title: Identification of full-sized forms of salivary (S-type) cystatins (cystatin SN, cystatin SA, cystatin S, and two phosphorylated forms of cystatin S) in human whole saliva and determination of phosphorylation sites of cystatin S.
abstract: Our recent work on the gene structures for human salivary (S-type) cystatins [Saitoh, E. et al. (1987) Gene 61, 329-338] has suggested that the structures of cystatins which we determined previously at the protein level lack N-terminal peptide portions of the full-sized intact forms. In the present study, attempts were made to isolate full-sized S-type cystatins by introducing methanol fractionation into the purification steps to suppress the enzymatic activity present in saliva. Full-sized cystatin SN and two phosphorylated forms of full-sized cystatin S were thus isolated. Analysis of one fraction indicated that this was a mixture of full-sized cystatin SA and non-phosphorylated cystatin S. The phosphorylation sites of cystatin S were determined to be Ser-Ser-Ser1(P)-Lys-Glu-Glu- for monophosphorylated cystatin S and Ser1(P)-Ser-Ser3(P)-Lys-Glu-Glu- for diphosphorylated cystatin S. Immunoblotting analysis with anti-cystatin S antiserum revealed that tears and seminal plasma also contained S-type cystatins, but diphosphorylated cystatin S was detected neither in tears nor in seminal plasma and no cystatin SN was found in seminal plasma. These data indicate that S-type cystatins are secreted into the oral cavity without significant degradation in salivary glands or ducts and that they are expressed tissue specifically.
authors.affiliation: Nippon Dental University Junior College at Niigata.
authors.forename: S
authors.initials: S
authors.lastname: Isemura
authors.forename: E
authors.initials: E
authors.lastname: Saitoh
authors.forename: K
authors.initials: K
authors.lastname: Sanada
authors.forename: K
authors.initials: K
authors.lastname: Minakata
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D015891:Cystatins / Q000302:isolation & purification*; D006801:Humans; D015151:Immunoblotting; D008252:Macaca fascicularis; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000302:isolation & purification; D010766:Phosphorylation; D010768:Phosphoserine / Q000032:analysis; D012463:Saliva / Q000737:chemistry*; D055333:Salivary Cystatins; D012661:Semen / Q000737:chemistry; D012689:Sequence Homology, Nucleic Acid; D013666:Tears / Q000737:chemistry
chemical_list: D000596:Amino Acids; C525171:CST1 protein, human; C525172:CST2 protein, human; C525173:CST4 protein, human; D015891:Cystatins; D010748:Phosphopeptides; D055333:Salivary Cystatins; D010768:Phosphoserine