trypsinogen and Autolysis

Acute pancreatitis (AP) is an important cause of morbidity and mortality worldwide and the annual incidence appears to be increasing. It presents as a mild self-limiting illness in 80% of patients. However, one-fifth of these develop a severe complicated life-threatening disease requiring intensive and prolonged therapeutic intervention. Alcohol and gallstone disease remain the commonest causes of AP but metabolic abnormalities, obesity and genetic susceptibility are thought be increasingly important aetiological factors. The prompt diagnosis of AP and stratification of disease severity is essential in directing rapid delivery of appropriate therapeutic measures. In this review, the range of diagnostic and prognostic assays, severity scoring systems and radiological investigations used in current clinical practice are described, highlighting their strengths and weaknesses. Increased understanding of the complex pathophysiology of AP has generated an array of new potential diagnostic assays and these are discussed. The multidisciplinary approach to management of severe pancreatitis is outlined, including areas of controversy and novel treatments.

Topics: Acute Disease; Alcohol Drinking; Amylases; Autolysis; Gallstones; Genetic Predisposition to Disease; Humans; Hypercalcemia; Hyperlipidemias; Lipase; Obesity; Pancreatitis; Prognosis; Trypsin; Trypsinogen

This study attempts to identify the biochemical alterations in human cationic trypsinogen and trypsin caused by the hereditary pancreatitis-associated mutations Arg117-->His and Asn21-->Ile.. Recombinant wild-type and mutant human cationic trypsinogens were expressed in Escherichia coli and purified to homogeneity, and trypsin autolysis and trypsinogen autoactivation were characterized.. Both mutations significantly enhanced the autoactivation of human cationic trypsinogen. In addition, the Arg117-->His mutation inhibited autocatalytic inactivation of trypsin, while the Asn21-->Ile mutation had no such effect.. The findings support the notion that enhanced trypsinogen activation in the pancreas is the common initiating step in hereditary pancreatitis, whereas trypsin stabilization plays a role in cases associated with the Arg117-->His mutation.

Topics: Autolysis; Catalysis; Enzyme Activation; Gene Expression Regulation; Genetic Vectors; Humans; Kinetics; Pancreatitis; Recombinant Proteins; Trypsin; Trypsinogen

Since the identification in 1996 of a "gain of function" missense mutation, R122H, in the cationic trypsinogen gene (PRSS1) as a cause of hereditary pancreatitis, continued screening of this gene in both hereditary and sporadic pancreatitis has found more disease-associated missense mutations than expected. In addition, functional analysis has yielded interesting findings regarding their underlying mechanisms resulting in a gain of trypsin. A critical review of these data, in the context of the complicated biogenesis and complex autoactivation and autolysis of trypsin(ogen), highlights that PRSS1 mutations cause the disease by various mechanisms depending on which biochemical process they affect. The discovery of these mutations also modifies the classical perception of the disease and, more importantly, reveals fascinating new aspects of the molecular evolution and normal physiology of trypsinogen. First, activation peptide of trypsinogen is under strong selection pressure to minimize autoactivation in higher vertebrates. Second, the R122 primary autolysis site has further evolved in mammalian trypsinogens. Third, evolutionary divergence from threonine to asparagine at residue 29 in human cationic trypsinogen provides additional advantage. Accordingly, we tentatively assign, in human cationic trypsinogen, the strongly selected activation peptide as the first-line and the R122 autolysis site as the second-line of the built-in defensive mechanisms against premature trypsin activation within the pancreas, respectively, and the positively selected asparagine at residue 29 as an "amplifier" to the R122 "fail-safe" mechanism.

Topics: Amino Acid Sequence; Animals; Autolysis; Binding Sites; Enzyme Activation; Enzyme Stability; Evolution, Molecular; Humans; Molecular Sequence Data; Mutation, Missense; Pancreatitis; Selection, Genetic; Trypsin; Trypsinogen

N-terminal sequences play crucial roles in regulating expression, translation, activation and enzymatic properties of proteins. To reduce cell toxicity of intracellular trypsin and increase secretory expression, we developed a novel auto-catalyzed strategy to produce recombinant trypsin by engineering the N-terminus of mature Streptomyces griseus trypsin (SGT). The engineered N-terminal peptide of SGT was composed of the thioredoxin, glycine-serine linker, His6-tag and the partial bovine trypsinogen pro-peptide (DDDDK). Furthermore, we constructed a variant TLEI with insertion of the artificial peptide at N-terminus and site-directed mutagenesis of the autolysis residue R145. In fed-batch fermentation, the production of extracellular trypsin activity was significantly improved to 47.4 ± 1.2 U·ml(-1) (amidase activity, 8532 ± 142.2 U·ml(-1) BAEE activity) with a productivity of 0.49 U·ml(-1)·h(-1), which was 329% greater than that of parent strain Pichia pastoris GS115-SGT. This work has significant potential to be scaled-up for microbial production of SGT. In addition, the N-terminal peptide engineering strategy can be extended to improve heterologous expression of other toxic enzymes.

Topics: Amino Acid Sequence; Animals; Autolysis; Batch Cell Culture Techniques; Biocatalysis; Cattle; Kinetics; Molecular Dynamics Simulation; Mutagenesis, Site-Directed; Peptides; Pichia; Protein Engineering; Protein Structure, Tertiary; Recombinant Fusion Proteins; Streptomyces griseus; Trypsin; Trypsinogen