COMMUNICATION J. Biochem. 96,1311-1314 (1984) Cystatin S : A Cysteine Proteinase Inhibitor of Human Saliva Satoko ISEMURA,* Eiichi SAITOH,* Seiki ITO,** Mamoru ISEMURA,*** and Kazuo SANADA* *Department of Oral Biochemistry , Niigata Faculty, Nippon Dental University. Niigata 951, **First Department of Internal Medicine, Niigata University School of Medicine, Niigata 951, and ***Department of Biochemistry, Tohoku University School of Medicine, Sendai, Miyagi 980 Received for publication, July 12, 1984 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. How ever, 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. We have recently isolated an acidic protein, SAP-1, 3.4.22.3], bromelain [EC 3.4.22.4], and clostripain with a molecular weight of 12,552 and PI 4.68 [EC 3.4.22.8] from Sigma; cathepsin C [dipeptidyl from human whole saliva, and determined its peptidase I, EC 3.4.14.1] from Serva; trypsin [EC amino acid sequence (1). This protein has se 3.4.21.4] and chymotrypsin [EC 3.4.21.1] from quence homology of 54% with human y-trace, the Worthington Biochemical Inc. Cathepsin B [EC cysteine proteinase inhibitor first found in cere 3.4.22.1] was prepared from porcine liver accord brospinal fluid (2, 3), and of 41 % with cystatin, ing to the method described by Takahashi et al. the cysteine proteinase inhibitor from egg-white (6). (4, 5). The high degree of structural similarity The activities of papain, ficin, bromelain, and has suggested that SAP-1 may be active in inhibit cathepsin B were measured with N-benzoyl-DL ing certain cysteine proteinases. In the present - arginine p-nitroanilide as a substrate according to work, we have found that is the case. We also the method described by Barrett (7). Cathepsin demonstrate that the serous cells of human parotid C was assayed by quantifying glycine released from and submaxillary glands are positively stained by the substrate Gly-Gly-Gly (Protein Research the indirect immunofluorescence method using the Foundation, Osaka). Trypsin and chymotrypsin antiserum raised against SAP-1. were assayed by the method of Schwert and Take Whole saliva was collected and SAP-1 was naka (8), and clostripain by the method of Mitchell isolated as described previously (1). The follow and Harrington (9). One unit (U) of enzyme was ing enzymes were obtained from the commercial defined as the activity hydrolyzing 1 ƒÊmol of the sources indicated: papain [EC 3.4.22.2], ficin [EC substrate per min under the conditions used. Vol. 96, No. 4, 1984 1311 1312 COMMUNICATION For the inhibition assay, papain, ficin, bro melain, clostripain, trypsin, and chymotrypsin were separately preincubated with varying amounts of SAP-1 in 500 ƒÊd of 0.05 M Tris-HCl buffer (pH 8.0) at 25•Ž for 5 min. In the cases of cathepsins B and C, preincubation with SAP-1 was performed in 500 ƒÊl of 0.05 M phosphate buffer (pH 6.5) at 25•Ž for 5 min and in 200,ƒÊl of 0.15 M acetate buffer (pH 4.7) containing 2.5 mm cysteine and 10 mm NaCl at 40•Ž for 5 min, respectively. The enzyme activity was then measured according to the method described above. In the case of ca Fig. 1. Inhibitory effect of SAP-1 on cysteine pro thepsin C 5 µl of 0.1 M Gly-Gly-Gly was added to teinases. Enzymes were preincubated for 5 min with the preincubated mixture of the enzyme and SAP varying amounts of the inhibitor and then the enzyme 1, and then the whole mixture was incubated for activities were determined as described in the text. 30 min at 40•Ž. The reaction was stopped by the Total reaction volumes were 2,050ƒÊl for assay of ca thepsin B, papain ficin and bromelain, or 205 ƒÊl for addition of 0.02% of monoiodoacetic acid in a that of cathepsin C. The enzymes used were 14.2 mU sampling buffer (2 ml) used for the amino acid of papain (•œ), 8.7 mU of ficin (•¢), 5.6 mU of bro analysis. Released glycine was quantified with a melain (•¡), 12.9 mU of cathepsin B (•›), and 1.7 mU Waters amino acid analysis system. of cathepsin C (•£). Results are expressed as % of SAP-1 did not show any activity inhibiting the residual activity of I mU of each enzyme and the serine proteinases, trypsin (1.69 U, 100 jig), and plotted against the amounts of the inhibitor. No in chymotrypsin (0.22 U, 100 jig) up to the amount hibition was observed when 0.7 U of clostripain, 1.69 U of 222 ,ƒÊg of SAP-1. The cysteine proteinases, of trypsin, or 0.22 U of chymotrypsin was tested. cathepsin B (12.9 mU) and clostripain (0.7 U 100 ƒÊ g) were not affected by SAP-1 (222 ƒÊg), either The cysteine proteinase inhibitors display con (Fig. 1). However, papain and ficin were inhib siderable variety in their relative activities from ited strongly, and bromelain and cathepsin C enzyme to enzyme on which they act (13, 16). partially (Fig. 1). The amounts of SAP-1 required As shown above, cystatin S did not inhibit cathep for 50% inhibition of the enzyme activity of 1 mU sin B. Thus cystatin S may be distinguished from of papain (2.18 jig), ficin (6.61 ƒÊg), and bromelain egg-white cystatin and human cystatins A, B, and (117.9 ƒÊg) were 0.8, 1.2, and 9.5 ƒÊg, respectively. C which inhibit human cathepsin B, although the When SAP-1 was reduced and S-carboxymeth possibility cannot be excluded that the difference ylated as described previously (1), the papain in may be due to the use of cathepsin B of different hibitory activity was lost completely. Thus the species. Therefore, the variety of inhibitions by conformation of the protein maintained by the inhibitors constituting a cystatin family appears disulfide bridges must be important for the ac to be much wider than has been considered (3). tivity. When the initial velocity of the reaction After the present work was completed, a paper with papain under various substrate concentrations appeared describing the presence of multiple forms in the presence or absence of SAP-1 was studied, of cysteine proteinase inhibitors in human saliva a Lineweaver-Burk plot demonstrated that SAP-I and salivary glands (17). Therefore, cystatin S is inhibited the enzyme non-competitively. considered to represent one of the several salivary These findings clearly indicate that SAP-1 is cysteine proteinase inhibitors, and the first with a a cysteine proteinase inhibitor. Several kinds of known amino acid sequence among them. cysteine proteinase inhibitor are now known (2 In the following experiments, sections of vari 5, 10-16) and some of them have been grouped ous human tissues were examined for the presence into a cystatin family (3). Because of the simi of cystatin S antigen. The rabbit antiserum larities in the amino acid sequence (1) and activity against cystatin S was prepared by the conven as revealed here between SAP-1 and cystatins, we tional method (16). The antiserum thus prepared propose the name cystatin S for SAP-l. formed a single precipitin line with cystatin S and J. Biochem. CYSTATIN S 1313 Fig. 2. Immunohistochemical detection of the cystatin S antigen in human tissues . Serial sections of parotid glands (A) or submaxillary glands (B) were stained with the anti-cystatin S antiserum (a), with the antiserum which had been absorbed with cystatin S (b), or with hematoxylin-eosin (c) (•~45 for (A) and •~90 for (B)) . The serous cells were positively stained. whole saliva, but not with human serum in an was not detected in these sections. The results Ouchterlony double immunodiffusion system. of the immunohistochemical examination may ex Histochemical detection by an indirect immu clude the possibility that cystatin S is the product nofluorescence technique and by hematoxylin of bacteria or exfoliated epithelial cells which are eosin staining were performed according to the also present in whole saliva. method described previously (18). Figure 2 shows Although the biological role of cystatin S is immunohistochemical evidence for the presence of not clear at present, these findings suggest that the cystatin S antigen in the serous cells of pa salivary glands, ducts or saliva may contain en rotid and submaxillary glands, suggesting that these zymes which are under regulation by the cysteine cells are responsible for its production. Sections proteinase inhibitor. It is also possible that the of pancreas and bronchea were also examined, inhibitor may function as protection for oral tis because these tissues have been shown to contain sues and proteins by inhibiting the enzymes pro the other salivary component, proline-rich peptide duced by oral bacteria. P-C (18, 19). However , the cystatin S antigen Vol. 96, No. 4, 1984 1314 COMMUNICATION 11. Takio, K., Kominami, E., Wakamatsu, N., Katsu numa, N., and Titani, K. (1983) Biochem. Biophys. REFERENCES Res. Common. 115, 902-908 1. Isemura, S., Saitoh, E., & Sanada, K. (1984) J. 12.