Chem Comp & Funct
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Transcript of Chem Comp & Funct
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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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Dennis E. Lopatin, Ph.D.Dennis E. Lopatin, Ph.D.Page no.Page no. 22
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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Dennis E. Lopatin, Ph.D.Dennis E. Lopatin, Ph.D.Page no.Page no. 33
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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Dennis E. Lopatin, Ph.D.Dennis E. Lopatin, Ph.D.Page no.Page no. 44
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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Dennis E. Lopatin, Ph.D.Dennis E. Lopatin, Ph.D.Page no.Page no. 55
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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Dennis E. Lopatin, Ph.D.Dennis E. Lopatin, Ph.D.Page no.Page no. 66
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,
StatherinsStatherins
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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Dennis E. Lopatin, Ph.D.Dennis E. Lopatin, Ph.D.Page no.Page no. 77
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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Dennis E. Lopatin, Ph.D.Dennis E. Lopatin, Ph.D.Page no.Page no. 88
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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Dennis E. Lopatin, Ph.D.Dennis E. Lopatin, Ph.D.Page no.Page no. 99
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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Dennis E. Lopatin, Ph.D.Dennis E. Lopatin, Ph.D.Page no.Page no. 1010
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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Dennis E. Lopatin, Ph.D.Dennis E. Lopatin, Ph.D.Page no.Page no. 1111
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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Dennis E. Lopatin, Ph.D.Dennis E. Lopatin, Ph.D.Page no.Page no. 1212
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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Dennis E. Lopatin, Ph.D.Dennis E. Lopatin, Ph.D.Page no.Page no. 1414
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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Dennis E. Lopatin, Ph.D.Dennis E. Lopatin, Ph.D.Page no.Page no. 1515
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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Dennis E. Lopatin, Ph.D.Dennis E. Lopatin, Ph.D.Page no.Page no. 1616
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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Dennis E. Lopatin, Ph.D.Dennis E. Lopatin, Ph.D.Page no.Page no. 1717
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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Dennis E. Lopatin, Ph.D.Dennis E. Lopatin, Ph.D.Page no.Page no. 1818
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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Dennis E. Lopatin, Ph.D.Dennis E. Lopatin, Ph.D.Page no.Page no. 1919
StatherinsStatherins
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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Dennis E. Lopatin, Ph.D.Dennis E. Lopatin, Ph.D.Page no.Page no. 2020
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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Dennis E. Lopatin, Ph.D.Dennis E. Lopatin, Ph.D.Page no.Page no. 2121
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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Dennis E. Lopatin, Ph.D.Dennis E. Lopatin, Ph.D.Page no.Page no. 2222
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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Dennis E. Lopatin, Ph.D.Dennis E. Lopatin, Ph.D.Page no.Page no. 2323
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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Dennis E. Lopatin, Ph.D.Dennis E. Lopatin, Ph.D.Page no.Page no. 2424
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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Dennis E. Lopatin, Ph.D.Dennis E. Lopatin, Ph.D.Page no.Page no. 2525
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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Dennis E. Lopatin, Ph.D.Dennis E. Lopatin, Ph.D.Page no.Page no. 2626
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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Dennis E. Lopatin, Ph.D.Dennis E. Lopatin, Ph.D.Page no.Page no. 2727
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