Human Pancreatic Secretory Trypsin Inhibitor Research Articles

Pancreatic secretory trypsin inhibitor gene is highly expressed in the liver of adult-onset type II citrullinemia

Deficiency of argininosuccinate synthetase (ASS) causes citrullinemia. Type II citrullinemia is found in most patients with adult-onset citrullinemia in Japan, and ASS is deficient specifically in the liver. Previous studies have shown that the decrease of hepatic ASS activity is caused by a decrease in enzyme protein with normal kinetic properties and that there are no apparent abnormalities in the amount, translational activity, and nucleotide sequence of hepatic ASS mRNA. Recent results of homozygosity testing indicate that the primary defect of type II citrullinemia is not within the ASS gene locus. In this present work, to understand the pathogenesis and pathophysiology of type II citrullinemia, we have characterized the alterations of gene expression in the liver of type II patients using the recently developed mRNA differential display method. Some cDNA bands expressed differently in type II citrullinemia patients and control were selected, cloned, and sequenced. Nucleotide sequence analysis and homology searching revealed an interesting clone which has 99% homology with the human pancreatic secretory trypsin inhibitor (hPSTI). Northern blot and RT-PCR analyses showed that the expression of hPSTI mRNA increased significantly in the liver of all type II patients tested. Furthermore, the concentration of hPSTI protein was found to be higher in the liver of type II citrullinemia than in control. These results suggest that hPSTI may be related to the primary defect of type II citrullinemia and may be useful as a diagnostic marker, although the detailed mechanism of the high expression of hPSTI mRNA in type II liver is not yet known.

Highly effective protease inhibitors from variants of human pancreatic secretory trypsin inhibitor (hPSTI): an assessment of 3-D structure-based protein design.

The results of a protein design project are used to compare different predictive strategies with respect to protein-protein interactions. We have been able to generate variants of human pancreatic secretory trypsin inhibitor (hPSTI) optimized with respect to the affinity and specificity for human leukocyte elastase relative to trypsin and chymotrypsin, and in particular chymotrypsin. The extremely strong and specific human leukocyte elastase inhibitors were thus developed in three rounds of mutagenesis and two rounds of 3-D modelling; only 24 variants in total were synthesized, although variations at seven different amino acid positions were involved (i.e. from 20(7) possible variants). An excellent elastase inhibitor could be designed with the minimum of two amino acid exchanges. The value of structural modelling and actual structure determination is discussed in the light of the experimental results of the designed protein variants and the results of tertiary structure determinations of the free variant and the inhibitor-protease complex. Particular reference is given to the strategy to be followed in protein design projects in general and to the development of protease inhibitors in particular.

Human Pancreatic Secretory Trypsin Inhibitor in the Assessment of the Severity of Acute Pancreatitis

We assessed the ability of serum human pancreatic secretory trypsin inhibitor (hPSTI) to establish the severity of acute pancreatitis and compared it in this respect to that of serum C-reactive protein (CRP). Of 26 patients studied with acute pancreatitis, 16 had mild pancreatitis, and 10, severe disease. Initial studies were performed at onset of the disease in 20 patients, on the second day of illness in two, and on the third day of illness in the remaining four. In all, serum hPSTI and CRP concentrations were determined on admission and daily for the following 5 days using commercial kits; Ranson's score was evaluated within the first 48 h of admission. Sixty-three healthy subjects and 31 patients with nonpancreatic acute abdomen were also studied. Values of 70 ng/ml for serum hPSTI and 10 mg/dl for serum CRP were taken as limits to distinguish severe from mild-to-moderate acute pancreatitis. When assessed within the first 24 h of pain, serum hPSTI correctly classified 71% of the patients with severe acute pancreatitis, whereas serum CRP did so for 29%. In subsequent days, the two markers showed a similar sensitivity in predicting severe acute pancreatitis. Serum hPSTI and CRP were alike in excluding a diagnosis of severe acute pancreatitis. Ranson's score correctly identified 50% of patients with severe illness and 63% of patients with mild pancreatitis. This study indicates that, when assessed within 24 h of pain onset, serum hPSTI is a better predictor of the severity of acute pancreatitis than serum CRP or Ranson's criteria.

Overexpression of pancreatic secretory trypsin inhibitor in pancreatic cancer

Four clones of pancreatic secretory trypsin inhibitor (PSTI)-overexpressing cells (TF-PANC clones 1, 6, 8, and 36) were established to evaluate the physiological function of PSTI secreted by cancer cells, by means of introducing a PSTI-expression vector (pRSV-PSTI) into the human pancreatic adenocarcinoma cell line (PANC-1). No obvious changes were observed in the histological features of these transplanted tumors in nude mice, in the growth of TF-PANC and PANC-1, or that of 3T3 fibroblasts when cocultured with them. Addition of recombinant human PSTI to the cultured media resulted in no increase in proliferation of fibroblasts (3T3 and WI-38) or of four pancreatic cancer cell lines (PANC-1, CAPAN-I, MIAPaCa-2, and Hs766T). These results suggest that the estimation of tumor-bearing PSTI as a paracrine or autocrine growth factor in recent studies should be given careful consideration.

Solution Structure of a Variant of Human Pancreatic Secretory Trypsin Inhibitor Determined by Nuclear Magnetic Resonance Spectroscopy

The structure of a variant of human pancreatic secretory trypsin inhibitor (PSTI) has been determined by 1H nuclear magnetic resonance (n.m.r.) spectroscopy and a combination of distance geometry and molecular dynamics simulations. After complete assignment of the 1H signals, the nuclear Overhauser data imply the existence of a rather well-determined tertiary structure stabilized by a central α-helix and a short three-stranded β-sheet. The tertiary structure of the amino terminus and of the loop 11-17 could not be defined by n.m.r. data, suggesting a high flexibility in these areas. As the crystal structures of two complexes of human PSTI variants and that of an uncomplexed variant are also known a comparison of the PSTI tertiary structure in solution and in the crystal is now possible.

Quantification of pancreatic secretory trypsin inhibitor in colonic carcinoma and normal adjacent colonic mucosa.

To measure the content of immunoreactive human pancreatic secretory trypsin inhibitor (irPSTI) in colonic carcinoma and adjacent normal colonic mucosa. From a stable hybridoma cell line producing monoclonal antibodies specific for human PSTI, a specific enzyme linked immunosorbent assay (ELISA) for human PSTI was developed. In a precipitation assay system these antibodies bound human PSTI in a dose-dependent manner. The specimens were obtained from resectional surgery. The content of irPSTI was 19.9 micrograms/g protein (0.55 micrograms/g tissue wet weight) in colonic carcinoma. In adjacent normal colonic mucosa 43.6 micrograms/g protein (1.12 micrograms/g tissue wet weight) was shown. The enzymatic degradation of surrounding tissue necessary for tumour cell invasion could be facilitated by this relative deficit of the inhibitor in infiltrative carcinoma.

Characterization of recombinant human and rat pancreatic phospholipases A 2 secreted from Saccharomyces cerevisiae: Difference in proteolytic processing

An expression plasmid for human pancreatic phospholipase A 2 in Saccharomyces cerevisiae was constructed by insertion of cDNA encoding its preprophospholipase A 2 into a yeast expression vector pAM82. The resulting product secreted in the yeast culture medium was mainly prophospholipase A 2, which was the same as the natural proenzyme in all aspects examined, including the higher order structure. However, when the rat preprophospholipase A 2 cDNA was manipulated in the same manner, the active phospholipase A 2 of the intact mature form was secreted with the proenzyme being hardly detected in the medium. This unexpected favorable result would occur due to cleavage of rat phospholipase A 2 pro-peptide by a trypsin-like proteinase in S. cerevisiae. Based on this finding, we constructed a plasmid carrying the sequence coding for the prepro-peptide of rat pancreatic phospholipase A 2 behind the PHO5 promoter in the pAM82 vector, which leads to the secretion of heterologous proteins as their mature form. The use of this plasmid led to secretion of biologically active human pancreatic secretory trypsin inhibitor and a glutamic acid-specific endopeptidase from Staphylococcus aureus ATCC 12600, which are eukaryote and prokaryote proteins, respectively, in the culture medium of S. cerevisiae.

Measurements of exocrine proteins in the pig pancreas using microdialysis.

The authors adapted microdialysis for the study of intrapancreatic exocrine proteins. Immunoreactive cationic and anionic trypsinogen were continuously measured after the insertion of one probe inside the pig pancreas and another placed under the peritoneum on the anterior surface of the gland. The concentration initially declined in the dialysate from the intrapancreatic probe, followed by an increasing tendency until 120 minutes after the insertion, after which a decline to a relative steady state after four to six hours was observed. A dialysate with a lower concentration of enzymes was obtained from the probe on the pancreatic surface. In order to study the turnover of an exogenously administered substance, a microdialysis probe was inserted into the pancreas and human recombinant pancreatic secretory trypsin inhibitor was injected into the pancreatic duct. The inhibitor was cleared from the pancreas with an intrapancreatic half-life of approximately 45 minutes. Membrane function, measured in vitro before and after use in vivo, was not significantly affected after seven hours in the pig pancreas.

Is human pancreatic secretory trypsin inhibitor a cholecystokinin (CCK)-releasing-peptide in man?

Is human pancreatic secretory trypsin inhibitor a cholecystokinin (CCK)-releasing-peptide in man?

Three-dimensional structure of a recombinant variant of human pancreatic secretory trypsin inhibitor (Kazal type)

A modified version of the human pancreatic trypsin inhibitor (PSTI), generated in a protein-design project, has been crystallized in spacegroup P4 3 with lattice constants a = 40.15 A ̊ , c = 33.91 A ̊ . The structure has been solved by molecular replacement. Refinement of the structure by simulated annealing and conventional restrained leastsquares yielded for 8.0 to 2.3 Å data a final R-value of 19.1%. Differences to the known structures of porcine PSTI complexed with trypsinogen and modified human PSTI complexed with chymotrypsinogen occur at the flexible N-terminal part of the molecule. These differences are influenced by crystal packing, as are low temperature factors for the binding loop. The geometry of the binding loop is similar to the complexed structures.

Secretion of correctly processed and folded pancreatic secretory trypsin inhibitor by Bacillus subtilis

We constructed a plasmid, designated pNPP126, containing a DNA sequence encoding a fusion protein composed of Bacillus amyloliquefaciens neutral protease prepeptide (signal peptide) and human pancreatic secretory trypsin inhibitor (hPSTI), where the mature hPSTI is accurately fused to the 3′-terminal of the prepeptide coding region. It was observed that the strain Bacillus subtilis MT600 harboring pNPP126 could secrete a trypsin inhibitory activity into the culture medium. The N-terminal amino acid sequence, the amino acid composition and the stoichiometry of the purified hPSTI produced by B. subtilis were the same as those of natural hPSTI, indicating that the transformant B. subtilis MT600 (pNPP126) could efficiently secrete the correctly processed and folded hPSTI into the culture medium.

Changes in plasma and duodenal cholecystokinin concentrations after pancreatic duct occlusion in rats

The changes in plasma and duodenal cholecystokinin (CCK) concentrations after pancreatic duct occlusion were examined in rats. The rats were sacrificed 1, 3, 7, 10, 14, and 30 days after occlusion of the duct. Histological examination showed acute inflammation on days 1 and 3 after duct occlusion, interstitial fibrosis and regenerative changes on days 7, 10, and 14, and pancreatic atrophy on day 30. The plasma CCK concentration increased from 0.45 pM to 2.0 pM after the occlusion and then remained high throughout the observation period. In contrast to the stable increase in plasma CCK concentration, the CCK content in the duodenum increased on days 1 and 3, decreased on day 7, increased on day 10, reaching over the control level on day 14, and then returned to the control level on day 30. Administration of boiled and 10-fold concentrated rat pancreatic juice or human pancreatic secretory trypsin inhibitor for seven days after pancreatic duct occlusion reversed the decrease in duodenal CCK content. The major molecular forms of duodenal CCK were CCK-8, -33, and -58. These results indicate that (1) basal plasma CCK concentration did not reflect the duodenal CCK content, (2) duodenal CCK content was well correlated with a decrease in inflammation in the pancreas, and (3) a nonenzymatic component in the pancreatic juice reversed the decrease in duodenal CCK content and body weight caused by pancreatic duct occlusion.

Protective Effect of Human Pancreatic Secretory Trypsin Inhibitor on Cerulein-lnduced Acute Pancreatitis in Rats

We examined the protective effect of human pancreatic secretory trypsin inhibitor (PSTI), a specific trypsin inhibitor secreted from pancreatic acinar cells into the pancreatic duct, on cerulein-induced acute pancreatitis in conscious rats. The protective effect of human PSTI-RS, an analogue of PSTI with Arg-44 to Ser substitution which has a longer half-life in vitro, was also examined. Intraperitoneal administration of a pharmacological dose of cerulein to conscious rats induced acute pancreatitis, characterized by light microscopy as cellular disorganization of the acini and interstitial edema. Intravenous infusion of human PSTI (10, 50 or 250 micrograms/rat/h) into rats with cerulein-induced acute pancreatitis decreased their pancreatic wet weight and plasma amylase concentration. It also caused a dose-dependent decrease in vacuoles in acinar cells and interstitial edema. Human PSTI-RS, which has a longer half-life in vivo, was more effective than native PSTI at the same dose rate (10 micrograms/rat/h) in reducing pancreatitis. These results suggest that human PSTI may have a beneficial effect on acute pancreatitis.

Three-dimensional structure of the complexes between bovine chymotrypsinogen A and two recombinant variants of human pancreatic secretory trypsin inhibitor (Kazal-type)

Variants of the human pancreatic secretory trypsin inhibitor (PSTI) have been created during a protein design project to generate a high-affinity inhibitor with respect to some serine proteases other than trypsin. Two modified versions of human PSTI with high affinity for chymotrypsin were crystallized as a complex with chymotrypsinogen. Both crystallize isomorphously in space group P4 12 12 with lattice constants a = 84.4 A ̊ , c = 86.7 A ̊ and diffract to 2·3 Å resolution. The structure was solved by molecular replacement. The final R-value after refinement with 8·0 to 2·3 Å resolution data was 19·5% for both complexes after inclusion of about 50 bound water molecules. The overall three-dimensional structure of PSTI is similar to the structure of porcine PSTI in the trypsinogen complex (1TGS). Small differences in the relative orientation of the binding loop and the core of the inhibitors indicate flexible adaptation to the proteases. The chymotrypsinogen part of the complex is similar to chymotrypsin. After refolding induced by binding of the inhibitor the root-mean-square difference of the active site residues A186 to A195 and A217 to A222 compared to chymotrypsin was 0·26 Å.

A new method for building protein conformations from sequence alignments with homologues of known structure

We describe a largely automatic procedure for building protein structures from sequence alignments with homologues of known structure. This procedure uses simple rules by which multiple sequence alignments can be translated into distance and chirality constraints, which are then used as input for distance geometry calculations. By this means one obtains an ensemble of conformations for the unknown structure that are compatible with the rules employed, and the differences among these conformations provide an indication of the reliability of the structure prediction. The overall approach is demonstrated here by applying it to several Kazal-type trypsin inhibitors, for which experimentally determined structures are available. On the basis of our experience with these test problems, we have further predicted the conformation of the human pancreatic secretory trypsin inhibitor, for which no experimentally determined structure is presently available.

Immunohistochemical demonstration of pancreatic secretory trypsin inhibitor in normal and neoplastic colonic mucosa.

Specimens of normal and neoplastic colonic mucosa from 52 patients were analysed by immunohistochemistry using a monospecific polyclonal antiserum against human pancreatic secretory trypsin inhibitor (PSTI). In normal colonic mucosa PSTI was found in the goblet cells in the basal parts of the crypts. In adenomas of tubular, villous, and tubulo-villous types PSTI was also found in the upper parts of the polyps, usually occurring in the regeneration zone. There was a more intense staining reaction in polyps with increased atypia. Carcinomas of different types and of various grades of differentiation and of in situ type did not contain PSTI. These findings indicate that PSTI could be a marker for adenomatous rather than carcinomatous epithelium in the colon. Furthermore, the absence of the inhibitor in malignant cells might facilitate tissue invasion by malignant cells because of deficient protease inhibition.

Identification and characterization of receptors specific for human pancreatic secretory trypsin inhibitor.

Specific binding sites for human pancreatic secretory trypsin inhibitor (PSTI) on 3T3 Swiss albino cells were studied using radioiodinated recombinant PSTI. Some ion species, pH, and temperature significantly influenced the binding of 125I-PSTI. Kinetic studies showed that the binding of 125I-PSTI to 3T3 Swiss albino cells reached the maximum level within 120 min at 4 degrees C, with a slow dissociation rate. The half-maximal inhibition (ID50) of 125I-PSTI binding by unlabeled PSTI occurred at 1.0 x 10(-10) M. On Scatchard analysis of the competitive binding data, linear plots indicated a single class of receptors with high affinity (Kd = 5.3 x 10(-10) M) on 3T3 Swiss albino cells, the number of receptors being 5,400 per cell. Treatment of surface-bound radiolabeled PSTI with a chemical crosslinker (disuccinimidyl suberate) led to the identification of a membrane polypeptide of Mr 140,000 to which PSTI was crosslinked. The formation was inhibited by an excess amount of unlabeled PSTI in a dose-dependent manner. The binding of 125I-PSTI to 3T3 Swiss albino cells was competitively inhibited by unlabeled PSTI but not by other peptide hormones, such as epidermal growth factor (EGF), bovine fibroblast growth factor, insulin-like growth factor, transforming growth factor alpha, platelet-derived growth factor, and tumor necrosis factor, indicating the presence of receptors specific for PSTI. Various protease inhibitors had no or only a little effect, and mercaptoethanol and dithiothreitol strongly decreased the binding of 125I-PSTI. Incubation at 37 degrees C resulted in rapid internalization of cell-bound 125I-PSTI, followed by the appearance of trichloroacetic acid-soluble 125I-radioactivity in the culture medium, due to degradation of internalized PSTI. In addition, PSTI stimulated [3H]thymidine incorporation into DNA on 3T3 Swiss albino cells in a dose-dependent manner. The combined addition of PSTI and EGF stimulated [3H]thymidine incorporation to an extent greater than that seen with either agent alone. These results indicated that the biological effect of PSTI was mediated by high affinity plasma membrane receptors, which were not a cell-surface proteinase(s). Specific binding of 125I-PSTI was noted with the following cells: WI-38, 3T3 Swiss albino, HUVE, BDC-1, and H4-II-E-C3.

Reevaluation of serum pancreatic secretory trypsin inhibitor (PSTI) as a diagnostic marker with monoclonal antibody-based tow-site enzyme immunoassay.

After reexamining the validity of our EIA method for human pancreatic secretory trypsin inhibitor (PSTI), we measured PSTI levels in sera of patients with various diseases. Patients with acute pancreatitis or chronic disease during recrudescence showed an elevated PSTI level in their blood circulation, whereas patients with chronic pancreatitis during remission showed values within the normal range. Alpha-amylase activity in serum, one of the important markers used in the diagnosis of pancreatic diseases, was not meaningfully correlated with PSTI level. Patients suffering from various tumors also showed an elevated level of serum PSTI. A curative resection of the pancreas decreased the serum PSTI level of the patient to below one half of the pre-operation value, indicating that PSTI in the circulation originated mostly from the pancreas. However, patients having had other tissues resected showed a transient elevation of PSTI levels in their sera. From these results, PSTI in the circulation would appear to be one of the acute-phase reactants delivered from unclarified tissue(s) and/or organ(s).

Site-directed mutagenesis of human pancreatic secretory trypsin inhibitor.

Arg-42 or Lys-43 or Arg-44 of human pancreatic secretory trypsin inhibitor (PSTI) was replaced by Thr or Ser by site-directed mutagenesis, and the inactivation rates of the mutants after mixing with human trypsin were compared with that of the natural form. The inactivation rate decreased for one mutant (Arg-44----Ser), whereas no change was observed for another (Arg-42----Thr) and an increase was observed for a third (Lys-43----Thr). Kinetic studies on the interactions between human trypsin and synthetic peptides, comprising the regions of Phe39-Ser47 of the respective PSTI species, showed that human trypsin cleaved the Arg42-Lys43 bond preferentially to the Arg44-Gln45 bond. However, it is cleavage of the latter bond that is thought to cause inactivation of human PSTI. These results suggest that the Arg44-Gln45 bond of human PSTI is responsible for its inhibitory activity, and inactivation of human PSTI is probably caused by deletion of the dipeptide Lys43-Arg44.

Membrane and Adsorption Processes in the Purification of the Human Pancreatic Secretory Trypsin Inhibitor (hPSTI)

AbstractThis paper describes a process for the purification of hPSTI from a recombinant Escherichia coli. The E. coli strain carries the hPSTI‐gene on a multicopy plasmid with the ompA‐leader‐sequence, which results in the secretion of hPSTI into the medium. The separation of the cells and the prepurification were carried out either by ultrafiltration alone, by a combination of cross‐flow microfiltration and ultrafiltration, or by a combination of flocculation and ultrafiltration. The ultrafiltration was followed by three chromatographic steps. After the final purification step hPSTI was homogeneous as judged by SDS‐PAGE (silver stain). Process scale up should be relatively simple.