Salivary Proteins

DENT 5302
Topics in Dental Biochemistry
Dr. Joel Rudney
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
 Biotène™ (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

 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, extra-
parotid 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 histatins

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

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
 Biotène™ 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