Mass Spectrometric Identification of Key Proteolytic Cleavage Sites in Statherin Affecting Mineral Homeostasis and Bacterial Binding Domains

• Published in: Journal of proteome research Vol. 9; no. 10; pp. 5413 - 5421
• Main Authors: Helmerhorst, Eva J, Traboulsi, Georges, Salih, Erdjan, Oppenheim, Frank G
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 01.10.2010
• Subjects: Amino Acid Sequence; Arginine - metabolism; Bacteria - metabolism; Binding Sites; Chlorides - chemistry; Chromatography, High Pressure Liquid - methods; Chromatography, Liquid; Glycine - metabolism; Homeostasis; Humans; Hydrolysis; Mass Spectrometry - methods; Minerals - metabolism; Molecular Sequence Data; Parotid Gland - metabolism; Peptides - metabolism; Phenylalanine - metabolism; Protein Binding; Saliva - metabolism; Salivary Proteins and Peptides - chemistry; Salivary Proteins and Peptides - isolation & purification; Salivary Proteins and Peptides - metabolism; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry; Tyrosine - metabolism; Zinc Compounds - chemistry; saliva; oral; enzymatic; statherin; fragmentation
• DOI: 10.1021/pr100653r
• ISSN: 1535-3893

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 high 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 3.6× faster in whole saliva than in whole saliva supernatant. The main and primary cleavage sites were located in the N-terminal half of statherin, specifically after Arg9, Arg10, and Arg13; after Phe14 and Tyr18; and after Gly12, Gly15, Gly17 and Gly19 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 lubrication and inhibition of primary 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.