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Hsienbin Huang - One of the best experts on this subject based on the ideXlab platform.
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purification and characterization of a Chymotrypsin inhibitor from the venom of ophiophagus hannah king cobra
Biochemical and Biophysical Research Communications, 2001Co-Authors: Longsen Chang, Charling Chung, Hsienbin HuangAbstract:Abstract A Chymotrypsin inhibitor from the venom of Ophiophagus hannah was isolated by a combination of ion-exchange chromatography and reverse phase HPLC. Amino acid sequence analysis revealed that this protein consists of 58 amino acids, six of these being cysteine residues and is highly homologous to Kunitz-type protease inhibitors. ESI-mass spectrum showed that the protein had a mass of 6493, which is in agreement with that predicted from its primary structure. In contrast to P1 Leu, Met, Phe, Trp, and Tyr appearing in other Chymotrypsin inhibitors, a P1 Asn in the novel inhibitor may cause a weak binding (Ki = 3.52 μM) with Chymotrypsin. Phylogenetic analysis suggests that the functional variations of the Chymotrypsin inhibitor and other Kunitz-type inhibitors probably distinguish from dendrotoxins by accelerated evolution.
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purification and characterization of a Chymotrypsin inhibitor from the venom of ophiophagus hannah king cobra
Biochemical and Biophysical Research Communications, 2001Co-Authors: Longsen Chang, Charling Chung, Hsienbin HuangAbstract:Abstract A Chymotrypsin inhibitor from the venom of Ophiophagus hannah was isolated by a combination of ion-exchange chromatography and reverse phase HPLC. Amino acid sequence analysis revealed that this protein consists of 58 amino acids, six of these being cysteine residues and is highly homologous to Kunitz-type protease inhibitors. ESI-mass spectrum showed that the protein had a mass of 6493, which is in agreement with that predicted from its primary structure. In contrast to P1 Leu, Met, Phe, Trp, and Tyr appearing in other Chymotrypsin inhibitors, a P1 Asn in the novel inhibitor may cause a weak binding (Ki = 3.52 μM) with Chymotrypsin. Phylogenetic analysis suggests that the functional variations of the Chymotrypsin inhibitor and other Kunitz-type inhibitors probably distinguish from dendrotoxins by accelerated evolution.
Miklos Sahintoth - One of the best experts on this subject based on the ideXlab platform.
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determinants of Chymotrypsin c cleavage specificity in the calcium binding loop of human cationic trypsinogen
FEBS Journal, 2012Co-Authors: Andras Szabo, Miklos SahintothAbstract:The pancreatic serine protease Chymotrypsin C (CTRC) cleaves the Leu81–Glu82 peptide bond in the calcium-binding loop of human cationic trypsinogen and thereby promotes its degradation. This serves as a protective mechanism against ectopic trypsinogen activation in the pancreas. In the present study, we demonstrate that cleavage of the Leu81–Glu82 peptide bond by CTRC is highly specific, and other human pancreatic Chymotrypsins (CTRB1, CTRB2 and CTRL1) and elastases (ELA2A, ELA3A and ELA3B) do not catalyze this reaction. To elucidate the mechanistic basis for CTRC specificity, we surveyed the primary (P1) cleavage preference of these pancreatic proteases on peptide substrates. We found that CTRC cleaved after a P1 Leu with at least tenfold higher catalytic efficiency than other enzymes tested. To assess extended sub-site interactions, we introduced Ala mutations into human cationic trypsinogen at the P3, P1′ P3′ and P4′ amino acid positions, where P1–P1′ corresponds to Leu81–Glu82. Interestingly, CTRC-mediated cleavage was stimulated threefold by mutation E82A and unaffected by mutations E79A and N84A, but all three mutations compromised specificity and resulted in increased cleavage by ELA2A. Mutation E85A decreased CTRC cleavage by twofold. Remarkably, other Chymotrypsins and elastases did not cleave human cationic trypsinogen even with the L81F or L81A mutations, which introduced favorable P1 residues for these enzymes. We conclude that specific cleavage of the Leu81–Glu82 peptide bond in human cationic trypsinogen by CTRC is primarily determined by its distinctively high activity on leucyl peptide bonds, with the P1′ Glu82, P3′ Asn84 and P4′ Glu85 residues serving as additional specificity determinants.
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high affinity small protein inhibitors of human Chymotrypsin c ctrc selected by phage display reveal unusual preference for p4 acidic residues
Journal of Biological Chemistry, 2011Co-Authors: Andras Szabo, Evette S Radisky, David Heja, David Szakacs, Katalin Zboray, Katalin A Kekesi, Miklos Sahintoth, Gabor PalAbstract:Human Chymotrypsin C (CTRC) is a pancreatic protease that participates in the regulation of intestinal digestive enzyme activity. Other Chymotrypsins and elastases are inactive on the regulatory sites cleaved by CTRC, suggesting that CTRC recognizes unique sequence patterns. To characterize the molecular determinants underlying CTRC specificity, we selected high affinity substrate-like small protein inhibitors against CTRC from a phage library displaying variants of SGPI-2, a natural Chymotrypsin inhibitor from Schistocerca gregaria. On the basis of the sequence pattern selected, we designed eight inhibitor variants in which amino acid residues in the reactive loop at P1 (Met or Leu), P2′ (Leu or Asp), and P4′ (Glu, Asp, or Ala) were varied. Binding experiments with CTRC revealed that (i) inhibitors with Leu at P1 bind 10-fold stronger than those with P1 Met; (ii) Asp at P2′ (versus Leu) decreases affinity but increases selectivity, and (iii) Glu or Asp at P4′ (versus Ala) increase affinity 10-fold. The highest affinity SGPI-2 variant (KD 20 pm) bound to CTRC 575-fold tighter than the parent molecule. The most selective inhibitor variant exhibited a KD of 110 pm and a selectivity ranging from 225- to 112,664-fold against other human Chymotrypsins and elastases. Homology modeling and mutagenesis identified a cluster of basic amino acid residues (Lys51, Arg56, and Arg80) on the surface of human CTRC that interact with the P4′ acidic residue of the inhibitor. The acidic preference of CTRC at P4′ is unique among pancreatic proteases and might contribute to the high specificity of CTRC-mediated digestive enzyme regulation.
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Chymotrypsin c is a co activator of human pancreatic procarboxypeptidases a1 and a2
Journal of Biological Chemistry, 2011Co-Authors: Richard Szmola, Andras Szabo, Melinda Bence, Andrea Carpentieri, Catherine E Costello, John Samuelson, Miklos SahintothAbstract:Human digestive carboxypeptidases CPA1, CPA2, and CPB1 are secreted by the pancreas as inactive proenzymes containing a 94–96-amino acid-long propeptide. Activation of procarboxypeptidases is initiated by proteolytic cleavage at the C-terminal end of the propeptide by trypsin. Here, we demonstrate that subsequent cleavage of the propeptide by Chymotrypsin C (CTRC) induces a nearly 10-fold increase in the activity of trypsin-activated CPA1 and CPA2, whereas CPB1 activity is unaffected. Other human pancreatic proteases such as Chymotrypsin B1, Chymotrypsin B2, Chymotrypsin-like enzyme-1, elastase 2A, elastase 3A, or elastase 3B are inactive or markedly less effective at promoting procarboxypeptidase activation. On the basis of these observations, we propose that CTRC is a physiological co-activator of proCPA1 and proCPA2. Furthermore, the results confirm and extend the notion that CTRC is a key regulator of digestive zymogen activation.
Richard Szmola - One of the best experts on this subject based on the ideXlab platform.
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Chymotrypsin c is a co activator of human pancreatic procarboxypeptidases a1 and a2
Journal of Biological Chemistry, 2011Co-Authors: Richard Szmola, Andras Szabo, Melinda Bence, Andrea Carpentieri, Catherine E Costello, John Samuelson, Miklos SahintothAbstract:Human digestive carboxypeptidases CPA1, CPA2, and CPB1 are secreted by the pancreas as inactive proenzymes containing a 94–96-amino acid-long propeptide. Activation of procarboxypeptidases is initiated by proteolytic cleavage at the C-terminal end of the propeptide by trypsin. Here, we demonstrate that subsequent cleavage of the propeptide by Chymotrypsin C (CTRC) induces a nearly 10-fold increase in the activity of trypsin-activated CPA1 and CPA2, whereas CPB1 activity is unaffected. Other human pancreatic proteases such as Chymotrypsin B1, Chymotrypsin B2, Chymotrypsin-like enzyme-1, elastase 2A, elastase 3A, or elastase 3B are inactive or markedly less effective at promoting procarboxypeptidase activation. On the basis of these observations, we propose that CTRC is a physiological co-activator of proCPA1 and proCPA2. Furthermore, the results confirm and extend the notion that CTRC is a key regulator of digestive zymogen activation.
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Chymotrypsin c caldecrin promotes degradation of human cationic trypsin identity with rinderknecht s enzyme y
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Richard SzmolaAbstract:Digestive trypsins undergo proteolytic breakdown during their transit in the human alimentary tract, which has been assumed to occur through trypsin-mediated cleavages, termed autolysis. Autolysis was also postulated to play a protective role against pancreatitis by eliminating prematurely activated intrapancreatic trypsin. However, autolysis of human cationic trypsin is very slow in vitro, which is inconsistent with the documented intestinal trypsin degradation or a putative protective role. Here we report that degradation of human cationic trypsin is triggered by Chymotrypsin C, which selectively cleaves the Leu81-Glu82 peptide bond within the Ca2+ binding loop. Further degradation and inactivation of cationic trypsin is then achieved through tryptic cleavage of the Arg122-Val123 peptide bond. Consequently, mutation of either Leu81 or Arg122 blocks Chymotrypsin C-mediated trypsin degradation. Calcium affords protection against Chymotrypsin C-mediated cleavage, with complete stabilization observed at 1 mM concentration. Chymotrypsin C is highly specific in promoting trypsin degradation, because Chymotrypsin B1, Chymotrypsin B2, elastase 2A, elastase 3A, or elastase 3B are ineffective. Chymotrypsin C also rapidly degrades all three human trypsinogen isoforms and appears identical to enzyme Y, the enigmatic trypsinogen-degrading activity described by Heinrich Rinderknecht in 1988. Taken together with previous observations, the results identify Chymotrypsin C as a key regulator of activation and degradation of cationic trypsin. Thus, in the high Ca2+ environment of the duodenum, Chymotrypsin C facilitates trypsinogen activation, whereas in the lower intestines, Chymotrypsin C promotes trypsin degradation as a function of decreasing luminal Ca2+ concentrations.
Andras Szabo - One of the best experts on this subject based on the ideXlab platform.
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determinants of Chymotrypsin c cleavage specificity in the calcium binding loop of human cationic trypsinogen
FEBS Journal, 2012Co-Authors: Andras Szabo, Miklos SahintothAbstract:The pancreatic serine protease Chymotrypsin C (CTRC) cleaves the Leu81–Glu82 peptide bond in the calcium-binding loop of human cationic trypsinogen and thereby promotes its degradation. This serves as a protective mechanism against ectopic trypsinogen activation in the pancreas. In the present study, we demonstrate that cleavage of the Leu81–Glu82 peptide bond by CTRC is highly specific, and other human pancreatic Chymotrypsins (CTRB1, CTRB2 and CTRL1) and elastases (ELA2A, ELA3A and ELA3B) do not catalyze this reaction. To elucidate the mechanistic basis for CTRC specificity, we surveyed the primary (P1) cleavage preference of these pancreatic proteases on peptide substrates. We found that CTRC cleaved after a P1 Leu with at least tenfold higher catalytic efficiency than other enzymes tested. To assess extended sub-site interactions, we introduced Ala mutations into human cationic trypsinogen at the P3, P1′ P3′ and P4′ amino acid positions, where P1–P1′ corresponds to Leu81–Glu82. Interestingly, CTRC-mediated cleavage was stimulated threefold by mutation E82A and unaffected by mutations E79A and N84A, but all three mutations compromised specificity and resulted in increased cleavage by ELA2A. Mutation E85A decreased CTRC cleavage by twofold. Remarkably, other Chymotrypsins and elastases did not cleave human cationic trypsinogen even with the L81F or L81A mutations, which introduced favorable P1 residues for these enzymes. We conclude that specific cleavage of the Leu81–Glu82 peptide bond in human cationic trypsinogen by CTRC is primarily determined by its distinctively high activity on leucyl peptide bonds, with the P1′ Glu82, P3′ Asn84 and P4′ Glu85 residues serving as additional specificity determinants.
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high affinity small protein inhibitors of human Chymotrypsin c ctrc selected by phage display reveal unusual preference for p4 acidic residues
Journal of Biological Chemistry, 2011Co-Authors: Andras Szabo, Evette S Radisky, David Heja, David Szakacs, Katalin Zboray, Katalin A Kekesi, Miklos Sahintoth, Gabor PalAbstract:Human Chymotrypsin C (CTRC) is a pancreatic protease that participates in the regulation of intestinal digestive enzyme activity. Other Chymotrypsins and elastases are inactive on the regulatory sites cleaved by CTRC, suggesting that CTRC recognizes unique sequence patterns. To characterize the molecular determinants underlying CTRC specificity, we selected high affinity substrate-like small protein inhibitors against CTRC from a phage library displaying variants of SGPI-2, a natural Chymotrypsin inhibitor from Schistocerca gregaria. On the basis of the sequence pattern selected, we designed eight inhibitor variants in which amino acid residues in the reactive loop at P1 (Met or Leu), P2′ (Leu or Asp), and P4′ (Glu, Asp, or Ala) were varied. Binding experiments with CTRC revealed that (i) inhibitors with Leu at P1 bind 10-fold stronger than those with P1 Met; (ii) Asp at P2′ (versus Leu) decreases affinity but increases selectivity, and (iii) Glu or Asp at P4′ (versus Ala) increase affinity 10-fold. The highest affinity SGPI-2 variant (KD 20 pm) bound to CTRC 575-fold tighter than the parent molecule. The most selective inhibitor variant exhibited a KD of 110 pm and a selectivity ranging from 225- to 112,664-fold against other human Chymotrypsins and elastases. Homology modeling and mutagenesis identified a cluster of basic amino acid residues (Lys51, Arg56, and Arg80) on the surface of human CTRC that interact with the P4′ acidic residue of the inhibitor. The acidic preference of CTRC at P4′ is unique among pancreatic proteases and might contribute to the high specificity of CTRC-mediated digestive enzyme regulation.
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Chymotrypsin c is a co activator of human pancreatic procarboxypeptidases a1 and a2
Journal of Biological Chemistry, 2011Co-Authors: Richard Szmola, Andras Szabo, Melinda Bence, Andrea Carpentieri, Catherine E Costello, John Samuelson, Miklos SahintothAbstract:Human digestive carboxypeptidases CPA1, CPA2, and CPB1 are secreted by the pancreas as inactive proenzymes containing a 94–96-amino acid-long propeptide. Activation of procarboxypeptidases is initiated by proteolytic cleavage at the C-terminal end of the propeptide by trypsin. Here, we demonstrate that subsequent cleavage of the propeptide by Chymotrypsin C (CTRC) induces a nearly 10-fold increase in the activity of trypsin-activated CPA1 and CPA2, whereas CPB1 activity is unaffected. Other human pancreatic proteases such as Chymotrypsin B1, Chymotrypsin B2, Chymotrypsin-like enzyme-1, elastase 2A, elastase 3A, or elastase 3B are inactive or markedly less effective at promoting procarboxypeptidase activation. On the basis of these observations, we propose that CTRC is a physiological co-activator of proCPA1 and proCPA2. Furthermore, the results confirm and extend the notion that CTRC is a key regulator of digestive zymogen activation.
Longsen Chang - One of the best experts on this subject based on the ideXlab platform.
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purification and characterization of a Chymotrypsin inhibitor from the venom of ophiophagus hannah king cobra
Biochemical and Biophysical Research Communications, 2001Co-Authors: Longsen Chang, Charling Chung, Hsienbin HuangAbstract:Abstract A Chymotrypsin inhibitor from the venom of Ophiophagus hannah was isolated by a combination of ion-exchange chromatography and reverse phase HPLC. Amino acid sequence analysis revealed that this protein consists of 58 amino acids, six of these being cysteine residues and is highly homologous to Kunitz-type protease inhibitors. ESI-mass spectrum showed that the protein had a mass of 6493, which is in agreement with that predicted from its primary structure. In contrast to P1 Leu, Met, Phe, Trp, and Tyr appearing in other Chymotrypsin inhibitors, a P1 Asn in the novel inhibitor may cause a weak binding (Ki = 3.52 μM) with Chymotrypsin. Phylogenetic analysis suggests that the functional variations of the Chymotrypsin inhibitor and other Kunitz-type inhibitors probably distinguish from dendrotoxins by accelerated evolution.
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purification and characterization of a Chymotrypsin inhibitor from the venom of ophiophagus hannah king cobra
Biochemical and Biophysical Research Communications, 2001Co-Authors: Longsen Chang, Charling Chung, Hsienbin HuangAbstract:Abstract A Chymotrypsin inhibitor from the venom of Ophiophagus hannah was isolated by a combination of ion-exchange chromatography and reverse phase HPLC. Amino acid sequence analysis revealed that this protein consists of 58 amino acids, six of these being cysteine residues and is highly homologous to Kunitz-type protease inhibitors. ESI-mass spectrum showed that the protein had a mass of 6493, which is in agreement with that predicted from its primary structure. In contrast to P1 Leu, Met, Phe, Trp, and Tyr appearing in other Chymotrypsin inhibitors, a P1 Asn in the novel inhibitor may cause a weak binding (Ki = 3.52 μM) with Chymotrypsin. Phylogenetic analysis suggests that the functional variations of the Chymotrypsin inhibitor and other Kunitz-type inhibitors probably distinguish from dendrotoxins by accelerated evolution.