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

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

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

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

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

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

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

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

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

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

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

14. Statherin up close z Multiple gene family z Small tyrosine-rich phosphoproteins z Negatively charged Ca 2+ binding N-terminal y Two phosphoserines - additional negative charges y Maintains Ca 2+ balance, strongly prevents precipitation z Binds tooth surfaces and changes conformation y C-terminal rich in "bulky" tyrosines y Lubrication of tooth surfaces (pellicle) y Adherence of Actinomyces species (pellicle)

15. Acidic PRP up close z Multiple gene family z Proline-rich phosphoproteins y Negatively charged Ca 2+ binding N-terminal y Two phosphoserines - additional negative charges y Ca 2+ balance, strongly prevents precipitation z Binds tooth surfaces and changes conformation y C-terminal rich in "bulky" prolines y Adherence of Streptococcus species (pellicle) z Proteases cleave N-terminal from C-terminal y Free C-terminal binds tannins; blocks bacterial adhesion

16. Histatins up close z Multigene family - largest is phosphoprotein, others not z Small peptides after proteolysis y Positive charge - histidine-rich z Microbial cell damage - antibacterial and anti-fungal y Also Ca 2+ balance, tannin binding, protease inhibitor z Clinical interest - very safe - easy to make y Early trials with histatin rinses and gels x Some benefit in experimental gingivitis model No oral hygiene for a month y No trials with caries, periodontitis, or candidiasis patients yet

17. Current Products z Products with added lysozyme, lactoferrin, peroxidase z All influence aggregation/adherence, plus unique effects y Px enzyme - bacterial H 2 O 2 + saliva SCN - > OSCN - x OSCN - inhibits/kills bacteria x Removing H 2 O 2 may protect soft tissues y Lz enzyme cleaves bacterial cell walls > lysis x Also positive charge effects similar to histatins y Lf sequesters iron from some microbes, but not all x Unsaturated Lf is independently bactericidal z Clinical interest - can be purified from cow's milk y Biotène™ toothpaste, rinses, gum, dry mouth gels x Minor to minimal benefit in published clinical trials

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