Molecular characterization of trypsinogens and development of trypsinogen gene expression and tryptic activities in grass carp (Ctenopharyngodon idellus) and topmouth culter (Culter alburnus)
21
Citation
52
Reference
10
Related Paper
Citation Trend
Keywords:
Grass carp
Trypsin,one member of the serine protease family,plays important roles in animal protein digestion.It is synthesized and secreted as proenzyme trypsinogen,and removed the N-terminal activation peptide by enterokinase converting to active form in the intestine.The active trypsin can specifically cleavage at the peptide bond on the carboxyl side of basic L-amino acids such as arginine or lysine residuse.For studying the protein structure and physiological funtions of trypsinogen in fish,we successfully got three trypsinogen cDNAs(zftry1a、zftry1b and zftry2) from zebrafish by RT-PCR and RACE.The results showed zftry1a and zftry1b consisted of 242 amino acids which contained a signal peptide(SP) of 15 amino acids and an activation peptide(AP) of five amino acids,LDDDK.zftry2 consisted of 247 amino acids which contained a SP of 15 amino acids and an AP of nine amino acids,APLGDDDDK.The alignment based on the amino acid sequences revealed that they had the conversed structural characteristics,such as the catalytic triad(His-57,Asp-102,and Ser-195),12 cysteines forming 6 disulfide bonds,Asp-189 at the bottom of the substrate-binding pocket and Gly-216 and Gly-226 lining the sides of the binding pocket,and so on.The results from isoelectric and phylogenetic analyses suggested that zftry1a and zftry1b were group with teleost anionic trypsinogen group I,while zftry2 was phylogenetically related to teleost cationic group II.Tissue expression pattern was similar to each other,and all trypsinogens were mainly expressed in the intestine.These results from zebrafish trypsinogens can provide the foundations for further study of its expression profiles,the molecular characteristics and evolution of fish trypsinogen.
Catalytic triad
Enteropeptidase
Cite
Citations (0)
Grass carp
Cite
Citations (21)
Trypsinogen is the inactive precursor of trypsin, a serine protease that cleaves proteins and peptides after arginine and lysine residues. In this study, human trypsinogen was used as a model protein to study the influence of electrostatic forces on protein–protein interactions. Trypsinogen is active only after its eight-amino-acid-long activation peptide has been cleaved off by another protease, enteropeptidase. Trypsinogen can also be autoactivated without the involvement of enteropeptidase. This autoactivation process can occur if a trypsinogen molecule is activated by another trypsin molecule and therefore is based on a protein–protein interaction. Based on a rational protein design based on autoactivation-defective guinea pig trypsinogen, several amino acid residues, all located far away from the active site, were changed to modify the surface charge of human trypsinogen. The influence of the surface charge on the activation pattern of trypsinogen was investigated. The autoactivation properties of mutant trypsinogen were characterized in comparison to the recombinant wild-type enzyme. Surface-charged trypsinogen showed practically no autoactivation compared to the wild-type but could still be activated by enteropeptidase to the fully active trypsin. The kinetic parameters of surface-charged trypsinogen were comparable to the recombinant wild-type enzyme. The variant with a modified surface charge compared to the wild-type enzyme showed a complete different activation pattern. Our study provides an example how directed modification of the protein surface charge can be utilized for the regulation of functional protein–protein interactions, as shown here for human trypsinogen.
Enteropeptidase
Cite
Citations (12)
Incubation at 37°C of human cationic trypsinogen purified by PAGE electrophoresis, results in development of proteolytic activity (enzyme Y) capable of rapidly degrading cationic and anionic trypsinogens to inert products. Enzyme Y appears to be a serine protease with a molecular weight of about 20 000 daltons and is different from any of the known pancreatic enzymes. The active enzyme may be derived from trypsinogen itself or a hitherto unrecognized precursor contaminating the trypsinogen fraction used in this work. Appearance of enzyme Y activity seems to be associated with the presence of traces of free trypsin. Enzyme Y possesses insignificant or no activity when tested with a variety of synthetic trypsin, chymotrypsin and other protease substrates. It is not inactivated by the specific trypsin and chymotrypsin inhibitors TLCK and TPCK, but its activity is reduced gradually by increasing concentrations of pancreatic secretory trypsin inhibitor. Ca2+ concentrations >3 mM strongly inhibit enzyme Y, and diisopropylfluorophosphate completely inactivates it. The enzyme is stable when incubated at pH 1.9 and 37°C for 30 min and its activity is not abolished by treatment with Hg2+. When added to pancreatic juice with low inhibitor content it causes rapid inactivation of zymogens without significant release of active enzymes or reduction of pancreatic trypsin inhibitor. Its physiological role may be perceived as a second line of defense against premature intrapancreatic activation of zymogens. Enzyme Y activity may be generated when trypsin inhibitor, the first line of defense, is sufficiently depleted by complex formation with inappropriately released trypsin to permit dissociation of a small amount of trypsin from this complex. This in turn may lead to activation of enzyme Y and inactivation of the zymogens of pancreatic proteases.
Zymogen
Trypsin inhibitor
Enteropeptidase
Cite
Citations (48)
Enteropeptidase
Cleavage (geology)
Cite
Citations (14)
High levels of immunoreactive cathodal trypsin are shown to be present in serum and peritoneal fluid in acute pancreatitis. The immunoreactive material is contained in two fractions with different molecular weights: free trypsinogen and trypsin in complex with protease inhibitors, mainly alpha1-antitrypsin. The presence of trypsin in complex with protease inhibitors indicates the formation of active trypsin during acute pancreatitis.
Peritoneal fluid
Cite
Citations (50)
Astacus leptodactylus
Protein primary structure
Cite
Citations (16)
The currently available radioimmunoassays of trypsin measure total immunoreactive trypsin (EC 3.1.1.7), which includes both trypsinogen and alpha 1-protease inhibitor-bound trypsin. Hitherto, the only way to differentiate these two forms of trypsin has been to fractionate them on a gel-filtration column. We describe here a solid-phase immunoenzymometric assay that rapidly measures the amount of cationic trypsin bound to alpha 1-protease inhibitor in the plasma of rats with experimental pancreatitis. The assay specifically measures this complex within the range from 0.2 to 5.0 ng without interference by high concentrations of free alpha 1-protease inhibitor. The high correlation (r = 0.985) of the values obtained by size fractionation and by this assay demonstrates the accuracy of the assay, which is the first single-tube method for determining this form of activated cationic trypsin in plasma.
Protease inhibitor (pharmacology)
Alpha (finance)
Trypsin inhibitor
Cite
Citations (6)
Deoxyribonucleases
Cite
Citations (3)