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 10 100 1000 10000
Size (kDa)
Page no. 3 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
Peroxidases Anti- Buffering
Bacterial
Cystatins, Amylases,
Mucins Anti- Mucins, Lipase
Viral Digestion
Salivary
Families
Anti- Mineral-
Fungal ization Cystatins,
Histatins
Histatins, Proline-
Lubricat-
Tissue ion &Visco- rich proteins,
Amylases, Coating elasticity Statherins
Cystatins, Mucins,
Proline-rich proteins, Statherins Mucins, 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 proline-
rich 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
» 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.

Page no. 8 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
Oral function
function Problem
Problem Protein
Protein function
function
•• Acts
Acts as
as an
an airway
airway •• Air-born
Air-born organisms
organisms •• Anti-bacterial
Anti-bacterial
•• Dehydration
Dehydration systems
systems
•• Water-retaining
Water-retaining
glycoproteins
glycoproteins
•• Speech
Speech •• Need
Need for
for lubrication
lubrication •• Lubrication
Lubrication system
system
•• Taste
Taste --
-- •• Gustin
Gustin
•• Entry-point
Entry-point for
for food
food •• Food-born
Food-born •• Anti-bacterial
Anti-bacterial
mastication,
mastication, organisms
organisms systems
systems
swallowing
swallowing •• Soft
Soft and
and hard
hard tissue
tissue •• Lubrication;
Lubrication;
abrasion
abrasion mucins,
mucins, statherin
statherin
•• Food
Food toxins
toxins •• Toxin-neutralization
Toxin-neutralization
Page no. 10 Dennis E. Lopatin, Ph.D.
 Salivary Protein Functions (cont’d)
Oral
Oral function
function Problem
Problem Protein
Protein function
•• Control
Control of
of in-
in- •• Colonization
Colonization & & •• Anti-bacterial
Anti-bacterial
digenous
digenous && in-
in- infection
infection systems
systems
vading
vading bacteria,
bacteria, •• Controlling
Controlling •• Immunoglobu-
Immunoglobu-
fungi
fungi and
and viruses
viruses pathogens
pathogens and
and lins,
lins, histatins,
histatins,
commensals
commensals glycoproteins,
glycoproteins,
•• Adhesion
Adhesion ofof bac-
bac- lysozyme,
lysozyme, sia-
sia-
teria
teria versus
versus their
their loperoxidase,
loperoxidase,
detection
detection lactoferrin
lactoferrin
•• Adhesion-
Adhesion-
modulating
modulating pro-
pro-
teins
teins

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

 Salivary Protein Functions (cont’d)
Oral
Oral function
function Problem
Problem Protein
Protein function
function
•• Digestion
Digestion __
__ •• Starch
Starch && fat
fat
hydrolysis:
hydrolysis:
amylase
amylase andand
lingual
lingual lipase
lipase
•• Protection
Protection && •• Toxins,
Toxins, •• Mucin-rich
Mucin-rich
repair
repair of
of soft
soft carcinogens,
carcinogens, protective
protective
tissues
tissues degradative
degradative barrier
barrier film
film
proteases
proteases •• Protease
Protease
inhibitors,
inhibitors,
cystatins,
cystatins, tissue
tissue
growth
growth factors
factors

Page no. 12 Dennis E. Lopatin, Ph.D.

 Salivary Protein Functions (cont’d)
Oral
Oral function
function Problem
Problem Protein
Protein function
function
•• Protection
Protection && re-re- •• Enamel
Enamel mineral
mineral •• Biologically
Biologically
pair
pair of
of hard
hard titis-
s- is is potentially
potentially controlled
controlled pro-
pro-
sues
sues soluble;
soluble; acid-
acid- tective
tective & & re-
re-
damaged
damaged pairative
pairative inor-
inor-
enamel
enamel requires
requires ganic
ganic environ-
environ-
remineralization
remineralization ment,ment, stabilized
stabilized
by
by statherin,
statherin,
acidic
acidic proline-
proline-
rich
rich and
and pellicle
pellicle
proteins
proteins
•• Pellicle
Pellicle forma-
forma- __
__ __
__
tion
tion
•• Plaque
Plaque acid
acid •• Plaque
Plaque pH
pH co
con-n- •• Basic
Basic amino
amino
formation
formation trol
trol acids
acids && peptides
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 negatively-
charged 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 glucosyl-
transferase
 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.