Characterization of a new cysteine proteinase inhibitor of human saliva, cystatin SN, which is immunologically related to cystatin S.

A new cysteine proteinase inhibitor, cystatin SN, was purified from human whole saliva by chromatography with DE32, Sephacryl S200, and CM-Sepharose CL6B. Cystatin SN is immunologically related to cystatin S and both inhibitors have a similar molecular mass of about 13 kDa. The new inhibitor, however, was clearly distinguished from cystatin S by its much higher pI value. These inhibitors showed similar inhibitory activity for ficin, but cystatin SN was a much better inhibitor for papain and dipeptidyl peptidase I. The amino acid sequence of cystatin SN deduced in the light of the known structure of cystatin S indicates that they have 10 different amino acid residues in the sequence comprising in total 113 residues.

Characterization and amino acid sequence of a new acidic cysteine proteinase inhibitor (cystatin SA) structurally closely related to cystatin S, from human whole saliva.

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.

Isolation and amino acid sequence of SAP-1, an acidic protein of human whole saliva, and sequence homology with human gamma-trace.

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.

The amino acid sequence of a salivary proline-rich peptide, P-C, and its relation to a salivary proline-rich phosphoprotein, protein C.

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.

Isolation and amino acid sequences of proline-rich peptides of human whole saliva.

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.

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.

Cystatin S: a cysteine proteinase inhibitor of human saliva.

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.

Biochemical and clinical factors influencing oral malodor in periodontal patients.

The amounts of volatile sulfur compounds (VSC) and methyl mercaptan/hydrogen sulfide ratio in mouth air from patients with periodontal involvement were 8 times greater than those of control subjects. Our studies demonstrated that, in patients with periodontal disease: 1) the concentration of disulfide, which is converted to VSC, increased in proportion to the total pocket depth; 2) 60% of the VSC was produced from the tongue surface; 3) the amount of tongue coating was 4 times greater than in control subjects; and 4) VSC production and the methyl mercaptan/hydrogen sulfide ratio of the tongue coating were increased. 2-Ketobutyrate, which is a byproduct of the metabolism of methionine to methyl mercaptan, was higher in the saliva of patients with periodontal disease. This implies that metabolism of methionine to methyl mercaptan increases in the oral cavity of patients with periodontal pockets. Since free L-methionine, rather than protein, is the main source for methyl mercaptan, we estimated the methionine supply from the gingival fluid into the oral cavity of patients with periodontal involvement. The results showed that the ratio of methionine to whole free amino acids was significantly higher than that of cysteine. Our studies suggest that not only microorganisms, but also the tongue coating and gingival fluid are factors which enhance VSC production in patients with periodontal disease.

Inhibition of calcium-carbonate precipitation by human salivary proline-rich phosphoproteins.

The effects of the proline-rich phosphoproteins (PRP) on the rate of precipitation of CaCO3 from a CaCO3-supersaturated solution were examined by recording the absorbance at 570 nm and the pH, when 20 mM CaCl2 was added to 20 mM NaHCO3, in the presence or absence of proteins. The PRP suppressed CaCO3 nucleation and exhibited inhibitory effects on CaCO3 precipitation under those conditions and under stimulated physiological conditions (final concentration of Ca2+ was 2 mM and that of HCO-3 60 mM, pH was 8.0 at 37 degrees C). PRP may be of biological significance in maintaining homeostasis of the buffering system of saliva, which is mainly composed of bicarbonate, and in preventing the formation of stones consisting of CaCO3 in the salivary ducts.

Effects of a two-phase oil-water mouthwash on halitosis.

Many oral microorganisms possess hydrophobic outer surfaces. A two-phase, oil-water mouthwash has, therefore, recently been developed to remove such oral microorganisms. The oil phase consists of olive oil and other essential oils. The aqueous phase includes cetylpyridinium chloride, which is a disinfectant that promotes the adhesion of microorganisms to oil droplets. This study determined the effects of this mouthwash on the production of volatile sulfide in vivo and in vitro. Neither rinsing with water nor brushing teeth decreased the concentration of sulfide in mouth air at 3.5 h after treatment. A reduction of only 30% of sulfide was observed when a commercial mouthwash was used. However, this study demonstrated that use of the two-phase mouthwash led to approximately 80% reduction of sulfide. Furthermore, volatile sulfide and 2-ketobutyrate productions from methionine in a saliva putrefaction system were completely inhibited by the two-phase mouthwash; and consumption of methionine was decreased by 65 percent. It is concluded that the two-phase mouthwash strongly inhibits the production of volatile sulfide.

Volatile sulfur compounds in mouth air from clinically healthy subjects and patients with periodontal disease.

Volatile sulfur compounds (VSC) in mouth air were estimated by gas chromatography. The amount of VSC and the methyl mercaptan/hydrogen sulfide ratio were significantly increased in patients with periodontal disease. These two parameters also increased in proportion to the bleeding index and probing depth. A study was also done on the effect of removal of tongue coating on VSC concentrations in mouth air from patients with periodontal involvement. VSC and the methyl mercaptan/hydrogen sulfide ratio were reduced to 49% and 35%, respectively, by removal of the tongue coating. The average amount of tongue coating removed from patients with periodontal disease was significantly higher than from controls (90.1 mg vs. 14.6 mg, p less than 0.01). Estimated production of VSC from tongue coating was 4 times higher than the control value, and the methyl mercaptan/hydrogen sulfide ratio was also markedly increased. However, a saliva putrefaction study suggested that saliva does not contribute to the elevated ratio of methyl mercaptan in mouth air. These results strongly suggest that, in addition to periodontal pockets, tongue coating has an important role in VSC production, in particular leading to an elevated concentration of methyl mercaptan, which is more pathogenic than hydrogen sulfide.

Cystatin superfamily. Evidence that family II cystatin genes are evolutionarily related to family III cystatin genes.

Human saliva contains at least three molecular species of cystatin S-type cysteine proteinase inhibitor (cystatin S, cystatin SN and cystatin SA), which have similar but distinct amino-acid sequences. The nucleotide sequences of the CST 1 gene for cystatin SN and the CST 2 gene for cystatin SA are highly homologous to each other and to the corresponding regions of the cDNA for cystatin C and the EcoRI-PstI fragment from the cystatin C gene. Three cystatin-like domains in the kininogen gene and the salivary cystatin genes share the same gene organizations. These data demonstrate that family II cystatin genes are evolutionarily related to family III cystatin genes.

Conformational study of the basic proline-rich polypeptides from human parotid saliva.

The conformational study of two basic proline-rich polypeptides from human parotid saliva, P--D and P--E of known primary structures, was performed by CD and 1H--n.m.r. spectra measurements. These polypeptides contain consecutive sequences of five prolyl residues in their amino acid sequences. The troughs in CD spectra of P--D and P--E were found at 202 and 201 nm, respectively. These wavelengths were different from the value of 206 nm of poly-L-proline form II conformation. In spite of this, the existence of poly-L-proline form II conformation was suggested in the structure of P--D, because the trough for a fragmental peptide of P--D containing five consecutive prolyl residues was found at 204 nm. No remarkable change was detected in CD and 1H--n.m.r. spectra of P--D and P--E in the range of pH 3.0-11.0. The result suggests that no folding of polypeptide which might be affected by ionic interaction exists in its structure.

Genetic polymorphisms of the CST2 locus coding for cystatin SA.

A new genetic polymorphism of cystatin SA has been identified in human submandibular-sublingual saliva by means of basic gel electrophoresis and immunoblotting with anti-cystatin S. Two proteins, SA1 and SA2, are given by two alleles of CST2, viz., CST2*1 and CST*2. Inheritance is controlled by two codominant alleles at an autosomal locus. This hypothesis is supported by studies of 16 families 32 children. Gene frequencies for CST2*1 and CST2*2 are 0.935 and 0.065, respectively (n = 341). Eighteen amino acids determined among 20 N-terminal residues of cystatin SA2 are identical with the sequence encoded by CST2. Three forms of cystatin S (mono-phosphorylated cystatin S, di-phosphorylated cystatin S, and non-phosphorylated cystatin S) are present in the 341 saliva samples tested.

Cyst-like lesion of a developing tooth induced by mandibular fracture.

A dentigerous cyst-like formation in the lower canine region caused by mandibular fracture in a 10-year-old boy is reported. His medical history revealed that he had been unconscious for about 2 weeks after traumatic head injuries sustained in a traffic accident, and a complicated mandibular fracture had been left untreated until his dentist diagnosed the lesion. Eleven months after trauma, a dentigerous cyst measuring 20 mm in diameter was found in the fracture area. The lesion was enucleated and the boy's postoperative recovery was uneventful. The mass completely enveloped the developing canine, and epithelial cells proliferated into the connective tissue. However, there was no distinct epithelial lining. Small round cell infiltrations and several vessels with thrombosis were noted in the cyst wall. The cause of cyst formation was considered to be infection of the canine tooth bud and the surrounding soft tissue.

[Salivary peptide P-C (III). Its relation to the pathogenesis of diabetes mellitus].

Previous immunohistochemical study showed that salivary peptide P-C like immunoreactivity, originally isolated from whole human saliva, was present not only in human salivary glands but also in human pancreatic B-cells. To elucidate the pathophysiological role of this peptide-like immunoreactivity in human pancreatic B-cells, immunohistochemical study using antisera against both insulin and peptide P-C was carried out on the paraffin embedded pancreatic tissues of 27 diabetic patients and 30 control subjects. Positive immunofluorescence due to insulin was detected in 96% of the diabetic pancreases and 100% of the controls. A pancreas of only one IDDM did not have any immunofluorescence due to insulin. Thus, no significant differences were seen in connection with the presence of insulin between pancreases of NIDDM and the controls, though the present study did not examine whether the number of pancreatic B-cells in the pancreas of NIDDM was the same as that in the controls or not. In contrast, positive immunofluorescence due to peptide P-C like immunoreactivity was demonstrated in only 41% of the diabetic pancreases but in 87% of the controls. In view of the fact that the diabetic pancreases and the controls were fixed and embedded into paraffin in the same department of pathology and under the same conditions, the negative finding for salivary peptide P-C like immunoreactive in the diabetic pancreas seems to result neither from the effects of fixatives nor the destructive postmortem effects of enzymes on the antigenicity of peptide P-C like immunoreactivity but from the decreased content of peptide P-C like immunoreactivity in the diabetic pancreas.(ABSTRACT TRUNCATED AT 250 WORDS)