Chemical composition and functions
of saliva
Page no. 1
Dennis E. Lopatin, Ph.D.
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)

Page no. 2
Dennis E. Lopatin, Ph.D.
Major salivary components
Histatins
Statherins
Lysozyme
Proline-rich proteins
Carbonic anhydrases
Amylases
Peroxidases
Lactoferrin
Mucin 2 (MG2)
sIgA
Mucin 1 (MG1)
1
Page no. 3
10
100
Size (kDa)
1000
10000
Dennis E. Lopatin, Ph.D.
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

Page no. 4
Dennis E. Lopatin, Ph.D.
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
Page no. 5
Dennis E. Lopatin, Ph.D.
Multifunctionality
Amylases, Cystatins,
Carbonic anhydrases,
Histatins, Mucins,
Histatins
AntiPeroxidases
Buffering
Bacterial
Amylases,
Cystatins,
Mucins, Lipase
AntiMucins
Digestion
Viral
Salivary
Families
MineralAntiization
Fungal
Cystatins,
Histatins
Histatins, ProlineLubricatrich proteins,
Tissue ion &ViscoStatherins
Coating elasticity
Amylases,
Cystatins, Mucins,
Mucins, Statherins
Proline-rich proteins, Statherins
Page no. 6
adapted from M.J. Levine, 1993
Dennis E. Lopatin, Ph.D.
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 prolinerich proteins
– moderate inhibitors such as histatins and cystatins
– weak inhibitors such as mucins and amylase
Page no. 7
Dennis E. Lopatin, Ph.D.
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
Page no. 8
» 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. Dennis E. Lopatin, Ph.D.
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
Page no. 9
Dennis E. Lopatin, Ph.D.
Salivary Protein Functions
Oral function
Problem
• Acts as an airway
• Air-born organisms
• Dehydration
• Speech
• Taste
Protein function
• Anti-bacterial
systems
• Water-retaining
glycopr oteins
• Need for lubrication • Lubrication system
--
• Entry-point for food • Food-born
mastication,
organisms
swallowing
• Soft and hard tissue
abrasion
• Food toxins
Page no. 10
• Gustin
• Anti-bacterial
systems
• Lubrication;
mucins, statherin
• Toxin-neutralization
Dennis E. Lopatin, Ph.D.
Salivary Protein Functions (cont’d)
Oral function
Problem
Protein function
• Control of indigenous & i nvading bacteria,
fungi and v iruses
• Colonization &
infection
• Controlling
pathogens and
commensals
• Adhesion of ba cteria versus their
detection
• Anti-bacterial
systems
• Immunoglob ulins, histatins,
glycoproteins,
lysozyme, sialoperoxidase,
lactoferrin
• Adhesionmodulating pr oteins
Page no. 11
Dennis E. Lopatin, Ph.D.
Salivary Protein Functions (cont’d)
Page no. 12
Oral function
• Digestion
Problem
__
• Protection &
repair of soft
tissues
• Toxins,
carcinogens,
degradative
proteases
Protein function
• Starch & fat
hydrolysis:
amylase and
lingual lipase
• Mucin-rich
protective
barrier film
• Protease
inhibitors,
cystatins, tissue
growth factors
Dennis E. Lopatin, Ph.D.
Salivary Protein Functions (cont’d)
Oral function
Problem
Protein function
• Protection & re- • Enamel mineral • Biologically
pair of hard tisis potentially
controlled prosues
soluble; acidtective & redamaged
pairative inorenamel requires
ganic environremineralization ment, stabilized
by statherin,
acidic prolinerich and pellicle
proteins
• Pellicle form a__
__
tion
• Plaque acid
• Plaque pH con- • Basic amino
formation
trol
acids & peptides
Page no. 13
Dennis E. Lopatin, Ph.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)
Page no. 14
Dennis E. Lopatin, Ph.D.
Mucin Functions

Tissue Coating
– 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
Page no. 15
Dennis E. Lopatin, Ph.D.
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
Page no. 16
Dennis E. Lopatin, Ph.D.
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?

Page no. 17
Dennis E. Lopatin, Ph.D.
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

Page no. 18
Dennis E. Lopatin, Ph.D.
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

Page no. 19
Dennis E. Lopatin, Ph.D.
Proline-rich Proteins (PRPs)
Like statherin, PRPs are also highly asymmetrical
 Inhibitors of calcium phosphate crystal growth
 Inhibition due to first 30 residues of negativelycharged amino-terminal end
 Present in the initially formed enamel pellicle and in
“mature” pellicles

Page no. 20
Dennis E. Lopatin, Ph.D.
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 glucosyltransferase
 Pellicle acts as a diffusion barrier, slowing both attacks
by bacterial acids and loss of dissolved calcium and
phosphate ions

Page no. 21
Dennis E. Lopatin, Ph.D.
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

Page no. 22
Dennis E. Lopatin, Ph.D.
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

Page no. 23
Dennis E. Lopatin, Ph.D.
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

Page no. 24
Dennis E. Lopatin, Ph.D.
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
Page no. 25
Dennis E. Lopatin, Ph.D.
These anti-microbial proteins will
be discussed in a later lecture
Secretory Immunoglobulins
 Lactoferrin
 Lysozyme
 Sialoperoxidase
 Cystatins
 Histatins

Page no. 26
Dennis E. Lopatin, Ph.D.
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
Page no. 27
Dennis E. Lopatin, Ph.D.