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.

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.