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Masanori Watanabe - One of the best experts on this subject based on the ideXlab platform.
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Enteropeptidase inhibitor SCO-792 effectively prevents kidney function decline and fibrosis in a rat model of chronic kidney disease.
Nephrology dialysis transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association, 2020Co-Authors: Yuko Katayama, Yusuke Moritoh, Jun Sugama, Tomohisa Suzuki, Yoshimasa Ishimura, Akihiro Kobayashi, Masanori WatanabeAbstract:Background Inhibiting Enteropeptidase, a gut serine protease regulating protein digestion, suppresses food intake and ameliorates obesity and diabetes in mice. However, the effects of Enteropeptidase inhibition on the kidney parameters are largely unknown. Here, we evaluated the chronic effects of an Enteropeptidase inhibitor, SCO-792, on kidney function, albuminuria, and kidney pathology in spontaneously hypercholesterolaemic (SHC) rats, a rat chronic kidney disease (CKD) model. Methods SCO-792, an orally available Enteropeptidase inhibitor, was administered (0.03% and 0.06% (w/w) in the diet) for five weeks to 20-week-old SHC rats showing albuminuria and progressive decline in glomerular filtration rate (GFR). The effects of SCO-792 and the contribution of amino acids to these effects were evaluated. Results SCO-792 increased the faecal protein content, indicating that SCO-792 inhibited Enteropeptidase in SHC rats. Chronic treatment with SCO-792 prevented GFR decline and suppressed albuminuria. Moreover, SCO-792 improved glomerulosclerosis and kidney fibrosis. Pair feeding with SCO-792 (0.06%) was less effective in preventing GFR decline, albuminuria, and renal histological damage than SCO-792 treatment, indicating the Enteropeptidase-inhibition-dependent therapeutic effects of SCO-792. SCO-792 did not affect the renal plasma flow, suggesting that its effect on GFR was mediated by an improvement in filtration fraction. Moreover, SCO-792 increased hydrogen sulphide production capacity, which has a role in tissue protection. Finally, methionine and cysteine supplementation to the diet abrogated SCO-792-induced therapeutic effects on albuminuria. Conclusions SCO-792-mediated inhibition of Enteropeptidase potently prevented GFR decline, albuminuria, and kidney fibrosis; hence, it may have therapeutic potential against CKD.
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Enteropeptidase inhibition improves obesity by modulating gut microbiota composition and enterobacterial metabolites in diet-induced obese mice
Pharmacological research, 2020Co-Authors: Jun Sugama, Hiroaki Yashiro, Kazue Tsuchimori, Yusuke Moritoh, Masanori WatanabeAbstract:Enteropeptidase is a transmembrane serine protease localized in the lumen of the duodenum that acts as a key enzyme for protein digestion. SCO-792 is an orally available Enteropeptidase inhibitor that has been reported to have therapeutic effects on obesity and diabetes in mice. However, the mechanism underlying the therapeutic effect of SCO-792 has not yet been fully elucidated. In this study, we evaluated the role of gut microbiota on SCO-792-induced body weight (BW) reduction in high-fat diet-induced obese (DIO) mice. Chronic administration of SCO-792 substantially decreased BW and food intake in DIO mice. While the pair-fed study uncovered food intake-independent mechanisms of BW reduction by SCO-792. Interestingly, antibiotics-induced microbiota elimination in the gut canceled SCO-792-induced BW reduction by nearly half without affecting the anorectic effect, indicating the involvement of gut microbiota in the anti-obesity mechanism that is independent of food intake reduction. Microbiome analysis revealed that SCO-792 altered the gut microbiota composition in DIO mice. Notably, it was found that the abundance of Firmicutes decreased while that of Verrucomicrobia increased at the phylum level. Increased abundance of Akkermansia muciniphila, a bacterium known to be useful for host metabolism, was observed in SCO-792-treated mice. Fecal metabolome analysis revealed increased amino acid levels, indicating gut Enteropeptidase inhibition. In addition, SCO-792 was found to increase the level of short-chain fatty acids, including propionate, and bile acids in the feces, which all help maintain gut health and improve metabolism. Furthermore, it was found that SCO-792 induced the elevation of colonic immunoglobulin A (IgA) concentration, which may maintain the microbiota condition, in DIO mice. In conclusion, this study demonstrates the contribution of microbiota to SCO-792-induced BW reduction. Enteropeptidase-mediated regulation of microbiota, enterobacterial metabolites, and IgA in the gut may coordinately drive the therapeutic effects of SCO-792 in obesity.
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Enteropeptidase inhibition improves kidney function in a rat model of diabetic kidney disease
Diabetes obesity & metabolism, 2020Co-Authors: Jun Sugama, Yusuke Moritoh, Yuko Katayama, Masanori WatanabeAbstract:AIM To examine the effects of an Enteropeptidase inhibitor, SCO-792, on kidney function in rats. MATERIALS AND METHODS The pharmacological effects of SCO-792 were evaluated in Wistar fatty (WF) rats, a rat model of diabetic kidney disease (DKD). RESULTS Oral administration of SCO-792 increased faecal protein content and improved glycaemic control in WF rats. SCO-792 elicited a rapid decrease in urine albumin-to-creatinine ratio (UACR). SCO-792 also normalized glomerular hyperfiltration and decreased fibrosis, inflammation and tubular injury markers in the kidneys. However, pioglitazone-induced glycaemic improvement had no effect on kidney variables. Dietary supplementation of amino acids (AAs), which bypass the action of Enteropeptidase inhibition, mitigated the effect of SCO-792 on UACR reduction, suggesting a pivotal role for Enteropeptidase. Furthermore, autophagy activity in the glomerulus, which is impaired in DKD, was elevated in SCO-792-treated rats. Finally, a therapeutically additive effect on UACR reduction was observed with a combination of SCO-792 with irbesartan, an angiotensin II receptor blocker. CONCLUSIONS This study is the first to demonstrate that Enteropeptidase inhibition is effective in improving disease conditions in DKD. SCO-792-induced therapeutic efficacy is likely to be independent of glycaemic control and mediated by the regulation of AAs and autophagy. Taken together with a combination effect of irbesartan, SCO-792 may be a novel therapeutic option for patients with DKD.
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Discovery and characterization of a small‐molecule Enteropeptidase inhibitor, SCO‐792
Pharmacology research & perspectives, 2019Co-Authors: Masako Sasaki, Ikuo Miyahisa, Sachiko Itono, Hiroaki Yashiro, Hideyuki Hiyoshi, Kazue Tsuchimori, Kenichi Hamagami, Yusuke Moritoh, Masanori Watanabe, Kimio TohyamaAbstract:Enteropeptidase, localized into the duodenum brush border, is a key enzyme catalyzing the conversion of pancreatic trypsinogen proenzyme to active trypsin, thereby regulating protein digestion and energy homeostasis. We report the discovery and pharmacological profiles of SCO-792, a novel inhibitor of Enteropeptidase. A screen employing fluorescence resonance energy transfer was performed to identify Enteropeptidase inhibitors. Inhibitory profiles were determined by in vitro assays. To evaluate the in vivo inhibitory effect on protein digestion, an oral protein challenge test was performed in rats. Our screen identified a series of Enteropeptidase inhibitors, and compound optimization resulted in identification of SCO-792, which inhibited Enteropeptidase activity in vitro, with IC 50 values of 4.6 and 5.4 nmol/L in rats and humans, respectively. In vitro inhibition of Enteropeptidase by SCO-792 was potentiated by increased incubation time, and the calculated K inact/KI was 82 000/mol/L s. An in vitro dissociation assay showed that SCO-792 had a dissociation half-life of almost 14 hour, with a calculated k off rate of 0.047/hour, which suggested that SCO-792 is a reversible Enteropeptidase inhibitor. In normal rats, a ≤4 hour prior oral dose of SCO-792 effectively inhibited plasma elevation of branched-chain amino acids in an oral protein challenge test, which indicated that SCO-792 effectively inhibited protein digestion in vivo. In conclusion, our new screen system identified SCO-792 as a potent and reversible inhibitor against Enteropeptidase. SCO-792 slowly dissociated from Enteropeptidase in vitro and inhibited protein digestion in vivo. Further study using SCO-792 could reveal the effects of inhibiting Enteropeptidase on biological actions.
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discovery and characterization of a small molecule Enteropeptidase inhibitor sco 792
Pharmacology Research & Perspectives, 2019Co-Authors: Masako Sasaki, Ikuo Miyahisa, Sachiko Itono, Hiroaki Yashiro, Hideyuki Hiyoshi, Kazue Tsuchimori, Kenichi Hamagami, Yusuke Moritoh, Masanori Watanabe, Kimio TohyamaAbstract:Enteropeptidase, localized into the duodenum brush border, is a key enzyme catalyzing the conversion of pancreatic trypsinogen proenzyme to active trypsin, thereby regulating protein digestion and energy homeostasis. We report the discovery and pharmacological profiles of SCO-792, a novel inhibitor of Enteropeptidase. A screen employing fluorescence resonance energy transfer was performed to identify Enteropeptidase inhibitors. Inhibitory profiles were determined by in vitro assays. To evaluate the in vivo inhibitory effect on protein digestion, an oral protein challenge test was performed in rats. Our screen identified a series of Enteropeptidase inhibitors, and compound optimization resulted in identification of SCO-792, which inhibited Enteropeptidase activity in vitro, with IC 50 values of 4.6 and 5.4 nmol/L in rats and humans, respectively. In vitro inhibition of Enteropeptidase by SCO-792 was potentiated by increased incubation time, and the calculated K inact/KI was 82 000/mol/L s. An in vitro dissociation assay showed that SCO-792 had a dissociation half-life of almost 14 hour, with a calculated k off rate of 0.047/hour, which suggested that SCO-792 is a reversible Enteropeptidase inhibitor. In normal rats, a ≤4 hour prior oral dose of SCO-792 effectively inhibited plasma elevation of branched-chain amino acids in an oral protein challenge test, which indicated that SCO-792 effectively inhibited protein digestion in vivo. In conclusion, our new screen system identified SCO-792 as a potent and reversible inhibitor against Enteropeptidase. SCO-792 slowly dissociated from Enteropeptidase in vitro and inhibited protein digestion in vivo. Further study using SCO-792 could reveal the effects of inhibiting Enteropeptidase on biological actions.
Yusuke Moritoh - One of the best experts on this subject based on the ideXlab platform.
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Enteropeptidase inhibitor SCO-792 effectively prevents kidney function decline and fibrosis in a rat model of chronic kidney disease.
Nephrology dialysis transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association, 2020Co-Authors: Yuko Katayama, Yusuke Moritoh, Jun Sugama, Tomohisa Suzuki, Yoshimasa Ishimura, Akihiro Kobayashi, Masanori WatanabeAbstract:Background Inhibiting Enteropeptidase, a gut serine protease regulating protein digestion, suppresses food intake and ameliorates obesity and diabetes in mice. However, the effects of Enteropeptidase inhibition on the kidney parameters are largely unknown. Here, we evaluated the chronic effects of an Enteropeptidase inhibitor, SCO-792, on kidney function, albuminuria, and kidney pathology in spontaneously hypercholesterolaemic (SHC) rats, a rat chronic kidney disease (CKD) model. Methods SCO-792, an orally available Enteropeptidase inhibitor, was administered (0.03% and 0.06% (w/w) in the diet) for five weeks to 20-week-old SHC rats showing albuminuria and progressive decline in glomerular filtration rate (GFR). The effects of SCO-792 and the contribution of amino acids to these effects were evaluated. Results SCO-792 increased the faecal protein content, indicating that SCO-792 inhibited Enteropeptidase in SHC rats. Chronic treatment with SCO-792 prevented GFR decline and suppressed albuminuria. Moreover, SCO-792 improved glomerulosclerosis and kidney fibrosis. Pair feeding with SCO-792 (0.06%) was less effective in preventing GFR decline, albuminuria, and renal histological damage than SCO-792 treatment, indicating the Enteropeptidase-inhibition-dependent therapeutic effects of SCO-792. SCO-792 did not affect the renal plasma flow, suggesting that its effect on GFR was mediated by an improvement in filtration fraction. Moreover, SCO-792 increased hydrogen sulphide production capacity, which has a role in tissue protection. Finally, methionine and cysteine supplementation to the diet abrogated SCO-792-induced therapeutic effects on albuminuria. Conclusions SCO-792-mediated inhibition of Enteropeptidase potently prevented GFR decline, albuminuria, and kidney fibrosis; hence, it may have therapeutic potential against CKD.
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Enteropeptidase inhibition improves obesity by modulating gut microbiota composition and enterobacterial metabolites in diet-induced obese mice
Pharmacological research, 2020Co-Authors: Jun Sugama, Hiroaki Yashiro, Kazue Tsuchimori, Yusuke Moritoh, Masanori WatanabeAbstract:Enteropeptidase is a transmembrane serine protease localized in the lumen of the duodenum that acts as a key enzyme for protein digestion. SCO-792 is an orally available Enteropeptidase inhibitor that has been reported to have therapeutic effects on obesity and diabetes in mice. However, the mechanism underlying the therapeutic effect of SCO-792 has not yet been fully elucidated. In this study, we evaluated the role of gut microbiota on SCO-792-induced body weight (BW) reduction in high-fat diet-induced obese (DIO) mice. Chronic administration of SCO-792 substantially decreased BW and food intake in DIO mice. While the pair-fed study uncovered food intake-independent mechanisms of BW reduction by SCO-792. Interestingly, antibiotics-induced microbiota elimination in the gut canceled SCO-792-induced BW reduction by nearly half without affecting the anorectic effect, indicating the involvement of gut microbiota in the anti-obesity mechanism that is independent of food intake reduction. Microbiome analysis revealed that SCO-792 altered the gut microbiota composition in DIO mice. Notably, it was found that the abundance of Firmicutes decreased while that of Verrucomicrobia increased at the phylum level. Increased abundance of Akkermansia muciniphila, a bacterium known to be useful for host metabolism, was observed in SCO-792-treated mice. Fecal metabolome analysis revealed increased amino acid levels, indicating gut Enteropeptidase inhibition. In addition, SCO-792 was found to increase the level of short-chain fatty acids, including propionate, and bile acids in the feces, which all help maintain gut health and improve metabolism. Furthermore, it was found that SCO-792 induced the elevation of colonic immunoglobulin A (IgA) concentration, which may maintain the microbiota condition, in DIO mice. In conclusion, this study demonstrates the contribution of microbiota to SCO-792-induced BW reduction. Enteropeptidase-mediated regulation of microbiota, enterobacterial metabolites, and IgA in the gut may coordinately drive the therapeutic effects of SCO-792 in obesity.
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Enteropeptidase inhibition improves kidney function in a rat model of diabetic kidney disease
Diabetes obesity & metabolism, 2020Co-Authors: Jun Sugama, Yusuke Moritoh, Yuko Katayama, Masanori WatanabeAbstract:AIM To examine the effects of an Enteropeptidase inhibitor, SCO-792, on kidney function in rats. MATERIALS AND METHODS The pharmacological effects of SCO-792 were evaluated in Wistar fatty (WF) rats, a rat model of diabetic kidney disease (DKD). RESULTS Oral administration of SCO-792 increased faecal protein content and improved glycaemic control in WF rats. SCO-792 elicited a rapid decrease in urine albumin-to-creatinine ratio (UACR). SCO-792 also normalized glomerular hyperfiltration and decreased fibrosis, inflammation and tubular injury markers in the kidneys. However, pioglitazone-induced glycaemic improvement had no effect on kidney variables. Dietary supplementation of amino acids (AAs), which bypass the action of Enteropeptidase inhibition, mitigated the effect of SCO-792 on UACR reduction, suggesting a pivotal role for Enteropeptidase. Furthermore, autophagy activity in the glomerulus, which is impaired in DKD, was elevated in SCO-792-treated rats. Finally, a therapeutically additive effect on UACR reduction was observed with a combination of SCO-792 with irbesartan, an angiotensin II receptor blocker. CONCLUSIONS This study is the first to demonstrate that Enteropeptidase inhibition is effective in improving disease conditions in DKD. SCO-792-induced therapeutic efficacy is likely to be independent of glycaemic control and mediated by the regulation of AAs and autophagy. Taken together with a combination effect of irbesartan, SCO-792 may be a novel therapeutic option for patients with DKD.
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Discovery and characterization of a small‐molecule Enteropeptidase inhibitor, SCO‐792
Pharmacology research & perspectives, 2019Co-Authors: Masako Sasaki, Ikuo Miyahisa, Sachiko Itono, Hiroaki Yashiro, Hideyuki Hiyoshi, Kazue Tsuchimori, Kenichi Hamagami, Yusuke Moritoh, Masanori Watanabe, Kimio TohyamaAbstract:Enteropeptidase, localized into the duodenum brush border, is a key enzyme catalyzing the conversion of pancreatic trypsinogen proenzyme to active trypsin, thereby regulating protein digestion and energy homeostasis. We report the discovery and pharmacological profiles of SCO-792, a novel inhibitor of Enteropeptidase. A screen employing fluorescence resonance energy transfer was performed to identify Enteropeptidase inhibitors. Inhibitory profiles were determined by in vitro assays. To evaluate the in vivo inhibitory effect on protein digestion, an oral protein challenge test was performed in rats. Our screen identified a series of Enteropeptidase inhibitors, and compound optimization resulted in identification of SCO-792, which inhibited Enteropeptidase activity in vitro, with IC 50 values of 4.6 and 5.4 nmol/L in rats and humans, respectively. In vitro inhibition of Enteropeptidase by SCO-792 was potentiated by increased incubation time, and the calculated K inact/KI was 82 000/mol/L s. An in vitro dissociation assay showed that SCO-792 had a dissociation half-life of almost 14 hour, with a calculated k off rate of 0.047/hour, which suggested that SCO-792 is a reversible Enteropeptidase inhibitor. In normal rats, a ≤4 hour prior oral dose of SCO-792 effectively inhibited plasma elevation of branched-chain amino acids in an oral protein challenge test, which indicated that SCO-792 effectively inhibited protein digestion in vivo. In conclusion, our new screen system identified SCO-792 as a potent and reversible inhibitor against Enteropeptidase. SCO-792 slowly dissociated from Enteropeptidase in vitro and inhibited protein digestion in vivo. Further study using SCO-792 could reveal the effects of inhibiting Enteropeptidase on biological actions.
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discovery and characterization of a small molecule Enteropeptidase inhibitor sco 792
Pharmacology Research & Perspectives, 2019Co-Authors: Masako Sasaki, Ikuo Miyahisa, Sachiko Itono, Hiroaki Yashiro, Hideyuki Hiyoshi, Kazue Tsuchimori, Kenichi Hamagami, Yusuke Moritoh, Masanori Watanabe, Kimio TohyamaAbstract:Enteropeptidase, localized into the duodenum brush border, is a key enzyme catalyzing the conversion of pancreatic trypsinogen proenzyme to active trypsin, thereby regulating protein digestion and energy homeostasis. We report the discovery and pharmacological profiles of SCO-792, a novel inhibitor of Enteropeptidase. A screen employing fluorescence resonance energy transfer was performed to identify Enteropeptidase inhibitors. Inhibitory profiles were determined by in vitro assays. To evaluate the in vivo inhibitory effect on protein digestion, an oral protein challenge test was performed in rats. Our screen identified a series of Enteropeptidase inhibitors, and compound optimization resulted in identification of SCO-792, which inhibited Enteropeptidase activity in vitro, with IC 50 values of 4.6 and 5.4 nmol/L in rats and humans, respectively. In vitro inhibition of Enteropeptidase by SCO-792 was potentiated by increased incubation time, and the calculated K inact/KI was 82 000/mol/L s. An in vitro dissociation assay showed that SCO-792 had a dissociation half-life of almost 14 hour, with a calculated k off rate of 0.047/hour, which suggested that SCO-792 is a reversible Enteropeptidase inhibitor. In normal rats, a ≤4 hour prior oral dose of SCO-792 effectively inhibited plasma elevation of branched-chain amino acids in an oral protein challenge test, which indicated that SCO-792 effectively inhibited protein digestion in vivo. In conclusion, our new screen system identified SCO-792 as a potent and reversible inhibitor against Enteropeptidase. SCO-792 slowly dissociated from Enteropeptidase in vitro and inhibited protein digestion in vivo. Further study using SCO-792 could reveal the effects of inhibiting Enteropeptidase on biological actions.
Miklós Sahin-tóth - One of the best experts on this subject based on the ideXlab platform.
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Evolution of Trypsinogen Activation Peptides
2013Co-Authors: Jian-min Chen, Zoltán Kukor, Cédric Le Maréchal, Miklós Tóth, À Laurent Tsakiris, Odile Raguénès, Jj Claude Férec, Miklós Sahin-tóthAbstract:The activation peptide of mammalian trypsinogens contains a highly conserved tetra-aspartate sequence (D19-D20-D21-D22) preceding the K23-I24 scissile peptide bond, which is hydrolyzed as the first step in the activation process. Here, we examined the evolution and function of trypsinogen activation peptides through integrating functional characterization of disease-associated mutations with comparative genomic analysis. Activation properties of three chronic pancreatitis-associated activation peptide mutants (the novel D19A and the previously reported D22G and K23R) were simultaneously analyzed, for the first time, in the context of recombinant human cationic trypsinogen. A dramatic increase in autoactivation of cationic trypsinogen was observed in all three mutants, with D22G and K23R exhibiting the most marked increases. The physiological activator Enteropeptidase activated the D19A mutant normally, activated the D22G mutant very poorly, and stimulated activation of the K23R mutant. The biochemical and structural data, taken together with a comprehensive sequence comparison, indicates that the tetra-aspartate sequence in mammalian trypsinogen activation peptides has evolved not only for optimal Enteropeptidase recognition in the duodenum but also for efficient inhibition of trypsinogen autoactivation within the pancreas. Moreover, the use of lysine instead of arginine at the P1 position of activation peptides also has an advantageous effect against trypsinogen autoactivation. Finally, fixed substitutions in the key residues of the trypsinogen activation peptide may suggest the evolution of new function
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The guinea pig pancreas secretes a single trypsinogen isoform, which is defective in autoactivation.
Pancreas, 2008Co-Authors: Béla Ózsvári, Péter Hegyi, Miklós Sahin-tóthAbstract:OBJECTIVES The aim of the present study was to purify and clone the trypsinogen isoforms from the guinea pig pancreas and characterize their activation properties. METHODS Trypsinogens from pancreatic homogenates were isolated by ecotin-affinity chromatography, followed by cation-exchange chromatography. Activation of trypsinogens was tested with Enteropeptidase, cathepsin B, and trypsin. Complementary DNAs for pretrypsinogens were cloned from total RNA after reverse transcription and polymerase chain reaction amplification. RESULTS Purification of trypsinogens yielded a single peak with an N-terminal amino-acid sequence of LPIDD. Cloning of pretrypsinogen cDNAs revealed 2 distinct but nearly identical isoforms. At the amino acid level, the only difference between the 2 isoforms is an Ala/Ser change at position 15 within the signal peptide. Thus, both cDNA variants give rise to the same mature trypsinogen upon secretion. Guinea pig trypsinogen is readily activated by Enteropeptidase and cathepsin B but exhibits essentially no autoactivation, under conditions where human cationic and anionic trypsinogens rapidly autoactivate. CONCLUSIONS The observations suggest that multiple trypsinogen isoforms and their ability to autoactivate are not required universally for normal digestive physiology in mammals. Furthermore, the inability of guinea pig trypsinogen to undergo autoactivation suggests that this species might be more resistant to pancreatitis than humans, where increased autoactivation of cationic trypsinogen mutants has been linked to hereditary pancreatitis.
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The Tetra-aspartate Motif in the Activation Peptide of Human Cationic Trypsinogen Is Essential for Autoactivation Control but Not for Enteropeptidase Recognition
The Journal of biological chemistry, 2005Co-Authors: Zsofia Nemoda, Miklós Sahin-tóthAbstract:Abstract The activation peptide of vertebrate trypsinogens contains a highly conserved tetra-aspartate sequence (Asp19-22 in humans) preceding the Lys-Ile scissile bond. A large body of research has defined the primary role of this acidic motif as a specific recognition site for Enteropeptidase, the physiological activator of trypsinogen. In addition, the acidic stretch was shown to contribute to the suppression of autoactivation. In the present study, we determined the relative importance of these two activation peptide functions in human cationic trypsinogen. Individual Ala replacements of Asp19-22 had minimal or no effect on trypsinogen activation catalyzed by human Enteropeptidase. Strikingly, a tetra-Ala19-22 trypsinogen mutant devoid of acidic residues in the activation peptide was still a highly specific substrate for human, but not for bovine, Enteropeptidase. In contrast, an intact Asp19-22 motif was critical for autoactivation control. Thus, single Ala mutations of Asp19, Asp20 and Asp21 resulted in 2-3-fold increased autoactivation, whereas the Asp22 → Ala mutant autoactivated at a 66-fold increased rate. These effects were multiplicative in the tri-Ala19-21 and tetra-Ala19-22 mutants. Structural modeling revealed that the conserved hydrophobic S2 subsite of trypsin and the unique Asp218, which forms part of the S3-S4 subsite, participate in distinct inhibitory interactions with the activation peptide. Finally, mutagenesis studies confirmed the significance of the negative charge of Asp218 in autoactivation control. The results demonstrate that in human cationic trypsinogen the Asp19-22 motif per se is not required for Enteropeptidase recognition, whereas it is essential for maximal suppression of autoactivation. The evolutionary selection of Asp218, which is absent in the large majority of vertebrate trypsins, provides an additional mechanism of autoactivation control in the human pancreas.
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Evolution of Trypsinogen Activation Peptides
Molecular biology and evolution, 2003Co-Authors: Jian-min Chen, Zoltán Kukor, Miklós Tóth, Odile Raguénès, Cédric Le Maréchal, Laurent Tsakiris, Claude Férec, Miklós Sahin-tóthAbstract:The activation peptide of mammalian trypsinogens contains a highly conserved tetra-aspartate sequence (D19-D20-D21-D22) preceding the K23-I24 scissile peptide bond, which is hydrolyzed as the first step in the activation process. Here, we examined the evolution and function of trypsinogen activation peptides through integrating functional characterization of disease-associated mutations with comparative genomic analysis. Activation properties of three chronic pancreatitis-associated activation peptide mutants (the novel D19A and the previously reported D22G and K23R) were simultaneously analyzed, for the first time, in the context of recombinant human cationic trypsinogen. A dramatic increase in autoactivation of cationic trypsinogen was observed in all three mutants, with D22G and K23R exhibiting the most marked increases. The physiological activator Enteropeptidase activated the D19A mutant normally, activated the D22G mutant very poorly, and stimulated activation of the K23R mutant. The biochemical and structural data, taken together with a comprehensive sequence comparison, indicates that the tetra-aspartate sequence in mammalian trypsinogen activation peptides has evolved not only for optimal Enteropeptidase recognition in the duodenum but also for efficient inhibition of trypsinogen autoactivation within the pancreas. Moreover, the use of lysine instead of arginine at the P1 position of activation peptides also has an advantageous effect against trypsinogen autoactivation. Finally, fixed substitutions in the key residues of the trypsinogen activation peptide may suggest the evolution of new functions unrelated to digestion, as found in the group III trypsinogens of cold-adapted fishes.
J. E. Sadler - One of the best experts on this subject based on the ideXlab platform.
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Activation of recombinant proEnteropeptidase by duodenase.
FEBS letters, 2000Co-Authors: T S Zamolodchikova, E A Sokolova, J. E. SadlerAbstract:Duodenase, a serine proteinase from bovine Brunner's (duodenal) glands that was predicted to be a natural activator of Enteropeptidase zymogen, cleaves and activates recombinant single-chain bovine proEnteropeptidase (kcat/Km = 2700 M(-1) s(-1)). The measured rate of proEnteropeptidase cleavage by duodenase was about 70-fold lower compared with the rate of trypsin-mediated cleavage of the zymogen. The role of duodenase is supposed to be the primary activator of proEnteropeptidase maintaining a certain level of active Enteropeptidase in the duodenum. A new scheme of proteolytic activation cascade of digestive proteases is discussed.
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Crystal structure of Enteropeptidase light chain complexed with an analog of the trypsinogen activation peptide.
Journal of molecular biology, 1999Co-Authors: Klaus Fütterer, Xinglong Zheng, Sergey Korolev, Kai Tan, Gabriel Waksman, J. E. SadlerAbstract:Enteropeptidase is a membrane-bound serine protease that initiates the activation of pancreatic hydrolases by cleaving and activating trypsinogen. The enzyme is remarkably specific and cleaves after lysine residues of peptidyl substrates that resemble trypsinogen activation peptides such as Val-(Asp)4-Lys. To characterize the determinants of substrate specificity, we solved the crystal structure of the bovine Enteropeptidase catalytic domain to 2.3 A resolution in complex with the inhibitor Val-(Asp)4-Lys-chloromethane. The catalytic mechanism and contacts with lysine at substrate position P1 are conserved with other trypsin-like serine proteases. However, the aspartyl residues at positions P2-P4 of the inhibitor interact with the enzyme surface mainly through salt bridges with the Nzeta atom of Lys99. Mutation of Lys99 to Ala, or acetylation with acetic anhydride, specifically prevented the cleavage of trypsinogen or Gly-(Asp)4-Lys-beta-naphthylamide and reduced the rate of inhibition by Val-(Asp)4-Lys-chloromethane 22 to 90-fold. For these reactions, Lys99 was calculated to account for 1.8 to 2.5 kcal mol(-1) of the free energy of transition state binding. Thus, a unique basic exosite on the Enteropeptidase surface has evolved to facilitate the cleavage of its physiological substrate, trypsinogen.
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Apical sorting of bovine Enteropeptidase does not involve detergent-resistant association with sphingolipid-cholesterol rafts.
The Journal of biological chemistry, 1999Co-Authors: Xinglong Zheng, J. E. SadlerAbstract:Abstract Enteropeptidase is a heterodimeric type II membrane protein of the brush border of duodenal enterocytes. In this location, Enteropeptidase cleaves and activates trypsinogen, thereby initiating the activation of other intestinal digestive enzymes. Recombinant bovine Enteropeptidase was sorted directly to the apical surface of polarized Madin-Darby canine kidney cells. Replacement of the cytoplasmic and signal anchor domains with a cleavable signal peptide (mutant proEnteropeptidase lacking the amino-terminal signal anchor domain (dSA-BEK)) caused apical secretion. The additional amino-terminal deletion of a mucin-like domain (HL-BEK) resulted in secretion both apically and basolaterally. Further deletion of the noncatalytic heavy chain (L-BEK) resulted in apical secretion. Thus Enteropeptidase appears to have at least three distinct sorting signals as follows: the light chain (L-BEK) directs apical sorting, addition of most of the heavy chain (HL-BEK) inhibits apical sorting, and addition of the mucin-like domain (dSA-BEK) restores apical sorting. Inhibition of N-linked glycosylation with tunicamycin or disruption of microtubules with colchicine caused L-BEK to be secreted equally into apical and basolateral compartments, whereas brefeldin A caused basolateral secretion of L-BEK. Full-length BEK was not found in detergent-resistant raft domains of Madin-Darby canine kidney cells or baby hamster kidney cells. These results suggest apical sorting of Enteropeptidase depends on N-linked glycosylation of the serine protease domain and an amino-terminal segment that includes an O-glycosylated mucin-like domain and three potential N-glycosylation sites. In contrast to many apically targeted proteins, Enteropeptidase does not form detergent-resistant associations with sphingolipid-cholesterol rafts.
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Bovine proEnteropeptidase is activated by trypsin, and the specificity of Enteropeptidase depends on the heavy chain.
The Journal of biological chemistry, 1997Co-Authors: Yuan X, Xinglong Zheng, J. E. SadlerAbstract:Enteropeptidase, also known as enterokinase, initiates the activation of pancreatic hydrolases by cleaving and activating trypsinogen. Enteropeptidase is synthesized as a single-chain protein, whereas purified Enteropeptidase contains a approximately 47-kDa serine protease domain (light chain) and a disulfide-linked approximately 120-kDa heavy chain. The heavy chain contains an amino-terminal membrane-spanning segment and several repeated structural motifs of unknown function. To study the role of heavy chain motifs in substrate recognition, secreted variants of recombinant bovine proEnteropeptidase were constructed by replacing the transmembrane domain with a signal peptide. Secreted variants containing both the heavy chain (minus the transmembrane domain) and the catalytic light chain (pro-HL-BEK (where BEK is bovine Enteropeptidase)) or only the catalytic domain (pro-L-BEK) were expressed in baby hamster kidney cells and purified. Single-chain pro-HL-BEK and pro-L-BEK were zymogens with extremely low catalytic activity, and both were activated readily by trypsin cleavage. Trypsinogen was activated efficiently by purified Enteropeptidase from bovine intestine (Km = 5.6 microM and kcat = 4.0 s-1) and by HL-BEK (Km = 5.6 microM and kcat = 2.2 s-1), but not by L-BEK (Km = 133 microM and kcat = 0.1 s-1); HL-BEK cleaved trypsinogen at pH 5.6 with 520-fold greater catalytic efficiency than did L-BEK. Qualitatively similar results were obtained at pH 8.4. In contrast to this striking difference in trypsinogen recognition, the small synthetic substrate Gly-Asp-Asp-Asp-Asp-Lys-beta-naphthylamide was cleaved with similar kinetic parameters by both HL-BEK (Km = 0.27 mM and kcat = 0.07 s-1) and L-BEK (Km = 0.60 mM and kcat = 0.06 s-1). The presence of the heavy chain also influenced the rate of reaction with protease inhibitors. Bovine pancreatic trypsin inhibitor preferred HL-BEK (initial Ki = 99 nM and final Ki* = 1.8 nM) over L-BEK (Ki = 698 nM and Ki* = 6.2 nM). Soybean trypsin inhibitor exhibited a reciprocal pattern, inhibiting L-BEK (Ki* = 1.6 nM), but not HL-BEK. These kinetic data indicate that the Enteropeptidase heavy chain has little influence on the recognition of small peptides, but strongly influences macromolecular substrate recognition and inhibitor specificity.
Mikhail P. Kirpichnikov - One of the best experts on this subject based on the ideXlab platform.
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New Strategy for High-Level Expression and Purification of Biologically Active Monomeric TGF-β1/C77S in Escherichia coli
Molecular Biotechnology, 2015Co-Authors: Yana V. Kim, Marine E. Gasparian, Dmitry A. Dolgikh, Eduard V. Bocharov, Rita V. Chertkova, Elena N. Tkach, Mikhail P. KirpichnikovAbstract:Mature transforming growth factor beta1 (TGF-β1) is a homodimeric protein with a single disulfide bridge between Cys77 on the respective monomers. The synthetic DNA sequence encoding the mature human TGF-β1/C77S (further termed TGF-β1m) was cloned into plasmid pET-32a downstream to the gene of fusion partner thioredoxin (Trx) immediately after the DNA sequence encoding Enteropeptidase recognition site. High-level expression (~1.5 g l^−1) of Trx/TGF-β1m fusion was achieved in Escherichia coli BL21(DE3) strain mainly in insoluble form. The fusion was solubilized and refolded in glutathione redox system in the presence of zwitterionic detergent CHAPS. After refolding, Trx/TGF-β1m fusion was cleaved by Enteropeptidase, and the carrier protein of TGF-β1m was separated from thioredoxin on Ni-NTA agarose. Separation of monomeric molecules from the noncovalently bounded oligomers was done using cation-exchange chromatography. The structure of purified TGF-β1m was confirmed by circular dichroism analysis. The developed technology allowed purifying biologically active tag-free monomeric TGF-β1m from bacteria with a yield of about 2.8 mg from 100 ml cell culture. The low-cost and easy purification steps allow considering that our proposed preparation of recombinant monomeric TGF-β1 could be employed for in vitro and in vivo experiments as well as for therapeutic intervention.
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Heterogeneous catalysis on the phage surface: Display of active human Enteropeptidase.
Biochimie, 2013Co-Authors: Marine E. Gasparian, Dmitry A. Dolgikh, T. V. Bobik, Yana V. Kim, Natalia A. Ponomarenko, Alexander G. Gabibov, Mikhail P. KirpichnikovAbstract:Abstract Enteropeptidase (EC 3.4.21.9) plays a key role in mammalian digestion as the enzyme that physiologically activates trypsinogen by highly specific cleavage of the trypsinogen activation peptide following the recognition sequence D 4 K. The high specificity of Enteropeptidase makes it a powerful tool in modern biotechnology. Here we describe the application of phage display technology to express active human Enteropeptidase catalytic subunits (L-HEP) on M13 filamentous bacteriophage. The L-HEP/C122S gene was cloned in the g3p-based phagemid vector pHEN2m upstream of the sequence encoding the phage g3p protein and downstream of the signal peptide-encoding sequence. Heterogeneous catalysis of the synthetic peptide substrate (GDDDDK-β-naphthylamide) cleavage by phage-bound L-HEP was shown to have kinetic parameters similar to those of soluble enzyme, with the respective K m values of 19 μM and 20 μM and k cat of 115 and 92 s −1 . Fusion proteins containing a D 4 K cleavage site were cleaved with phage-bound L-HEP/C122S as well as by soluble L-HEP/C122S, and proteolysis was inhibited by soybean trypsin inhibitor. Rapid large-scale phage production, one-step purification of phage-bound L-HEP, and easy removal of enzyme activity from reaction samples by PEG precipitation make our approach suitable for the efficient removal of various tag sequences fused to the target proteins. The functional phage display technology developed in this study can be instrumental in constructing libraries of mutants to analyze the effect of structural changes on the activity and specificity of the enzyme or generate its desired variants for biotechnological applications.
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Dissecting structural basis of the unique substrate selectivity of human Enteropeptidase catalytic subunit
Journal of biomolecular structure & dynamics, 2012Co-Authors: Valeriy G. Ostapchenko, Marine E. Gasparian, Dmitry A. Dolgikh, Yurij A. Kosinsky, Roman G. Efremov, Mikhail P. KirpichnikovAbstract:Enteropeptidase is a key enzyme in the digestion system of higher animals. It initiates enzymatic cascade cleaving trypsinogen activation peptide after a unique sequence DDDDK. Recently, we have found specific activity of human Enteropeptidase catalytic subunit (L-HEP) being significantly higher than that of its bovine ortholog (L-BEP). Moreover, we have discovered that L-HEP hydrolyzed several nonspecific peptidic substrates. In this work, we aimed to further characterize species-specific Enteropeptidase activities and to reveal their structural basis. First, we compared hydrolysis of peptides and proteins lacking DDDDK sequence by L-HEP and L-BEP. In each case human enzyme was more efficient, with the highest hydrolysis rate observed for substrates with a large hydrophobic residue in P2-position. Computer modeling suggested enzyme exosite residues 96 (Arg in L-HEP, Lys in L-BEP) and 219 (Lys in L-HEP, Gln in L-BEP) to be responsible for these differences in Enteropeptidase catalytic activity. Indeed, human-to-bovine mutations Arg96Lys, Lys219Gln shifted catalytic properties of L-HEP toward those of L-BEP. This effect was amplified in case of the double mutation Arg96Lys/Lys219Gln, but still did not cover the full difference in catalytic activities of human and bovine enzymes. To find a missing link, we studied monopeptide benzyl-arginine-β-naphthylamide hydrolysis. L-HEP catalyzed it with an order lower K (m) than L-BEP, suggesting the monopeptide-binding S1 site input into catalytic distinction between two Enteropeptidase species. Together, our findings suggest structural basis of the unique catalytic properties of human Enteropeptidase and instigate further studies of its tentative physiological and pathological roles.
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Strategy for improvement of Enteropeptidase efficiency in tag removal processes
Protein expression and purification, 2011Co-Authors: Marine E. Gasparian, M. L. Bychkov, Dmitry A. Dolgikh, Mikhail P. KirpichnikovAbstract:Abstract Enteropeptidase (synonym: enterokinase, EC 3.4.21.9) is a heterodimeric serine protease of the intestinal brush border that activates trypsinogen by highly specific cleavage of the trypsinogen activation peptide following the sequence (Asp) 4 -Lys. It has also great biotechnological interest because of the unique substrate specificity of the serine protease domain. The high degree of specificity exhibited by Enteropeptidase makes it a suitable reagent for cleaving recombinant proteins to remove affinity or other tags. However often unwanted cleavages elsewhere in the protein occurred during cleavage of fusions when high amount of enzyme is required. In this study we have improved the efficiency of fusion proteins cleavage by Enteropeptidase by substitution of the Lys residue by Arg in specific cleavage sequence (Asp) 4 -Lys. We have demonstrated that 3–6-fold lower amounts of the catalytic subunit of human and bovine Enteropeptidase is required for 95% cleavage of Trx/TRAIL and Trx/FGF-2 fusions with (Asp) 4 -Arg cleavage sequence in comparison to native sequence (Asp) 4 -Lys. As a result, reduced amount of non-specifically cleaved peptide fragments were observed during cleavage of (Asp) 4 -Lys/Arg mutated fusions. These findings overcome limitations of Enteropeptidase in tag removal processes during recombinant proteins purification and extend its commercial benefit in the biopharmaceutical industry.
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Expression, purification, and characterization of human Enteropeptidase catalytic subunit in Escherichia coli.
Protein expression and purification, 2003Co-Authors: Marine E. Gasparian, Dmitry A. Dolgikh, Valeriy G. Ostapchenko, Alexey A. Schulga, Mikhail P. KirpichnikovAbstract:Enteropeptidase (synonym:enterokinase, EC 3.4.21.9) is a heterodimeric serine protease of the intestinal brush border that activates trypsinogen by highly specific cleavage of the trypsinogen activation peptide following the sequence (Asp)(4)-Lys. The DNA sequence encoding the light chain (catalytic subunit) of human Enteropeptidase (GenBank Accession No. U09860) was synthesized from 26 oligonucleotides by polymerase chain reaction and cloned into plasmid pET-32a downstream to the gene of fusion partner thioredoxin immediately after the DNA sequence encoding Enteropeptidase recognition site. The fusion protein thioredoxin/human Enteropeptidase light chain was expressed in Escherichia coli BL21(DE3) strain in both soluble and insoluble forms. The soluble recombinant fusion protein failed to undergo autocatalytic cleavage and activation; however, autocatalytic cleavage and activation of recombinant human Enteropeptidase light chain (L-HEP) were achieved by solubilization and renaturation of the fusion protein from inclusion bodies and the active L-HEP was purified on agarose-linked soybean trypsin inhibitor. The purified L-HEP cleaved the synthetic peptide substrate Gly-Asp-Asp-Asp-Asp-Lys-beta-naphthylamide with kinetic parameters K(m)=0.16 mM and k(cat)=115 s(-1) and small ester Z-Lys-SBzl with K(m)=140 microM, k(cat)=133 s(-1). L-HEP associated with soybean trypsin inhibitor slowly and small ester Z-Lys-SBzl cleavage was inhibited with K(i)(*)=2.3 nM. L-HEP digested thioredoxin/human epidermal growth factor fusion protein five times faster than equal activity units of bovine recombinant light chain (EKMax, Invitrogen) at the same conditions.