FEBS 23240 FEBS Letters 466 (2000) 295^299 Activation of recombinant proenteropeptidase by duodenase Tatyana S. Zamolodchikovaa;*, Elena A. Sokolovaa, Deshun Lub, J. Evan Sadlerc aShemyakin and Ovchinnikov Institute of Bioorganic Chemistry, RAS, 16/10 Miklukho-Maklaya, 117871, Moscow V-437, Russia bCardiovascular Research Division, Eli Lilly and Company, Indianapolis, IN 46285, USA cHoward Hughes Medical Institute, Washington University School of Medicine, St. Louis, MO, USA Received 13 December 1999 Edited by Vladimir Skulachev to suggest that duodenase belongs to the group of processing Abstract Duodenase, a serine proteinase from bovine Brunner's (duodenal) glands that was predicted to be a natural activator of enzymes. It was supposed that duodenase is the primary acti- enteropeptidase zymogen, cleaves and activates recombinant vator of proenteropeptidase [7]. 31 Enteropeptidase is a intrinsic membrane protein of enter- single-chain bovine proenteropeptidase (kcat/Km =2700 M s31). The measured rate of proenteropeptidase cleavage by ocytes of the proximal small intestine. The enzyme plays the duodenase was about 70-fold lower compared with the rate of key role in the digestive cascade, cleaving the N-terminal ac- trypsin-mediated cleavage of the zymogen. The role of duodenase tivation peptide from trypsinogen to produce trypsin, thereby is supposed to be the primary activator of proenteropeptidase leading to subsequent activation of other pancreatic zymogens maintaining a certain level of active enteropeptidase in the by trypsin [2]. Enteropeptidase is synthesized as single chain duodenum. A new scheme of proteolytic activation cascade of precursor which apparently undergoes proteolytic processing. digestive proteases is discussed. The questions concerning the mechanism of enteropeptidase z 2000 Federation of European Biochemical Societies. activation and the identi¢cation of the enzyme or enzymes Key words: Duodenase; Proenteropeptidase activation; responsible for proenteropeptidase processing has been a fo- Serine protease; Duodenal mucosa cus of many investigations [8^12]. The exclusive localization of duodenase in the duodenum, its high content in this tissue and its potential role in initiating of the activation cascade of digestive hydrolases (activation of 1. Introduction proenteropeptidase) permits to speculate the importance of duodenase in the function of the duodenum. The duodenum is the central part of the digestive system. To prove the role of this enzyme, we studied activation of Normal function of the duodenum is important for health. the recombinant single-chain bovine enteropeptidase precur- Digestion in the duodenum depends on various enzymatic sor by duodenase. processes in which numerous enzymes of pancreatic (trypsin, chymotrypsins, carboxypeptidases, kallikrein and elastase) 2. Materials and methods and intestinal origin take part. Pancreatic enzymes are synthe- sized as inactive forms and activated in the duodenum by a 2.1. Materials proteolytic cascade [1,2]. After hydrolysis by pancreatic pro- Recombinant proenteropeptidase and duodenase were prepared as teinases dietary proteins are metabolized by peptidases of the described previously [5,12]. Native bovine enteropeptidase was kindly mucosal epithelial cells into amino acids, which are absorbed provided by Dr. A.G. Mikhaylova (Shemyakin and Ovchinnikov In- stitute of Bioorganic Chemistry, Moscow, Russia). The chromogenic through the epithelium [3]. substrate glutamyl-glycyl-arginine 4-nitroanilide (Glu-Gly-Arg-pNA) A new proteolytic enzyme, duodenase, was discovered by us was kindly provided by Prof. V.A. Tkachuk (Cardiology Centre, in the bovine duodenal mucosa [4,5]. The primary structure of Moscow, Russia). The enteropeptidase substrate glycyl-tetra-aspar- the enzyme is related to that of chymases from mast cells, tyl-lysine L-naphthylamide (Gly-(Asp)4-Lys-NA) was purchased granzymes from cytotoxic T-lymphocytes and cathepsin G from Bachem, Germany. Bovine trypsin was from Miles Lab. Re- search Products, Republic of South Africa. Chicken egg white ovo- from leukocytes [6]. Nevertheless, duodenase has no relation mucoid was from Serva, Germany. Soybean trypsin inhibitor and to haemopoietic cells. It is the ¢rst protease localized in se- bovine trypsinogen were purchased from Sigma, USA. cretory granules of epitheliocytes in the Brunner's (duodenal) glands presented in the duodenal mucosa [7]. The enzyme as 2.2. Determination of the concentration and purity of the enzyme preparations constituent of the gland secrete enters duodenal slit and this Concentration of active duodenase was determined using titration suggests duodenase functions extracellularly. Duodenase has with soybean trypsin inhibitor as described earlier [7]; trypsin solution unique amino acid residues within its substrate binding pocket was titrated with p-nitrophenyl-p'-guanidinobenzoate [13]. Enzymatic [6], determining unusual speci¢city of the enzyme. The sub- purity of duodenase was con¢rmed by SDS^PAGE using substrate gel strate speci¢city of duodenase is dual: trypsin-like and chy- with co-polymerized azocasein [14]. Purity of duodenase and trypsin preparations used in the study was con¢rmed additionally by compar- motrypsin-like. However, it hydrolyzes protein substrates ative study of their proteolytic action on melittin and proinsulin [15]. yielding large and stable fragments [5]. This fact allows us 2.3. Successive activation of zymogens The usual method for assaying enteropeptidase by activation of *Corresponding author. Fax (7)-95-3107007. trypsinogen and subsequent measuring of the generated trypsin [16] E-mail: [email protected] was modi¢ed in our study. Recombinant proenteropeptidase (15 nM) was incubated with duodenase (13 nM) in 10 mM Tris^HCl, pH 8.0 at Abbreviations: -pNA, 4-nitroanilide; -NA, L-naphthylamide 37³C. Samples (5 Wl) were withdrawn after certain time periods and 0014-5793 / 00 / $20.00 ß 2000 Federation of European Biochemical Societies. All rights reserved. PII: S0014-5793(00)01092-9 FEBS 23240 21-1-00 296 T.S. Zamolodchikova et al./FEBS Letters 466 (2000) 295^299 added to 245 Wl of a reaction mixture containing trypsinogen (0.4 WM), vation by trypsin was decreased but not eliminated. The in- ovomucoid (4 WM), and 0.05 M CaCl2 in 0.1 M Na^acetate bu¡er crease of the trypsin activity in the analyzed samples was (pH 5.0). The control sample contained proenteropeptidase, trypsino- gen, and ovomucoid in the same bu¡er. The reaction mixture was proportional to the time of incubation of duodenase with incubated for 30 min at 37³C, and then 25 Wl of 1 M HCl were added proenteropeptidase, that con¢rmed activation of proentero- to achieve a ¢nal pH of 2.0 in order to inactivate enteropeptidase and peptidase by duodenase (Fig. 1A). However, in this assay dissociate the trypsin^ovomucoid complex. The activity of the formed design we were unable to determine the rate of proenteropep- trypsin was determined spectrophotometrically at 405 nm from hy- drolysis of Glu-Gly-Arg-pNA at 37³C. Trypsin-containing sample tidase activation by duodenase with su¤cient accuracy. (5 Wl) was added to the solution of the substrate (0.8 mM; 500 Wl) in 0.1 M Tris^HCl (pH 8.0), containing 0.05 M CaCl2. The hydrolysis 3.2. Determining the kcat/Km value of proenteropeptidase rate was measured from the initial slope of the charted curve. activation by duodenase To exclude the confounding e¡ect of trypsin-mediated 2.4. Direct monitoring of enteropeptidase activity Proenteropeptidase (38 nM) and duodenase (13 nM) were incu- cleavage of proenteropeptidase, the activation of proentero- bated at 37³C in 30 mM Tris^HCl, pH 8.0. At certain times, samples peptidase by duodenase was studied using a sensitive £uores- were withdrawn, and the reaction was stopped by adding soybean cent enteropeptidase substrate Gly-(Asp)4-Lys-NA. The time trypsin inhibitor to a ¢nal duodenase/inhibitor ratio of 1/10. Entero- course of proenteropeptidase (70 nM) activation by 5 nM peptidase activity was determined using the enteropeptidase substrate duodenase (Fig. 1B) indicated that generation of active en- Gly-(Asp)4-Lys-NA with a Hitachi 650-60 (Japan) £uorescence spec- trophotometer at 37³C. The reaction mixture (300 Wl) contained 0.1 M teropeptidase was linear over 4 h, and about 20% of the zy- Tris^HCl (pH 8.0), 0.05 M CaCl2, and 0.1 mM substrate. The reac- mogen was converted into the active enzyme. When the duo- tion was initiated by adding samples containing the activated entero- denase/proenteropeptidase molar ratio was increased to 1:3 peptidase. The concentration of liberated L-naphthylamine was deter- (duodenase concentration was 13 nM and proenteropeptidase mined from the £uorescence increment (Vex = 330 nm and Vem = 410 nm). concentration was 38 nM), about 90% of the precursors were The concentration of recombinant proenteropeptidase was assumed activated within 3 h. No autocatalytic cleavage or activation to be equal to the concentration of active enteropeptidase released was observed in the absence of duodenase. after total activation of proenteropeptidase by trypsin [12]. The concentrations of proenteropeptidase tested (up to 70 The k /K value was determined at the concentrations of proen- cat m nM) were not su¤cient to allow determination of k and K teropeptidase and duodenase of 70 nM and 5 nM, respectively, using cat m for cleavage of the zymogen by duodenase. Because the con- the equation v =(kcat/Km)[E]0[S], when [S]IKm [17]. All steps were performed as described above. The concentration of duodenase-acti- centration of proenteropeptidase in our experiments was vated enteropeptidase was calculated using the rate of liberation of L- much smaller than the predicted Km value for the duodenase naphthylamine and kinetic parameters of Gly-(Asp)4-Lys-NA hydro- cleavage ( s 1 WM), it was possible to estimate only the k / lysis by native enteropeptidase at pH 8.0 [18]. cat Km value using the equation v =(kcat/Km)[E]0[S] (when 2.5. Activation of proenteropeptidase: analysis by SDS^PAGE [S]IKm) [17]. The calculated kcat/Km value for duodenase The reaction mixture (8.5 Wl) contained recombinant proenteropep- activation of proenteropeptidase was 2700 M31 s31.