Conformation and adsorption of enamel pellicle precursor proteins onto hydroxyapatite differing in carbonate content
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Within the oral cavity, a thin protein film forms on the tooth enamel surface by selective adsorption of salivary proteins, called the acquired enamel pellicle (AEP) (Dawes et al, 1963). It serves important functions in the oral cavity including lubrication (Tabak et al, 1982), regulation of oral microbial attachment (Gibbons et al, 1990) and enamel mineral homeostasis (Zahradnik et al, 1979). Dental enamel is comprised primarily of hydroxyapatite (HA), the chief inorganic constituent of teeth and bone. Besides the major constituents, Ca2+ and PO3-, dental enamel contains various cationic and anionic substitutions in the crystal lattice. Among these, carbonate represents the major trace substituent in dental enamel and amounts to 2-3 wt % (LeGeros et ai, 1971; Clasen and Ruyter, 1997). Carbonate when incorporated, has an enormous effect on the properties of the carbonated hydroxyapatite crystal (CHA) crystal. It limits the size of the growing crystal and thereby affects its morphology. lmportantly, the interference of carbonate in the crystalization process increases the solubility of the crystals (LeGeros al, 1967, 1971). ln the past, in vitro AEP studies have been based on the adsorption of individual or complex mixtures of salivary proteins (as in whole saliva) to various enamel prototypes such as intact human enamel (Al Hashimi et al, 1989), intact bovine enamel (Orstavik and Kraus, 1974), calcium fluoride treated enamel (Rykke et al, 1989), enamel powder (Pruitt et al, 1969), HA powder (Hay, 1967), HA discs (Vacca smith and Bowen, 2000) and HA plates (Shimomura, 1996). The most widely used material to study in vitro AEP is HA powder (McGouchey et al, 1966; Ericson et a1 1967, Armstrong, 1971, Hay 1967, 1973). ln view of the fact that carbonate has enormous structural and physicochemical effects on HA (LeGeros et al, 1991), and since CHA more closely resembles dental enamel than HA, studying the in vitro effect of carbonate inclusion on salivary protein binding is of primary importance and a first step towards understanding the effect of carbonate in tooth enamel on in vivo AEP formation. To our knowledge thhere are only a few studies that have explored the in vitro effect of carbonate inclusion in HA on protein binding to HA surfaces (Kandori et al, 1995, Takemoto et al, 2004) and none have used individual and complex mixtures of salivary proteins. The objective of this dissertation work was to synthesize HA with different levels of carbonate and to study the in vitro effect of carbonate inclusion on salivary protein adsorption. The latter objective was achieved by comparing the adsorption of established pellicle precursor proteins to the synthesized HA and CHA powders in binding assays and by conducting conformational and proteomic analyses of the adsorbed proteins. The results obtained showed that carbonate incorporated into the HA lattice has a definite in vitro influence on the binding of salivary proteins. Irrespective of the net charge (pI), the proteins examined (albumin, lysozyme, histatins and PRP-1), exhibited a lower number of binding sites (N) for CHA when compared to HA. With regard to the affinity (K) for crystal surfaces, basic proteins such as lysozyme and histatins tended to exhibit a lower affinity for CHA compared to HA, while the opposite was observed for acidic proteins, such as PRP-1 and albumin. This observation is best explained by the higher surface charge of CHA (+1.4 mV), compared to HA (-2.3 mV). PAGE analysis of the protein profiles of HA and CHA pellicles revealed little differences in protein adsorption between the two apatite prototypes. However, more sensitive proteomic analyses showed a slightly increased number of proteins binding to HA (45) when compared to CHA (39). Proteomic data also demonstrated a predominance of acidic proteins (~65%) in both HA and CHA pellicles. One of the acidic pellicle precursor proteins (PRP-1) was further subjected to conformational analysis using FTIR. These investigations have revealed for the first time that an acidic PRP molecule in solution exhibits polyproline II(PP II) conformation. It was also demonstrated that PRP-1 undergoes significant conformational changes upon binding to apatites (HA or CHA) which are best characterized by the loss of hydrated PP II structure and the adoption of anhydrous PP II conformation, [Beta]-sheets and [Beta]-turns. Such conformational changes may play important roles in the buildup of AEP in vivo, with respect to multiprotein adsorption and subsequent bacterial adhesion.
PLEASE NOTE: This work is protected by copyright. Downloading is restricted to the BU community: please click Download and log in with a valid BU account to access. If you are the author of this work and would like to make it publicly available, please contact firstname.lastname@example.org.Thesis (D.Sc.)--Boston University, Henry M. Goldman School of Dental Medicine, 2007 (Dept. of Periodontology and Oral Biology).Includes bibliographical references: leaves 104-124.
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