This document summarizes the major salivary enzymes and their functions. It begins by outlining the chronological discovery of salivary components starting in the 1950s. It then describes the current understanding of salivary proteins' structure-function relationships and conformational requirements. The document discusses the expected network action of the salivary defense system and lists the five primary defense networks involving proteins that agglutinate bacteria, lyse bacterial membranes, and have antifungal and antiviral properties. Finally, it provides details on specific salivary components like defensins, histatins, lactoferrin, and lysozymes that have antimicrobial functions.

1. Salivary enzymes & Their functions
Presented by
Roshni Maurya
Dept.of Pediatric & Preventive Dentistry

2. 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)

3. 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
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

4. EXPECTED NETWORK ACTION OF THE SALIVARY DEFENSE
SYSTEM
Salivary defense proteins may enrich concentrations to “efficient” levels in certain locations in
the oral cavity, notwithstanding that many of them are found in whole saliva in less than- efficient
concentrations.
In these locations, a “single-hit” type of action (one type of agent affects the target) of salivary
defense proteins may occur.
Salivary proteins(including cationic peptides) may also provide antimicrobial defense, even if
they are present in concentration that are lower than efficient.
In such case, a network type “multihit” approach may be expected. Several kinds of salivary
defense proteins affect the targeted microbe at the same time.
Although, a certain salivary protein may induce a “partial knockout” of targeted microbe only,
their network type “acting together” at same time may lead to efficient elimination of the target.

5. EXPECTED FIVE PRIMARY DEFENSE NETWORKS OF SALIVARY
PROTEINS
PROTEINS IN WHOLE SALIVA
 First network may be responsible for microbial agglutination and/or surface exclusion. This
may include those salivary proteins and peptides which bind bacteria (microbes) and also
bind to either oral surfaces or each other or both.
 2nd network may be expected to be responsible for lysis of microbial membranes. This
primarily targets bacteria, includes salivary cationic peptides and lysozyme.
 Third and Fourth networks may be responsible for antifungal and antiviral properties of
saliva respectively.
 Finally, a fifth, immune regulatory network of salivary proteins may be expected. This
includes all those salivary proteins exerting immune activator/modulator properties. It may
be important for fine-regulation of local action of mucosal immune system.

6. MAJOR SALIVARY COMPONENTS
Mucin 1 (MG1)
sIgA
Mucin 2 (MG2)
Lactoferrin
Peroxidases
Amylases
Carbonic anhydrases
Proline-rich proteins
Lysozyme
Statherins
Histatins
1 10 100 1000 10000
Size (kDa)

7. SALIVARY ANTIBODIES
MAJOR CLASSES OF SALIVARY ANTIBODIES
 Salivary antibodies act in first line of defense by performing immune exclusion of antigen in : saliva; mucus
layer on epithelial surfaces; acquired pellicle on tooth surface.
 They are constitutively excreted into saliva and oral cavity.
 2 major classes in human saliva :a) secretory IgA-its main source is salivary gland ;in less amt.secreted by
mucosal cell; ,is dimeric,may be polymeric.
 The other is IgG- monomeric,either serum derived or produced by local plasma cells.
 Although majority of Abs belong to IgA(90-98%); IgG (1-10%) classes small fractions of IgM ,IgE Abs are
present in saliva as well.
 Serum derived IgG and monomeric IgA enters oral cavity via: GCF(mainly); mucosal transudate;
ultrafiltration through salivary gland acini.

8. PRODUCTION OF SALIVARY SECRETORY ANTIBODY
 Intial stimulation of secretory Ig expressing B-cells takes place in MALT,such as GALT and
NALT.
 Salivary secretory namely sIgA and very small amt. of sIgM are produced by specific plasma cells
residing primarily in salviary gland storma and in small nos. in o.mucosa.
 The plasma cell release mainly dimeric; some polymeric IgA molecules and few pentameric IgM
molecules.
 These Ig are exported to saliva by acinar cells;ductal cells of minor and major sal.gland via a
common sal.gland epithelial transport mechanism.

9. ANTIGEN BINDING;AGGLUTINATION AND SURFACE EXCLUSION:
 Pri.function of sal.(secretory and other)Ig is to inactivate parasites-bacteria;fungi;viruses via binding/or
agglutination of such particles.
 Surface immune exclusion of pathogen within o.cavity via anchorage of secretory Ig- binding antigens to
the superficial mucous layer lining mucosal surface –another imp. mechanism to prevent invasion of
underlying tissues.
PHAGOCYTOSIS;ANTIGEN PRESENTATION;DEGRANULATION AND CYTOKINE PRODUCTION:
 Oral mucosal surface is extensively populated by antigen presenting cells(Langerhans and dendrictic
cells);there is a considerable flux of neutrophil granulocytes through gingival sulcus into the saliva even in
the healthy. As both dendrictic and neutro.graulocytes have immune activating IgA activating receptors on
their surface, premised immune –cell coupled defense functions of sal.IgA and others are likely to be active
to maintain immune surveillance.
ANTIBODY CATALYZED OZONE FORMATION:
 In the presence of reactive oxy.int.(ROI)- producing neutro.gran.;sal.Abs can catalyze ozone formation
leading to efficient microbial killing. During this process, Abs kill microbes by catalytically converting a
less toxic ROI to a mixture of hydrogen peroxide and ozone.

10. SALIVARY CHAPEROKINE HSP70/HSPAS
 Salivary glands are among the main sources of HSP70/HSPAs in saliva.Besides sal.glands other
imp.sources are mucosal cells,GCF,oral mucosal cells,intra oral bleeding(bleeding periodontal
pockets,wounds,ulcers)
BINDING OF BACTERIA,AGGLUTINATION AND SURFACE EXCLUSION:
 Sal.HSP70/HSPAs may entrap and agglutinate bacteria. Recent data has indicated that the this
proteins bind both gram+ve( Strepto.mutans;mitis) and gram-ve (E.coli) bacteria.
IMMUNOLOGICAL DEFENSE MECHANSIMS:
 3 major facets of immune activation are described:
First involves extracellular second involves complexes of third exert an opsonizing
HSP70/HSPA as an ancestral extracellular HSP70/HSPA proteins effect on bacteria activating
danger signal of cellular stress and other peptides. Is imp. In defense the killing activity of PMNS
death or lysis. Against bacteria, tumor cells, neutrophil granulocytes,
virus infected cells. Effective under inflammatory
conditions, in case of oral lesions

11. CATIONIC PEPTIDES
DEFENSINS
‘Prototype’of cationic peptides.
 Characterized by a “ hairpin-like” globular structure stabilized by three intramolecular
disulfide bridges linking six cysteine aminoacids.
 Based on pattern of cystein pairing , 2 types :-
 Alpha-defensins.
 Beta- defensins.
 Salivary Alpha-defensins (HNP1, HNP2, HNP3, HNP4) are produced by neutrophil
granulocytes .
 Salivary Beta- defensins (hBD1, hBD2, hBD3, hBD4) are produced by mucosal cells.
 Beside whole saliva, both Alpha- and Beta- defensins are also present in GCF. Both show
broad antibacterial activity.

12.  These absorb via electrostatic forces onto negatively-charged bacterial cell membrane leading
to their subsequent aggregation and integration into lipid bilayer. Hence,
Integration of Defensins into bacterial membranes.
causes
Formation of ion channels, transmembrane pore
results
membrane leakage and rupture
Destruction of bacteria
 Beside their broad antibacterial activity, they also exert antifungal and antiviral properties,
exert various immune activator and modulatory activities.

14. HISTATINS
 HISTATINS are small histidine-rich cationic peptide ranging in size from 7 to 38 aa.
 Secreted by parotid gland, sublingulal and submandibular glands.
 Imp. histatins (HRPs) are :histatin1; histatin2; histatin3; histatin5
 They exert broad spectrum antibacterial ;antifungal; antiviral properties.
 MOA is similar to Defensins. It also binds and complex Cu+2,Ni+2 ions leading to elimination of metal
ions and consequent inhibition of enzy.; their cofactors and microbial growth.
 Histatins (esp.5) exert efficient activities against fungi; mainly C albicans. It inhibits a trypsin like
protease of Bacteroides gingi valis.
 Histatins (esp.1) are incorporated into acquired pellicle on tooth surfaces and may play role in bacterial
colonization on tooth surfaces.
 Histatin1 was shown to competitively inhibit absorption of HMWGP-binding cariogenic
bacteria(i.e,S.mutans onto tooth surfaces).
 Histatins (esp.1;2;3)were identified as highly imp. for Wound closure stimulating factors of human saliva.

15. LACTOFERRIN
 It is an iron- binding cationic glycoprotein present in most exocrine secretions,including saliva.
 Major sources in saliva are sal. glands neutrophhil granulocytes entering oral cavity,mucosal epi.cells,GCF.
 It is active against bacteria,fungi,parasites,viruses.MOA is similar to cationic peptides.
 Besides,its cationic peptide activity,lactoferrin is a known scavenger of Fe+3 ions.It binds and sequestrates iron;
depriving micro.of iron i.e, essential for their growth.
 It binds bacterial fimbrial adhesins;inhibiting epi.adhesions of certain bacteria.
 Antiviral activity is due to binding and blocking of certain host cell GAGS used by viruses for adsorption,may
neutralise viruses by direct binding.
 Immune modulatory and anticancer activity of lactoferrin seems likely.
ADRENOMEDULLIN:
 A pluripotent hormone like cationic peptide of 52 AA.
 Present in GCF;glandular saliva, whole saliva,oral epithelial cells excrete adernomedullin into saliva.
 It is able to prevent bacterial growth (S.aureus) via generation of abnormal septum formation during cell
division.

16. CATHELICIDINS(LL-37)
 Human Cathelicidin is referred to as hCAP-18 and most important derivative referred as LL-37,(an α-
helix type cationic antimicrobial peptide).
 These are characterized by a conserved N-terminal domain that is proteolytically cleaved to generate the
active peptide.
 Salivary LL-37 is likely to originate primarily from neutrophil leukocytes, is present in GCF.
 MOA is similar to other cationic peptides .
 It binds and neutralises bacterial LPS.
 LL-37 and its derivatives are likely to play a role in re-epithelization of wounds and ulcers in oral cavity.
Exert immune activator and immune modular properties.
Secretory Leukocyte Proteinase Inhibitor (SLPI)
It is 107 aa serine protease inhibiters , controls excessive proteolysis caused by proteases of neutrophil granulocytes.
Hence, referred as antileukoprotease(ALP).
SLPI present in saliva is produced by keratinocytes of oral mucosa; neutrophil granulocytes entering oral cavity.
It is a non-glycosylated ,basic,single-chain,cysteine rich cationic polypeptide.
Being cationic in character, it acts as a cationic peptide. It exerts antimicrobial activity against both
bacteria(P.aeruginosa,S.aureus) and fungi(C.albicans), exerts antiviral properties .

17. DEFENSE PROTEINS OF SALIVA
Statherin
 It is a tyrosine-glutamine and proline rich phosphoprotein .
 It prevents precipitation crystallization of supersaturated calcium phosphate in ductal saliva and oral fluid.
 It binds hydroxypatite ,indicating a possible role in acquired pellicle and dental plague formation .
 It competitively inhibits absorption of HMWGPs to tooth surfaces and may inhibit adhesion of HMWGP-
binding cariogenic bacteria including S.mutans.
 Besides antibacterial properties, it induces transition of hyphae to yeast in C.albicans , indicating its
contribution to oral defense against fungi.
Salivary Agglutins(SAG, gp 340)
 SAG is a scavenger receptor cysteine-rich gp.
 It acts as a pattern recognition scavenger receptor and as such it binds a broad range of oral pathogens,
including bacteria and viruses.
 Also binds salivary proteins, including IgA and mucin MUC5b.
 Based on premised properties , SAG efficiently aggregates bacteria and viruses, significatly increases their
clearance from mouth towards the stomach, leading to yheir acidic digestion.

18. LYSOZYMES
 Salivary lysozyme is produced by salivary glands(highest level in sublingual saliva , by neutrophil granulocytes entering
mouth) ; also present in GCF.
 It exerts muramidaze activity via hydrolysis of B-1,4 , glycosidic bonds between N-acetylmuramic acid and N-acetyl-D-
glucosamine of bacterial cell wall peptidoglycan
 It mainly kills gram +ve bacteria that damages surface exposed peptidoglycan.
 Killing of bacteria by lysozyme is , in many cases, largely independent of its enzymatic activity. In these cases, membrane
permeabillzing property of lysozyme seems to be active against both gram +ve and gram-ve bacteria ; fungi.
 It exerts antiviral properties , may induce lysis of tumor cells.
 It binds bacterial LPS , a bacterial surface structure and bacterial toxin, frequently responsible for tissue destructive
inflammatory reactions .
 It can influence human granulocyte and lymphocyte function, may inactive viruses.
BPI, BPI-like and PLUNC proteins
 These proteins belong to lipid binding protein family.
 Primary sources of salivary BPI are neutrophil granolocytes; epithelial cells of oral
 mucosa.
 BPI exerts bactericidial, endotoxin neutralizing and opsonic properties.
 PLUNC proteins appear to bind bacterial LPS similar to BPI, they do not exert direct killing activity, are
bacteriostatic similar to PSP.
 Promotes agglutination of bacteria and modulate cytokine production.
 The most important represantative of BPI-like proteins in saliva is PSP(parotid secretory protein). It is secreted
by salivary glands keratinocytes of oral mucosa.

19.  PSP is bacteriostatic, bind bacterial LPS and promote agglutination of bacteria.
 Salivary PLUNC proteins are primarily produced by major an dminor salivary glands.
 There are 8 functional PLUNC in humans divided into 2 subgroups :-
a) Short-type S-PLUNC proteins :- (SPLUNC-1, SPLUNC-2, SPLUNC-3)
b) Long-type-PLUNC proteins :- (LPLUNC-7, LPLUNC-2, LPLUNC-3, LPLUNC-4, LPLUNC-6)
CYSTATINs
 Human cystatin gene family contain 14 genes from which 7 cystatins are present in saliva namely:-
 Cystatin-A, Cystatin-B, Cystatin-C, Cystatin-D, Cystatin-S, Cystatin-SA, Cystatin-SN.
 Highest concentration found in submandibular saliva but present in parotid saliva at a lower concentration
is present in GCF.
 They are cysteine protease inhibitors, blocking the action of endogenous, bacterial and parasitic protozoan
protease.
 Cystatin-C and Cystatin-S inhibits bacterial growth (P.gingivalis)
 Cystatin SN and Cystatin S are present in human acquired enamel pellicle, binds bacteria and bacterial
LPS.
 Expert direct immonomodulatory properties, exert certain antiviral effect.
 Both the peroxidase catalyze the oxidation of thiocyanat CSCN- by hydrogen peroxide, leading to
production of a much more bactericidial & fungicidal agent, namely hypothiocyanite (OSCN-)
 Function of salivary peroxidare seems to be facilitated by DUOX-2, a homologue of catalytic core (gp21)
of NADPH oxidases.

20.  α- Amylase
 Salivary amylase is a highly abundant protein in saliva. Highest conc. was found in saliva of parotid
and palatine minor salivary glands.
 Most widely known function-endoglicosidase activity. Splitting the α-1,4-glicosidic bindings of
several glycans, such as starch (amylopectin), amylase produces oligosaccharides (dextrin)
disaccharides (maltose, isomaltose) and monosaccharide glucose . Besides its enzymatic activity, it
also takes part in acquired pellicle formation on tooth surface .
 It also performs a direct inhibitory effect on the growth of certain bacteria. It binds bacterial LPS .
 Also exert virus inhibitory properties.

21. PROLINE RICH PROTEINS(PRPS)
 PRPs form a major portion of salivary proteins(20-30% of total).
 They are highly phosphorylated proteins. Major source are salivary glands, highest
concentration in parotid saliva.
 Acidic PRPs contain a longer and highly acidic N-terminal region and somewhat different
repeat sequence, compared with basic PRPs.
 Acidic PRPs binds bacteria, basic PRPs bind fungi whereas glycosylated PRPs bind bacteria
and viruses.

22. 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
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

23. 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
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

24.  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)
Functions
MUCINS
 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

25.  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
 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

26. CONCLUSION
 Whole saliva is a major determinant of the environment on all the oral surfaces.
It plays an imp.role in physico-chemical as well as immune defense of
o.mucosal surfaces .Saliva plays an imp.role in fine regulation of o.mucosal
immune reactions ,as well as in healing of several mucosal lesions,wounds and
ulcers.
 There are numerous defense proteins in saliva, some such as salivary
Igs&HSP70/HSPAs are involved in both innate and acquired immune
activation,whereas cationic peptides& other sal.defense proteins are primarliy
responsible for innate immunity.

27. MULTIFUNCTIONALITY
Salivary
Families
Anti-
Bacterial
Buffering
Digestion
Mineral-
ization
Lubricat-
ion &Visco-
elasticity
Tissue
Coating
Anti-
Fungal
Anti-
Viral
Carbonic anhydrases,
Histatins
Amylases,
Mucins, Lipase
Cystatins,
Histatins, Proline-
rich proteins,
Statherins
Mucins, Statherins
Amylases,
Cystatins, Mucins,
Proline-rich proteins,
Statherins
Histatins
Cystatins,
Mucins
Amylases,
Cystatins,
Histatins, Mucins,
Peroxidases
adapted from M.J. Levine, 1993

28. THANK YOU