Chemical composition and functions of saliva

Chronology of defining salivary components and functions Beginning in 1950’s whole saliva evaluated (antimicrobial properties, role in microbial attachment, mineralization, taste, lubrication) Secretions of major glands (parotid and submandibular/sublingual) In 1970’s individual components isolated and biochemically characterized In mid-1980’s beginning to map functional domains (peptide synthesis and recombinant approaches)

Major salivary components Histatins Statherins Lysozyme Proline-rich proteins Carbonic anhydrases Amylases Peroxidases Lactoferrin Mucin 2 (MG2) sIgA Mucin 1 (MG1) 1 10 100 1000 10000 Size (kDa)

Current concepts regarding the functional features of salivary macromolecules Recent structure/function studies have identified general principles regarding function Based on in vitro studies of purified molecules Additional studies required to evaluate concepts in situ

Conformational requirements Conformation or shape of a molecule is critical for its biological function Examples Proline-rich proteins interact with A. viscosus and St. gordonii only when adsorbed onto mineralized surface Statherins and histatins require -helical conformation Human salivary amylase require 5 inter-chain disulfide bonds

Multifunctionality Amylases, Cystatins, Carbonic anhydrases, Histatins, Mucins, Peroxidases Carbonic anhydrases, Histatins Anti- Bacterial Buffering Amylases, Mucins, Lipase Cystatins, Mucins Anti- Viral Digestion Salivary Families Mineral- ization Anti- Fungal Cystatins, Histatins, Proline- rich proteins, Statherins Histatins Lubricat- ion &Visco- elasticity Tissue Coating Amylases, Cystatins, Mucins, Proline-rich proteins, Statherins Mucins, Statherins adapted from M.J. Levine, 1993

Redundancy Saliva has built-in redundancy in regard to its protective functions. Example - Many salivary molecules can inhibit the precipitation of calcium phosphate salts. strong inhibitors such as statherin and acidic proline-rich proteins moderate inhibitors such as histatins and cystatins weak inhibitors such as mucins and amylase

Amphifunctionality A molecule may have both protective and detrimental properties - “double-edged sword”. May depend on molecule’s location or site of action Amylases In solution, they facilitate clearance of viridans streptococci Adsorbed to tooth surface, they can promote adherence of these bacteria and digest starch to dietary maltose and production of acid Statherin and acidic proline-rich proteins At enamel surface, they play an important role in mineralization by inhibiting the formation of primary and secondary calcium phosphate salts. When adsorbed to the enamel surface, they promote attachment of cariogenic microorganisms.

Complexing Functional relationships exist between different molecules in saliva Two types of complexing (covalent and non-covalent) homotypic (between similar molecules) heterotypic (between different molecules) Example: Mucins homotypic complexes necessary for lubrication and viscoelastic properties heterotypic complexes with sIgA, lysozyme and cystatins concentrate these anti-microbials at tissue interfaces

Salivary Protein Functions

Salivary Protein Functions (cont’d)

Salivary Protein Functions (cont’d)

Salivary Protein Functions (cont’d)

Mucins Lack precise folded structure of globular proteins Asymmetrical molecules with open, randomly organized structure Polypeptide backbone (apomucin) with CHO side-chains Side-chains may end in negatively charged groups, such as sialic acid and bound sulfate Hydrophillic, entraining water (resists dehydration) Unique rheological properties (e.g., high elasticity, adhesiveness, and low solubility) Two major mucins (MG1 and MG2)

Mucin Functions Tissue Coating Lubrication Protective coating about hard and soft tissues Primary role in formation of acquired pellicle Concentrates anti-microbial molecules at mucosal interface Lubrication Align themselves with direction of flow (characteristic of asymmetric molecules) Increases lubricating qualities (film strength) Film strength determines how effectively opposed moving surfaces are kept apart

Mucin Functions (cont’d) Aggregation of bacterial cells Bacterial adhere to mucins may result in surface attachment, or Mucin-coated bacteria may be unable to attach to surface Bacterial adhesion Mucin oligosaccharides mimic those on mucosal cell surface React with bacterial adhesins, thereby blocking them

Amylases Calcium metalloenzyme Hydrolyzes (1-4) bonds of starches such as amylose and amylopectin Several salivary isoenzymes Maltose is the major end-product (20% is glucose) “Appears” to have digestive function Why is it also present in tears, serum, bronchial, and male and female urogenital secretions? A role in modulating bacterial adherence?

Lingual Lipase Secreted by von Ebner’s glands of tongue Involved in first phase of fat digestion Hydrolyzes medium- to long-chain triglycerides Important in digestion of milk fat in new-born Unlike other mammalian lipases, it is highly hydrophobic and readily enters fat globules

Statherins Calcium phosphate salts of dental enamel are soluble under typical conditions of pH and ionic strength Supersaturation of calcium phosphates maintain enamel integrity Statherins prevent precipitation or crystallization of supersaturated calcium phosphate in ductal saliva and oral fluid Produced by acinar cells in salivary glands Also an effective lubricant

Proline-rich Proteins (PRPs) Like statherin, PRPs are also highly asymmetrical Inhibitors of calcium phosphate crystal growth Inhibition due to first 30 residues of negatively-charged amino-terminal end Present in the initially formed enamel pellicle and in “mature” pellicles

Role of PRPs in enamel pellicle formation Acquired enamel pellicle is 0.1-1.0 µm thick layer of macromolecular material on the dental mineral surface Pellicle is formed by selective adsorption of hydroxyapatite-reactive salivary proteins, serum proteins and microbial products such as glucans and glucosyl-transferase Pellicle acts as a diffusion barrier, slowing both attacks by bacterial acids and loss of dissolved calcium and phosphate ions

Remineralization of enamel and calcium phosphate inhibitors Early caries are repaired despite presence of mineralization inhibitors in saliva Sound surface layer of early carious lesion forms impermeable barrier to diffusion of high mol.wt. inhibitors. Still permeable to calcium and phosphate ions Inhibitors may encourage mineralization by preventing crystal growth on the surface of lesion by keeping pores open

Calculus formation and calcium phosphate inhibitors Calculus forms in plaque despite inhibitory action of statherin and PRPs in saliva May be due to failure to diffuse into calcifying plaque Proteolytic enzymes of oral bacteria or lysed leukocytes may destroy inhibitory proteins Plaque bacteria may produce their own inhibitors

Calcium phosphate precipitation inhibitors and plaque Statherin and PRPs might be expected to occur in plaque, have not been detected Plaque bacteria produce calcium phosphate inhibitors Might be necessary to prevent calcification of bacteria -- happens with dead cells Immobilized crystal growth inhibitors can function as nucleators of crystal growth Immobilization may occur in plaque, encouraging calculus formation

Interaction of oral bacteria with PRPs and other pellicle proteins Several salivary proteins appear to be involved in preventing or promoting bacterial adhesion to oral soft and hard tissues PRPs are strong promoters of bacterial adhesion Amino terminal: control calcium phosphate chemistry Carboxy terminal: interaction with oral bacteria Interactions are highly specific Depends on proline-glutamine carboxy-terminal dipeptide PRPs in solution do not inhibit adhesion of bacteria

These anti-microbial proteins will be discussed in a later lecture Secretory Immunoglobulins Lactoferrin Lysozyme Sialoperoxidase Cystatins Histatins

Summary - Clinical Highlights Understanding of salivary mechanisms at fundamental level a prerequisite for effective treatment of salivary gland dysfunctions modulation of bacterial colonization development of artificial saliva other “cutting edge” approaches to salivary dysfunctions and diseases