Salivary Proteins

DENT 5302
Topics in Dental Biochemistry
Dr. Joel Rudney

Supplemental reading
Rudney JD (2000). Saliva and Dental Plaque.
Adv Dent Res 14:29-39.
Lamkin MS, Oppenheim FG (1993). Structural
features of salivary function. Crit Rev Oral Biol
Med 4:251-259.

Clinical Importance
Demographic change - the number of elderly will increase
Implications:
Increases in diseases affecting salivary glands
Sjogren's syndrome, other autoimmune diseases,
Head and neck cancer (radiation therapy)
Increased use of medications with effects on saliva
Anticholinergic (antihistamines, antidepressants)
Reduced flow - indirect/direct effects on proteins
Beta adrenergic agonists and antagonists
Direct effects on protein synthesis/secretion
(asthma, hypertension, cardiovascular disease)

Salivary protein therapies

Current artificial salivas replace mainly fluids, ions
Genetically-engineered human salivary proteins soon
Raised from seed
Which ones go in artificial saliva? How much to add?
Already toothpastes/rinses containing saliva proteins
Biotne (peroxidase, lysozyme lactoferrin)
Histatin rinses/gels in trials
Clinicians will need to be able to evaluate new products

Origins of salivary proteins

Different secretory cells in different glands
Serous acinar - water, ions, proteins
Most in parotid, less in SM/SL
Mucus acinar - complex glycoproteins
Only SM/SL and minor glands
Different proteins emphasized in different glands
Duct cells also secrete proteins - differs among glands
Immune system cells contribute proteins
B cell product (S-IgA) translocated into ducts
Neutrophils - indirect leakage into gingival crevice
Leakage from gingival fluid contributes serum proteins (WS only)
Oral epithelial cells release surface proteins (whole saliva only)

Functions - Protect tissues

Protect oral surfaces by forming pellicle
Statherin, acidic proline-rich proteins, amylase, histatins,
cystatins, MUC7 mucin, lysozyme, albumin, carbonic anhydrase
Lubrication - oral surfaces must slide freely
Statherin, MUC5B mucin (also reflux protection)
Maintain saliva calcium in equilibrium with enamel
Saliva supersaturated with calcium and phosphate
Precipitation must be prevented
Statherin, aPRP, histatins, cystatins

Functions - Food processing

Initial breakdown of starches - Amylase
Binding/detoxification of dietary tannins
aPRP, basic PRP, histatins
Protein processing - Kallikrein and other proteases
Swallowing - MUC5B

Functions - Manage Microbes

Antimicrobial functions (bacteria, fungi, viruses)
Direct - cell killing - Histatins, lysozyme, amylase, MUC7,
lactoferrin, defensins, peroxidase
Indirect - Inhibition of infectivity, microbial metabolism,
bacterial/viral proteases - Lactoferrin, cystatins, histatins, basic
PRP, SLIPI, peroxidase, S-IgA
"Aggregation" - bind to microbes, clear by swallowing - MUC7,
lysozyme, lactoferrin, glcosylated PRP, parotid agglutinin, extraparotid glycoprotein, S-IgA

Functions - Microbes Manage

Microbial use of saliva proteins (coevolution)
Microbial adherence to pellicle proteins - Statherin, aPRP,
amylase, MUC5B, MUC7, lysozyme, lactoferrin, glcosylated
PRP, parotid agglutinin, extra-parotid glycoprotein, S-IgA,
peroxidase
Microbial metabolism of salivary proteins - MUC5B
Microbial use to metabolize host diet - Amylase

Complexity and Redundancy

Most saliva proteins have more than one function
Different domains on the same protein for different functions
Most saliva proteins cans be "amphifunctional
Some actions help host, others seem to help microbes
Also can be mediated by different domains
Many proteins share similar functions - redundancy
Multiple gene families
2-4 closely linked genes coding very similar proteins
aPRP, bPRP, gPRP, cystatins, histatins , amylase, MUCs
Multiple alleles for each gene
Unrelated proteins with the same function - backup systems?

Fragments and Complexes

Many salivary proteins are cleaved by proteases
During secretion or in the mouth
aPRP, bPRP, gPRP, histatins, S-IgA
Fragments may function differently than intact proteins
Proteins function differently together than they do alone
Lysozyme, lactoferrin, peroxidase
Salivary proteins bind in large heterotypic complexes
MUC5B, amylase, aPRP, S-IgA, peroxidase, lysozyme,
lactoferrin, statherin
Complexes function differently than component proteins

Pictures of proteins in pellicle

aPRP

statherin

histatins

Schupbach et. al. 2001, Eur J Oral Sci 109:60

Pictures of proteins in pellicle

statherin

Schupbach et. al. 2001, Eur J Oral Sci 109:60

histatins

Statherin up close
Multiple gene family
Small tyrosine-rich phosphoproteins
Negatively charged Ca2+ binding N-terminal
Two phosphoserines - additional negative charges
Maintains Ca2+ balance, strongly prevents precipitation
Binds tooth surfaces and changes conformation
C-terminal rich in "bulky" tyrosines
Lubrication of tooth surfaces (pellicle)
Adherence of Actinomyces species (pellicle)

Acidic PRP up close

Multiple gene family
Proline-rich phosphoproteins
Negatively charged Ca2+ binding N-terminal
Two phosphoserines - additional negative charges
Ca2+ balance, strongly prevents precipitation
Binds tooth surfaces and changes conformation
C-terminal rich in "bulky" prolines
Adherence of Streptococcus species (pellicle)
Proteases cleave N-terminal from C-terminal
Free C-terminal binds tannins; blocks bacterial adhesion

Histatins up close
Multigene family - largest is phosphoprotein, others not
Small peptides after proteolysis
Positive charge - histidine-rich
Microbial cell damage - antibacterial and anti-fungal
Also Ca2+ balance, tannin binding, protease inhibitor
Clinical interest - very safe - easy to make
Early trials with histatin rinses and gels
Some benefit in experimental gingivitis model
No oral hygiene for a month
No trials with caries, periodontitis, or candidiasis patients yet

Current Products
Products with added lysozyme, lactoferrin, peroxidase
All influence aggregation/adherence, plus unique effects
Px enzyme - bacterial H2O2 + saliva SCN- > OSCN OSCN- inhibits/kills bacteria
Removing H2O2 may protect soft tissues
Lz enzyme cleaves bacterial cell walls > lysis
Also positive charge effects similar to histatins
Lf sequesters iron from some microbes, but not all
Unsaturated Lf is independently bactericidal
Clinical interest - can be purified from cow's milk
Biotne toothpaste, rinses, gum, dry mouth gels
Minor to minimal benefit in published clinical trials

Future Prospects
Ideas about salivary protein function come from lab
Experimental models are greatly simplified
Change only one factor at a time
The mouth is an extremely complex environment
Difficult to isolate effects of single proteins
Redundancy may dilute the effects of supplements
We need to understand how different proteins work together
Supplements may need to be in the form of protein complexes