Chem Comp & Funct

8/6/2019 Chem Comp & Funct

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

Chemical composition and functionsChemical composition and functions

of salivaof saliva


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Chronology of defining salivaryChronology of defining salivary

components and functionscomponents and functions Beginning in 1950sBeginning in 1950s whole salivawhole saliva evaluatedevaluated

(antimicrobial properties, role in microbial attachment,(antimicrobial properties, role in microbial attachment,

mineralization, taste, lubrication)mineralization, taste, lubrication) Secretions ofSecretions ofmajor glandsmajor glands (parotid and(parotid and

submandibular/sublingual)submandibular/sublingual)

In 1970sIn 1970s individual componentsindividual components isolated andisolated and

biochemically characterizedbiochemically characterized

In midIn mid--1980s beginning to map1980s beginning to map functional domainsfunctional domains

(peptide synthesis and recombinant approaches)(peptide synthesis and recombinant approaches)


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M

ajor salivary componentsM

ajor salivary components

Mucin 1 (MG1)Mucin 1 (MG1)

sIgAsIgA

Mucin 2 (MG2)Mucin 2 (MG2)

LactoferrinLactoferrin

PeroxidasesPeroxidases

AmylasesAmylasesCarbonic anhydrasesCarbonic anhydrases

ProlineProline--rich proteinsrich proteins

LysozymeLysozyme

StatherinsStatherins

HistatinsHistatins

11 1010 100 100 1000 1000 10000 10000

Size (kDa)Size (kDa)


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Current concepts regarding the functionalCurrent concepts regarding the functionalfeatures of salivary macromoleculesfeatures of salivary macromolecules

Recent structure/function studies have identifiedRecent structure/function studies have identifiedgeneral principlesgeneral principles regarding functionregarding function

Based onBased on in vitroin vitro studies of purified moleculesstudies of purified molecules

Additional studies required to evaluate conceptsAdditional studies required to evaluate concepts

in situin situ


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Conformational requirementsConformational requirements

Conformation or shape of a molecule isConformation or shape of a molecule is criticalcritical

for its biological functionfor its biological function

ExamplesExamples ProlineProline--rich proteins interact withrich proteins interact with A. viscosusA. viscosus andandSt. gordoniiSt. gordonii only whenonly when adsorbedadsorbed onto mineralizedonto mineralized

surfacesurface

Statherins and histatins requireStatherins and histatins require EE--helicalhelical

conformationconformation

Human salivary amylase require 5 interHuman salivary amylase require 5 inter--chainchain

disulfide bondsdisulfide bonds


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MultifunctionalityMultifunctionality

SalivarySalivary

FamiliesFamilies

AntiAnti--

BacterialBacterialBufferingBuffering

DigestionDigestion

MineralMineral--

izationization

LubricatLubricat--ion &Viscoion &Visco--

elasticityelasticity

TissueTissue

CoatingCoating

AntiAnti--

FungalFungal

AntiAnti--

ViralViral

Carbonic anhydrases,Carbonic anhydrases,HistatinsHistatins

Amylases,Amylases,

Mucins, LipaseMucins, Lipase

Cystatins,Cystatins,

Histatins, ProlineHistatins, Proline--rich proteins,rich proteins,


Mucins, StatherinsMucins, Statherins

Amylases,Amylases,

Cystatins, Mucins,Cystatins, Mucins,

ProlineProline--rich proteins, Statherinsrich 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


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RedundancyRedundancy

Saliva has builtSaliva has built--inin redundancyredundancy in regard to itsin regard to its

protective functions.protective functions.

ExampleExample -- Many salivary molecules can inhibitMany salivary molecules can inhibitthe precipitation of calcium phosphate salts.the precipitation of calcium phosphate salts.

strong inhibitors such as statherin and acidic prolinestrong 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


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AmphifunctionalityAmphifunctionality

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

propertiesproperties -- doubledouble--edged swordedged sword..

May depend on moleculesMay depend on molecules locationlocation or site of actionor 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 theseAdsorbed to tooth surface, they can promote adherence of these

bacteria and digest starch to dietary maltose and production of acidbacteria and digest starch to dietary maltose and production of acid

Statherin and acidic prolineStatherin and acidic proline--rich proteinsrich proteins At enamel surface, they play an important role in mineralization byAt enamel surface, they play an important role in mineralization by

inhibiting the formation of primary and secondary calciuminhibiting the formation of primary and secondary calcium

phosphate salts. When adsorbed to the enamel surface, theyphosphate salts. When adsorbed to the enamel surface, they

promote attachment of cariogenic microorganisms.promote attachment of cariogenic microorganisms.


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ComplexingComplexing Functional relationships exist between differentFunctional relationships exist between different

molecules in salivamolecules in saliva

Two types of complexing (covalent and nonTwo types of complexing (covalent and non--covalent)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 andhomotypic complexes necessary for lubrication and

viscoelastic propertiesviscoelastic properties

heterotypic complexes with sIgA, lysozyme and cystatinsheterotypic complexes with sIgA, lysozyme and cystatins

concentrate these anticoncentrate these anti--microbials at tissue interfacesmicrobials at tissue interfaces


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Salivary Protein FunctionsSalivary Protein Functions

Oral function Pro blem 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

F

ood-bornorganisms Soft and hard tissue

abrasion Food toxins

A

nti-bacterialsystems Lubrication;

mucins, statherin Toxin-neutralization


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Salivary Protein Functions (contd)Salivary Protein Functions (contd)

Oral function Pro blem Protein function

Control of i n-digenous & i n-

vading bacteria,fungi and v iruses

Colonization &infection

Controllingpathogens andcommensals

Adhesion of ba c-teria versus theirdetection

Anti-bacterialsystems

Immunoglob u-lins, histatins,glycoproteins,lysozyme, sia-loperoxidase,lactoferrin

Adhesion-modulating pr o-teins


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Salivary Protein Functions (contd)Salivary Protein Functions (contd)

ral function Pro ble Protein function

igestion __ Starch athydrolysis:

amylase andlingual lipase

Protectionrepair o so ttissues

Toxins,carcinogens,degradativeproteases

Mucin-richprotective barrier ilm

Protease

inhibitors,cystatins, tissuegro th actors


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MucinsMucins

Lack precise folded structure of globular proteinsLack precise folded structure of globular proteins

Asymmetrical molecules with open, randomly organizedAsymmetrical molecules with open, randomly organized

structurestructure

Polypeptide backbone (apomucin) with CHO sidePolypeptide backbone (apomucin) with CHO side--chainschains SideSide--chains may end in negatively charged groups, such aschains 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)


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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 antiConcentrates anti--microbial molecules at mucosal interfacemicrobial molecules at mucosal interface

LubricationLubrication

Align themselves with direction of flow (characteristic ofAlign 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 movingFilm strength determines how effectively opposed moving

surfaces are kept apartsurfaces are kept apart


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Mucin Functions (contd)Mucin Functions (contd)

Aggregation of bacterial cellsAggregation of bacterial cells

Bacterial adhere to mucins may result in surfaceBacterial adhere to mucins may result in surface

attachment, orattachment, or MucinMucin--coated bacteria may be unable to attach to surfacecoated bacteria may be unable to attach to surface

Bacterial adhesionBacterial adhesion

Mucin oligosaccharides mimic those on mucosal cellMucin oligosaccharides mimic those on mucosal cell

surfacesurface

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


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AmylasesAmylases Calcium metalloenzymeCalcium metalloenzyme

HydrolyzesHydrolyzes EE(1(1--4) bonds of starches such as4) bonds of starches such as

amylose and amylopectinamylose and amylopectin

Several salivary isoenzymesSeveral salivary isoenzymes

Maltose is the major endMaltose is the major end--product (20% is glucose)product (20% is glucose)

Appears to have digestive functionAppears to have digestive function

Why is it also present in tears, serum, bronchial, andWhy is it also present in tears, serum, bronchial, andmale and female urogenital secretions?male and female urogenital secretions?

A role in modulating bacterial adherence?A role in modulating bacterial adherence?


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Lingual LipaseLingual Lipase

Secreted by von Ebners glands of tongueSecreted by von Ebners glands of tongue

Involved in first phase of fat digestionInvolved in first phase of fat digestion

Hydrolyzes mediumHydrolyzes medium-- to longto long--chain triglycerideschain triglycerides

Important in digestion of milk fat in newImportant in digestion of milk fat in new--bornborn

Unlike other mammalian lipases, it is highlyUnlike other mammalian lipases, it is highly

hydrophobic and readily enters fat globuleshydrophobic and readily enters fat globules


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Calcium phosphate salts of dental enamel are solubleCalcium 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 maintainSupersaturation of calcium phosphates maintain

enamel integrityenamel integrity

Statherins prevent precipitation or crystallization ofStatherins prevent precipitation or crystallization of

supersaturated calcium phosphate in ductal saliva andsupersaturated 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


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ProlineProline--rich Proteins (PRPs)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 negativelyInhibition due to first 30 residues of negatively--

charged aminocharged amino--terminal endterminal end

Present in the initially formed enamel pellicle and inPresent in the initially formed enamel pellicle and in

mature pelliclesmature pellicles


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Role of PRPs in enamel pellicleRole of PRPs in enamel pellicle

formationformation

Acquired enamel pellicle is 0.1Acquired enamel pellicle is 0.1--1.0 m thick layer of1.0 m thick layer of

macromolecular material on the dental mineral surfacemacromolecular material on the dental mineral surface

Pellicle is formed by selective adsorption ofPellicle is formed by selective adsorption ofhydroxyapatitehydroxyapatite--reactive salivary proteins, serum proteinsreactive salivary proteins, serum proteins

and microbial products such as glucans and glucosyland microbial products such as glucans and glucosyl--

transferasetransferase

Pellicle acts as a diffusion barrier, slowing both attacksPellicle acts as a diffusion barrier, slowing both attacks

by bacterial acids and loss of dissolved calcium andby bacterial acids and loss of dissolved calcium and

phosphate ionsphosphate ions


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Remineralization of enamel andRemineralization of enamel and

calcium phosphate inhibitorscalcium phosphate inhibitors Early caries are repaired despite presence ofEarly caries are repaired despite presence of

mineralization inhibitors in salivamineralization inhibitors in saliva

Sound surface layer of early carious lesion formsSound surface layer of early carious lesion formsimpermeable 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 preventingInhibitors may encourage mineralization by preventing

crystal growth on the surface of lesion by keepingcrystal growth on the surface of lesion by keeping

pores openpores open


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Calculus formation and calciumCalculus formation and calcium

phosphate inhibitorsphosphate inhibitors

Calculus forms in plaque despite inhibitory action ofCalculus forms in plaque despite inhibitory action of

statherin and PRPs in salivastatherin and PRPs in saliva

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

plaqueplaque

Proteolytic enzymes of oral bacteria or lysedProteolytic 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


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Calcium phosphate precipitationCalcium phosphate precipitation

inhibitors and plaqueinhibitors and plaque Statherin and PRPs might be expected to occur inStatherin 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 bacteriaMight be necessary to prevent calcification of bacteria ----

happens with dead cellshappens with dead cells

Immobilized crystal growth inhibitors can function asImmobilized crystal growth inhibitors can function as

nucleators of crystal growthnucleators of crystal growth

Immobilization may occur in plaque, encouragingImmobilization may occur in plaque, encouraging

calculus formationcalculus formation


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Interaction of oral bacteria withInteraction of oral bacteria with

PRPs and other pellicle proteinsPRPs and other pellicle proteins SeveralSeveral salivary proteins appear to be involved insalivary proteins appear to be involved in

preventing or promoting bacterial adhesion to oral softpreventing or promoting bacterial adhesion to oral soft

and hard tissuesand hard tissues

PRPs are strongPRPs are strong promoterspromoters of bacterial adhesionof 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 highlyInteractions are highly specificspecific Depends on prolineDepends on proline--glutamine carboxyglutamine carboxy--terminal dipeptideterminal dipeptide

PRPs in solution do not inhibit adhesion of bacteriaPRPs in solution do not inhibit adhesion of bacteria


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These antiThese anti--microbial proteins willmicrobial proteins will

be discussed in a later lecturebe discussed in a later lecture

Secretory ImmunoglobulinsSecretory Immunoglobulins

LactoferrinLactoferrin

LysozymeLysozyme

SialoperoxidaseSialoperoxidase

CystatinsCystatins

HistatinsHistatins


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SummarySummary -- Clinical HighlightsClinical Highlights

Understanding of salivary mechanisms atUnderstanding of salivary mechanisms at

fundamental level a prerequisite forfundamental level a prerequisite for

effective treatment of salivary glandeffective treatment of salivary glanddysfunctionsdysfunctions

modulation of bacterial colonizationmodulation of bacterial colonization

development of artificial saliva other cuttingdevelopment of artificial saliva other cutting

edge approaches to salivary dysfunctions andedge approaches to salivary dysfunctions anddiseasesdiseases

Chem Comp & Funct

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