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

J Biochem. 1991 Oct;110(4):648-54. doi: 10.1093/oxfordjournals.jbchem.a123634.


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.

Publication types

  • Comparative Study

MeSH terms

  • Amino Acid Sequence
  • Amino Acids / analysis
  • Animals
  • Cystatins / isolation & purification*
  • Humans
  • Immunoblotting
  • Macaca fascicularis
  • Molecular Sequence Data
  • Phosphopeptides / isolation & purification
  • Phosphorylation
  • Phosphoserine / analysis
  • Saliva / chemistry*
  • Salivary Cystatins
  • Semen / chemistry
  • Sequence Homology, Nucleic Acid
  • Tears / chemistry


  • Amino Acids
  • CST1 protein, human
  • CST2 protein, human
  • CST4 protein, human
  • Cystatins
  • Phosphopeptides
  • Salivary Cystatins
  • Phosphoserine