organic components of saliva Flashcards
what is salivary protein content
1 - 6 g/L
varies according to flow rate + contribution from diff salivary glands (ie parotid saliva has higher protein conc than sublingual secretions)
what is protein conc of saliva compared to plasma
much lower (<1/10) than of plasma
how many different proteins have been identified to date
50
what are the broad categories of proteins according to their functions
1) those with lubricating properties
2) antimicrobial
3) mineral binding
4) some = more than one of above
what are salivary mucins
- high molecular weight glycoproteins (glycosylated proteins)
- all contain O- + N-linked sugars
- more than 40% (up to 80%) carb on weight basis
- 25% of ALL salivary proteins
why are salivary mucins important
1) lubricants - protect hard and soft tissue
2) lubricant properties directly related to structure
carbs are added to proteins by covalent attachment to amino acid side chains
what types of attachment are found and what are they
2 types
- O- and N- linkages
are are O-linked
MOST ABUNDANT
added to serine (SER) or threonine (THR) amino acid residues in the mucin polypep chain via their side chains which BOTH terminate w a hydroxyl group (so dehydration + lose H2O)
are are N-linked
attached to the amide nitrogen of the side chain of asparagine (ASN) residues w/in the polypep chain
carbohydrate is only added to ASN side chains where it occurs as part of the sequence (asparagine -> other amino acid -> threonine or serine)
what is the name of a peptide sequence which tells enzymes to add a modification to the amino acid
consensus sequence
what is the addition of carbohydrates to amino acid residues an example of
POST-TRANSLATION MODIFICATION
occurs aft polypeptide chain has been synthesised via enzymes in the ER and golgi
give an example of a molecule which may be O or N linked
acetyl galactose amine
N-linked GlcNAc
O-linked GalNAc
what is the common core structure of N-linked oligosaccharides
- 3 mannose
- 2 N-acetylglucosamine residues
further sugars attach to this common core (to mannose)
give an example of an N-linked oligosaccharide
Sialic acid (N-acetylneuraminate)
- freq part of the oligosaccharide structure in salivary mucins
- presence of substituted carbs confer a -ve charge on salivary mucins resulting in an extended structure for the proteins in solution due to the repulsion of charges
what do mucin monomers comprise
1) carboxyl + amino terminal domains (rich in cysteine (CYS) residues)
2) CYS residues capable of forming covalent cross links (bc sulphurdryl (SH) side chains) so 1+ of these monomeric structures can be linked together in a linear fashion to form mucin oligomers (happens in salivary mucin MG1)
3) large central portion of the molecule = multiple repeats of 10-80 amino acid residue seqs (up to ½ of amino acids are serine OR threonine so area becomes saturated w 100s of O linked oligosaccharides)
4) N-linked oligosaccharides found on mucins BUT less abundantly
what 2 types of mucins does saliva contain
MG1
MG2
MG = mucous glycoprotein
differentially expressed in major and minor salivary glands
describe MG1
- oligomeric structure
- high molecular weight (>1MDa) bc several mucin monomers covalently attached by S-S bonds in a linear array
- mixture of 3 diff gene products
- forms complexes w other salivary components / proteins (amylases, PRPs, statherin, histatins, anti-microbial peptides)
- so binds to human defence peptides produced by oral epithelium to protect against microorganisms
describe MG2
- derived from a single gene (so is a single monomer)
- low molecular weight (250kDa)
- binds oral pathogens and yeast (candida albicans)
- purified MG2 binds to oral streptococci (caries) and actinobacillus actinmycetemcomitans (perio disease)
why is the molecular weight of MG2 still relatively large
compared to avg serum protein ie albumin - 65,000 kDa
list the functions of salivary mucins
1) protect oral surfaces against dessication (form selectively permeable diffusion barrier between underlying surfaces + external environment)
2) lubricate hard and soft tissues (protect from mechanical damage - so facilitates speech + swallowing)
3) provide physical barrier to microbial colonisation
4) regulate other salivary proteins + peptides
5) anti-microbial + anti-fungal
6) direct bacterial colonisation of tooth and soft tissue (as they can bind to these surfaces)
what is the name of statherin derived from
- greek “to stabilise”
what is statherin
- produce by acinar cells
- small protein (<5kDa)
- unique amino acid primary seq (proline (PRO) rich)
- high -ve charge as asymmetrically distributed
- important component of salivary pellicle (thin protein rich deposit covering tooth surface)
what does statherin do
- protect against ectopic calcification in oral cavity bc whole statherin molecule inhibits de novo (spontaneous) precipitation of HAP SO prevents unwanted deposition of HAP mineral (which would cause calculus, tartar etc)
- binds to HAP mineral + inhibits crystal (secondary) growth of HAP
- amino acid residues 1-6 binds Ca preventing spontaneous precipitation of HAP = so binds to HAP by 2 possible sites (whole statherin molecule + amino acid residues 1-6)
explain the primary structure of statherin
- asymmetrical distribution of -ve charge
- amino terminal highly charged w (5 consecutive amino acids w -vely charged side chains at physiological pH) and this part of molecule (amino acid residues 1-6) bind Ca tightly
explain the secondary structure of statherin
- proline (PRO) rich precluding alpha helix structures found in many other proteins
- whole of the statherin molecule binds to existing HAP mineral (+ inhibits further crystal growth)
what are proline rich peptides (PRP)
- secondary structure rich in amino acid proline
- 40% of their amino acid residues are proline
what proportion of total salivary protein do PRPs comprise
10 - 40%
50mg / 100mL saliva
- rich in submandibular gland secretions (and those of trachea and pharynx)
PRPs are
POLYMORPHIC (derived from a group of genes situated on chromosome 12)
how many PRPs have been identified so far
6
share sequence homology (closely related in terms of their primary structure suggesting imp biological function preserved through evolution)
proteolytically processed in the salivary glands generating a large number of related proteins in the PRP family
what properties do the primary structure of PRPs have
- high amount of -ve charge at amino terminal (distributed asymmetrically)
- acidic
what are the functions of PRPs
1) strongly adsorbed to HAP + inhibit HAP growth (residues 1-30)
2) interact w oral microorganisms (-vely charged NH2 terminal binds existing HAP and inhibits its growth + COOH terminal interacts w bacterial cell walls)
3) highly selective - exclusion of pathogens
BUT
4) may inhibit remineralisation
what does the interaction of the COOH terminal of PRPs suggest it may have a role in
excluding pathogens from the dental surfaces by binding to the mineral + interacting specifically w benign microorganisms favouring their colonisation of the tooth surfaces over that of pathogens
what are histatins
- small defense peptides rich in histidine (amino acid)
- pK of ring = 6
- small (<3KDa)
- highly conserved
- bind to HAP
how is histidine present in PRPs and what does this mean
imidazole ring side chain
so capable of being protonated via the nitrogen of the ring
what is the conc of histatins in saliva and how many different histatins are present in saliva
- 3mg / 100mL in parotid / submandibular
- at least 6
what charge is the unprotonated form of histidine
+1
properties of histidine at pH 6
1) 1/2 of chains protonated (+1)
2) 1/2 neutral
what other amino acids do histatins contain and what are their properties
- arginine ARG
- lysine LYS
- +vely charged side chains (at physiological pH)
- means histatins are basic proteins
- these 2 give histatin its basic character
why are histatins different from statherins, PRPs etc
basic proteins, +vely charged @ physiological pH (not acidic ie -vely charged)
what is the function of histatins
1) buffer potential as pKa of side chain is close to neutral (BUT low conc in saliva)
2) potent inhibitor of candida albicans
3) some activity against S. mutans
4) histatin 1 binds Ca+ preventing HAP precipitation
5) found in pellicle proteins on tooth surface
what are immunoglobulins (ANTIBODIES)
- proteins of the immune system (defense against pathogens)
how many classes of immunoglobulins exist, how many are in saliva, name them
5
3 (IgM, IgG, IgA, )
differ in roles and structures
explain the structure of
a) IgM
b) IgG
c) IgA
all share a basic monomeric structure based upon a Y shaped molecule comprising 4 polypeptide chains
a) pentameric structure made up of 5 y shaped molecules
b) single one of the y shaped molecules
c) dimer with 2 y shaped molecules attached end to end by a small polypep chain
where are the 3 salivary immunoglobulins secreted
IgA = secreted directly into saliva via gland epithelial cells of salivary gland
IgM + IgG = from GCF
what are the functions of the 3 salivary immunoglobulins
1) bacterial agglutination = recognise invading pathogens, bind to them + eliminate them from the body
2) specific Ab-Ag targeting (ie against bacterial adhesins, glucosyl transferases) = v specific in binding capabilities + so target specific bacterial proteins only
3) when bind to their targets (antigens) they form precipitate structure by cross linking / bridging between 1+ bacteria (agglutination) = bacterial immunoglobulin complex can then be swallowed + removed
explain IgA
- most important and abundant
- during transport through epithelial cells it picks up an additional polypeptide chain called the secretory component
what does the polypeptide chain / secretory component of IgA do
- wraps round the molecule
- confers protection for IgA in hostile environment of the mouth
- this form of IgA is therefore called SECRETED IgA (SIgA)
why do we called salivary IgA SIgA
to differentiate it from the form circulating in the blood and tissue fluid which does NOT contain the secretory component
where else is SIgA found and why is it important
- tears + gut secretions + urinogenital tract
- 1st line of defense for the immune system
explain IgM + IgG
- present in trace amounts only in saliva
- may arise as contaminants from the GCF
what is GCF
GINGIVAL CREVICULAR FLUID
- outflow of fluids that exudes @ the gingival margin
- serum derived exudate
- seeps into mouth esp where gum inflammation is present
which enzymes are present in saliva
1) salivary amylase (ptyalin)
2) lysozyme
3) salivary / silo peroxidase
lesser roles
- Acid phosphatases
- Esterases
- Glucuronidases
- Carbonic anhydrase
what is salivary amylase (ptyalin)
- produced in 1+ form
- Ca2+ dependent
- Cl- activated
- 5 isoenzymes
- conc varies with flow rate but signif amnts in stimulated saliva (0.5 g/L)
- most of it (1/3) is secreted by parotid
- binds to HAP + some microrganisms
- closely related to pancreatic amylase
what does salivary amylase (ptyalin) catalyse
hydrolytic cleavage of alpha 1-4 glycosidic linkages (NOT alpha 1-2 or 1-6 = inactive against these)
- so highly specific
- active against long chain glucose polymers (ie those in starch) + breaks them into maltose units but NOT active against maltose itself
- cannot break down sucrose OR complex branched polysaccharides in plaque)
functions of salivary amylase (ptyalin)
- CHO (carbs) digestion as catalyses hydrolysis of alpha 1-4 glycosidic bonds between glucose residues in maltose
- interacts w oral microorganisms
- detected in carious lesions of enamel
in what other secretions is amylase found
tears serum bronchial urinogenital unlikely to have digestive function here so its full roles = unknown
what is lysozyme
- antimicrobial enzyme
- originates from striated duct
- 1 of 1st enzymes to be fully characterised
- neuraminidase enzyme found in many other exocrine secretions
what does lysozyme catalyse
- hydrolysis of alpha 1-4 linkages between n-acetylmuramic acid and N-acetylglucosamine in bacterial cell walls
- lysis of bacterial cell walls = antimicrobial
functions of lysozyme
- co-operate with SIgA - lyses bacterial cells which have already been recognised + tagged with bound SIgA (incs specificity of its effects against particular bacterial species)
- binds to HAP (+ is 1 of the proteins found in the salivary pellicle but not known if its antimicrobial activity is effective when bound to hap)
- cause aggregation of some bacterial species resulting in agglutination
how do bacteria confer resistance against lysozyme
protective coating on cell wall
restricts access to target substrate in the cell wall
what is weight of salivary peroxidase // silo peroxidase (sp)
Mr = 80 KDa
what does salivary peroxidase (sp) cataylse
conversion of HYDROGEN PEROXIDE (from bacterial metabolism) and THIOCYNATE (from diet ie cabbage, fruit, cig smoke) to HYPOTHIOCYANITE ION and water
what is the equation for salivary peroxidase (sp) enzyme action
H2O2 + SCN- -> OSCN + H2O
reactants = toxic
oscn = antibacterial
SO not only removes H2O2 which would be a source of free O2 radicals (damaging to soft tissues) and thiocyanite (toxic to cells)
BUT ALSO generates a powerful antimicrobial agent
functions of salivary peroxidase (sp)
1) important detoxifier - must remove H2O2 to prevent free radical formation = toxic to bacteria via their inhibitory effects on bacterial metabolic enzymes used in energy production
2) role in protecting soft tissue = due to role of O2 free radicals in connective tissue destruction (ie in perio disease)
what has evidence suggested about pts with low salivary peroxidase (sp) levels
more susceptible to perio breakdown (bc H2O2 generates O2 free radicals)
what is lactoferrin
- salivary antimicrobial protein
what is the main function of lactoferrin
- sequesters / binds ferric ions (Fe3+) which are essential bacterial nutrients
- so antimicrobial = makes iron unavailable for bacterial use
what other functions does lactoferrin have
- direct bactericidal effect on some microorganisms inc s. mutans strains
what are the issues with lactoferrin
- susceptible to degradation by certain bacterial proteases
- 1 type of bacteria (a spirokete of the treponema family) can transport lactoferrin into its cell + use the bound iron for its own nutritional purposes
what are cystatins
- small proteins
- ubiquitous throughout all bodily fluids and tissues
- several different, closely related types all present in saliva (similar protective function in oral environment inhibiting bacterial proteases so protecting soft tissues of mouth)
what are the functions of cystatins
1) bind to HAP + found in salivary pellicle
2) bind to + inhibit cysteine proteases
3) protect against acid erosion
what has lds research suggested about cystatins
- protective role during an acid erosive challenge by remaining bound to + protecting the tooth surface
what are some small molecules present in saliva useful as, which molecules?
useful pH rise factors
urea and sialin
what do pH rise factors do
metabolised by plaque microorganisms to produce ammonia increasing pH and neutralising plaque acid
what is urea
- carbon bonded to 2 amine groups and 1 oxygen
- end product of the metabolic breakdown of proteins
what is sialin
GLY - GLY - LYS - ARG
- 4 amino acid tetrapeptide
what is a biproduct of sialin metabolism
PUTRESCINE (derived from arginine) which contributes to halitosis
