Salivary enzymes & Their functions

Presented by Roshni Maurya

Dept.of Pediatric & Preventive Dentistry

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)

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

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.

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.

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 10000Size (kDa)

SALIVARY ANTIBODIESMAJOR 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.

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.

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.

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

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.

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.

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.

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.

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 .

DEFENSE PROTEINS OF SALIVAStatherin

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.

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.

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.

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.

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.

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

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

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

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

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.

MULTIFUNCTIONALITY

SalivaryFamilies

Anti-Bacterial Buffering

Digestion

Mineral-ization

Lubricat-ion &Visco-elasticity

TissueCoating

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

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