biochemical composition of human saliva in relation

BIOCHEMICAL COMPOSITION OF HUMAN SALIVA IN RELATION TO OTHER MUCOSAL FLUIDS LeonCP.M.Schenkels Enno C.I. Veerman Arie V. Nieuw Amerongen

Department of Oral Biochemistry, Academic Centre for Dentistry Amsterdam (AQA), Vrije Universiteit, Van der Boechorststraat 7, NL-1081 BT Amsterdam, The Netherlands

ABSTRACT: This paper describes several salivary components and their distribution in other mucosal secretions. Histatins are polypeptides which possess exceptional anti-fungal and anti-bacterial activities, but are nevertheless present only in saliva. Proline-rich proteins (PRPs) are members of a closely related family, of which the acidic PRPs are found solely in saliva, whereas the basic PRPs are also found in other secretions. Mucins are a group of glycoproteins that contribute to the visco-elastic character of the mucosal secretions. Despite the similarities in their structure and behavior, mucins have distinct tissue distributions and amino acid sequences. Other salivary proteins are present in one or more mucosal secretions. Lysozyme is an example of a component belonging to an ancient self-defense system, whereas secretory immunoglobulin A (slgA) is the secreted part of a sophisticated adaptive immune system. Cystatins are closely related proteins which belong to a multigene family. a-Amylase is a component that is believed to play a specific role in digestion, but is nevertheless present in several body fluids. Kallikrein and albumin are components of blood plasma. But whereas albumin diffuses into the different mucosal secretions, kallikrein is secreted specifically by the mucosal glands. The presence of these proteins specifically in saliva, or their distribution in other mucosal secretions as well, may provide important clues with respect to the physiology of those proteins in the oral cavity.

Keywords. Cystatins, histatins, mucins, proline-rich proteins, salivary proteins.

Introduction

T

he function of mucosal fluids is the subject of many investigations. These mucosal fluids cover the whole body surface, and therefore play an important role in the physiology of an organism. However, studies often focus on a particular body site and as a result, information on the composition of mucosal secretions has been scattered over many different scientific fields. This review will deal with the biochemical composition of human saliva in relation to other mucosal secretions.

Saliva and Other Mucosal Secretions Saliva is the fluid present in the oral cavity and is produced by different salivary glands. Its functional importance is evident in patients with reduced salivary flow (hyposalivation or xerostomia). A lack of saliva results in, e.g., a painful tongue and mucosa, problems with taste, swallowing, chewing, and phonation, tooth decay and tooth loss, and increased risk for infection (Table 1) (Vissink, 1985; Foxetal, 1985; Mandel, 1989; Axell, 1992).

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This lack of saliva can be caused by several circumstances, including aging (in medicated individuals), disease (Sjogren's syndrome), radiotherapy of the headneck region, and/or certain medications (e.g., antihypertensives, antidepressants, and antihistamines) (Valdez and Fox, 1993). Saliva belongs to a large group of mucosal fluids (e.g., tear fluid, nasal mucus, bronchial mucus, gastric mucus, colonic mucus, seminal plasma, cervical mucus, sweat) which have in common the wetting of the body surface. Several specific and essential processes take place on the body surfaces, e.g., exchange of gases, intake of nutrition, and excretion of waste products. These tissues are also vulnerable to all types of chemical, mechanical, and microbial attacks. Depending on the requirement of a particular body site, the composition of each secretion will be tuned appropriately to meet its protective and physiologic roles. Therefore, in some respects, the secretion composition will be site-specific in order to fulfill a particular function (respiration, digestion). On the other hand, mucosal secretions share a general function in the protection of epithelial tissue

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• proteins not originating from secretory glands, but from other sources like blood plasma (albumin, Zn-alpha2-glycoprotein). In addition, Table 3 summarizes details on the molecular biology of these proteins. While salivary proteins will not be discussed in detail, they will be reviewed in relation to the other mucosal secretions: first, proteins that are unique to saliva; and second, those that are common for mucosal secretions.

TABLE 1 Functions of Human Salivary Secretions Protective Functions Tissue coating (mucosal and tooth pellicle) Lubrication } Visco-elastic properties Humidification Remineralization of the teeth Host Defense Functions Immunological activity Anti-bacteria I activity Anti-viral activity Anti-fungal activity Digestion Digestive enzymes Bolus formation Taste

SALIVARY P R O T E I N S WITH A R E S T R I C T E D

DISTRIBUTION: HlSTATINS AND ACIDIC PROLINE-RICH PROTEINS Histatins

against external harmful effects. Principally, protection is achieved by the physical movement of exocrine glandular secretions that mechanically entrap and effectively remove many potentially harmful micro-organisms and compounds (Ericson et al, 1975; Mandel, 1979; Mandel and Ellison, 1985; Ligtenberg et al, 1992). Bacteria cluster by interaction with host components and form aggregates that are easily washed away. Components can also bind to microorganisms and thereby inhibit their adhesion to the mucosal surface (Williams and Gibbons, 1975). In addition, antibacterial mechanisms can influence the metabolism of bacteria by bacteriolysis, membrane damage, inhibition of growth, and cell killing (Lumikari et al, 1991). It is likely that the sharing of these general needs of mucosal surfaces for protection is the main reason that mucosal secretions also share a variety of protective components.

Salivary Components Present in Other Mucosal Fluids Similarities are found not only in the architecture of the different glands and the major functions of mucosal secretions, but also in the composition of the secretions. Table 2 shows the distribution of several salivary proteins in various body fluids. Based on distribution (saliva-specific or ubiquitous) and origin (salivary glands or external sources), salivary proteins can be classified into three groups: • proteins such as histatins and acidic prolinerich proteins which are present only in saliva; • proteins that are present in several different body fluids (lysozyme, mucins, and immunoglobulins), including saliva; or 162

Histatins are a family of related neutral and basic histidine-rich peptides which are secreted mainly in parotid saliva and, to a lesser extent, in submandibular saliva (Oppenheim et al, 1988; Sabatini et al, 1989). Twelve salivary histatins have been isolated from human saliva, and their primary structures have been determined. At least two histatins (histatin 1 and 3) originate from two different gene loci, while the others are products of posttranslational proteolysis (Oppenheim, 1989; Sabatini and Azen, 1989; Table 3). Spectroscopic analysis provides evidence that the candidacidal domain of histatins is structurally flexible, adopting a helical structure upon binding to membranes (Raj et al, 1994). Histatins possess antimicrobial properties against a few strains of Streptococcus mutans

(MacKay et al,

1984) and

inhibit

hemagglutination of the periopathogen Porphyrornonas gingivalis (Nishikita et al, 1989; Murakami et al, 1992). In addition, histatins neutralize the endotoxic lipopolysaccharides located in the outer membranes of Gram-negative bacteria, which may be an important part of the host's defense system (Sugiyama, 1993). Moreover, they are potent inhibitors of the growth and germination of Candida albicans, having an efficacy comparable with those of the synthetic antibiotics, imidazole and clotrimazole (Xu etal, 1991). It is assumed that these bactericidal and fungicidal effects occur through binding of the positively charged histatins to biological membranes, which subsequently results in a disruption of the membrane architecture and alteration in its permeability. In addition to antimicrobial activities, histatins are involved in functions specific for the oral cavity, such as formation of the acquired pellicle and participation in the mineralization dynamics of oral fluids (Oppenheim, 1989). Another biological role of histatins in the oral cavity is the inhibition of the release of histamine from mast cells, suggesting that they play a role in oral inflammation (Sugiyama et al, 1990). Despite their antimicrobial properties, which would favor a broad localization of histatins in fluids moistening epithelial surfaces, histatins have thus far been

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demonstrated only in secretions bathing the oral cavity. Thus, it would seem that the delicate mucosal epithelial tissues of the oral cavity, which are vulnerable to mechanical and microbial injuries, require more protection than the mechanically stronger keratinized skin tissue, despite the fact that both sites are exposed to microbial attacks. In this respect, it is interesting to note that granular glands present in amphibian skin also produce basic antimicrobial and membrane-active peptides: magainins (Jacob and Zasloff, 1994). Magainin-like substances have been discovered in human submandibular and labial minor salivary glands, but the exact nature of these peptides has not yet been elucidated (Wolff et al, 1990). Proline-rich proteins The human salivary proline-rich proteins (PRPs) are a heterogeneous group of proteins that comprise about 70% of the parotid proteins. They are characterized by a predominance of the amino acids proline, glycine, and glutamic acid/glutamine (a total of 80% of all amino acids). PRPs are classified into three groups: acidic (MW 16 kDa), basic (MW 6-9 kDa), and glycosylated (MW 36 kDa; Bennick, 1982). These PRPs are coded by a multigene family of 6 genes (Table 3), resulting in more than 20 PRPs by both differential RNA splicing and proteolytic cleavages after secretion (Maeda et al, 1985). The observation that a few genes result in a much larger number of protein species is also true for histatins. Interestingly, the activity of the processed PRPs and processed histatins remains, or is even enhanced. As can be noticed from Table 2, acidic PRPs are expressed only by the salivary glands, whereas the basic forms of PRPs are also found in other secretions (Sabatini et al, 1989). This specific distribution of acidic PRPs is striking because acidic, basic, and glycosylated PRP species are closely related in their amino acid sequence and their chromosomal localization (Table 3). Still, the functions attributed to the different types of PRPs are very distinct. The acidic PRPs bind Ca++ with a strength that indicates that they may be important in pellicle formation and in maintaining supersaturation of ionic calcium in relation to phosphate ions in saliva (Bennick et al, 1983). Therefore, the acidic PRPs may be of biological significance in maintaining the calcium homeostasis of saliva and in preventing the formation of salivary stones (Saitoh et al, 1985). In addition, acidic PRPs inhibit apatitic crystal growth, suggesting that, when adsorbed on the tooth surface, they block specific mineral growth sites (Aoba et al, 1984). Glycosylated basic PRPs (PRGs) function as masticatory lubricants (Hatton et al, 1985) and have also been shown to interact with several types of micro-organisms such as Fusobacterium nucleatum (Gillece-Castro et al, 1991). In addition, the PRPs are thought to serve as a defense mechanism against dietary tannins by forming precipitates, which reduce harmful effects of tannins (Mehansho

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et al, 1987). It has been suggested that PRPs are involved in the bitter taste sensation (Azen et al, 1990). In conclusion, acidic PRPs, which are involved in typical oral processes like mineral homeostasis and neutralization of toxic substances in the diet, are present only in salivary secretions. However, the broader localization of basic PRPs (in saliva, in nasal and bronchial mucus) would point to a more general protective function for this group of proline-rich proteins. SALIVARY PROTEINS PRESENT IN OTHER FLUIDS

Statherins Statherin is a low-molecular-weight acidic protein consisting of 43 amino acids. It is secreted by the parotid, submandibular, and von Ebner's salivary glands, but is not present in labial saliva (Hay et al, 1984; Azen et al, 1990). Evidence has been presented that statherin, together with the acidic PRPs, plays a role in the calcium homeostasis of saliva (Hay et al, 1986; Raj et al, 1992). Statherin inhibits the spontaneous precipitation of calcium phosphate and calcium carbonate salts, a function in which the first 18 amino acids appear to be essential (Schwartz et al, 1992). Furthermore, statherin bound to the enamel surface can inhibit crystal growth of hydroxyapatite. For this function, the first 6 amino acids of the polypeptide are essential. Statherin adsorbed onto hydroxyapatite can promote adherence of a few oral bacteria, such as P. gingivalis and Actinornyces viscosus (Amano

et al, 1994). This property is not exhibited when statherin is in a soluble state. As a component of oral pellicles, statherin may contribute to boundary lubrication of oral surfaces, i.e., a lubrication that is mediated by a macromolecular layer of proteins (Douglas etal. 1991). In addition to salivary secretions, statherin is also present in tear fluid and nasal and bronchial mucus. The role of statherin in these fluids is not clear, but it is tempting to speculate that it also prevents excessive precipitation of calcium salts in these fluids. Mucins Mucins are proteins that give the typical visco-elastic character to all the mucosal secretions (Table 2) (Balmer and Hirsch, 1978; Waterman et al, 1988; Veerman et al, 1989; Van der Reijden et al, 1993). They are defined as a distinct group of glycoconjugates, differing structurally from serum glycoproteins and proteoglycans (Strous and Dekker, 1992). Mucins are highly glycosylated proteins with sugars constituting from 50% up to 90% of the dry weight of the molecule. The oligosaccharide side-chains vary in length from 1 to more than 20 sugar residues, mostly attached by O-glycosidic linkages of N-acetylgalactosamine to serine or threonine (Klein et al, 1992). The biochemical and functional properties of mucins are mainly determined by the terminal residues, particularly


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TABLE 2 Distribution of Salivary Proteins in Several Body Fluids Saliva 0

Tear

Fluidb

Nasal Mucusc

Bronchial Mucusd

Seminal Plasmae

Cervical Mucus*

Blood

Sweats

Plasmah

Mucins Acidic PRPs a-Amylase Basic PRPs Basic PRG Secretory IgA Cystatins Statherin igG EP-GP VEGh Hi statins Lysozyme Kallikrein Lactoferrin Lactoperoxidase Haptocorrin B-Microseminoprotein IgM Albumin Zn-a2 Glycoprotein

- not detected; + < 1 % of the total protein amount, or detected but not estimated; ++ between 1 % and 5% of the total protein amount; +++ between 5% and 15% of the total protein amount; ++++ > 15% of the total protein amount; empty spaces indicate "not determined".

References: (a) Abrahamson etai, 1986; Kamboh and Ferrell, 1986; Nieuw Amerongen, 1994; Page etai, 1990; Sabatini etai, 1989; Schenkels ef ai, 1991; Tonnesen ef ai, 1990; Weiber ef ai, 1991. (b) Abrahamson etai, 1986; Fullard and Snyder, 1990; Kamboh and Ferrell, 1986; Kuizenga etai, 1990; Raphael etai, 1989; Sabatini ef ai, 1989; Schenkels ef ai, 1991; Tonnesen ef ai, 1990; Weiber ef ai, 1991. (c) Abrahamson ef ai, 1986; Foster ef ai, 1991; Getchell and Mellert, 1991; Hegnoj ef ai, 1985; Kaliner, 1991; Mestecky and McGhee, 1987; Raphael ef ai, 1989; Sabatini ef ai, 1989; Schenkels ef ai, 1991; Tachibana ef ai, 1986; Tonnesen ef ai, 1990. (d) Hayashi etai, 1986; Hegnoj etai, 1985; Raphael etai, 1989; Sabatini etai, 1989; Schenkels etai, 1991; Van-Seuningen etai, 1992. (e) Abrahamson ef ai, 1986; Eckersall and Beeley, 1981; Schenkels ef ai, 1991; Weiber ef ai, 1990. (f) Hayashi ef ai, 1986; Hegnoj ef ai, 1985; Pruitt ef ai, 1990; Salas-Herrera ef ai, 1991; Schenkels ef ai, 1991. (g) Dupuy ef ai, 1990; Kamboh and Ferrell, 1986; Nakayashiki, 1990; Poblete ef ai, 1991; Schenkels ef ai, 1991; Yokozeki ef ai, 1991. (h) Abrahamson etai, 1986; Kamboh and Ferrell, 1986; Mestecky and McGhee, 1987; Raphael etai, 1989; Schenkels etai, 1991; Hitzig, 1977.

sialic acid, sulphate, or fucose. The protein backbone is built up of tandem repeats, and the predominant amino acids are serine, threonine, alanine, and proline. These tandem repeats are heavily glycosylated and are therefore insensitive to several proteases (Rose, 1992; Verma and Davidson, 1994; Wu et ai, 1994). Mucins therefore are able to protect the underlying tissue against proteolytic

164

attack by micro-organisms. The N-terminal and C-terminal ends of the protein backbone have a more globular protein structure, with several cysteine residues involved in molecular complex formation (Strous and Dekker, 1992). In general, the physiological functions of the mucins include, among others, cytoprotection, lubrication, pro-

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tection against dehydration, and maintenance of viscoelasticity in secretions (Levine et al, 1987; Tabak, 1991). These functions of mucins are performed in part by forming heterotypic complexes with other salivary proteins, e.g., slgA and albumin (Levine et al, 1987). Mucins also promote the clearance of various bacteria by masking their surface adhesins, a factor which inhibits bacterial colonization (Koop et al, 1990; Toribara et al, 1991). Mucins also play a role in mucosal surface coating and in the adherence of the microbial flora (Tabak and Bowen, 1989). The visco-elastic properties of mucins play a role in lubrication, and are considered to be an important characteristic of mucins (Gans et al, 1990; Van der Reijden et al, 1993). The capacity of mucins to protect epithelial surfaces depends largely on their high content of oligosaccharides and their ability to form a gel layer together with other salivary proteins (Bradway et al, 1992). Changes in amounts and characteristics of mucins lead to altered rheological behavior. In addition, structural changes, particularly in the oligosaccharide moiety of secretory mucins, are used as markers for malignancy in several lesions (Gendler et al, 1991). Human saliva contains two saliva-specific types of mucins—namely, low- and high-molecular-weight mucin glycoproteins, MG2 (150-200 kDa) and MG1 (> 1000 kDa), respectively—which are structurally and functionally distinct (Loomis et al, 1987). MG1 adheres to the tooth surface, thereby forming a barrier against acidic attacks (Nieuw Amerongen et al, 1987). There is little evidence for MG1 interaction with bacteria except Haemophilus paminfluenzae (Veerman et al, 1995), whereas MG2 binds to a large number of different micro-organisms (Stinson et al, 1982; Levine et al, 1987; Biesbrocketa!., 1991; Ligtenbergeta/., 1992; Scannapieco and Levine, 1993), including Candida albicans (Hoffman and Haidaris, 1993) and Actinobacillus actinomycetemcomitans (Groenink eta/., 1995). The gene of MG2 has recently been cloned (Bobek et al, 1993). Up to now, 7 mucin genes have been identified, and these are summarized in Table 3. Surprisingly, neither the amino acid sequences nor the chromosomal localization of the known mucin genes shows any similarity to each other (Verma and Davidson, 1994). Initially, expression of these genes was thought to be tissue-specific. However, it is becoming evident that mucin genes can be expressed at multiple sites (Audie et al, 1993). For instance, the submandibular gland has been shown to express the MUC5B gene, originally isolated from tracheobronchial tissue (Audie et al, 1993). Further research is needed to reveal which genes for mucins are expressed in the salivary glands, and whether MG1 and/or MG2 can also be found in other secretions. Thus, mucins form a very heterogeneous group of proteins, present at very specific locations. Despite their genetic diversity in structure and physiology, different

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mucin species are comparable and serve essentially the same functions. Cystatins Animal tissues and body fluids contain various kinds of endogenous proteinase inhibitors to regulate their protein metabolism or to protect tissues from proteolytic attacks by bacteria or viruses (Abrahamson, 1988). Cystatins belong to the class of cysteine proteinase inhibitors (Barrett, 1987). Like mucins, cystatins constitute a group of proteins present in all mucosal secretions. Cystatins belong to a heterogeneous family of proteins with a conserved consensus in their active site (QXVXG motif and an essential G in the N-terminal region). The superfamily of cystatins is subdivided into three main families (Table 3). Of these, family II cystatins in particular are secreted in different mucosal secretions. The genes encoding family II cystatins are all localized on chromosome 20 (Fong et al, 1991) and are clustered on one locus on 20pl 1 (Saitoh et al, 1991; Schnittger et al, 1993), illustrating the close genetic relationship between the members of this cystatin family. The concentrations of cystatins vary greatly between the different fluids (Abrahamson et al, 1986). The predominant inhibitor in cerebrospinal fluid, seminal plasma, and in milk is cystatin C, whereas the cystatins S, SA, and SN dominate in saliva and tears. Kininogen is the most abundant in blood plasma, synovial fluid, and amniotic fluid. Based on the different inhibition constants of the cystatins, it is suggested that cystatin C is the controlling inhibitor for cathepsin B (the predominant human cysteine proteinase) in almost every fluid, aided by cystatin S group in saliva and tears. Cystatins A and B do not participate in the control of cathepsin B. The latter cystatins may have a physiological role in controlling exogeneous (e.g., bacterial, viral) cysteine proteinases. Thus, all types of cystatins are present in the various mucosal secretions, but their concentrations vary, depending on the specific site. Although their role as a proteinase inhibitor is wellestablished, the physiological significance of cystatins in exocrine secretions remains to be fully explained. Nevertheless, as mentioned previously with respect to the control of exogenous proteinases, cystatins may also regulate the activity of cathepsins, liberated during inflammatory reactions, e.g., in gingivitis and periodontitis (Ichimura et al, 1992; Henskens et al, 1994). It has been shown that cystatins are important in the inhibition of several viruses, presumably by blocking necessary cysteine proteinases (Korant et a!., 1985; Bjorck et al, 1990). Another function of cystatins is the control of the proliferation and invasion of tumor cells (Sloane and Honn, 1984). Typical salivary functions have also been ascribed to cystatins. They have been reported to bind to hydroxyapatite (Rathman et al, 1989), and therefore may play a role


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TABLE 3 Genes Encoding Salivary Components and Homologues Protein (s)

Gene

Chromosomal Localization

Remarks

References

Mucins

MUC1 MUC2 MUC3 MUC4 MUC5 MUC6 MUC7

lq21-24

Ilpl5.5 7q22 3q29 Ilpl5 Ilpl5.5 4

epithelial cells (Episialin) gastro-intestinal, prostatic intestinal tracheobronchial tracheobronchial gastric salivary (MG2)

Dracopoli etai, 1991 Xuefa/,,1992 Fox ef ai, 1992 Nguyen etaL, 1991 Nguyen ef a/., 1990 Toribara ef a/., 1993 Bobek ef a/., 1993

Acidic Proline-rich Proteins (aPRP)

PRH1 PRH2

12pl3.2 12pl3.2

specific for saliva

Kim ef a/., 1990

a-Amylase

AMY1A/B AMYlc AMY2A/B AMYP1

Ip21 Ip21 1P21

saliva

Grootefa/., 1989 Dracopoli etai, 1991

Basic Proline-rich Proteins (bPRP)

PRB1 PRB2 PRB4

12pl3.2 12pl3.2 12pl3.2

Basic Proline-rich Glycoprotein (PRG)

PRB3

12pl3.2

Immunoglobulin A

IGHA

14q32.33

Bengerefa/., 1991

J chain

IGJ

4q21

Maxefa/., 1986

Secretory component

SC or PIGR

Iq31-q42

identical to transmembrane SC or poly Ig receptor

Krajciefa/., 1991

Cystatins

Family 1

STF1

3cen-q21

Cystatin A

Hsiehefa/., 1991

Family II

CST1 CST2 CST3 CST4 CST5 CSTP1 CSTP2

20p11.2

Cystatin SN Cystatin SA Cystatin C Cystatin S Cystatin D Cystatin pseudogene

Saitoh ef al, 1987 Schnittger ef a/., 1993

KNG

3q26-qter

Statherin

STATH

4ql 1-13

Hi statins

HTN1 HTN3

4ql2.q21 4ql2-q21

Lysozyme

LYZ

12

Kallikreins

KLK1 APS KLK2

19ql3.2-ql3.4 19ql3.2-ql3.4 19ql3.2-ql3.4

Family III

166

pancreas pseudogene

Kim ef a/., 1990

basic proline-rich glycoprotein

20pl1.2 20pl 1.2 20pll.2 20pll.21 20pl 1.2 20pl 1.2

Kim ef a/., 1990

Cystatin pseudogene

Kininogen

Cheung etai., 1992 Sabatini ef ai, 1993

specific for saliva

Sabatini and Azen, 1989

Peters ef a/., 1989 tissue Kallikrein human glandular Kallikrein-1 prostate-specific antigen

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Riegman ef a/., 1992

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in acquired pellicle formation. On the other hand, cystatins have been shown to inhibit hydroxyapatite crystal growth (lohnsson et al, 1991). OC-AMYLASE

Amylase (46-60 kDa) is an abundant salivary component. It was first described 150 years ago (Leuchs, 1831) under the name "diastase". It is an enzyme (EC 3.2.1.1.) that catalyzes the hydrolysis of oc(l,4)glycosidic binding between glucose residues of polysaccharides like starch, glycogen, and dextrins. a-Amylase is found in animals as distinct as insects and mammals (Karn and Malacinski, 1978), and it is especially found in those with starch or glycogens as part of their diet. Human amylase exists in two forms, produced mainly in the pancreas and in the salivary glands. These two forms have 97% amino acid homology. Salivary and pancreatic amylase have been shown to be the products of two closely related but different genetic loci, AMY1 and AMY2, respectively (Table 3). In addition to saliva, amylase has been demonstrated in virtually every mucosal fluid of the human body, e.g., tears, semen, bronchial mucus, etc. This ubiquitous distribution of amylase is difficult to reconcile with its single function of digestion. It would thus be appropriate to seek other functions of amylase, in line with the concept of multifunctional saliva proteins (Levine, 1993). In this respect, Mellersh etal. (1979) have reported that amylase displays growth-inhibitory activity against Neisseria gonorrhoeae. More recently, specific binding of salivary amylase to S. gordonii has been demonstrated by various investigators (Douglas, 1983; Scannapieco et al, 1989), although this binding appears to be more for the benefit of the guest, rather than for the host (bacterium-bound amylase allows the microbe to utilize starch as a carbohydrate source (Douglas et al, 1992). Studies to elucidate the role of amylase in other secretory fluids (tears, seminal fluid, sweat, bronchial mucus) have not been reported. Secretory Immunoglobulin A (slgA) slgA is a member of the adaptive immune response (Mestecky and McGhee, 1987). slgA is the predominant molecule especially designed for secretions, and thus it is the predominant immunoglobulin of the mucosal immune system. In addition, the slgA production (± 66 mg/kg/day) is greater than that of the other isotypes of immunoglobulins (Kraehenbuhl and Neutra, 1992). This high production is necessary to cover the epithelial surfaces of the body. slgA is synthesized by specific cells, B lymphocytes, which are distributed throughout the body. The complete slgA molecule is made up of two four-chain units of IgA, one secretory component (MW 70 kDa), and one joining (I) chain (MW 15 kDa). In contrast, the serum IgA exists largely as a monomer. A siterestricted slgA-associated response can be induced by

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local stimulation of mucosal membranes and secretory glands with antigens. However, local stimulation is not the only pathway that leads to an effective induction of a secretory immune response. Secretions of glands that are anatomically remote from the site of immunization, such as mammary, salivary, and lacrimal glands, can contain slgA antibodies to antigens encountered through the respiratory or gastro-intestinal tracts. Following this pathway, IgA-producing cells are induced by the common mucosal immune response, consisting of lymphoid tissues concentrated in special structures, such as the Peyer's patches. An immune response results in a pattern of relatively specific re-circulation of lymphoid cells from these specialized structures to the mucosa. This process is known as mucosal homing. The slgA-associated response, induced by the latter pathway, initiates a general response of the mucosal immune system. The protective role of slgA has been demonstrated in several experimental systems. slgA can neutralize antigens (from viruses, toxins, and enzymes; Tomasi, 1983). It has been proposed that slgA may interact in concert with the innate immune factors (e.g., lysozyme, lactoperoxidase, lactoferrin; Rudney and Smith, 1985). The effects of slgA deficiency on man are still controversial. About 30% of the patients suffering from this deficiency are affected by upper respiratory tract infections, including sinusitis (Kaliner, 1991). In contrast, no correlation has been found between slgA deficiency and periodontal diseases, possibly due to compensation by normal or even elevated IgG and IgM levels (Norhagen-Engstrom et al 1992; Kirstila et al, 1994). The correlation between caries and levels of salivary IgA antibodies to the Streptococcus rnutans group is still unclear (Hocini et al, 1993). Lysozyme Lysozyme (EC 3.2.1.17; MW 14 kDa), also called muramidase, was first recognized by Fleming in 1922 for its antibacterial effect (Fleming and Allison, 1922). It is a widely distributed enzyme occurring in many human secretions (Table 2), as well as in various other vertebrates and invertebrates (bacteria, phages, and plants). Because of its widespread occurrence, lysozyme is considered to belong to a primitive defense system, also known as the innate immune system. Lysozyme has been the subject of numerous investigations, and several properties of this protein have been elucidated, e.g., its phylogeny, structure, catalytic mechanism, immunology, structurefunction relationships, and genetics. It is generally accepted that the physiological significance of lysozyme lies in its anti-bacterial properties. These properties were first ascribed to the enzymatic activity of lysozyme, which is able to cleave |3(l-4)-glycosidic bonds between muramic acid and N-acetylglucosamine residues in the peptidoglycan of the bacterial cell wall. Although the


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majority of bacterial species are not directly lysed upon exposure to lysozyme, their cell walls become weakened to such an extent that subsequent addition of anions or detergent results in immediate lysis (Goodman et al, 1981; Cho et al, 1982; Pollock et al, 1987) . In addition, data have been presented indicating that binding of heat-inactivated lysozyme to bacterial cells is sufficient to induce killing and lysis. Several theories have been put forward to explain the non-enzymatic bactericidal activity of lysozyme, e.g., it has been proposed that binding of lysozyme might activate bacterial autolysins (Laible and Germaine, 1985). Other mechanisms proposed are bacterial aggregation (Pollock et al, 1976), inhibition of bacterial adherence (Iacono et al, 1980), or inhibition of bacterial metabolism (Twetman et al, 1986). Thus, although the bactericidal role of lysozyme is undisputed, the precise mechanism is still unknown. Extra-Parotid GlycoProtein (EP-GP) This acidic salivary glycoprotein of low molecular weight, 18-20 kDa, was originally isolated from submandibularsublingual saliva and has been shown to have a strong affinity to hydroxyapatite (Rathman et al, 1989). EP-GP can be localized in the submandibular glands only in the serous acinar cells and is absent from the parotid gland (Rathman et al, 1990). Based on these data, it was presumed that EP-GP would have a specific function in the oral cavity, e.g., as a component of the dental pellicle. However, because identical proteins were detected in a number of other secretion fluids, e.g., tears, nasal mucus, and seminal plasma, this idea was abandoned (Schenkels et al, 1991). This was further strengthened by the finding that this protein, in vitro as well as in vivo, binds to several oral (e.g., Streptococcus salivarius) and nonoral bacteria (e.g., Staphylococci) (Schenkels etal, 1993). Its polypeptide chain appears to be biochemically identical to that of Secretory Actin Binding Protein (SABP) in seminal plasma, Prolactin-Inducible Protein (PIP), and Gross Cystic Disease Fluid Protein-15 (GCDFP-15) (Schenkels et al, 1994). However, these proteins can be distinguished from each other by differences in the degree of glycosylation. The function of EP-GP and its homologues has not yet been elucidated. However, the fact that, both in vitro and in vivo, this protein binds to oral streptococci leads one to suggest a role for this protein in the modulation of oral microflora. Furthermore, it is noteworthy that an acidic protein of 20 kDa is also present in the secretion fluid of the Von Ebner's salivary gland in the tongue, designated VEGh (Blaker et al., 1993). Although biochemically it has a number of properties in common with EP-GP, they are unrelated. VEGh appears to be a member of the superfamily of the lipocalins, which are also present in different body fluids.

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Kallikrein Kallikreins (EC 3.4.21.8) are a group of serine proteases that are found in glandular cells, neutrophils, and biological fluids. The best-known activity of these enzymes is the cleavage of kininogen into kinin. Man has 3-5 genes for kallikrein: hRKALL, hGGK-1, and PSA (clustered on 19ql3.3-13.4). Glandular (tissue) kallikrein is found in a variety of tissues and biological fluids, including saliva (Mann et al, 1980). Kallikrein has a well-documented role in blood coagulation, via the activation of the Hageman factor. However, specific functions for this enzyme in secretions have not yet been determined, although in saliva some hydrolysis of proline-rich proteins occurs by kallikrein (Bennick, 1982). It has been implicated in the regulation of local blood flow in salivary glands (Berg et al, 1989), the processing of polypeptide hormones such as epidermal growth factor (Issaksson et al, 1987), in ion transport in epithelial cells (Lewis and Alles, 1986), and neutrophil chemotaxis (Modeer, 1977). Marked increases in salivary kallikrein have been observed following restriction of dietary sodium (Horwitz et al, 1982). In addition, higher levels of kallikrein have been observed in subjects with tumors of both oral and non-oral origin when compared with control subjects (Jenzanoeta/., 1986; Arnold etal, 1989). Haptocorrin Haptocorrin is an acidic glycoprotein (MW 60-80 kDa) that is present as a minor component in blood and other body fluids. It binds cobalamine (Vitamin B12) but should be distinguished from two other vitamin B12binding proteins: the intrinsic factor and transcobalamine. Of these three cobalamine-binding proteins, only haptocorrin has been detected in saliva, in which the concentration is at least three-fold higher than in serum. The highest concentrations have been detected in tears and nasal secretion (Tonnesen et al, 1990). In human salivary glands, it has been localized only in mucous acinar cells and in intercalated duct cells (Nexo et al, 1985), from which it can be released via |3-adrenergic receptor stimulation. In common with Zn-oc2-glycoprotein and EP-GP, haptocorrin can be visualized by iso-electric focusing as a ladder of 4-6 isoproteins, differing from each other by about 0.2 pH-units—a pattern which is different in the various mucous fluids. Apparently, all three glycoproteins are glycosylated in a way that is characteristic for a gland type. In addition to the involvement of haptocorrin in making available vitamin B12 to an organism, it has been suggested to play a role in the defense against micro-organisms (Nexo et al, 1985). jHVHCROSEMINOPROTEIN

)3-microseminoprotein is one of the major proteins in seminal plasma and is an example of a protein to which


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a tissue-specific function had first been attributed because of its presumed specific localization in prostate secretions. Originally, this 11-kDa protein, consisting of 94 amino acids, was isolated from human semen as a factor inhibiting the pituitary release of follitropin. However, since highly purified preparations of the protein were devoid of such activity, this function was questioned. Moreover, in later studies it was shown that the same protein was present in several other tissues and secretory fluids of the human body (Table 2), including bronchial secretions, even at a concentration comparable with that of seminal plasma, thereby suggesting a more general function (Weiber et al, 1990). For example, because of its presence in various amounts in mucous secretions, it has been hypothesized that it may be a factor that modifies the properties of mucus, such as the viscosity and its ability to adhere to surfaces of mucous membranes. Similarly, it has been proposed that |3microseminoprotein has a protective function for the mucins by acting as a physiological inhibitor of endogenous mucin-degrading enzymes from leucocytes, or by acting as an anti-bacterial agent. To our knowledge, these claims, although interesting, have never been experimentally substantiated. PROTEINS NOT ORIGINATING FROM SECRETORY GLANDS

Albumin Albumin is the most abundant protein present in serum plasma, constituting from 55 to 62% of the total serum proteins. It is an acidic (pi 4.9) 67.5-kDa protein with affinity for a broad spectrum of components, e.g., water, Ca++, Na+, K+, fatty acids, bilirubin, hormones, and drugs. In several glandular salivas, albumin has been detected as a minor component (Rathman et al, 1989; Sweeney and Beeley, 1990). Albumin concentrations in saliva and other mucosal secretions reflect a passive contribution of serum-derived protein, which may be caused by epithelial inflammation. Changes in albumin content of saliva can be used as a diagnostic tool for certain diseases like chronic pancreatitis (Durr et al, 1982) and stomatitis in cancer therapy patients (Izutzu et al, 1981). In the saliva of orally healthy individuals, albumin can be detected in only very small amounts (Henskens et al, 1993). Salivary albumin concentrations are significantly increased in individuals with gingivitis or periodontitis in comparison with healthy subjects (Henskens et al, 1993). Saliva-specific functions have also been described for albumin. When hydroxyapatite is exposed to whole saliva, it is rapidly bound by this mineral (Rolla et al, 1983; Rathman et al, 1989). Accordingly, albumin has been described as a component of the acquired pellicle (Eggen and Rolla, 1984). Albumin is also found in a complexed form with prolinerich glycoprotein (PRG), and this complex, as a pellicle

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constituent, appears to play an effective role in lubrication of oral tissue surfaces (Hatton et al, 1985). Zinc-0C2~Glycoprotein Zinc-oc2-Glycoprotein (Zn-a2-GP) was detected in serum for the first time in 1961. It consists of a single polypeptide chain of 41 kDa, contains 18% carbohydrate (Kamboh and Ferrell, 1986), and gets its name because it precipitates zinc ions. Different isoforms of Zinc~cc2-glycoprotein are present as a minor component in several body fluids, e.g., serum, sweat, tears, and saliva. By isoelectric focusing, a ladder of protein bands was observed which could be reduced by sialidase treatment to one major band with a pi of 5.2. Apparently, one and the same polypeptide chain can be glycosylated differently in the various tissues. In pregnant women, Zn-oc2-glycoprotein was found to have a higher content of sialic acid. Although it has been suggested to play a role as a carrier protein for the nephritogenic renal glycoprotein, its biological function is still unknown.

Conclusions An overview of the composition of glandular secretions reveals that the proteins that are genuinely specific for saliva are few. Most salivary components are also present in other mucosal secretions, and in turn can be subdivided into two groups. One group consists of similar components encoded by identical genes (lysozyme, slgA). The second group consists of components that are not identical but are structurally or genetically related, e.g., cystatins, mucins, oc-amylase, kallikreins. This divergence may be important to induce molecular adjustments to the physical environment of the specific secretion. Divergence, on the other hand, may function as a playground for evolutionary experiments on secretory proteins. The distribution of the salivary components also has consequences for the physiological roles they play in the oral cavity. Proteins present in more than one secretion consequently have functions common to mucosal secretions, as for instance, protection. On the other hand, one might expect that a protein that is present only in saliva will have functions that are specific for saliva. The only saliva-specific proteins appear to be histatins and acidic PRPs. Consequently, these proteins presumably have a predominant function that is unique to the oral cavity. Other components will serve mainly in the common functions of mucosal secretions. Additionally, many components described here share two principles that can be applied to functions of the mucosal secretions: First, many secretory proteins share more than one function, consistent with the finding that, in several proteins, functional modular units can be distinguished (Baron et al, 1991); and second, mucosal components tend to complex with each other, thus forming new functional aggregates— for example, in pellicles (Levine, 1993).


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REFERENCES

Abrahamson M (1988). Human cysteine proteinase inhibitors: Isolation, physiological importance, inhibitory mechanism, gene structure and relation to hereditary cerebral haemorrhage. Scand J Clin Lab Invest 48(Suppl 191):21-31. Abrahamson M, Barrett AJ, Salvesen G, Grubb A (1986). Isolation of six cysteine proteinase inhibitors from human urine; their physicochemical and enzyme kinetic properties and concentrations in biological fluids. J Bio! Chern 261:11282-11289. Amano A, Sojar HT, Lee J-Y, Sharma A, Levine MJ, Genco RJ (1994). Salivary receptors for recombinant fimbrillin of Porphyromonas gingivalis. Infect \mmun 62:3372-

3380. Aoba T, Moreno EC, Hay DI (1984). Inhibition of apatite crystal growth by the amino-terminal segment of human salivary acidic proline-rich proteins. Calcif Tissue Int 36:651-658. Arnold WH, Bartholmes P, Senkel H, Roodenburg JLN, Roder R (1989). Kallikrein activity in saliva of patients with inflammatory hyperplastic diseases of the oral mucous membrane. Adv Exp Ued Biol 247(B):385-387. Audie JP, Janin A, Porchet N, Copin MC, Gosselin B, Aubert JP (1993). Expression of human mucin genes in respiratory, digestive and reproductive tracts ascertained by in situ hybridization. I Histochem Cytochem 41:1479-1485. Axell T (1992). The oral mucosa as a mirror of general health or disease. Scand J Dent Res 100:9-16. Azen EA, Hellekant G, Sabatini LM, Warner TF (1990). mRNAs for PRPs, statherin and histatins in Von Ebner's gland tissues. J Dent Res 69:1724-1730. Balmer RT, Hirsch SR (1978). The non-Newtonian behaviour of human saliva. AICHE Symp Ser Biorheol 74:125-129. Baron M, Norman DG, Campbell ID (1991). Protein modules. Trends Biochem Sci 16:13-17.

Barrett AJ (1987). The cystatins: a new class of peptidase inhibitors. Trends Biochem Sci 12:193-196. Benger JC, Teshima I, Walter MA, Brubacher MG, Daouk GH, Cox DW (1991). Localization and genetic linkage of the human immunoglobulin heavy chain genes and the creatine kinase brain (CKB) gene: Identification of a hot spot for recombination. Genomics 9:614-622. Bennick A (1982). Salivary proline-rich proteins. Mol Cell Biochem 45:83-99. Bennick A, Chau G, Goodlin R, Abrams S, Tustian D, Madapallimattam G (1983). The role of human salivary acidic proline-rich proteins in the formation of acquired dental pellicle in vivo and their fate after adsorption to the human enamel surface. Arch Oral Biol 28:19-27. Berg T, Carretero OA, Scicli AG, Tilley B, Stewart JW (1989). Role of kinin in regulation of rat submandibu-

170

lar gland blood flow. Hypertension 14:73-80. Biesbrock AR, Reddy MS, Levine MJ (1991). Interaction of a salivary mucin-secretory immunoglobulin A complex with mucosal pathogens. Infect \mmun 59:3492-3497. Bjorck L, Grubb A, Kjellen L (1990). Cystatin C, a proteinase inhibitor, blocks replication of herpes simplex virus. } Virol 64:941-943. Blaker M, Kock K, Ahlers C, Buck F, Schmale H (1993). Molecular cloning of human Von Ebner's gland protein, a member of the lipocalin superfamily highly expressed in lingual salivary glands. Biochem Biophys Acta 1172:131-137. Bobek LA, Tsai H, Biesbrock AR, Levine MJ (1993). Molecular cloning, sequence, and specificity of expression of the low molecular weight human salivary mucin (MUC7). J Biol Chem 268:20563-20569. Bradway SD, Bergey EJ, Scannapieco FA, Ramasubbu N, Zawacki S, Levine MJ (1992). Formation of salivarymucosal pellicle: the role of transglutaminase. Biochem J 284:557-564. Cheung PP, Cannizzaro LA, Colman RW (1992). Chromosomal mapping of human kininogen gene (KNG) to 3q26-qter. Cytogenet Cell Genet 59:24-26. Cho MI, Holt SC, Iacono VJ, Pollock JJ (1982). Effects of lysozyme and inorganic anions on the morphology of Streptococcus mutans BHT—Electron-microscopic examination. J Bacteriol 151:1498-1507. Douglas CWI (1983). The binding of human salivary ccamylase by oral strains of streptococcal bacteria. Arch Oral Biol 28:567-583. Douglas CWI, Heath J, Gwynn JP (1992). Enzymatic activity of salivary glands when bound to the surface of oral streptococci. FEMS Microbiol Lett 92:193-198. Douglas WH, Reeh ES, Ramasubbu N, Raj PA, Bhandary KK, Levine MJ (1991). Statherin—A major boundary lubricant of human saliva. Biochem Biophys Res Commun 180:91-97. Dracopoli NC, O'Connell P, Eisner TI, Lalouel JM, White RL, Buetow KH, et al. (1991). The CEPH consortium linkage map of human chromosome 1. Genomics 9:686-700. Dupuy P, LePendu J, Jothy S, Wilkinson RD (1990). Characterization of a monoclonal antibody against a mucin-type glycoprotein in human sweat. Hybridoma 9:589-596. Durr GHK, Amouric M, Bode C, Sarles H, Figarella C (1982). Salivary secretion in chronic pancreatitis with special reference to albumin and lactoferrin. Digestion 24:87-93. Eckersall PD, Beeley JA (1981). Human saliva and semen: Evidence for proteins common to both which do not occur in serum. Clin Sci 60:179-184. Eggen KH, Rolla G (1984). Further information on the composition of the acquired enamel pellicle. In: Cariology today. Guggenheim B, editor. Basel: Karger, pp. 109-118.


6(2):161-175 (1995)

Ericson T, Pruitt K, Wedel H (1975). The reaction of salivary substances with bacteria. J Oral Pathol Ued 4:307323. Fleming A, Allison VD (1922-23). Observations on a bacteriolytic substance (lysozyme) found in secretions and tissues. Br J Exp Pathol 252-260. Fong D, Chan MM-Y, Hsieh W-T (1991). Gene mapping of human cathepsins and cystatins. Biorned Biochim Ada 50:595-598. Foster ID, Getcher ML, Getcher TV (1991). Identification of sugar residues in secretory glycoconjugates of olfactory mucosae using lectin histochemistry. Anat Rec 229:525-544. Fox MF, Lahbib F, Pratt W, Attwood J, Gum I, Kim Y, et al. (1992). Regional localization of the intestinal mucin gene MUC3 to chromosome 7q22. Ann Hum Genet 56(Pt4):281-287. Fox PC, Van der Ven PF, Sonies BC, Weiffenbach JM, Baum BJ (1985). Xerostomia: Evaluation of a symptom with increasing significance. J Am Dent Assoc 110:519-525. Fullard RJ, Snyder C (1990). Protein levels in nonstimulated and stimulated tears of normal human subjects. Invest Ophthalmol Vis Sri 31:1119-1126.

Gans RF, Watson GE, Tabak LA (1990). A new assessment in vitro of human salivary lubrication using a compliant substrate. Arch Oral Biol 35:487-492. Gendler S), Spicer AP, Lalani EN, Duhig T, Peat N, Burchell J, et al. (1991). Structure and biology of a carcinoma-associated mucin, MUC1. Am Rev Respir Dis 144:S42-S47. Getchell ML, Mellert TK (1991). Olfactory mucus secretion. In: Smell and taste in health and disease. Getchell TV, Bartoshuk LM, Doty RL, Snow JB, editors. New York: Raven Press, pp. 83-95. Gillece-Castro BL, Prakobphol A, Burlingame AL, Leffler H, Fisher SJ (1991). Structure and bacterial receptor activity of a human salivary proline-rich glycoprotein. I BiolChem 266:17358-17368. Goodman H, Pollock JJ, Katona LI, Iacono VI, Cho M, Thomas E (1981). Lysis of Streptococcus mutans by hen egg white lysozyme and inorganic sodium salts. I Bacteriol 146:764-774. Groenink ], Ligtenberg AJM, Veerman EG, Nieuw Amerongen AV (1995). Interaction of the salivary lowmolecular weight mucin (MG2) with Actinobacillus actinomycetemcomitans. Infect \rnrnun (submitted). Groot PC, Bleeker MJ, Pronk JC, Arwert F, Mager WH, Planta RJ, et al. (1989). The human oc-amylase multigene family consists of haplotypes with variable numbers of genes. Genomics 5:29-42. Hatton MN, Loomis RE, Levine MI, Tabak LA (1985). Masticatory lubrication. The role of carbohydrate in the lubricating property of a salivary glycoproteinalbumin complex. Biochem J 230:817-820.

6(2): 161-1 75 (1995)

Hay DI, Smith DJ, Schluckebier SK, Moreno EC (1984). Relationship between concentration of human salivary statherin and inhibition of calcium phosphate precipitation in stimulated human parotid saliva. I Dent Res 63:857-863. Hay DI, Schluckebier SK, Moreno EC (1986). Saturation of human salivary secretions with respect to calcite and inhibition of calcium carbonate precipitation by salivary constituents. Calcif Tissue \nt 39:151-160. Hayashi Y, Fukayama M, Koike M, Nakayama T (1986). Amylase in human lungs and the female genital tract. Histochemical and immunohistochemical localization. Histochemistry 85:491-496. Hegnoj J, Ove B, Muckadell S (1985). An enzyme linked immunosorbent assay for measurements of lactoferrin in duodenal aspirates and other biological fluids. Scand J Clin Lab Invest 45:489.

Henskens YMC, Velden U van der, Veerman ECI, Nieuw Amerongen AV (1993). Protein, albumin and cystatin concentrations in saliva of healthy subjects and of patients with gingivitis or periodontitis. I Periodont Res 28:43-48. Henskens YMC, Veerman ECI, Mantel MS, Velden U van der, Nieuw Amerongen AV (1994). Cystatins SandC in human whole saliva and in glandular salivas in periodontal health and disease. I Dent Res 73:1606-1614. Hitzig WH (1977). Plasmaproteine, Pathophysiologie und Klinik. Berlin, Heidelberg, New-York: Springer-Verlag. Hocini H, Iscaki S, Bouvet JP, Pillot I (1993). Unexpectedly high levels of some presumably protective secretory immunoglobulin A antibodies in dental plaque bacteria in salivas of both caries-resistant and caries-susceptible subjects. Infect \mmun 61:3597-3604. Hoffman MP, Haidaris CG (1993). Analysis of Candida albicans adhesion to salivary mucins. Infect \mmun 61:19401949. Horwitz D, Proud D, Lawton WJ, Yates KN, Highet P, Pisano JJ, et al. (1982). Effects of restriction of sodium or restriction of fludrocortisone on parotid salivary kallikrein in man. I Lab Clin Ued 100:146-154. Hsieh WT, Fong D, Sloane BF, Golembieski W, Smith DI (1991). Mapping of the gene for human cysteine proteinase inhibitor Stefin A, STF1, to chromosome 3cen-q21. Genomics 9:207-209. Iacono VI, MacKay BJ, Dirienzo S, Pollock JJ (1980). Selective anti-bacterial properties of lysozyme for oral microorganisms. Infect \mmun 29:623-632. Ichimura E, Imura K, Hara Y, Kato Y, Kato I (1992). Cystatin activity in gingival crevicular fluid from periodontal disease patients measured by a new quantitative analysis method. I Periodont Res 27M 19-125. Issaksson PJ, Dunbar JC, Bradshaw RA (1987). Role of glandular kallikreins as growth factor processing enzymes: Structural and evolutionary considerations. I Cell Biochem 33:65-75.


171

Izutzu KT, Truelove EL, Bleyer WA, Andersen WM, Schubert MM, Rice JC (1981). Whole saliva as an indicator of stomatitis in cancer therapy patients. Cancer 48:1450-1454. Jacob L, Zasloff M (1994). Potential therapeutic applications of magainins and other antimicrobial agents of animal origin. In: Antimicrobial peptides. CIBA Foundation Symposium 186. Chichester: J. Wiley and Sons, pp. 197-223. Jenzano JW, Courts NF, Timko DA, Lundblad RL (1986). Levels of glandular kallikrein in whole saliva obtained with solid tumors remote from the oral cavity. I Dent Res 65:67-70. Johnsson M, Richardson CF, Bergey El, Levine MI, Nancollas GH (1991). The effects of human salivary cystatins and statherin on hydroxyapatite crystallization. Arch Oral Biol 36:631-636. Kaliner MA (1991). Human nasal respiratory secretions and host defense. Am Rev Resp Dis 144:S52-S56. Kamboh MI, Ferrell RE (1986). Genetic studies of lowabundance human plasma proteins. I. Microheterogeneity of zinc-oc2-glycoprotein in biological fluids. Biochem Gen 24:849-857. Karn RC, Malacinski GM (1978). The comparative biochemistry, physiology, and genetics of animal oc-amylase. Adv Comp Physiol Biochem 7:1-103.

Kim HS, Smithies O, Maeda N (1990). A physical map of the human salivary proline-rich protein gene cluster covers over 700 kbp of DNA. Genomics 6:260-267. Kirstila V, Tenovuo J, Ruuskanen O, Nikoskelainen J, Irjala K, Vilja P (1994). Salivary defense factors and oral health in patients with common-variable immunodeficiency. J din Immunol 14:229-236. Klein A, Carnoy CH, Wieruszeski JM, Strecker G, Strang AM, van Halbeek H, et al. (1992). The broad diversity of neutral and sialylated oligosaccharides derived from human salivary mucins. Biochem 31:6152-6165. Koop HM, Valentijn-Benz M, Nieuw Amerongen AV, Roukema PA, Graaff J de (1990). Aggregation of oral bacteria by salivary mucins in comparison to salivary and gastric mucins of animal origin. Ant van Leeuwenhoek 58:255-263. Korant BD, Brzin J, Turk V (1985). Cystatin, a protein inhibitor of cysteine proteinases, alters viral protein cleavages in infected human cells. Biochem Biophys Res Commun 127:1072-1076. Kraehenbuhl J-P, Neutra MT (1992). Molecular and cellular basis of immune protection of mucosal surfaces. Physiol Rev 72:853-879. Krajci P, Grzeschik KH, Geurts van Kessel AH, Olaisen B, Brandtzaeg P (1991). The human transmembrane secretory component (poly-Ig receptor): Molecular cloning, restriction fragment length polymorphism and chromosomal sublocalization. Hum Genet 87:642-648. Kuizenga A, Van Agtmaal J, Van Haeringen NJ, Kijlstra A

172

(1990). Sialic acid in human tear fluid. Exp Eye Res 50:45-50. Laible NJ, Germaine GR (1985). Bactericidal activity of human lysozyme, muramidase-inactive lysozyme, and cationic polypeptides against Streptococcus sanguis and Streptococcus faecalis-. Inhibition by chitin oligosaccharides. Infect \mmun 48:720-728. Leuchs E (1831). Ueber die Verzuckerung des Starkmehls durch Speichel. Arch Gesammte Naturl 3:105-107. Levine MJ (1993). Salivary macromolecules: a structure/function synopsis. Ann NY Acad Sci 694:11-16. Levine MJ, Reddy MS, Tabak LA, Loomis RE, Bergey EJ, Jones PC, et al. (1987). Structural aspects of salivary glycoproteins. J Dent Res 66:436-441. Lewis SA, Alles WP (1986). Urinary kallikrein: a physiological regulator of epithelial Na+ absorption. Proc Natl Acad Sci USA 83:5345-5348. Ligtenberg AJM, Walgreen-Weterings E, Veerman EG, de Soet II, de Graaff J, Nieuw Amerongen AV (1992). Influence of saliva on the aggregation and adherence of Streptococcus gordonii HG222. Infect \mmun 60:3878-

3884. 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. Lumikari M, Soukka T, Nurmio S, Tenovuo JO (1991). Inhibition of the growth of Streptococcus mutans, Streptococcus sobrinus and Lactobacillus casei by oral per-

oxidase systems in human saliva. Arch Oral Biol 36:155-160. MacKay BJ, Denepitiya L, Iacona VJ, Krost SB, Pollock JJ (1984). Growth inhibitory and bactericidal of human parotid salivary histidine-rich polypeptides on Streptococcus mutans. Infect \mmun 44:695-701. 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. Mandel ID (1979). In defense of the oral cavity. In: Saliva and dental caries (Spec Suppl, Microbiology Abstracts). Kleinberg I, Ellison SA, Mandel ID, editors. Washington, DC: Information Retrieval, Inc., pp. 473-491. Mandel ID (1989). The role of saliva in maintaining oral homeostasis. J Am Dent Assoc 119:298-304. Mandel ID, Ellison SA (1985). The biological significance of the nonimmunoglobulin defense factors. In: The lactoperoxidase system: chemistry and biological significance. Pruitt KM, Tenovuo JO, editors. Basel, New York: Marcel Dekker Inc., pp. 1-14. Mann K, Lipp B, Grunst J, Giger R, Karl HI (1980). Determination of kallikrein by radioimmunoassay in human body fluids. Agents Actions 10:329-334. Max EE, McBride OW, Morton CC, Robinson MA (1986).


6(2):161-175 (1995)

Human I chain gene: Chromosomal localization and associated restriction fragment length polymorphisms. Proc Natl head Sci USA 83:5592-5596. Mehansho H, Butler LG, Carlson DM (1987). Dietary tannins and salivary proline-rich proteins: Interactions, induction and defense mechanisms. Ann Rev Nutr 7:423-440. Mellersh A, Clark A, Hafiz S (1979). Inhibition of Neisseria gonorrhoeae by human normal saliva. Br J Venereal Diseases 55:20-23. Mestecky I, McGhee JR (1987). Immunoglobulin A (IgA): Molecular and cellular interactions involved in IgA biosynthesis and immune response. Adv Immunol 40:153-245. Modeer T (1977). Human saliva kallikrein. Biological properties of saliva kallikrein isolated by use of affinity chromatography. Ada Odontol Scand 35:23-29. Murakami Y, Tamagawa H, Shizukuishi S, Tsunemitsu A, Akimoto S (1992). Biological role of an arginineresidue present in histidine-rich peptide which inhibits hemagglutination of Porphyromonas gingivalis. FEMS Uicrobiol Lett 98:201-204. Nakayashiki N (1990). Sweat protein components tested by SDS-polyacrylamide gel electrophoresis followed by immunoblotting. Tohoku J Exp Med 161:25-31. Nexo E, Hansen M, Seier Poulsen S, Skov Olsen P (1985). Characterization and immunohistochemical localization of rat salivary cobalamin-binding protein and comparison with human salivary haptocorrin. Biochim Biophys Ada 838:264-269. Nguyen VC, Aubert IP, Gross MS, Porchet N, Degand P, Frezal I (1990). Assignment of human tracheobronchial mucin gene(s) to Ilpl5 and a tracheobronchial mucin-related sequence to chromosome 13. Hum Genet 86:167-172. Nguyen VC, Aubert IP, Gross MS, Porchet N, Degand P, Bernheim A (1991). Tracheobronchial mucin 4 (MUC4) gene: Assignment to 3q29 and polymorphism of VNTR type. Cytogenet Cell Genet 58:1879-1880. Nieuw Amerongen AV (1994). Speeksel en Mondgezondheid (Saliva and oral health) (monograph). Amsterdam: VU-Uitgeverij. Nieuw Amerongen AV, Oderkerk CH, Driessen AA (1987). Role of mucins from human whole saliva in the protection of tooth enamel against demineralization in vitro. Caries Res 21:297-309.

Nishikita M, Kanehira T, Oh H, Tani H, Tazaki M, Kuboki Y (1989). Salivary histatin as an inhibitor of a protease by the oral bacterium Bacteroides gingivalis. Biochem Biophys Res Commun 174:625-630.

Norhagen-Engstrom G, Engstrom P-E, Hammarstrom L, Smith CIE (1992). Oral condition in individuals with selective immunoglobulin A deficiency and common variable immunodeficiency. I Periodontol 62:984-989. Oppenheim FG (1989). Salivary histidine-rich proteins.

6(2):161-175 (1995)

In: Human saliva: Clinical chemistry and microbiology. Tenovuo JO, editor. Boca Raton: CRC Press, pp. 151-160. Oppenheim FG, Xu T, McMillan FM, Levitz SM, Diamond RD, Offner GD, et al. (1988). Histatins, a novel family of histidine-rich proteins in human parotid secretion. I Biol Chem 263:7472-7477. Page DJ, Gilbert RJ, Bowen WH, Stephen KW (1990). Concentration of antimicrobial proteins in human saliva. The effect of long-term usage of a zinc-containing dentifrice on the protein composition of stimulated saliva from 198 children. Caries Res 24:216-219. Peters CW, Kruse U, Pollwein R, Grzeschik KH, Sippel AE (1989). The human lysozyme gene. Sequence organization and chromosomal localization. Ear ] Biochem 182:507-516. Poblete MT, Reynolds NJ, Figueroa CD, Burton JL, MullerEsterl W, Bhoola KD (1991). Tissue kallikrein and kininogen in human sweat glands and psoriatic skin. BrjDermatol 124:236-241. Pollock II, Iacono VI, Goodman Bicker H, MacKay BJ, Katona LI, Taichman LB, et al. (1976). The binding, aggregation and lytic properties of lysozyme. In: Proceedings: Microbial aspects of dental caries (Spec Suppl, Microbiology Abstracts). Stiles HM, Loesche WJ, O'Brien TC, editors. Washington, DC: Information Retrieval Inc., pp. 325-352. Pollock II, Lotardo S, Gavai R, Grossbard BL (1987). Lysozyme-protease-inorganic monovalent anion lysis of oral bacterial strains in buffers and stimulated whole saliva. I Dent Res 66:467-474. Pollock II, Santarpia RP, Heller HM, Xu L, Lai K, Fuhrer J, et al. (1992). Determination of salivary anticandidal activities in healthy adults and patients with AIDS—A pilot study. J AIDS 5:610-618. Pruitt KM, Kamau DN, Miller K, Mansson-Rahemtulla B, Rahemtulla F (1990). Quantitative, standardized assays for determining the concentrations of bovine lactoperoxidase, human salivary peroxidase, and human myeloperoxidase. Anal Biochem 191:278-286. Raj PA, Edgerton M, Levine MI (1990). Salivary histatin 5: dependence of sequence, chain length, and helical conformation for candidacidal activity. I Biol Chem 265:3898-3905. Raj PA, Johnsson M, Levine MI, Nancollas GH (1992). Salivary statherin. J Biol Chem 267:5968-5976. Raj PA, Soni S-D, Levine M) (1994). Membrane-induced helical conformation of an active candidacidal fragment of salivary histatins. I Biol Chem 269:9610-9619. Raphael GD, Jeney EV, Baraniuk IN, Kim I, Meredith SD, Kaliner MA (1989). Pathophysiology of rhinitis. Lactoferrin and lysozyme in nasal secretions. I Clin Invest 84:1528-1535. Rathman WM, Van Zeijl M), Van den Keijbus PAM, Bank RA, Veerman ECI, Nieuw Amerongen AV (1989).


173

Isolation and characterization of three non-mucinous human salivary proteins with affinity for hydroxyapatite. I Biol Buccale 17:199-208. Rathman WM, van den Keijbus PAM, van Zeijl MI, Dopp EA, Veerman ECI, Nieuw Amerongen AV (1990). Characterization of monoclonal antibodies to human salivary (glyco)proteins. Cellular localization of mucin cystatin-like 14 kD protein and 20 kD glycoprotein in the human submandibular gland. J Biol Buccale 18:19-27. Riegman PH, Vliestra RV, Suurmeijer L, Cleutjens CB, Trapman I (1992). Characterization of the human kallikrein locus. Genomics 14:6-11. Rolla G, Ciarda IE, Bowen WH (1983). Identification of IgA, IgG, lysozyme, albumin, oc-amylase and glucosyltransferase in the protein layer adsorbed to hydroxyapatite from whole saliva. Scand J Dent Res 91:186-190. Rose MC (1992). Mucins: structure, function, and role in pulmonary diseases. Am I Physiol 263:L413-L429. Rudney ID, Smith QI (1985). Relationships between levels of lysozyme, lactoferrin, salivary peroxidase, and secretory immunoglobulin A in stimulated parotid saliva. Infect \mmun 49:469-475. Sabatini LM, Azen EA (1989). Histatins, a family of histidine-rich proteins, are encoded by at least two loci, HIS1 and HIS2. Biochem Biophys Res Commun 160:495-502. Sabatini LM, Warner TF, Saitoh E, Azen EA (1989). Tissue distribution of RNA's for cystatins, histatins, statherin, and proline-rich salivary proteins in humans and macaques. J Dent Res 68:1138-1145. Sabatini LM, Oka T, Azen EA (1993). Nucleotide sequence analysis of human salivary protein genes HIS 1 and HIS 2 and evolution of the STATH/HIS gene family. Mol BiolEvol 10:497-511. Saitoh E, Isemura S, Sanada K (1985). Inhibition of calcium-carbonate precipitation by human salivary proline-rich phosphoproteins. Arch Oral Biol 30:641-643. Saitoh E, Kim HS, Smithies O, Maeda N (1987). Human cysteine-proteinase inhibitors: Nucleotide sequence analysis of three members of the cystatin gene family. Gene 61:329-338. Saitoh E, Isemura S, Sanada K, Ohnishi K (1991). The human cystatin gene family: Cloning of three members and evolutionary relationship between cystatins and Bowman-Birk type proteinase inhibitors. Biomed BiochimActa 50:599-605. Salas-Herrera IG, Pearson RM, Turner P (1991). Quantitation of albumin and alpha-1-acid glycoprotein in human cervical mucus. Hum Exp Toxicol 10:137-139. Scannapieco FA, Levine MJ (1993). Salivary mucins and dental plaque formation. In: Cariology for the nineties. Bowen WH, Tabak LA, editors. Rochester: University of Rochester Press, pp. 85-105. Scannapieco FA, Bergey EJ, Reddy MS, Levine MJ (1989). Characterization of salivary a-amylase binding to Streptococcus sanguis. Infect Immun 57:2853-2863.

174

Schenkels LCPM, Rathman WM, Veerman ECI, Nieuw Amerongen AV (1991). Detection of proteins related to a salivary glycoprotein (EP-GP). Concentrations in human secretions (saliva, sweat, tears, nasal mucus, cerumen, seminal plasma). Bio! Chem Woppe Seyler 372:325-329. Schenkels LCPM, Ligtenberg AIM, Veerman ECI, Nieuw Amerongen AV (1993). Interaction of the salivary glycoprotein EP-GP with the bacterium Streptococcus salivarius HB. I Dent Res 72:1559-1565. Schenkels LCPM, Schaller J, Walgreen-Weterings E, Schadee-Eestermans IL, Veerman ECI, Nieuw Amerongen AV (1994). Identity of human extra parotid glycoprotein (EP-GP) with secretory actin binding protein (SABP) and its biological properties. Biol Chem Woppe Seyler 375:609-615.

Schnittger S, Gopal Rao WN, Abrahamson M, Hansman I (1993). Cystatin C (CST3), the candidate gene for hereditary cystatin C amyloid angiopathy (HCCAA) and other members of the cystatin gene family are clustered on chromosome 20pl 1.2. Genomics 16:50-55. Schwartz SS, Hay DI, Schluckebier SK (1992). Inhibition of calcium phosphate precipitation by human salivary statherin: structure-activity relationships. Calcif Tissue \nt 50:511-517. Sloane BF, Honn KV (1984). Cysteine proteinases and metastasis. Cancer Metastasis 3:249-263. Stinson MW, Levine MJ, Cavese JM, Prakobphol A, Murray PA, Tabak LA, et al. (1982). Adherence of Streptococcus sanguis to salivary mucin bound to glass. J Dent Res 61:1390-1393. Strous GJ, Dekker J (1992). Mucin-type glycoproteins. Crit Rev Biochem Mol Biol 27:57-92.

Sugiyama K (1993). Anti-lipopolysaccharide activity of histatins, peptides from human saliva. Experientia 49:1095-1097. Sugiyama K, Suzuki Y, Furuta H (1985). Isolation and characterization of histamine-releasing peptides from human parotid saliva. Life Sci 37:475-480. Sugiyama K, Ogino T, Ogata K (1990). Rapid purification and characterization of histatins (histidine-rich polypeptides) from human whole saliva. Arch Oral Biol 35:415-419. Sweeney D, Beeley JA (1990). An enzyme-linked immunoassay for human salivary albumin. Arch Oral Biol 35:741-746. Tabak LA (1991). Genetic control of salivary mucin formation. In: Frontiers of oral biology. Vol. 8. Ferguson DB, editor. Basel: Karger, pp. 77-94. Tabak LA, Bowen WH (1989). Roles of saliva (pellicle), diet, and nutrition on plaque formation. J Dent Res 68:1560-1566. Tachibana M, Morioka H, Machino M, Tanimura F, Mizukoshi O (1986). Amylase secretion by nasal glands—an immunohistochemical study. Ann Otol


6(2):161-175 (1995)

Rhinol Laryngol 95:284-287.

Tomasi TB (1983). Mechanisms of immune regulation at mucosal surfaces. Rev Infect Dis 5(Suppl 4):784-792. Tonnesen P, Thim L, Nex0 E (1990). Epidermal growth factor and haptocorrin in nasal secretion. Scand I Clin Lab Invest 50:187-194. Toribara NW, Gum JR. Culhane PJ, Lagace RE, Hicks JW, Petersen GM, et al. (1991). MUC-2 human small intestinal mucin gene structure—repeated arrays and polymorphism. I Clin Invest 88:1005-1013. Toribara NW, Roberton AM, Ho SB, Kuo WL. Gum E, Hicks JW, et al. (1993). Human gastric mucin. Identification of a unique species by expression cloning. ) Bio! Chem 268:5879-5885. Twetman S, Lindqvest L, Sund M-L (1986). Effect of human lysozyme on 2-deoxyglucose uptake by Streptococcus mutans and other oral micro-organisms. J Dent Res 20:223-229. Valdez 1H, Fox PC (1993). Diagnosis and management of salivary dysfunction. Crit Rev Oral Med 4:271-277. Van der Reijden WA, Veerman EC1, Nieuw Amerongen AV (1993). Shear rate dependent viscoelastic behavior of human glandular salivas. Biorheology 30:141-152. Van-Seuningen I, Houdret N, Hayem A, Davril M (1992). Strong ionic interactions between mucins and two basic proteins, mucus proteinase inhibitor and lysozyme, in human bronchial secretions. \nt ] Biochem 24:303-311. Veerman ECI, Valentijn-Benz M, Nieuw Amerongen AV (1989). Viscosity of human salivary mucins: Effect of pH and ionic strength and role of sialic acid. I Biol Buccale 17:297-306. Veerman ECI, Ligtenberg AJM, Schenkels LCPM, Walgreen-Weterings E, Nieuw Amerongen AV (1995). Binding of high-molecular-weight salivary mucins (MG1) to Hemophilusparainfluenzae.} Dent Res 74:351-357.

6(2):161-175 (1995)

Verma M, Davidson EA (1994). Glycopinious mini-review. Mucin genes: structure, expression and regulation. Glycoconjugate J 11:172-179.

Vissink A (1985). Xerostomia. Development, properties and application of a mucin containing saliva substitute (dissertation). Groningen, the Netherlands: University of Groningen. Waterman HA, Blom C, Holterman HJ, 's-Gravenmade EJ, Mellema M (1988). Rheological properties of human saliva. Arch Oral Biol 33:589-596. Weiber H, Andersson C, Murne A, Rannevik G, Landstrom C, Lilja H, et al. (1990). |3-Seminoprotein is not a prostate-specific protein. Its identification in mucous glands and secretions. Am J Pathol 137:593-603. Williams RC, Gibbons RJ (1975). Inhibition of streptococcal attachment to receptors on human epithelial cells by antigenically similar salivary glands. Infect \mmun 11:711-718. Wolff A, Moreira JE, Bevins CL, Hand AR, Fox PC (1990). Magainin-like immunoreactivity in human submandibular and labial salivary glands. I Histochem Cytochem 38:1531-1534. Wu AM, Csako G, Herp A (1994). Structure, biosynthesis, and function of salivary mucins. Mo! Cell Biochem 137:39-55. Xu G, Huan L, Khatri I, Sajjan US, McCool D, Wang D, et al. (1992). Human intestinal mucin-like protein (MLP) is homologous with rat MLP in the C-terminal region, and is encoded by a gene on chromosome Ilpl5.5. Biochem Biophys Res Commun 183:821-828. Xu T, Levitz SM, Diamond RD, Oppenheim FG (1991). Anticandidal activity of major human salivary histatins. Infect Imrnun 59:2549-2554. Yokozeki H, Hibino T, Takemura T, Sato K (1991). Cysteine proteinase inhibitor in eccrine sweat is derived from sweat gland. Am J Physiol 260:R314-R320.


175