Salivary cystatin SA-III, a potential precursor of the acquired enamel pellicle, is phosphorylated at both its amino- and carboxyl-terminal regions.
Cystatin SA-III was purified from human submandibular/sublingual glandular secretions by adsorption to hydroxyapatite, gel filtration chromatography, and reversed-phase HPLC. The amino acid sequence of its amino-terminus was deduced by sequential Edman degradation and found to be identical to the first 10 residues of cystatin HSP-12. The purified protein was digested with endoproteinase Asp-N and the digestion products were subjected to fast atom bombardment mass spectroscopy. m/z values corresponding to 12 peptides were aligned to the sequence of cystatin S preceded by the eight-residue amino-terminal peptide detected in HSP-12. This process resulted in the assignment of peptides corresponding with 118 out of the 121 amino acid residues predicted from the nucleotide sequence for cystatin SA-III. In order to align several peptides, it was necessary to substitute four residues of phosphoserine for four residues of serine. Fast atom bombardment mass spectrometry and additional Edman degradation procedures localized the phosphate moieties to Ser-3, Ser-99, Ser-112, and Ser-116. This is the first report of the structure of cystatin SA-III deduced by amino acid sequencing techniques and indicates the sites of phosphoserine within the molecule. Based on these assignments, cystatin SA-III is unique among salivary proteins in that it possesses phosphate groups at its amino-terminus as well as its carboxyl-terminus.
Lamkin
M S
MS
Department of Periodontology and Oral Biology, Boston University Goldman School of Graduate Dentistry, Massachusetts 02118.
Jensen
J L
JL
Setayesh
M R
MR
Troxler
R F
RF
Oppenheim
F G
FG
D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D015891:Cystatins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000302:isolation & purification*; D044622:Dental Pellicle; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D010766:Phosphorylation; D012463:Saliva / Q000378:metabolism*; D055333:Salivary Cystatins; D012689:Sequence Homology, Nucleic Acid; D016339:Spectrometry, Mass, Fast Atom Bombardment; D013361:Sublingual Gland / Q000378:metabolism*; D013363:Submandibular Gland / Q000378:metabolism*
C525172:CST2 protein, human; C525173:CST4 protein, human; D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D010446:Peptide Fragments; D055333:Salivary Cystatins