. J Dent Res 79:732–739.
• Isemura S (2000). Nucleotide sequence of gene PBII encoding salivary proline-rich protein P-B. J Biochem 127:393–398.[Abstract/Free Full Text]
• Isemura S, Saitoh E, Sanada K (1982). Fractionation and characterization of basic proline-rich peptides of human parotid saliva and the amino acid sequence of proline-rich peptide P-E. J Biochem (Tokyo) 91:2067–2075.[Abstract/Free Full Text]
• Isemura S, Saitoh E, Sanada K (1984a). Isolation and amino acid sequence of SAP-1, an acidic protein of human whole saliva, and sequence homology with human gamma-trace. J Biochem (Tokyo) 96:489–498.[Abstract/Free Full Text]
• Isemura S, Saitoh E, Ito S, Isemura M, Sanada K (1984b). Cystatin S: a cysteine proteinase inhibitor of human saliva. J Biochem (Tokyo) 96:1311–1314.[Abstract/Free Full Text]
• Isemura S, Saitoh E, Sanada K (1986). Characterization of a new cysteine proteinase inhibitor of human saliva, cystatin SN, which is immunologically related to cystatin S. FEBS Lett 198:145–149.[CrossRef][Medline] [Order article via Infotrieve]
• Isemura S, Saitoh E, Sanada K (1987). Characterization and amino acid sequence of a new acidic cysteine proteinase inhibitor (cystatin SA) structurally closely related to cystatin S, from human whole saliva. J Biochem (Tokyo) 102:693–704.[Abstract/Free Full Text]
• Isemura S, Saitoh E, Sanada K, Minakata K (1991). Identification of full-sized forms of salivary (S-type) cystatins (cystatin SN, cystatin SA, cystatin S, and two phosphorylated forms of cystatin S) in human whole saliva and determination of phosphorylation sites of cystatin S. J Biochem (Tokyo) 110:648–654.[Abstract/Free Full Text]
• Jensen JL, Lamkin MS, Troxler RF, Oppenheim FG (1991). Multiple forms of statherin in human salivary secretions. Arch Oral Biol 36:529–534.[CrossRef][Medline] [Order article via Infotrieve]
• Karn RC, Shulkin JD, Merritt AD, Newell RC (1973). Evidence for post-transcriptional modification of human salivary amylase (amyl) isozymes. Biochem Genet 10:341–350.[Medline] [Order article via Infotrieve]
• Karn RC, Rosenblum BB, Ward JC, Merritt AD, Shulkin JD (1974). Immunological relationships and post-translational modification of human salivary amylase (Amy) and pancreatic amylase (Amy) isozymes. Biochem Genet 12:485–499.[Medline] [Order article via Infotrieve]
• Karn RC, Goodman PA, Yu PL (1985). Description of a genetic polymorphism of a human proline-rich salivary protein, Pc, and its relationship to other proteins in the salivary protein complex (SPC). Biochem Genet 23:37–51.[Medline] [Order article via Infotrieve]
• Kauffman DL, Keller PJ (1979). The basic proline-rich proteins in human parotid saliva from a single subject. Arch Oral Biol 24:249–256.[CrossRef][Medline] [Order article via Infotrieve]
• Kauffman DL, Watanabe S, Evans JR, Keller PJ (1973). The existence of glycosylated and non-glycosylated forms of human submandibular amylase. Arch Oral Biol 18:1105–1111.[Medline] [Order article via Infotrieve]
• Kauffman D, Wong R, Bennick A, Keller P (1982). Basic proline-rich proteins from human parotid saliva: complete covalent structure of protein IB-9 and partial structure of protein IB-6, members of a polymorphic pair. Biochemistry 21:6558–6562.
• Kauffman D, Hofmann T, Bennick A, Keller P (1986). Basic proline-rich proteins from human parotid saliva: complete covalent structures of proteins IB-1 and IB-6. Biochemistry 25:2387–2392.
• Kauffman DL, Bennick A, Blum M, Keller PJ (1991). Basic proline-rich proteins from human parotid saliva: relationships of the covalent structures of ten proteins from a single individual. Biochemistry 30:3351–3356.[CrossRef][Medline] [Order article via Infotrieve]
• Keller PJ, Kauffman DL, Allan BJ, Williams BL (1971). Further studies on the structural differences between the isoenzymes of human parotid-amylase. Biochemistry 10:4867–4874.[Medline] [Order article via Infotrieve]
• Kornfeld R, Kornfeld S (1985). Assembly of asparagine-linked oligosaccharides. Annu Rev Biochem 54:631–664.[CrossRef][Medline] [Order article via Infotrieve]
• Kousvelari EE, Baratz RS, Burke B, Oppenheim FG (1980). Immunochemical identification and determination of proline-rich proteins in salivary secretions, enamel pellicle, and glandular tissue specimens. J Dent Res 59:1430–1438.
• Koyama I, Komine S, Yakushijin M, Hokari S, Komoda T (2000). Glycosylated salivary alpha-amylases are capable of maltotriose hydrolysis and glucose formation. Comp Biochem Physiol B Biochem Mol Biol 126:553–560.[Medline] [Order article via Infotrieve]
• Lamkin MS, Oppenheim FG (1993). Structural features of salivary function. Crit Rev Oral Biol Med 4:251–259.[Free Full Text]
• Lamkin MS, Jensen JL, Setayesh MR, Troxler RF, Oppenheim FG (1991). Salivary cystatin SA-III, a potential precursor of the acquired enamel pellicle, is phosphorylated at both its amino- and carboxyl-terminal regions. Arch Biochem Biophys 288:664–670.[CrossRef][Medline] [Order article via Infotrieve]
• Lasa M, Chang PL, Prince CW, Pinna LA (1997). Phosphorylation of osteopontin by Golgi apparatus casein kinase. Biochem Biophys Res Commun 240:602–605.[CrossRef][Medline] [Order article via Infotrieve]
• Leach SA (1963). Release and breakdown of sialic acid from human salivary mucin and its role in the formation of dental plaque. Nature 199:486–487.[Medline] [Order article via Infotrieve]
• Levine MJ, Weill JC, Ellison SA (1969). The isolation and analysis of a glycoprotein from parotid saliva. Biochim Biophys Acta 188:165–167.[Medline] [Order article via Infotrieve]
• Levine MJ, Herzberg MC, Levine MS, Ellison SA, Stinson MW, Li HC, et al. (1978). Specificity of salivary-bacterial interactions: role of terminal sialic acid residues in the interaction of salivary glycoproteins with Streptococcus sanguis and Streptococcus mutans. Infect Immun 19:107–115.[Abstract/Free Full Text]
• Levine MJ, Reddy MS, Tabak LA, Loomis RE, Bergey EJ, Jones PC, et al. (1987a). Structural aspects of salivary glycoproteins. J Dent Res 66:436–441.
• Levine MJ, Aguirre A, Hatton MN, Tabak LA (1987b). Artificial salivas: present and future. J Dent Res 66(Spec Iss):693–698.[Medline] [Order article via Infotrieve]
• Ligtenberg AJ, Veerman EC, Nieuw Amerongen AV (2000). A role for Lewis A antigens on salivary agglutinin in binding to Streptococcus mutans. Antonie Van Leeuwenhoek 77:21–30.[CrossRef][Medline] [Order article via Infotrieve]
• Loomis RE, Prakobphol A, Levine MJ, Reddy MS, Jones PC (1987). Biochemical and biophysical comparison of two mucins from human submandibular-sublingual saliva. Arch Biochem Biophys 258:452–464.[CrossRef][Medline] [Order article via Infotrieve]
• Lu Y, Bennick A (1998). Interaction of tannin with human salivary proline-rich proteins. Arch Oral Biol 43:717–728.[CrossRef][Medline] [Order article via Infotrieve]
• Lyons KM, Azen EA, Goodman PA, Smithies O (1988). Many protein products from a few loci: assignment of human salivary proline-rich proteins to specific loci. Genetics 120:255–265.[Abstract/Free Full Text]
• Madapallimattam G, Bennick A (1990). Phosphopeptides derived from human salivary acidic proline-rich proteins. Biological activities and concentration in saliva. Biochem J 270:297–304.[Medline] [Order article via Infotrieve]
• Maeda N (1985). Inheritance of the human salivary protein-rich proteins: a reinterpretation in terms of six loci forming two subfamilies. Biochem Genet 23:455–464.[CrossRef][Medline] [Order article via Infotrieve]
• Maeda N, Kim HS, Azen EA, Smithies O (1985). Differential RNA splicing and post-translational cleavages in the human salivary proline-rich protein gene system. J Biol Chem 260:11123–11130.[Abstract/Free Full Text]
• Mahdavi J, Sonden B, Hurtig M, Olfat FO, Forsberg L, Roche N, et al. (2002). Helicobacter pylori SabA adhesin in persistent infection and chronic inflammation. Science 297:573–578.[Abstract/Free Full Text]
• Makinen KK (1966). Studies on oral enzymes. I. Fractionation and characterization of aminopeptidases of human saliva. Acta Odontol Scand 24:579–604.[Medline] [Order article via Infotrieve]
• McBride BC, Gisslow MT (1977). Role of sialic acid in saliva-induced aggregation of Streptococcus sanguis. Infect Immun 18:35–40.[Abstract/Free Full Text]
• Minaguchi K, Madapallimattam G, Bennick A (1988). The presence and origin of phosphopeptides in human saliva. Biochem J 250:171–177.[Medline] [Order article via Infotrieve]
• Minakata K, Asano M (1984). New protein inhibitors of cysteine proteinases in human saliva and salivary glands. Hoppe Seylers Z Physiol Chem 365:399–403.[Medline] [Order article via Infotrieve]
• Murray PA, Prakobphol A, Lee T, Hoover CI, Fisher SJ (1992). Adherence of oral streptococci to salivary glycoproteins. Infect Immun 60:31–38.[Abstract/Free Full Text]
• Nagata K, Nakao M, Shibata S, Shizukuishi S, Nakamura R, Tsunemitsu A (1983). Purification and characterization of galactosephilic component present on the cell surfaces of Streptococcus sanguis. ATCC 10557. J Periodontol 54:163–172.[Medline] [Order article via Infotrieve]
• Nishide T, Nakamura Y, Emi M, Yamamoto T, Ogawa M, Mori T, et al. (1986). Primary structure of human salivary alpha-amylase gene. Gene 41:299–304.[CrossRef][Medline] [Order article via Infotrieve]
• Norderhaug IN, Johansen FE, Schjerven H, Brandtzaeg P (1999). Regulation of the formation and external transport of secretory immunoglobulins. Crit Rev Immunol 19:481–508.[Medline] [Order article via Infotrieve]
• Offner GD, Troxler RF (2000). Heterogeneity of high-molecular-weight human salivary mucins. Adv Dent Res 14:69–75.[Abstract/Free Full Text]
• Oho T, Yu H, Yamashita Y, Koga T (1998). Binding of salivary glycoprotein-secretory immunoglobulin A complex to the surface protein antigen of Streptococcus mutans. Infect Immun 66:115–121.[Abstract/Free Full Text]
• Oppenheim FG, Yang YC, Diamond RD, Hyslop D, Offner GD, Troxler RF (1986). The primary structure and functional characterization of the neutral histidine-rich polypeptide from human parotid secretion. J Biol Chem 261:1177–1182.[Abstract/Free Full Text]
• Oppenheim FG, Xu T, McMillian FM, Levitz SM, Diamond RD, Offner GD, et al. (1988). Histatins, a novel family of histidine-rich proteins in human parotid secretion. Isolation, characterization, primary structure, and fungistatic effects on Candida albicans. J Biol Chem 263:7472–7477.[Abstract/Free Full Text]
• Oppenheim FG, Salih E, Siqueira WL, Zhang W, Helmerhorst EJ (2007). Salivary proteome and its genetic polymorphisms. Ann NY Acad Sci 1098:22–50.[CrossRef][Medline] [Order article via Infotrieve]
• Ornstein L (1964). Disc electrophoresis. I. Background and theory. Ann NY Acad Sci 121:321–349.[Medline] [Order article via Infotrieve]
• Payne JB, Iacono VJ, Crawford IT, Lepre BM, Bernzweig E, Grossbard BL (1991). Selective effects of histidine-rich polypeptides on the aggregation and viability of Streptococcus mutans and Streptococcus sanguis. Oral Microbiol Immunol 6:169–176.[Medline] [Order article via Infotrieve]
• Perinpanayagam HE, Van Wuyckhuyse BC, Ji ZS, Tabak LA (1995). Characterization of low-molecular-weight peptides in human parotid saliva. J Dent Res 74:345–350.
• Perlitsh MJ, Glickman I (1966). Salivary neuraminidase. I. The presence of neuraminidase in human saliva. J Periodontol 37:368–373.[Medline] [Order article via Infotrieve]
• Prakobphol A, Murray PA, Fisher SJ (1987). Bacterial adherence on replicas of sodium dodecyl sulfate-polyacrylamide gels. Anal Biochem 164:5–11.[CrossRef][Medline] [Order article via Infotrieve]
• Prakobphol A, Leffler H, Fisher SJ (1993). The high-molecular-weight human mucin is the primary salivary carrier of ABH, Le(a), and Le(b) blood group antigens. Crit Rev Oral Biol Med 4:325–333.[Abstract/Free Full Text]
• Prakobphol A, Thomsson KA, Hansson GC, Rosen SD, Singer MS, Phillips NJ, et al. (1998). Human low-molecular-weight salivary mucin expresses the sialyl Lewisx determinant and has L-selectin ligand activity. Biochemistry 37:4916–4927.[CrossRef][Medline] [Order article via Infotrieve]
• Prakobphol A, Tangemann K, Rosen SD, Hoover CI, Leffler H, Fisher SJ (1999). Separate oligosaccharide determinants mediate interactions of the low-molecular-weight salivary mucin with neutrophils and bacteria. Biochemistry 38:6817–6825.
• Prakobphol A, Xu F, Hoang VM, Larsson T, Bergstrom J, Johansson I, et al. (2000). Salivary agglutinin, which binds Streptococcus mutans and Helicobacter pylori, is the lung scavenger receptor cysteine-rich protein gp-340. J Biol Chem 275:39860–39866.[Abstract/Free Full Text]
• Prakobphol A, Boren T, Ma W, Zhixiang P, Fisher SJ (2005). Highly glycosylated human salivary molecules present oligosaccharides that mediate adhesion of leukocytes and Helicobacter pylori. Biochemistry 44:2216–2224.[CrossRef][Medline] [Order article via Infotrieve]
• Raj PA, Edgerton M, Levine MJ (1990). Salivary histatin 5: dependence of sequence, chain length, and helical conformation for candidacidal activity. J Biol Chem 265:3898–3905.[Abstract/Free Full Text]
• Raj PA, Johnsson M, Levine MJ, Nancollas GH (1992). Salivary statherin. Dependence on sequence, charge, hydrogen bonding potency, and helical conformation for adsorption to hydroxyapatite and inhibition of mineralization. J Biol Chem 267:5968–5976.[Abstract/Free Full Text]
• Ramachandran P, Boontheung P, Xie Y, Sondej M, Wong DT, Loo JA (2006). Identification of N-linked glycoproteins in human saliva by glycoprotein capture and mass spectrometry. J Proteome Res 5:1493–1503.[CrossRef][Medline] [Order article via Infotrieve]
• Ramasubbu N, Reddy MS, Bergey EJ, Haraszthy GG, Soni SD, Levine MJ (1991). Large-scale purification and characterization of the major phosphoproteins and mucins of human submandibular-sublingual saliva. Biochem J 280(Pt 2):341–352.[Medline] [Order article via Infotrieve]
• Reddy MS, Levine MJ, Tabak LA (1982). Structure of the carbohydrate chains of the proline-rich glycoprotein from human parotid saliva. Biochem Biophys Res Commun 104:882–888.[Medline] [Order article via Infotrieve]
• Reddy MS, Levine MJ, Prakobphol A (1985). Oligosaccharide structures of the low-molecular-weight salivary mucin from a normal individual and one with cystic fibrosis. J Dent Res 64:33–36.
• Royle L, Roos A, Harvey DJ, Wormald MR, van Gijlswijk-Janssen D, Redwan el-RM, et al. (2003). Secretory IgA N- and O-glycans provide a link between the innate and adaptive immune systems. J Biol Chem 278:20140–20153.[Abstract/Free Full Text]
• Ruhl S, Sandberg AL, Cisar JO (2004). Salivary receptors for the proline-rich protein-binding and lectin-like adhesins of oral actinomyces and streptococci. J Dent Res 83:505–510.
• Rundegren J, Arnold RR (1987a). Differentiation and interaction of secretory immunoglobulin A and a calcium-dependent parotid agglutinin for several bacterial strains. Infect Immun 55:288–292.[Abstract/Free Full Text]
• Rundegren JL, Arnold RR (1987b). Bacteria-agglutinating characteristics of secretory IgA and a salivary agglutinin. Adv Exp Med Biol 216(B):1005–1013.
• Rydberg EH, Sidhu G, Vo HC, Hewitt J, Cote HC, Wang Y, et al. (1999). Cloning, mutagenesis, and structural analysis of human pancreatic alpha-amylase expressed in Pichia pastoris. Protein Sci 8:635–643.[Medline] [Order article via Infotrieve]
• Sabatini LM, Azen EA (1989). Histatins, a family of salivary histidine-rich proteins, are encoded by at least two loci (HIS1 and HIS2). Biochem Biophys Res Commun 160:495–502.[CrossRef][Medline] [Order article via Infotrieve]
• Sabatini LM, Ota T, Azen EA (1993). Nucleotide sequence analysis of the human salivary protein genes HIS1 and HIS2, and evolution of the STATH/HIS gene family. Mol Biol Evol 10:497–511.[Abstract]
• Saitoh E, Isemura S, Sanada K (1983a). Complete amino acid sequence of a basic proline-rich peptide, P-D, from human parotid saliva. J Biochem (Tokyo) 93:495–502.[Abstract/Free Full Text]
• Saitoh E, Isemura S, Sanada K (1983b). Complete amino acid sequence of a basic proline-rich peptide, P-F, from human parotid saliva. J Biochem (Tokyo) 93:883–888.[Abstract/Free Full Text]
• Saitoh E, Isemura S, Sanada K (1983c). Further fractionation of basic proline-rich peptides from human parotid saliva and complete amino acid sequence of basic proline-rich peptide P-H. J Biochem (Tokyo) 94:1991–1999.[Abstract/Free Full Text]
• Saitoh E, Isemura S (1993). Molecular biology of human salivary cysteine proteinase inhibitors. Crit Rev Oral Biol Med 4:487–493.[Abstract/Free Full Text]
• Saitoh E, Isemura S, Sanada K, Kim HS, Smithies O, Maeda N (1988). Cystatin superfamily. Evidence that family II cystatin genes are evolutionarily related to family III cystatin genes. Biol Chem Hoppe Seyler 369(Suppl):191–197.[Medline] [Order article via Infotrieve]
• Saitoh E, Isemura S, Sanada K, Ohnishi K (1991). Cystatins of family II are harboring two domains which retain inhibitory activities against the proteinases. Biochem Biophys Res Commun 175:1070–1075.[CrossRef][Medline] [Order article via Infotrieve]
• Scannapieco FA (1994). Saliva-bacterium interactions in oral microbial ecology. Crit Rev Oral Biol Med 5:203–248.[Abstract/Free Full Text]
• Scannapieco FA, Bergey EJ, Reddy MS, Levine MJ (1989). Characterization of salivary alpha-amylase binding to Streptococcus sanguis. Infect Immun 57:2853–2863.[Abstract/Free Full Text]
• Schlesinger DH, Hay DI (1977). Complete covalent structure of statherin, a tyrosine-rich acidic peptide which inhibits calcium phosphate precipitation from human parotid saliva. J Biol Chem 252:1689–1695.[Abstract/Free Full Text]
• Shomers JP, Tabak LA, Levine MJ, Mandel ID, Hay DI (1982). Properties of cysteine-containing phosphoproteins from human submandibular-sublingual saliva. J Dent Res 61:397–399.
• Soares RV, Siqueira CC, Bruno LS, Oppenheim FG, Offner GD, Troxler RF (2003). MG2 and lactoferrin form a heterotypic complex in salivary secretions. J Dent Res 82:471–475.
• Soares RV, Lin T, Siqueira CC, Bruno LS, Li X, Oppenheim FG, et al. (2004). Salivary micelles: identification of complexes containing MG2, sIgA, lactoferrin, amylase, glycosylated proline-rich protein and lysozyme. Arch Oral Biol 49:337–343.[Medline] [Order article via Infotrieve]
• Srivastava RA (1991). Effect of glycosylation of bacterial amylase on stability and active site conformation. Indian J Biochem Biophys 28:109–113.[Medline] [Order article via Infotrieve]
• Staab JF, Bradway SD, Fidel PL, Sundstrom P (1999). Adhesive and mammalian transglutaminase substrate properties of Candida albicans Hwp1. Science 283:1535–1538.[Abstract/Free Full Text]
• Stubbs M, Chan J, Kwan A, So J, Barchynsky U, Rassouli-Rahsti M, et al. (1998). Encoding of human basic and glycosylated proline-rich proteins by the PRB gene complex and proteolytic processing of their precursor proteins. Arch Oral Biol 43:753–770.[CrossRef][Medline] [Order article via Infotrieve]
• Tabak LA, Levine MJ, Mandel ID, Ellison SA (1982). Role of salivary mucins in the protection of the oral cavity. J Oral Pathol 11:1–17.[Medline] [Order article via Infotrieve]
• Tabak LA, Levine MJ, Jain NK, Bryan AR, Cohen RE, Monte LD, et al. (1985). Adsorption of human salivary mucins to hydroxyapatite. Arch Oral Biol 30:423–427.[Medline] [Order article via Infotrieve]
• Tamaki N, Tada T, Morita M, Watanabe T (2002). Comparison of inhibitory activity on calcium phosphate precipitation by acidic proline-rich proteins, statherin, and histatin-1. Calcif Tissue Int 71:59–62.[Medline] [Order article via Infotrieve]
• Taylor AK, Wall R (1988). Selective removal of alpha heavy-chain glycosylation sites causes immunoglobulin A degradation and reduced secretion. Mol Cell Biol 8:4197–4203.[Abstract/Free Full Text]
• Ten Hagen KG, Fritz TA, Tabak LA (2003). All in the family: the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases. Glycobiology 13:1R–16R.[Abstract/Free Full Text]
• Thomsson KA, Prakobphol A, Leffler H, Reddy MS, Levine MJ, Fisher SJ, et al. (2002). The salivary mucin MG1 (MUC5B) carries a repertoire of unique oligosaccharides that is large and diverse. Glycobiology 12:1–14.[Abstract/Free Full Text]
• Troxler RF, Offner GD, Xu T, Vanderspek JC, Oppenheim FG (1990). Structural relationship between human salivary histatins. J Dent Res 69:2–6.
• van der Hoeven JS, van den Kieboom CW, Camp PJ (1990). Utilization of mucin by oral Streptococcus species. Antonie Van Leeuwenhoek 57:165–172.[CrossRef][Medline] [Order article via Infotrieve]
• Vitorino R, Lobo MJ, Duarte JA, Ferrer-Correia AJ, Domingues PM, Amado FM (2004). Analysis of salivary peptides using HPLC-electrospray mass spectrometry. Biomed Chromatogr 18:570–575.[CrossRef][Medline] [Order article via Infotrieve]
• Walz A, Stuhler K, Wattenberg A, Hawranke E, Meyer HE, Schmalz G, et al. (2006). Proteome analysis of glandular parotid and submandibular-sublingual saliva in comparison to whole human saliva by two-dimensional gel electrophoresis. Proteomics 6:1631–1639.[CrossRef][Medline] [Order article via Infotrieve]
• Weerapana E, Imperiali B (2006). Asparagine-linked protein glycosylation: from eukaryotic to prokaryotic systems. Glycobiology 16:91R–101R.[Abstract/Free Full Text]
• Williams-Ashman HG, Canellakis ZN (1980). Transglutaminase-mediated covalent attachment of polyamines to proteins: mechanisms and potential physiological significance. Physiol Chem Phys 12:457–472.[Medline] [Order article via Infotrieve]
• Wilmarth PA, Riviere MA, Rustvold DL, Lauten JD, Madden TE, David LL (2004). Two-dimensional liquid chromatography study of the human whole saliva proteome. J Prot Res 3:1017–1023.
• Wold AE, Mestecky J, Tomana M, Kobata A, Ohbayashi H, Endo T, et al. (1990). Secretory immunoglobulin A carries oligosaccharide receptors for Escherichia coli type 1 fimbrial lectin. Infect Immun 58:3073–3077.[Abstract/Free Full Text]
• Wong RS, Bennick A (1980). The primary structure of a salivary calcium-binding proline-rich phosphoprotein (protein C), a possible precursor of a related salivary protein A. J Biol Chem 255:5943–5948.[Abstract/Free Full Text]
• Wong RS, Hofmann T, Bennick A (1979). The complete primary structure of a proline-rich phosphoprotein from human saliva. J Biol Chem 254:4800–4808.[Abstract/Free Full Text]
• Wu Z, Lee S, Abrams W, Weissman D, Malamud D (2006). The N-terminal SRCR-SID domain of gp-340 interacts with HIV type 1 gp120 sequences and inhibits viral infection. AIDS Res Hum Retroviruses 22:508–515.[CrossRef][Medline] [Order article via Infotrieve]
• Xu L, Lal K, Santarpia RP 3rd, Pollock JJ (1993). Salivary proteolysis of histidine-rich polypeptides and the antifungal activity of peptide degradation products. Arch Oral Biol 38:277–283.[CrossRef][Medline] [Order article via Infotrieve]
• Xu T, Levitz SM, Diamond RD, Oppenheim FG (1991). Anticandidal activity of major human salivary histatins. Infect Immun 59:2549–2554.[Abstract/Free Full Text]
• Yamashita K, Tachibana Y, Nakayama T, Kitamura M, Endo Y, Kobata A (1980). Structural studies of the sugar chains of human parotid alpha-amylase. J Biol Chem 255:5635–5642.[Abstract/Free Full Text]
• Yan Q, Bennick A (1995). Identification of histatins as tannin-binding proteins in human saliva. Biochem J 311(Pt 1):341–347.[Medline] [Order article via Infotrieve]
• Yao Y, Lamkin MS, Oppenheim FG (1999). Pellicle precursor proteins: acidic proline-rich proteins, statherin, and histatins, and their cross -linking reaction by oral transglutaminase. J Dent Res 78:1696–1703.
• Yao Y, Lamkin MS, Oppenheim FG (2000). Pellicle precursor protein crosslinking characterization of an adduct between acidic proline-rich protein (PRP-1) and statherin generated by transglutaminase. J Dent Res 79:930–938.
• Yao Y, Berg EA, Costello CE, Troxler RF, Oppenheim FG (2003). Identification of protein components in human acquired enamel pellicle and whole saliva using novel proteomics approaches. J Biol Chem 278:5300–5308.[Abstract/Free Full Text]
• Young A, Rykke M, Rõlla G (1999). Quantitative and qualitative analyses of human salivary micelle-like globules. Acta Odontol Scand 57:105–110.[Medline] [Order article via Infotrieve]

Journal of Dental Research, Vol. 86, No. 8, 680-693 (2007)
DOI: 10.1177/154405910708600802


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