Age-Dependent Modifications of the Human Salivary Secretory Protein Complex Tiziana Cabras, § Elisabetta Pisano, † Roberto Boi, ‡ Alessandra Olianas, § Barbara Manconi, § Rosanna Inzitari, ‡,⊥ Chiara Fanali, ‡,⊥ Bruno Giardina, ‡,⊥,| Massimo Castagnola, ‡,⊥,| and Irene Messana* ,§ Dipartimento di Scienze Applicate ai Biosistemi, Sezione di Biochimica e Biologia Molecolare, Universita ` di Cagliari, Cagliari, Italy, Dipartimento di Chirurgia e Scienze Odontostomatologiche, Universita ` di Cagliari, Cagliari, Italy, Istituto di Biochimica e Biochimica Clinica, Facolta ` di Medicina, Universita ` Cattolica, Rome, Italy, Istituto per la Chimica del Riconoscimento Molecolare, Consiglio Nazionale delle Ricerche (C.N.R.), Rome, Italy, and Istituto Scientifico Internazionale “Paolo VI”, Rome, Italy Received March 4, 2009 Physiological variability of the naturally occurring, human salivary secretory peptidome was studied as a function of age. The qualitative and quantitative changes occurring in the secretion of proteins/ peptides specific to the oral cavity (i.e., basic salivary proline-rich proteins, salivary acidic proline-rich phosphoproteins, statherin, proline-rich peptide P-B, salivary cystatins, and histatins) were investigated by high-performance liquid chromatography-electrospray ionization-mass spectrometry in 67 subjects aged between 3 and 44 years. Subjects were divided into five age groups: group A, 8 donors, 3-5 years; group B, 11 donors, 6-9 years; group C, 20 donors, 10-12 years; group D, 15 donors, 13-17 years; group E, 13 donors, 24-44 years. Basic salivary proline-rich proteins, almost undetectable in the 3-5 and 6-9 years groups, reached salivary levels comparable to that of adults (24-44 years) around puberty. Levels of peptide P-D, basic peptide P-F, peptide P-H, peptide P-J (a new basic salivary proline-rich protein characterized in this study), and basic proline-rich peptide IB-1 were significantly higher in the 10-12-year-old group than in the 3-5-year-old group, whereas the increase of proline-rich peptide II-2 was significant only after the age of 12 years. The concentration of salivary acidic proline-rich phosphoproteins, histatin-3 1/24, histatin-3 1/25, and monophosphorylated and diphosphorylated cystatin S showed a minimum in the 6-9-year-old group. Finally, the histatin-1 concentration was significantly higher in the youngest subjects (3-5 years) than in the other groups. Keywords: human saliva • age • salivary proline-rich proteins • histatins • proline-rich peptide P-B • statherin • salivary cystatins Introduction The multifunctional nature of saliva is related to its hetero- geneous composition, consisting of a complex mixture of proteins, peptides (comprised of enzymes and hormones), sugars, lipids, metal and nonmetal ions, and other compo- nents. 1 The salivary proteome consists of components specifically secreted by major and minor salivary glands and components originating from other sources, such as the gingival crevicular fluid, desquamated epithelial cells, plasma exudation, and host oral flora. 2 Several proteomic studies evidenced this complexity, disclosing the presence in human saliva of more than 1400 different peptides and proteins. 3-7 The major components secreted by the salivary glands and distinctive of human saliva can be grouped into a few families: histatins (Hst), statherin, proline-rich peptide P-B (P-B), S-type cystatins, acidic proline- rich phosphoproteins (aPRPs), basic salivary proline-rich pro- teins (bPRPs), basic glycosylated proline-rich proteins, amy- lases, and mucins. 2 These salivary protein families are characterized by elevated genetic polymorphism as well as several pre- and postsecretory post-translational modifications, such as glycosylation, phosphorylation, transglutamination, sulfation, and proteolytic cleavages. 2,5 Controversial findings are reported in the literature regarding the relationship between the age and the flow rate and composition of saliva in adults and children. In adults, secretion rates of unstimulated whole saliva were found to be negatively correlated with age both in males and females 8,9 as well as females alone. 10 Conversely, no significant influence of age on the stimulated salivary flow rate was observed in a cross- * To whom correspondence should be addressed. Phone: +39-070- 6754520. Fax: +39-070-6754523. E-mail: imessana@unica.it. § Dipartimento di Scienze Applicate ai Biosistemi, Sezione di Biochimica e Biologia Molecolare, Universita ` di Cagliari. † Dipartimento di Chirurgia e Scienze Odontostomatologiche, Universita ` di Cagliari. ‡ Istituto di Biochimica e Biochimica Clinica, Facolta ` di Medicina, Universita ` Cattolica. ⊥ Istituto per la Chimica del Riconoscimento Molecolare, Consiglio Nazionale delle Ricerche. | Istituto Scientifico Internazionale “Paolo VI”. 10.1021/pr900212u CCC: $40.75  XXXX American Chemical Society Journal of Proteome Research XXXX, xxx, 000 A Downloaded by CILEA CONSORTIA ITALY on July 13, 2009 Published on June 26, 2009 on http://pubs.acs.org | doi: 10.1021/pr900212u