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PubMed1985

Cholecystokinin-induced secretion and synthesis of amylase and cationic trypsinogen by pancreatic acini isolated from rats given an ethanol diet.

Effects of chronic alcohol intake on secretion and synthesis of amylase and cationic trypsinogen (CT) were studied with pancreatic acini isolated from male Sprague-Dawley rats fed a Lieber-DeCarli ethanol or control diet for 30 days. Pancreatic acini were incubated in a media containing increasing concentrations of cholecystokinin octapeptide (CCK-8: 0-1000pM) followed by addition of [3H]leucine. Amylase and CT secreted in the media and those labeled in acini were quantitated. Basal and CCK-stimulated secretion of CT was not different in alcoholic and control groups. On the other hand, a dose-response curve of CCK-stimulated amylase secretion from alcoholic acini was markedly reduced with both basal and maximal secretion decreased to only 40% of controls. Basal incorporation of [3H]leucine into amylase was reduced by 70% in alcoholic acini compared to controls while that into CT was not different in the two groups. CCK-8 exhibited a biphasic effect on [3H]leucine incorporation into both enzymes in alcoholic acini: low concentrations of CCK-8 (less than 100pM) increased the incorporation whereas high concentrations (greater than 100pM) decreased it. However, in control acini, CCK-8 induced progressive suppression of the incorporation into these enzymes, the pattern of which was similar to that previously observed in fasted rats (Am. J. Physiol. 241:G116-G112, 1981). This difference in the response pattern resulted in significantly higher rates of CCK-8 induced incorporation into CT in alcoholic acini. These results suggest that the differences observed may possibly be attributable not only to alcohol intake but also to the differences in carbohydrate intake and in temporal patterns of diet consumption.

Amylases↗
PubMed1994

Primary structure of aspergillopepsin I deduced from nucleotide sequence of the gene and aspartic acid-76 is an essential active site of the enzyme for trypsinogen activation.

The coding region of the aspergillopepsin I (EC 3.4.23.18) gene occupies 1340 base pairs of the genomic DNA and is separated into four exons by three introns. The predicted amino-acid sequence of aspergillopepsin I consists of 325 residues and is 32% and 27% homologous with those of human pepsin and calf chymosin. The cDNA of the gene prepared from mRNA has been cloned and expressed in yeast cells. To identify the residue of the substrate binding pocket in determining the specificity of aspergillopepsin I towards basic substrates, this residue was replaced with a serine residue by site-directed mutagenesis. The mutation is a single amino-acid change, Asp-76 converted to Ser-D76S, in the enzyme. The striking feature of this is that only the trypsinogen activating activity was destroyed. We therefore concluded that Asp-76 is the binding site towards basic substrates.

Amino Acid Sequence↗
PubMed1978

Demonstration of human pancreatic anionic trypsinogen in normal serum by radioimmunoassay.

A specific radioimmunoassay for human pancreatic anionic trypsin has been developed. The trypsin employed as radioiodinated tracer in the assay was inactivated with tosyl-L-lysine chloromethyl ketone in order to prevent binding of the tracer to the serum inhibitors alpha1-antitrypsin and alpha2-macroglobulin. A normal serum level of immunoreactive anionic trypsin of 5.45 ng/ml was determined. The results of experiments in which serum was fractionated by Sephadex G-200 gel filtration suggest that essentially all of the immunoreactive material in normal human serum is trypsinogen. This finding implies that a small fraction of the zymogens synthesized in the pancreas are released directly into the circulation.

Anions↗
PubMed2006

Conformational changes of bovine beta-trypsin and trypsinogen induced by divalent ions: an energy-dispersive X-ray diffraction and functional study.

The radius of gyration (R(g)) of bovine trypsinogen and beta-trypsin was measured by an energy-dispersive X-ray technique as a function of Ca(2+) or SO(4)(2-) concentration; these results have been supplemented with measurements of association equilibrium constants of Ca(2+) to its binding site(s) on both serine proteases and some of their adducts (with an effector and/or an inhibitor). As a whole, all information reported in the present work demonstrates that: (i) the strains exerted by different ions on these proteases produce diverse structural modifications; and (ii) at least in the case of Ca(2+), the changes in R(g) can be ascribed to the direct interaction of the binding site(s) on the protein matrix with the cation.

Animals↗
PubMed2003

Structural and evolutionary consequences of unpaired cysteines in trypsinogen.

Vertebrate trypsins usually contain six disulfide bonds but human trypsin 1 (PRSS1) contains only five and human trypsin 2 (PRSS2) contains only four. To elucidate possible evolutionary pathways leading to the loss of disulfide bonds, we have constructed mutants lacking one or two cysteines of four disulfide bonds (C22-C157, C127-C232, C136-C201, and C191-C220) in rat anionic trypsinogen and followed their expression in the periplasm of Escherichia coli. When both cysteines of any of the above-mentioned disulfide bonds were replaced by alanines we found, as expected, proteolytically active enzymes. In the case of C127-C232 (missing from both human trypsins) and C191-C220 both single mutants gave active enzymes although their yield was significantly reduced. In contrast, only one of the single mutants of disulfide bonds C22-C157 and C136-C201 (missing from human trypsin 2) was expressed in E. coli. In the case of these disulfide bonds, we obtained no expression when the solvent accessible molecular surface of the free cysteine residue was the smaller one, indicating that a buried unpaired cysteine was more deleterious than one on the surface of the molecule.

Base Sequence↗
PubMed2005

Cationic trypsinogen mutations and pancreatitis.

The discovery of PRSS 1 mutations in hereditary pancreatitis and analysis of how the genotype affects the presentation and progression of hereditary pancreatitis has led to a better understanding of the pathophysiology of the disease. Patients with hereditary pancreatitis present with symptoms at an early age and have a significant lifetime risk for the development of endocrine and exocrine insufficiency, albeit at a later stage than patients with either idiopathic or alcoholic chronic pancreatitis. There are distinct phenotypic differences between hereditary pancreatitis and with other types of pancreatitis. As many as 80% of patients with symptomatic hereditary pancreatitis have an underlying causative PRSS1 mutation; there are, however, few significant phenotypic differences between these PRSS1 mutations. TheR122H mutation is the most common PRSS1 mutation observed, and patients with the R122H mutation present earlier. This, however, does not necessarily translate into a more aggressive disease with respect to complications of chronic pancreatitis. Indeed, the age of presentation of symptoms may be a poor surrogate for predicting outcome, as inherited disorders of trypsinogen may cause subclinical attacks of pancreatitis, which ultimately lead to pancreatic destruction and dysfunction. All patients, irrespective of whether they carry a PRSS1 mutation, are at significant risk of developing pancreatic ductal adenocarcinoma. The risk appears to be insignificant below the age of 40 years, but it increases incrementally thereafter. Significantly, the risk of pancreatic cancer is not related to PRSS1 mutation type and does not appear to be related to the mode of inheritance. The role of SPINK1 mutations in modifying the expression of PRSS1mutations is unclear but appears to be of clinical importance. It is unlikely that they act as causative mutations per se, at least in the Western form of the disease. Additionally, they do not appear to have an impact on the penetrance of PRSS1 gene mutations in hereditary pancreatitis.

Chronic Disease↗
PubMed2004

Cationic trypsinogen mutations and pancreatitis.

The discovery of PRSS1 mutations in hereditary pancreatitis and analysis of how the genotype affects the presentation and progression of hereditary pancreatitis has led to a better understanding of the pathophysiology of the disease. Patients with hereditary pancreatitis present with symptoms at an early age and have a significant lifetime risk for the development of endocrine and exocrine insufficiency, albeit at a later stage than patients with either idiopathic or alcoholic chronic pancreatitis. There are distinct phenotypic differences between hereditary pancreatitis and with other types of pancreatitis. As many as 80% of patients with symptomatic hereditary pancreatitis have an underlying causative PRSS1 mutation; there are, however, few significant phenotypic differences between these PRSS1 mutations. The R122H mutation is the most common PRSS1 mutation observed, and patients with the R122H mutation present earlier. This, however, does not necessarily translate into a more aggressive disease with respect to complications of chronic pancreatitis. Indeed, the age of presentation of symptoms may be a poor surrogate for predicting outcome, as inherited disorders of trypsinogen may cause subclinical attacks of pancreatitis, which ultimately lead to pancreatic destruction and dysfunction. All patients, irrespective of whether they carry a PRSS1 mutation, are at significant risk of developing pancreatic ductal adenocarcinoma. The risk appears to be insignificant below the age of 40 years, but it increases incrementally thereafter. Significantly, the risk of pancreatic cancer is not related to PRSS1 mutation type and does not appear to be related to the mode of inheritance. The role of SPINK1 mutations in modifying the expression of PRSS1 mutations is unclear but appears to be of clinical importance. It is unlikely that they act as causative mutations per se, at least in the Western form of the disease. Additionally, they do not appear to have an impact on the penetrance of PRSS1 gene mutations in hereditary pancreatitis.

Humans↗
PubMed2005

Combining immunoreactive trypsinogen and pancreatitis-associated protein assays, a method of newborn screening for cystic fibrosis that avoids DNA analysis.

OBJECTIVES: To evaluate the performance of a strategy in which, after immunoreactive trypsinogen (IRT) determination, genetic analysis is replaced by a biological test, the pancreatitis-associated protein (PAP) enzyme-linked immunosorbent assay (ELISA). STUDY DESIGN: The French newborn screening program includes cystic fibrosis (CF) screening by the IRT/CFTR mutation strategy. PAP was assayed on screening cards, in parallel with IRT, in all newborns from 5 French regions (n = 204,749). Analysis of PAP values in CF and non-CF newborns with elevated IRT allowed direct comparison between the current strategy and the proposed IRT/PAP strategy. RESULTS: A protocol in which newborns with IRT >50 ng/mL and PAP >1.8 ng/mL and those with IRT >100 ng/mL and PAP >1.0 ng/mL are directly recalled for sweat testing would have the same performance as the IRT/CFTR mutation strategy. CONCLUSIONS: The IRT/PAP strategy is an alternative for CF newborn screening, which avoids the drawbacks of genetic analysis and is cheaper and easier to implement than the current IRT/CFTR mutation strategy.

Antigens, Neoplasm↗
PubMed2005

Two-tiered immunoreactive trypsinogen-based newborn screening for cystic fibrosis in Colorado: screening efficacy and diagnostic outcomes.

OBJECTIVE: To examine immunoreactive trypsinogen (IRT)-based screening for cystic fibrosis (CF) for recall rate, genotype distribution, and "borderline" sweat test results. STUDY DESIGN: CF newborn screening in Colorado began in 1982, and >1,153,000 infants were screened through 2002 with an IRT-based screen (IRT/IRT). RESULTS: We have identified 313 infants with CF, giving an overall incidence of 1 in 3684 and a Hispanic incidence of 1 in 6495. Fifty-five infants with meconium ileus (17.6%) were excluded from analysis. Fourteen infants with false-negative results were identified (5.4%). The average recall rate was 0.6%, with a positive predictive value of 4.7%. Ninety-three percent of the infants had at least 1 DeltaF508 mutation, and 98% of the infants had at least 1 mutation from the American College of Medical Genetics recommended panel. Six infants had hypertrypsinogenemia and borderline results on sweat tests (30-60 mmol/L). Increased variability in sweat chloride levels were seen in these infants compared with infants with homozygous DeltaF508. Three children with initial borderline results on sweat tests had CF diagnosed. CONCLUSIONS: The recall and false-negative rates of our IRT/IRT CF screening program are reported. Additionally, genotypes of the patients identified mirror the CF population genotypes, reflecting similar disease severity in the screened population. Finally, infants with persistent hypertrypsinogenemia and borderline sweat test results need long-term follow-up.

Chlorides↗
PubMed2006

Genetic and physiologic correlates of longitudinal immunoreactive trypsinogen decline in infants with cystic fibrosis identified through newborn screening.

OBJECTIVES: To characterize the time course and physiologic significance of decline in serum immunoreactive trypsinogen (IRT) levels in infants with cystic fibrosis (CF) by mode of diagnosis and genotype, and to examine IRT heritability. STUDY DESIGN: We studied longitudinal IRT measurements in 317 children with CF. We developed statistical models to describe IRT decline. Pancreatic disease severity (Mild or Severe) was assigned using CF genotype and was confirmed in 47 infants through fat malabsorption studies. RESULTS: Infants with severe disease exhibited IRT decline with non-detectable levels typically seen by 5 years of age. Infants with mild disease exhibited a decline in the first 2 years, asymptomatically approaching a level greater than published norms. IRT and fecal fat were inversely correlated. IRT values in infants with meconium ileus (MI) were significantly lower than newborn-screened infants at birth. The high proportion of shared variation in predicted IRT values among sibling pairs with severe disease suggests that IRT is heritable. CONCLUSIONS: IRT declines characteristically in infants with CF. Lower IRT values in newborns with MI suggest increased pancreatic injury. Furthermore, IRT is heritable among patients with severe disease suggesting genetic modifiers of early CF pancreatic injury. This study demonstrates heritability of a statistically modeled quantitative phenotype.

Biomarkers↗
PubMed1990

Kinetics of the trypsinogen activation by enterokinase and trypsin.

A global kinetic analysis of the mechanisms of the trypsinogen activation by enterokinase and trypsin is presented. The kinetic equations of both the transient-phase and the steady-state of these mechanisms are presented. In addition, we here derive the corresponding kinetic equations for the case in which the condition of rapid equilibrium prevails and we propose a kinetic data analysis. The significance of this approach to the treatment of other zymogen activation processes is discussed.

Animals↗
PubMed2000

Early trypsinogen activation in acute pancreatitis.

This article discusses zymogen activation within the acinar cell. The authors review advances in respect to the cellular mechanism involved in a premature intrapancreatic protease activation. Critical factors that determine the onset of premature protease activation appear to be the molecular structure of trypsinogen, the presence or absence of functionally intact lysosomal hydrolases, the pH in intracellular compartments, and the calcium signaling cascade in the pancreatic acinar cell.

Calcium Signaling↗
PubMed1987

Monoclonal antibody-based enzyme immunoassay for trypsinogen in neonatal screening for cystic fibrosis.

An enzyme immunoassay (EIA) with monoclonal antibodies against human trypsinogen in neonatal blood-spots has been evaluated for screening for neonatal cystic fibrosis (CF). In a retrospective study, 36 of 39 CF samples were distinguished from controls matched for age and storage time. 7 infants with CF were detected in 16,500 infants screened in a prospective study. The EIA is quicker and less labour intensive than conventional assays for the detection of immunoreactive trypsin and may have further advantages of specificity and sensitivity for monitoring the release of pancreatic zymogens in CF.

Antibodies, Monoclonal↗
PubMed2001

[Evaluation of 47,213 infants in neonatal screening for cystic fibrosis, using pancreatitis-associated protein and immunoreactive trypsinogen assays].

OBJECTIVES: The increasing evidence of the benefits of neonatal screening for cystic fibrosis (CF) indicates that this procedure could soon be implemented throughout France. The screening strategy currently used involves the detection of infants with elevated levels of immunoreactive trypsinogen (IRT) (approximately 1% of the population), followed by the detection of CFTR gene mutations. However, genetic analysis has certain drawbacks, the most important of which being the management of heterozygotes, and in France the requirement by law of previous informed consent. In cases of CF, pancreatic alterations are already present in utero. A previous study has demonstrated the value of pancreatitis-associated protein (PAP) as a screening test for CF, and has indicated that a feasible two-stage strategy could involve the following: 1) selection of infants with elevated PAP levels; 2) in this group of infants, subsequent detection of those with elevated IRT levels for direct CF diagnosis by the sweat test thereby avoiding the use of genetic analysis. The study aim was to evaluate this strategy in a large number of neonates. METHODS AND RESULTS: The aforementioned strategy was evaluated in a prospective study involving 47,213 infants in the Provence region of France. In infants with a PAP > 7.5 ng/mL (1.28%), 176 had an elevated IRT level > 700 ng/mL (0.37%). In this limited population sample (0.37% of the total), the sweat test diagnosed five cases of CF. A sixth case involving the monozygous twin of an infant with diagnosed CF remained undetected, probably because of a registration error. Genetic analysis confirmed the diagnosis, and also detected another case in an infant with two CFTR mutations but with a normal phenotype at 20 months of age. As the observed incidence was similar to that which had previously been reported, and as no further case was subsequently detected two years after the end of the study, this indicated that the sensitivity of this screening strategy was satisfactory. Its specificity makes the direct diagnosis of CF cases by the sweat test feasible, without further selection by genetic analysis. CONCLUSION: The PAP/IRT technique for CF detection seems to be suitable for mass screening, without the drawbacks of genetic testing.

Acute-Phase Proteins↗
PubMed2003

"Loss of function" mutations in the cationic trypsinogen gene (PRSS1) may act as a protective factor against pancreatitis.

Several genetic factors have been well known to predispose one to chronic pancreatitis (CP). However, little is known about the genetic factors that may provide a protective effect against the disease. Having found a nonsense mutation (c.111C>A; Y37X) and a splicing mutation (IVS2+1G>A) in the cationic trypsinogen gene (protease, serine, 1; PRSS1) in alcoholics without the development of CP, but not in alcoholics with CP and patients with hereditary or idiopathic CP, we propose that while "gain of function" mutations in the PRSS1 gene predispose one to pancreatitis, "loss of function" mutations in the gene may protect one against the disease.

Adult↗
PubMed1995

cDNA sequence and chromosomal localization of human enterokinase, the proteolytic activator of trypsinogen.

Enterokinase is a serine protease of the duodenal brush border membrane that cleaves trypsinogen and produces active trypsin, thereby leading to the activation of many pancreatic digestive enzymes. Overlapping cDNA clones that encode the complete human enterokinase amino acid sequence were isolated from a human intestine cDNA library. Starting from the first ATG codon, the composite 3696 nt cDNA sequence contains an open reading frame of 3057 nt that encodes a 784 amino acid heavy chain followed by a 235 amino acid light chain; the two chains are linked by at least one disulfide bond. The heavy chain contains a potential N-terminal myristoylation site, a potential signal anchor sequence near the amino terminus, and six structural motifs that are found in otherwise unrelated proteins. These domains resemble motifs of the LDL receptor (two copies), complement component Clr (two copies), the metalloprotease meprin (one copy), and the macrophage scavenger receptor (one copy). The enterokinase light chain is homologous to the trypsin-like serine proteinases. These structural features are conserved among human, bovine, and porcine enterokinase. By Northern blotting, a 4.4 kb enterokinase mRNA was detected only in small intestine. The enterokinase gene was localized to human chromosome 21q21 by fluorescence in situ hybridization.

Amino Acid Sequence↗
PubMed1999

Activity-stability considerations of trypsinogen during spray drying: effects of sucrose.

The preparation and processing of protein pharmaceuticals into powders may impose significant stresses that could perturb and ultimately denature them. In many cases their stabilization through added excipients is necessary to yield native and active proteins. In this study, the effect of spray drying on the structure and activity of a model protein (trypsinogen) was investigated. In the absence of excipients, spray drying resulted in small losses of its enzymatic activity. Protein conformational rearrangements in the solid state (observed via FTIR) and irreversible aggregation (upon reconstitution) constituted the major degradation pathways. The irreversible unfolding in the solid state was also confirmed by solution calorimetric studies that indicated a decreased thermal stability of the spray-dried protein after reconstitution. The presence of sucrose, a thermal and dehydration stress stabilizer, induced a concentration-dependent protective effect. Protein protection was afforded even at low carbohydrate concentrations, while at specific mass ratios (sucrose-to-protein = 1:1) complete activity preservation was achieved. However, at the high end of sucrose concentrations, a small destabilization was evident, indicating that excluded volume effects may be undesirable during preparation of protein microparticles via spray drying. The profile of both the protein conformational changes and thermal stability in the solid state closely followed that of the incurred activity losses, indicating that protein stabilization during dehydration is crucial during processing of these polypeptides.

Chemistry, Pharmaceutical↗