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Salivary Proteins

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.

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Salivary Proteins

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  1. Salivary Proteins DENT 5302 Topics in Dental Biochemistry Dr. Joel Rudney

  2. 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.

  3. 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)

  4. 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

  5. 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)

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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?

  11. 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

  12. Pictures of proteins in pellicle aPRP statherin histatins Schupbach et. al. 2001, Eur J Oral Sci 109:60

  13. Pictures of proteins in pellicle statherin histatins Schupbach et. al. 2001, Eur J Oral Sci 109:60

  14. 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)

  15. 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

  16. 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

  17. 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

  18. 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

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