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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.

Three basic proline-rich peptides were newly isolated from human parotid saliva, and designated as P-G, P-H, and P-I. The amino acid sequence of P-H was determined to be Ser-Pro-Pro-Gly-Lys-Pro-Gln-Gly-Pro-Pro-Gln-Gln-Glu-Gly-Asn-Asn- Pro-Gln-Gly-Pro-Pro-Pro-Pro-Ala-Gly-Gly-Asn-Pro-Gln-Gln-Pro-Gln-Ala-Pro-Pro- Ala-Gly-Gln-Pro-Gln-Gly-Pro-Pro-Arg-Pro-Pro-Gln-Gly-Gly-Arg-Pro-Ser-Arg-Pro- Pro-Gln by conventional methods. The amino terminal ten residues of P-H were the same as those of proline-rich peptides P-D, P-E, and P-F reported previously. Comparison of the amino acid sequences between P-H and P-D revealed that there are two deletion parts and several amino acid substitutions in the sequence of P-H. Homology between P-H and P-D was as high as 70%.

A basic proline-rich peptide, P-C, was isolated from human whole saliva and its amino acid sequence was determined to be Gly-Arg-Pro-Gln-Gly-Pro-Pro-Gln-Gln-Gly-Gly-His-Gln-Gln-Gly-Pro-Pro-Pro-Pro-Pro-Pro-Gly-Lys-Pro-Gln-Gly-Pro-Pro-Pro-Gln-Gly-Gly-Arg-Pro-Gln-Gly-Pro-Pro-Gln-Gly-Gln-Ser-Pro-Gln. P-C was also found to be present in stimulated parotid saliva. The sequence determined appears to correspond to the C-terminal 44 residues of a proline-rich phosphoprotein, Protein C, the partial sequence of which has been elucidated, and suggests that P-C might be derived from Protein C.

A low-molecular-weight acidic protein was isolated from human whole saliva by DE32 column chromatography and designated as SAP-1. The amino acid sequence was determined by conventional methods to be (sequence in text). The protein consisted of 113 residues and the calculated molecular weight was 12,552. Computer analysis revealed the presence of 54% sequence homology between SAP-1 and gamma-trace, a basic microprotein present in cerebrospinal fluid and in urine of patients with renal failure.

A cysteine proteinase inhibitor (designated as cystatin SA) was isolated from human whole saliva by procedures including chromatography on DE 32 and DEAE-Sepharose CL-6B. The amino acid sequence determined by conventional methods showed sequence homology of 90 and 87% as compared with the sequences of cystatin S and cystatin SN, respectively, both of which are salivary inhibitors characterized previously. The new inhibitor consisted of 117 residues and had a pI value of 4.3. Cystatin SA inhibited ficin and papain more strongly than cystatin S or cystatin SN did. It also exhibited inhibitory activity toward dipeptidyl peptidase I but the activity was much weaker than those toward ficin and papain.

Basic proline-rich peptides of human parotid saliva were fractionated and characterized. The amino acid sequence of one of the purified peptides, P-E, was determined to be (formula: see text). The results demonstrate the repetitiveness of the partial sequence within the molecule and the occurrence of structures common to those of other salivary polypeptides such as P-C and Protein C.

Three basic peptides with extremely high proline contents were isolated from human whole saliva. The amino acid sequences of two of these proline-rich peptides comprising 57 and 38 residues were determined by conventional methods. The sequence suggested that the smaller peptide was derived from the larger one and also revealed the occurrence of characteristic repeating units within the molecules. The present study is the first to describe this structural feature of proline-rich proteins or peptides.

An acidic protein of human saliva, which we named SAP-1 previously, is now shown to be an inhibitor of several cysteine proteinases. The protein inhibited papain and ficin strongly, and stem bromelain and bovine cathepsin C partially. However, it did not inhibit either porcine cathepsin B or clostripain. The mode of the inhibition of papain was found to be non-competitive. The name cystatin S has been proposed for this salivary protein in view of the similarities in activity and structure to other cysteine proteinase inhibitors such as chicken egg-white cystatin and human cystatins A, B, and C. The cystatin S antigen was detected immunohistochemically in the serous cells of human parotid and submaxillary glands.

A bone Gla-containing protein (osteocalcin) of cat has been isolated and the complete primary structure has been determined to be YLAPGLGAOAPYPDPLXPKRXICXLNPDCDELADHIGFQDAYRRFYGTV. The protein consists of 49 amino acid residues (Mr. 5,641) containing three Gla residues and a single disulfide bond. Although the C-terminal 30 residues (20th to 49th) of the sequences of cow, monkey, and human osteocalcins are identical, three amino acid substitutions occur at positions 22, 40, and 48 in the case of cat. These substitutions can be explained by single point mutations.