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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 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, 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- > OSCNOSCN- 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
