Mass spectrometric identification of key proteolytic cleavage sites in statherin affecting mineral homeostasis and bacterial binding domains
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Human salivary statherin inhibits both primary and secondary calcium phosphate precipitation and, upon binding to hydroxyapatite, associates with a variety of oral bacteria. These functions, crucial in the maintenance of tooth enamel integrity, are located in defined regions within the statherin molecule. Proteases associated with saliva however, cleave statherin effectively, and it is of importance to determine how statherin functional domains are affected by these events. Statherin was isolated from human parotid secretion by zinc precipitation and purified by reversed-phase hig performance liquid chromatography (RP-HPLC). To characterize the proteolytic process provoked by oral proteases, statherin was incubated with whole saliva and fragmentation was monitored by RP-HPLC. The early formed peptides were structurally characterized by reversed phase liquid chromatography electrospray-ionization tandem mass spectrometry. Statherin was degraded 3.6x faster in whole saliva than in whole saliva supernatant. The main and primary cleavage sites were located in N-terminal half of statherin, specifically after Arg(9), Argl (1O), and Ang(13); after Phe(14 )and Tyr(18); and after Gly(12), Gly(15), Gly(17) and GLY(19) while the C-terminal half of statherin remained intact. Whole saliva protease activities separated the charged N-terminus from the hydrophobic C-terminus, negatively impacting on full length statherin functions comprising enamel protection and inhibition of primary and secondary calcium phosphate precipitation. Cryptic epitopes for bacterial binding residing in the C-terminal domain were likewise affected. The full characterization of the statherin peptides generated facilitates the elucidation of their novel functional roles in the oral and gastro-intestinal environment.
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 (MSD) --Boston University, Henry M. Goldman School of Dental Medicine, 2011 (Department of Periodontology and Oral Biology).Includes bibliography: leaves 45-48.
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