Dennis E. Lopatin, Ph.D. Page no. 1 Chemical composition and functions of saliva.

Dennis E. Lopatin, Ph.D.Dennis E. Lopatin, Ph.D.Page no. Page no. 11

Chemical composition and functions Chemical composition and functions of salivaof saliva


Chronology of defining salivary Chronology of defining salivary components and functionscomponents and functions

Beginning in 1950’s Beginning in 1950’s whole salivawhole saliva evaluated evaluated (antimicrobial properties, role in microbial attachment, (antimicrobial properties, role in microbial attachment, mineralization, taste, lubrication)mineralization, taste, lubrication)

Secretions of Secretions of major glandsmajor glands (parotid and (parotid and submandibular/sublingual)submandibular/sublingual)

In 1970’s In 1970’s individual componentsindividual components isolated and isolated and biochemically characterizedbiochemically characterized

In mid-1980’s beginning to map In mid-1980’s beginning to map functional domainsfunctional domains (peptide synthesis and recombinant approaches)(peptide synthesis and recombinant approaches)


Major salivary componentsMajor salivary components

Mucin 1 (MG1)Mucin 1 (MG1)sIgAsIgA

Mucin 2 (MG2)Mucin 2 (MG2)LactoferrinLactoferrin

PeroxidasesPeroxidasesAmylasesAmylases

Carbonic anhydrasesCarbonic anhydrasesProline-rich proteinsProline-rich proteins

LysozymeLysozymeStatherinsStatherins

HistatinsHistatins

11 1010 100100 10001000 1000010000

Size (kDa)Size (kDa)


Current concepts regarding the functional Current concepts regarding the functional features of salivary macromoleculesfeatures of salivary macromolecules

Recent structure/function studies have identified Recent structure/function studies have identified general principlesgeneral principles regarding function regarding function

Based on Based on in vitroin vitro studies of purified moleculesstudies of purified molecules Additional studies required to evaluate concepts Additional studies required to evaluate concepts

in situin situ


Conformational requirementsConformational requirements Conformation or shape of a molecule is Conformation or shape of a molecule is criticalcritical

for its biological functionfor its biological function ExamplesExamples

– Proline-rich proteins interact with Proline-rich proteins interact with A. viscosus A. viscosus and and St. gordonii St. gordonii only when only when adsorbedadsorbed onto mineralized onto mineralized surfacesurface

– Statherins and histatins require Statherins and histatins require -helical-helical conformationconformation

– Human salivary amylase require 5 inter-chain Human salivary amylase require 5 inter-chain disulfide bondsdisulfide bonds


MultifunctionalityMultifunctionality

SalivarySalivaryFamiliesFamilies

Anti-Anti-BacterialBacterial

BufferingBuffering

DigestionDigestion

Mineral-Mineral-izationization

Lubricat-Lubricat-ion &Visco-ion &Visco-elasticityelasticity

TissueTissueCoatingCoating

Anti-Anti-FungalFungal

Anti-Anti-ViralViral

Carbonic anhydrases,Carbonic anhydrases,HistatinsHistatins

Amylases,Amylases,Mucins, LipaseMucins, Lipase

Cystatins,Cystatins,Histatins, Proline-Histatins, Proline-rich proteins,rich proteins,StatherinsStatherins

Mucins, StatherinsMucins, Statherins

Amylases,Amylases,Cystatins, Mucins, Cystatins, Mucins, Proline-rich proteins, StatherinsProline-rich proteins, Statherins

HistatinsHistatins

Cystatins,Cystatins,MucinsMucins

Amylases, Cystatins,Amylases, Cystatins,Histatins, Mucins,Histatins, Mucins,PeroxidasesPeroxidases

adapted from M.J. Levine, 1993adapted from M.J. Levine, 1993


RedundancyRedundancy

Saliva has built-in Saliva has built-in redundancyredundancy in regard to its in regard to its protective functions.protective functions.

Example - Many salivary molecules can inhibit Example - Many salivary molecules can inhibit the precipitation of calcium phosphate salts.the precipitation of calcium phosphate salts.– strong inhibitors such as statherin and acidic proline-strong inhibitors such as statherin and acidic proline-

rich proteinsrich proteins– moderate inhibitors such as histatins and cystatinsmoderate inhibitors such as histatins and cystatins– weak inhibitors such as mucins and amylaseweak inhibitors such as mucins and amylase


AmphifunctionalityAmphifunctionality A molecule may have both protective and detrimental A molecule may have both protective and detrimental

properties - “properties - “double-edged sworddouble-edged sword”.”. May depend on molecule’s May depend on molecule’s locationlocation or site of action or site of action

– AmylasesAmylases» In solution, they facilitate clearance of viridans streptococciIn solution, they facilitate clearance of viridans streptococci

» Adsorbed to tooth surface, they can promote adherence of these bacteria and Adsorbed to tooth surface, they can promote adherence of these bacteria and digest starch to dietary maltose and production of aciddigest starch to dietary maltose and production of acid

– Statherin and acidic proline-rich proteinsStatherin and acidic proline-rich proteins» At enamel surface, they play an important role in mineralization by At enamel surface, they play an important role in mineralization by

inhibiting the formation of primary and secondary calcium phosphate salts. inhibiting the formation of primary and secondary calcium phosphate salts. When adsorbed to the enamel surface, they promote attachment of When adsorbed to the enamel surface, they promote attachment of cariogenic microorganisms.cariogenic microorganisms.


ComplexingComplexing Functional relationships exist between different Functional relationships exist between different

molecules in salivamolecules in saliva Two types of complexing (covalent and non-covalent)Two types of complexing (covalent and non-covalent)

– homotypic (between similar molecules)homotypic (between similar molecules)– heterotypic (between different molecules)heterotypic (between different molecules)

Example: MucinsExample: Mucins– homotypic complexes necessary for lubrication and homotypic complexes necessary for lubrication and

viscoelastic propertiesviscoelastic properties– heterotypic complexes with sIgA, lysozyme and cystatins heterotypic complexes with sIgA, lysozyme and cystatins

concentrate these anti-microbials at tissue interfacesconcentrate these anti-microbials at tissue interfaces


Salivary Protein FunctionsSalivary Protein Functions

Oral function Problem Protein function

• Acts as an airway • Air-born organisms• Dehydration

• Anti-bacterialsystems

• Water-retainingglycoproteins

• Speech • Need for lubrication • Lubrication system

• Taste -- • Gustin

• Entry-point for foodmastication,swallowing

• Food-bornorganisms

• Soft and hard tissueabrasion

• Food toxins


• Lubrication;mucins, statherin

• Toxin-neutralization

Oral function Problem Protein function

• Acts as an airway • Air-born organisms• Dehydration


• Water-retainingglycoproteins

• Speech • Need for lubrication • Lubrication system

• Taste -- • Gustin

• Entry-point for foodmastication,swallowing

• Food-bornorganisms

• Soft and hard tissueabrasion

• Food toxins


• Lubrication;mucins, statherin

• Toxin-neutralization


Salivary Protein Functions (cont’d)Salivary Protein Functions (cont’d)

Oral function Problem Protein function• Control of in-

digenous & in-vading bacteria,fungi and viruses

• Colonization &infection

• Controllingpathogens andcommensals

• Adhesion of bac-teria versus theirdetection


• Immunoglobu-lins, histatins,glycoproteins,lysozyme, sia-loperoxidase,lactoferrin

• Adhesion-modulating pro-teins

Oral function Problem Protein function• Control of in-

digenous & in-vading bacteria,fungi and viruses

• Colonization &infection

• Controllingpathogens andcommensals

• Adhesion of bac-teria versus theirdetection


• Immunoglobu-lins, histatins,glycoproteins,lysozyme, sia-loperoxidase,lactoferrin

• Adhesion-modulating pro-teins


Salivary Protein Functions (cont’d)Salivary Protein Functions (cont’d)

Oral function Problem Protein function• Digestion __ • Starch & fat

hydrolysis:amylase andlingual lipase

• Protection &repair of softtissues

• Toxins,carcinogens,degradativeproteases

• Mucin-richprotectivebarrier film

• Proteaseinhibitors,cystatins, tissuegrowth factors

Oral function Problem Protein function• Digestion __ • Starch & fat

hydrolysis:amylase andlingual lipase

• Protection &repair of softtissues

• Toxins,carcinogens,degradativeproteases

• Mucin-richprotectivebarrier film

• Proteaseinhibitors,cystatins, tissuegrowth factors


Salivary Protein Functions (cont’d)Salivary Protein Functions (cont’d)Oral function Problem Protein function• Protection & re-

pair of hard tis-sues

• Enamel mineralis potentiallysoluble; acid-damagedenamel requiresremineralization

• Biologicallycontrolled pro-tective & re-pairative inor-ganic environ-ment, stabilizedby statherin,acidic proline-rich and pellicleproteins

• Pellicle forma-tion

__ __

• Plaque acidformation

• Plaque pH con-trol

• Basic aminoacids & peptides

Oral function Problem Protein function• Protection & re-

pair of hard tis-sues

• Enamel mineralis potentiallysoluble; acid-damagedenamel requiresremineralization

• Biologicallycontrolled pro-tective & re-pairative inor-ganic environ-ment, stabilizedby statherin,acidic proline-rich and pellicleproteins

• Pellicle forma-tion

__ __

• Plaque acidformation

• Plaque pH con-trol

• Basic aminoacids & peptides


MucinsMucins Lack precise folded structure of globular proteinsLack precise folded structure of globular proteins Asymmetrical molecules with open, randomly organized Asymmetrical molecules with open, randomly organized

structurestructure Polypeptide backbone (apomucin) with CHO side-chainsPolypeptide backbone (apomucin) with CHO side-chains Side-chains may end in negatively charged groups, such as Side-chains may end in negatively charged groups, such as

sialic acid and bound sulfatesialic acid and bound sulfate Hydrophillic, entraining water (resists dehydration)Hydrophillic, entraining water (resists dehydration) Unique rheological properties (e.g., high elasticity, Unique rheological properties (e.g., high elasticity,

adhesiveness, and low solubility)adhesiveness, and low solubility) Two major mucins (MG1 and MG2)Two major mucins (MG1 and MG2)


Mucin FunctionsMucin Functions Tissue CoatingTissue Coating

– Protective coating about hard and soft tissuesProtective coating about hard and soft tissues– Primary role in formation of acquired pelliclePrimary role in formation of acquired pellicle– Concentrates anti-microbial molecules at mucosal interfaceConcentrates anti-microbial molecules at mucosal interface

LubricationLubrication– Align themselves with direction of flow (characteristic of Align themselves with direction of flow (characteristic of

asymmetric molecules)asymmetric molecules)– Increases lubricating qualities (film strength)Increases lubricating qualities (film strength)– Film strength determines how effectively opposed moving Film strength determines how effectively opposed moving

surfaces are kept apartsurfaces are kept apart


Mucin Functions (cont’d)Mucin Functions (cont’d)

Aggregation of bacterial cellsAggregation of bacterial cells– Bacterial adhere to mucins may result in surface Bacterial adhere to mucins may result in surface

attachment, orattachment, or– Mucin-coated bacteria may be unable to attach to surfaceMucin-coated bacteria may be unable to attach to surface

Bacterial adhesionBacterial adhesion– Mucin oligosaccharides mimic those on mucosal cell Mucin oligosaccharides mimic those on mucosal cell

surfacesurface– React with bacterial adhesins, thereby blocking themReact with bacterial adhesins, thereby blocking them


AmylasesAmylases Calcium metalloenzymeCalcium metalloenzyme Hydrolyzes Hydrolyzes (1-4) bonds of starches such as amylose and (1-4) bonds of starches such as amylose and

amylopectinamylopectin Several salivary isoenzymesSeveral salivary isoenzymes Maltose is the major end-product (20% is glucose)Maltose is the major end-product (20% is glucose) ““Appears” to have digestive functionAppears” to have digestive function Why is it also present in tears, serum, bronchial, and male and Why is it also present in tears, serum, bronchial, and male and

female urogenital secretions?female urogenital secretions? A role in modulating bacterial adherence?A role in modulating bacterial adherence?


Lingual LipaseLingual Lipase

Secreted by von Ebner’s glands of tongueSecreted by von Ebner’s glands of tongue Involved in first phase of fat digestionInvolved in first phase of fat digestion Hydrolyzes medium- to long-chain triglyceridesHydrolyzes medium- to long-chain triglycerides Important in digestion of milk fat in new-bornImportant in digestion of milk fat in new-born Unlike other mammalian lipases, it is highly Unlike other mammalian lipases, it is highly

hydrophobic and readily enters fat globuleshydrophobic and readily enters fat globules


StatherinsStatherins Calcium phosphate salts of dental enamel are soluble Calcium phosphate salts of dental enamel are soluble

under typical conditions of pH and ionic strengthunder typical conditions of pH and ionic strength Supersaturation of calcium phosphates maintain Supersaturation of calcium phosphates maintain

enamel integrityenamel integrity Statherins prevent precipitation or crystallization of Statherins prevent precipitation or crystallization of

supersaturated calcium phosphate in ductal saliva and supersaturated calcium phosphate in ductal saliva and oral fluidoral fluid

Produced by acinar cells in salivary glandsProduced by acinar cells in salivary glands Also an effective lubricantAlso an effective lubricant


Proline-rich Proteins (PRPs)Proline-rich Proteins (PRPs)

Like statherin, PRPs are also highly asymmetricalLike statherin, PRPs are also highly asymmetrical Inhibitors of calcium phosphate crystal growthInhibitors of calcium phosphate crystal growth Inhibition due to first 30 residues of negatively-Inhibition due to first 30 residues of negatively-

charged amino-terminal endcharged amino-terminal end Present in the initially formed enamel pellicle and in Present in the initially formed enamel pellicle and in

“mature” pellicles“mature” pellicles


Role of PRPs in enamel pellicle Role of PRPs in enamel pellicle formationformation

Acquired enamel pellicle is 0.1-1.0 µm thick layer of Acquired enamel pellicle is 0.1-1.0 µm thick layer of macromolecular material on the dental mineral surfacemacromolecular material on the dental mineral surface

Pellicle is formed by selective adsorption of Pellicle is formed by selective adsorption of hydroxyapatite-reactive salivary proteins, serum proteins hydroxyapatite-reactive salivary proteins, serum proteins and microbial products such as glucans and glucosyl-and microbial products such as glucans and glucosyl-transferasetransferase

Pellicle acts as a diffusion barrier, slowing both attacks Pellicle acts as a diffusion barrier, slowing both attacks by bacterial acids and loss of dissolved calcium and by bacterial acids and loss of dissolved calcium and phosphate ionsphosphate ions


Remineralization of enamel and Remineralization of enamel and calcium phosphate inhibitorscalcium phosphate inhibitors

Early caries are repaired despite presence of Early caries are repaired despite presence of mineralization inhibitors in salivamineralization inhibitors in saliva

Sound surface layer of early carious lesion forms Sound surface layer of early carious lesion forms impermeable barrier to diffusion of high mol.wt. impermeable barrier to diffusion of high mol.wt. inhibitors.inhibitors.

Still permeable to calcium and phosphate ionsStill permeable to calcium and phosphate ions Inhibitors may encourage mineralization by preventing Inhibitors may encourage mineralization by preventing

crystal growth on the surface of lesion by keeping pores crystal growth on the surface of lesion by keeping pores openopen


Calculus formation and calcium Calculus formation and calcium phosphate inhibitorsphosphate inhibitors

Calculus forms in plaque despite inhibitory action of Calculus forms in plaque despite inhibitory action of statherin and PRPs in salivastatherin and PRPs in saliva

May be due to failure to diffuse into calcifying May be due to failure to diffuse into calcifying plaqueplaque

Proteolytic enzymes of oral bacteria or lysed Proteolytic enzymes of oral bacteria or lysed leukocytes may destroy inhibitory proteinsleukocytes may destroy inhibitory proteins

Plaque bacteria may produce their own inhibitorsPlaque bacteria may produce their own inhibitors


Calcium phosphate precipitation Calcium phosphate precipitation inhibitors and plaqueinhibitors and plaque

Statherin and PRPs might be expected to occur in Statherin and PRPs might be expected to occur in plaque, have not been detectedplaque, have not been detected

Plaque bacteria produce calcium phosphate inhibitorsPlaque bacteria produce calcium phosphate inhibitors Might be necessary to prevent calcification of bacteria Might be necessary to prevent calcification of bacteria

-- happens with dead cells-- happens with dead cells Immobilized crystal growth inhibitors can function as Immobilized crystal growth inhibitors can function as

nucleators of crystal growthnucleators of crystal growth Immobilization may occur in plaque, encouraging Immobilization may occur in plaque, encouraging

calculus formationcalculus formation


Interaction of oral bacteria with Interaction of oral bacteria with PRPs and other pellicle proteinsPRPs and other pellicle proteins

SeveralSeveral salivary proteins appear to be involved in salivary proteins appear to be involved in preventing or promoting bacterial adhesion to oral soft preventing or promoting bacterial adhesion to oral soft and hard tissuesand hard tissues

PRPs are strong PRPs are strong promoterspromoters of bacterial adhesion of bacterial adhesion– Amino terminal: control calcium phosphate chemistryAmino terminal: control calcium phosphate chemistry– Carboxy terminal: interaction with oral bacteriaCarboxy terminal: interaction with oral bacteria

Interactions are highly Interactions are highly specificspecific– Depends on proline-glutamine carboxy-terminal dipeptideDepends on proline-glutamine carboxy-terminal dipeptide– PRPs in solution do not inhibit adhesion of bacteriaPRPs in solution do not inhibit adhesion of bacteria


These anti-microbial proteins These anti-microbial proteins will be discussed in a later lecturewill be discussed in a later lecture

Secretory ImmunoglobulinsSecretory Immunoglobulins LactoferrinLactoferrin LysozymeLysozyme SialoperoxidaseSialoperoxidase CystatinsCystatins HistatinsHistatins


Summary - Clinical HighlightsSummary - Clinical Highlights

Understanding of salivary mechanisms at Understanding of salivary mechanisms at fundamental level a prerequisite for fundamental level a prerequisite for – effective treatment of salivary gland effective treatment of salivary gland

dysfunctionsdysfunctions– modulation of bacterial colonizationmodulation of bacterial colonization– development of artificial saliva other “cutting development of artificial saliva other “cutting

edge” approaches to salivary dysfunctions and edge” approaches to salivary dysfunctions and diseasesdiseases

Dennis E. Lopatin, Ph.D. Page no. 1 Chemical composition and functions of saliva.

Documents

Transcript of Dennis E. Lopatin, Ph.D. Page no. 1 Chemical composition and functions of saliva.