SYNTHESIS AND FUNCTION OF COMPOSITE SALIVARY MOLECULES

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The overall goal of this subproject is to begin a "new generation" of studies based upon information which our laboratory has gained over the last decade on the structure/function relationships of several groups of salivary molecules. This information has permitted us to assign specific functions to various structural domains on human salivary mucins, the proline-rich glycoprotein and cysteine-containing phosphoproteins. The oligosaccharide units of the major proline-rich glycoprotein (PRG) and the low molecular weight human salivary mucin (MG2) have been shown to specifically interact with sialic acid binding adhesins on the surface of streptococcus sanguis and exhibit lubricating activity. The cysteine-containing phosphoproteins (CCP's) have been shown to bind to hydroxyapatite (OHAp) and modulate mineralization processes. However, the precise structural domain(s) of CCPs which participates in these functions remains an objective of this subproject. We will utilize our current information to begin the preparation and in vitro testing of chimeric salivary molecules of a bicomposite and a tricomposite nature. Bicomposite molecules will consist of the carbohydrate units of the PRG or MG2 covalently coupled to intact "carrier" molecules such as albumin and the neutral cysteine-containing phosphoprotein (CCPl). Next, we will prepare a more advanced series of bicomposite molecules comprised of the OHAp binding domains of albumin or CCPl covalently coupled to the carbohydrate units of PRG or MG2. Finally, we will prepare tricomposite molecules comprised of the carbohydrate units of PRG and MG2 coupled to intact albumin or CCPl. Carbohydrate units (as glycopeptides) will be coupled to peptide moieties by enzymatic (transglutaminase) or chemical (reductive amidation) methods. This will enable us to prepare a population of molecules which contains a varying number of carbohydrate units. This population of neoglycoproteins will allow us to examine the effect of density and number of carbohydrate units in mediating streptococcal interactions and lubrication. The technologies which we will employ to prepare neoglycoproteins include characterization of complex carbohydrates, automated peptide sequencing, and automated peptide synthesis techniques. The information obtained could be of paramount importance in the eventual production of replacement therapies (artificial salivas) for therapeutic use by individuals with salivary dysfunction or occlusal abrasion. In summary, this subproject gives us the opportunity to explore the potential value of "composite or custom" salivary molecules designed for a patient's individual need.