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The Role of Glandular Kallikrein in the Formation of a Salivary Proline-Rich Protein a by Cleavage of a Single Bond in Salivary Protein C

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The Role of Glandular Kallikrein in the Formation of a Salivary Proline-Rich Protein a by Cleavage of a Single Bond in Salivary Protein C Biochem. J. (1983) 211, 35-44 35 Printed in Great Britain The role of glandular kallikrein in the formation of a salivary proline-rich protein A by cleavage of a single bond in salivary protein C Raymond S. C. WONG, George MADAPALLIMATTAM and Anders BENNICK Department ofBiochemistry, University ofToronto, Toronto, Ontario, Canada M5S 1A8 (Received 29 July 1982/Accepted 11 November 1982) An enzyme was purified from human parotid saliva that can cleave a single arginine-glycine peptide bond between residues 106 and 107 in human salivary proline-rich protein C, hereby giving rise to another proline-rich protein A, which is also found in saliva. The enzyme was purified 2400-fold. It cleaved salivary protein C at the rate of 59 ug of protein/h per pg of enzyme and had amino acid composition, molecular weight and inhibition characteristics similar to those reported for human salivary kallikrein. Confirmation that the enzyme was kallikrein was demonstrated by its kinin-generating ability. Histochemical evidence indicates that a post-synthetic cleavage of protein C by kallikrein would have to take place during passage of saliva through the secretory ducts. In secreted saliva, cleavage of salivary protein C can only be observed after 72 h incubation. In addition, there is no effect of salivary flow rate on the relative amounts of proteins A and C in saliva. On the basis of the experimental observations, it is proposed that in vivo it is unlikely that kallikrein secreted from ductal cells plays a significant role in converting protein C into protein A. Human parotid saliva and submaxillary saliva bonds in salivary protein A. A similar enzyme may contain a group of related acidic proline-rich be present in the human, because Karn et al. (1979) proteins that constitute approx. 28% of total protein found that human parotid saliva contains a factor in parotid saliva (Bennick & Cannon, 1978). The that converts salivary protein C into a component amino acid sequences of two of the major compo- with the same electrophoretic mobility as salivary nents, salivary proteins A and C, have been protein A. Karn et al. (1979) therefore suggested determined (Wong et al., 1979; Wong & Bennick, that protein A arose by a post-translational 1980). The sequence of salivary protein A was modification of protein C. An attempt in our identical with the N-terminal 106 residues of salivary laboratory to identify a proteinase in a homogenate protein C, which consists of a single polypeptide of human parotid gland capable of converting chain of 150 amino acids. This suggests that salivary salivary protein C into protein A was unsuccessful, protein A may be formed by a post-synthetic although degradation of salivary protein C was seen cleavage of salivary protein C at the arginine- (Wong & Bennick, 1980). Such an enzyme might be glycine bond in position 106-107. This is supported detected in saliva, being secreted either from the by the observation that human parotid saliva acinar cells together with proline-rich proteins or contains a peptide with amino acid sequence from another glandular source. Initial experiments in identical with the sequence of residues 107-150 in our laboratory suggested the presence of a pro- salivary protein C (Isemura et al., 1980). Such a teinase that cleaves salivary protein C between peptide was also isolated in the process of sequenc- residues 106 and 107. It was therefore decided to ing salivary protein C and was named peptide CTZ purify and characterize the enzyme and to evaluate (Wong & Bennick, 1980). In a previous study if it could be responsible for the presence in saliva of (Wong & Bennick, 1980) a proteinase isolated from protein A by cleavage of protein C subsequent to its mouse submaxillary gland (Schenkein et al., 1977) biosynthesis. cleaved salivary protein C only between residues 106 and 107. The enzyme did not break any peptide Experimental Abbreviations used: Tos-Arg-OMe, Na-tosyl-L- Materials arginine methyl ester; Bz-Arg-OEt, Na-benzoyl-L-arginine Tos-Arg-OMe, Bz-Arg-OEt, phenylmethane- ethyl ester. sulphonyl fluoride, N-ethylmaleimide, EDTA, Vol. 211 36 R. S. C. Wong, G. Madapallimattam and A. Bennick EGTA, soya-bean trypsin inhibitor, pancreatic column were assayed for proteolytic activity and trypsin inhibitor, bradykinin, diethylstilboestol, pooled. An affinity column was prepared by coupl- Sepharose 4B and molecular-weight markers for ing 20mg of pancreatic trypsin inhibitor to a 10ml sodium dodecyl sulphate/polyacrylamide-gel electro- volume of packed Sepharose 4B activated with phoresis were supplied by Sigma Chemical Co. CNBr as described by Parikh et al. (1974). The Dialysis tubing of 2000-M, cut-off was obtained column (1.5 cm x 6cm) was equilibrated with 0.1M- from Spectrum Medical Industries, and UM05 Tris/HCl buffer, pH 8.6, and the proteinase purified ultrafiltration membranes were from Amicon. by gel filtration was applied. The column was Sephadex G-50 was purchased from Pharmacia, developed with equilibration buffer until unbound and Bio-Gel P-2 from Bio-Rad Laboratories. CNBr proteins had emerged and the enzyme was then was obtained from Eastman. All other reagents were eluted with 0.1 M-sodium acetate buffer, pH 3.3. The of analytical grade. enzyme was freeze-dried and desalted on a Bio-Gel P-2 column equilibrated with 0.125 M-NH4HCO3 Collection ofparotid saliva and further purified by preparative polyacrylamide- Parotid saliva was collected from a single donor in gel electrophoresis (Lee et al., 1982) with a 100ml volumes as described by Bennick & Connell discontinuous buffer system (Davis, 1964) for (1971). separation of the components at pH 8.3. The gel was removed from the tube after Purification ofsalivaryproteins electrophoresis and the part containing the enzyme Salivary proteins A and C were purified as was cut out and re-loaded in the tube. The location described previously (Bennick, 1975, 1977). of the enzyme in the gel had been determined in pre- liminary experiments. The gel tube that was placed Preparation ofantiserum on top of a Bio-Gel P-2 column (1.5cm x 8.0 cm) Antiserum to salivary protein C was prepared as equilibrated in 0.1 M-Tris/HCl buffer, pH 8.6, was described by Bennick (1977). filled with 0.01 M-Tris/HCl buffer, pH 8.6. Electrodes were placed in the buffer and in the Bio-Gel P-2 Assaysforproteinase activity column outlet. After electrophoretic migration of the For routine assays hydrolysis of the arginine enzyme into the Bio-Gel P-2 resin, the polyacryl- esters Tos-Arg-OMe and Bz-Arg-OEt was deter- amide-gel tube was removed and the Bio-Gel P-2 mined as described by Hummel (1959) and Schwert column was developed with 0.1M-Tris/HCl buffer, & Takenaka (1955) respectively. pH8.6. The enzyme was freeze-dried, and salt and impurities were removed on another Bio-Gel P-2 Demonstration of proteolytic activity in human column equilibrated with 0.125 M-NH4HCO2. parotid saliva that cleaves proline-rich protein C By chromatography of human parotid saliva on Cleavage ofproline-rich proteins byparotid salivary CM-cellulose (CM-32), a number of fractions were proteinase previously obtained (Bennick, 1975). They were A 1 pg portion of purified proteinase was incu- assayed for proteinase activity, and the active bated with 615 pg of protein C or 425 pg of protein fractions were pooled and concentrated. A 200,u1 A in 0.125M-NH4HCO3 containing 0.02% NaN3 at sample of this salivary fraction was added to 501 of 230C for 69h. The digest was fractionated on the 1 M-Tris/HCl buffer, pH 8.5, that also contained Sephadex G-50 column (0.9cm x 118cm) that had 39,ug of proline-rich protein C labelled with 14C by been calibrated with the salivary proteins. Fractions reductive methylation (Wong & Bennick, 1980). with elution positions identical with those of protein The sample was incubated for 16.5h at 230C. The C, protein A or peptide CTZ were pooled and incubation mixture was fractionated on a Sephadex freeze-dried. Samples of these fractions were used G-50 column (0.9cmx 118cm) that had been for amino acid analysis and for determination of the calibrated with salivary proteins A and C and N-terminus (Percy & Buchwald, 1972). From the peptide CTZ. The radioactivities of 400 p1 samples amino acid composition and the absorption at of each fraction were counted on a liquid- 220nm of the sample of peptide CTZ an absorption scintillation counter. coefficient A -%,22o = 1.0 was found. To investigate the rate of cleavage, 665pug Purification ofparotid salivary proteinase samples of protein C were incubated with 0.2,pg of Parotid saliva (500ml) was dialysed against parotid proteinase in 0.1 ml of 0.125M-NH4HCO3 distilled water overnight at 30C and concentrated to containing 0.02% NaN3 at 370C for 8, 16, 24 and 85 ml by ultrafiltration. The concentrated saliva was 40h. The digests were fractionated on the calibrated applied to a Sephadex G-50 column (5cm x 60 cm) Sephadex G-50 column (0.9cm x 118cm) and the that was equilibrated and developed with 0.1 M- degree of degradation of protein C was measured Tris/HCl buffer, pH 8.6. Fractions eluted from the from the amount of peptide CTZ generated. 1983 Cleavage of salivary proline-rich proteins by kallikrein 37 Characterization oftheparotid salivaryproteinase immunoelectrophoresis (Bennick et al., 1982), which Estimations of the purity and molecular weight of results in a pattern of two peaks of immuno- the enzyme were done by sodium dodecyl sulphate/ precipitate. The major acidic proline-rich proteins in polyacrylamide-gel electrophoresis as described by the donor (A.
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