Identification of 'cystic fibrosis protein' as a complex of two calcium-binding proteins present in human cells of myeloid origin.

Cystic fibrosis protein is a serum protein characterized by a pI close to 8.4 and present with a higher concentration in serum and plasma of cystic fibrosis carriers than in controls. This protein was found immunologically indistinguishable from the cystic fibrosis antigen isolated from granulocytes and presenting a sequence analogous to that of MRP-8, a calcium-binding protein expressed in the myeloid cell lineage. Using antibodies directed against MRP-8 and its closely associated calcium-binding protein, MRP-14, we demonstrate here that cystic fibrosis protein purified from serum is a complex of the two proteins MRP-8 and MRP-14.

Isolation and characterization of colipase from porcine and human pancreatic juice by immunoaffinity chromatography.

Pure colipase was prepared by immunoaffinity chromatography from porcine and human pancreatic juice. A single form of the porcine colipase was obtained, having the structural and biological properties of previously characterized porcine procolipase A. Two forms of activated colipase (N-terminal Gly) were isolated from human pancreatic juice by the same procedure. The existence of two forms of activated colipase might arise from rapid activation of a precursor form of human colipase during collection of the pancreatic juice.

Evidence for a functional role of the cholecystokinin-B/gastrin receptor in the human fetal and adult pancreas.

Gastrin (G) and cholecystokinin (CCK) are gastrointestinal neuropeptides that are released into circulation during a meal. G is also transiently expressed during embryogenic and early ontogenic development of the pancreas and is believed to act on islet-cell development. Both peptides act on pancreatic endocrine function; however, the effects are dependent on the species and on cellular and molecular underlying mechanisms that remain poorly characterized. Since CCK-B/G subtype receptor is predominant over the CCK-A subtype in the human pancreas, we hypothesized that it could be expressed by islet cells. Here we present reverse transcription-polymerase chain reaction and immunohistochemistry data demonstrating that the CCK-B/G receptor is expressed in islet cells and that islet glucagon-producing cells are the major site of CCK-B/G receptor expression in adult and fetal pancreas. Moreover, G immunoreactivity was detected in the fetal human pancreas at embryogenic week 22. G- and CCK-stimulated glucagon are released from purified human islets. Concentration of CCK and G eliciting a half-maximal level of glucagon secretion were 13 +/- 6 and 8 +/- 5 pmol/l, respectively. Maximal glucagon secretion was achieved in the presence of 30 pmol/l peptides and was similar to that obtained in the presence of 10 mmol/l L-arginine (1.6 pmol x ml(-1) x 90 min(-1)). The nonpeptide antagonist of the CCK-B/G receptor, RPR-101048, fully inhibited CCK- and G-stimulated glucagon secretion at 100 nmol/l concentration. These data are consistent with the view that the CCK-B/G receptor is involved in glucose homeostasis in adult humans and mediates the autocrine effects of G on islet differentiation and growth in the fetal pancreas.

Crystal structure of human trypsin 1: unexpected phosphorylation of Tyr151.

The X-ray structure of human trypsin 1 has been determined in the presence of diisopropyl-phosphofluoridate by the molecular replacement method and refined at a resolution of 2.2 A to an R-factor of 18%. Crystals belong to the space group P4, with two independent molecules in the asymmetric unit packing as crystallographic tetramers. This study was performed in order to seek possible structural peculiarities of human trypsin 1, suggested by some striking differences in its biochemical behavior as compared to other trypsins of mammalian species. Its fold is, in fact, very similar to those of the bovine, rat and porcine trypsins, with root-mean-square differences in the 0.4 to 0.6 A range for all 223 C alpha positions. The most unexpected feature of the human trypsin 1 structure is in the phosphorylated state of tyrosine residue 151 in the present X-ray study. This feature was confirmed by mass spectrometry on the same inhibited sample and also on the native enzyme. This phosphorylation strengthens the outstanding clustering of highly negative or highly positive electrostatic surface potentials. The peculiar inhibitory behaviour of pancreatic secretory trypsin inhibitors of the Kazal type on this enzyme is discussed as a possible consequence of these properties. A charged surface loop has also been interpreted as an epitope site recognised by a monoclonal antibody specific to human trypsin 1.

Targeting of CFTR protein is linked to the polarization of human pancreatic duct cells in culture.

A relationship between targeting of the protein CFTR (Cystic Fibrosis Transmembrane conductance Regulator) and cellular polarization has been observed in various types of epithelial cells. However, there are no reports on this in human exocrine pancreatic cells, which are functionally altered in patients with cystic fibrosis. The expression of CFTR and its targeting to apical plasma membranes was investigated during growth and polarization of human ductal pancreatic cancerous Capan-1 cells. Despite their neoplastic origin, the cancerous pancreatic duct cells of the Capan-1 line secrete Cl- and HCO3- ions. We showed by electron microscopy, impregnation of cells with tannin and freeze-fracture that these cells become polarized during growth in culture, and are joined by tight junctions. The expression of CFTR and the various stages in its anchorage to membranes was followed using a specific polyclonal antibody, ECL-885, directed against a synthetic peptide mimicking one of the extracellular loops of CFTR. Qualitative and quantitative confocal microscopic studies showed that: (i) the expression of CFTR was constant during growth, irrespective of cellular conformation, (ii) the number of cells presenting CFTR anchored to membranes increased with time in culture, (iii) the rise in membrane-bound CFTR-immunoreactivity accompanied the polarization of the cells, (iv) CFTR anchored to plasma membranes was distributed regularly over the surface of non-polarized cells, but was localized only at the apical membranes of the polarized cells. Moreover, patch-clamp studies indicated the presence of few Cl- cAMP-dependent conductance CFTR channels on unpolarized cells, and a larger number of CFTR channels on the apical plasma membranes of polarized cells. These results indicated that the anchorage of a functional CFTR to the plasma membrane is progressive and occurs in step with polarization of these human pancreatic duct cells in culture. We suggest that the targeting of CFTR to the apical membranes is directly linked to the process of cellular polarization.

Characterization of trypsinogens 1 and 2 in two human pancreatic adenocarcinoma cell lines; CFPAC-1 and CAPAN-1.

Proteins with trypsin-like immunoreactivity (first detected by a specific immunoenzymatic assay) were isolated from CAPAN-1 and CFPAC-1 cell culture-conditioned media by chromatography on an immunoadsorbent prepared with a polyclonal antibody directed against trypsin 1. The adsorbed proteins were devoid of free trypsin activity but trypsin activity was present after enterokinase activation demonstrating that the immunoreactive trypsin present in cell supernatants corresponds to trypsinogens. When characterised by Western blotting using a monoclonal antibody directed against human trypsin 1 two protein bands corresponding to trypsinogen 1 (23 kDa) and trypsinogen 2 (25 kDa) gave a positive reaction. These results demonstrate the presence of trypsinogens 1 and 2 in CAPAN-1 and CFPAC-1 cells and in their culture-conditioned media.

Direct double antibody sandwich immunoassay for Pseudomonas aeruginosa elastase.

A direct sandwich enzyme immunoassay was developed in order to quantify Pseudomonas aeruginosa elastase. As a solid phase the wells of a microtitre plate were coated with specific IgG and horseradish peroxidase labelled IgG was used as the second antibody. The detection limit of the assay was 0.26 ng/ml and a good agreement was found with elastolytic activity determined using elastin-Congo red. This assay was simple, specific, sensitive and reproducible, and permits the determination of low levels of elastase.

Overexpression of the reg gene in non-obese diabetic mouse pancreas during active diabetogenesis is restricted to exocrine tissue.

We demonstrated pancreatic reg gene overexpression in non-obese diabetic (NOD) mice during active diabetogenesis. The aim of this study was to determine in which part of the pancreas (endocrine and/or exocrine) the gene(s) and the protein(s) were expressed and if their localization changed with progression of the disease. In situ hybridization analysis and immunocytochemical studies were carried out on pancreas of female and male NOD mice. Both develop insulitis but diabetes develops only in females and in males only when treated by cyclophosphamide. Our results show that whatever the age, sex, and presence of insulitis and/or diabetes, the expression of reg mRNAs and of the corresponding protein(s) was restricted to exocrine tissue. Moreover, reg remains localized in acinar cells in the two opposite situations of (a) cyclophosphamide-treated males in a prediabetic stage presenting a high level of both insulin and reg mRNAs, and (b) the overtly diabetic females with no insulin but a high level of reg mRNA. These findings suggest that overexpression of the reg gene(s) might represent a defense of the acinar cell against pancreatic aggression.

Immunocytochemical demonstration of a pancreatic secretory protein of unknown function in human duodenum.

We have previously isolated from human pancreatic juice a secretory glycoprotein of 19 KD (P19), devoid of known enzymatic activity. P19 gave by proteolysis a protein of 14 KD (P14), at first named protein X and also called pancreatic thread protein or pancreatic stone protein. Specific rabbit immunosera prepared against P19 and P14 were applied to localize these proteins in human small intestine. By comparison, antibodies directed against some human pancreatic enzymes (amylase, lipase, chymotrypsin, trypsinogen 1, trypsinogen 2, and trypsin 1) were also tested. Positive immunoreactivity was observed on Paneth cells with antisera directed against trypsinogens, trypsin 1, and P19-related proteins. In addition, antisera directed against P19-related proteins stained the columnar cells located in the crypts of Lieberkühn. These original findings are a further indication of the resemblance between Paneth and pancreatic acinar cells but show that their functional analogy is only partial. On the other hand, the presence of P19-related proteins on non-mature columnar cells suggests that this differential distribution is a consequence of differentiation.

Expression of REG protein during cell growth and differentiation of two human colon carcinoma cell lines.

We localized REG protein in Paneth cells and nonmature columnar cells of the human small intestinal crypts and speculated that this protein was associated with growth and/or differentiation. The aim of this study was to determine whether REG protein is present in two human colon cancer cell lines that exhibit enterocytic differentiation after confluence and to investigate changes in the level of its expression during growth and differentiation. Results were compared to those obtained on cells that remain undifferentiated. Western immunoblotting and immunofluorescence demonstrated the presence of REG protein in the three cell lines. With the antisera against human REG protein, the staining was diffusely spread throughout the cytoplasm at Day 2, and after Days 3-4 it appeared to have migrated to cell boundaries. After confluence, we observed only a punctate staining array along cell boundaries, which disappeared at Day 15. REG mRNA expression was demonstrated by RTPCR and REG mRNA hybridization until Day 13, but not after, in the three cell types. REG protein may be involved in cellular junctions. Its presence appears to be associated with the cell growth period and the protein must be downregulated when growth is achieved and differentiation is induced.

Sexual dimorphism of pancreatic gene expression in normal mice.

A differential pancreatic behavior observed between male and female mice in diabetes and pancreatitis led us to study the gene and protein expressions of endocrine and exocrine pancreatic proteins in normal mice. We compared the levels of expression of six pancreatic genes and of four of the corresponding proteins in male and female mice OF1. Amylase gene expression was found to be significantly higher in females than in males, whereas trypsinogen and lipase gene expression were significantly lower. For chymotrypsinogen, reg, and insulin the differences were not significant. This sexual dimorphism did not exist in rat pancreas, where no gender difference was observed. After characterization of mice enzymes by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and antibodies directed to the closely related human pancreatic enzymes, we have compared the levels of these proteins in mice pancreatic homogenates. No significant difference was observed between males and females at the level of protein expression. These data suggest a hormonal sexual difference in the regulation of pancreatic protein synthesis at the pre- and posttranscriptional levels in normal mice, which may play a role in the development of mice pancreatic diseases.

Reg protein: a potential beta-cell-specific growth factor?

Beta-cell regeneration in adult pancreas is usually considered to be limited. However, various animal models suggest that this tissue is still capable of regeneration under certain conditions. Reg protein could be responsible for this replicative process. The reg gene codes for a 166 amino-acid protein usually synthesized and secreted by pancreatic acinar cells but expressed in islet beta cells during experimental regenerative processes in animals (90% pancreatectomy + nicotinamide, or insulinoma tumor removal in rats, or the "wrapping pancreas model" in the hamster). In addition, recombinant rat reg protein can stimulate beta-cell replication in vivo and in vitro. In animal models of Type 1 diabetes mellitus, reg gene overexpression occurs during active phases of diabetogenesis and could be a defence mechanism. During human pancreatic development, reg gene is expressed at an early stage but is not associated with the expression of other pancreatic genes. Conversely, gene expression for reg and insulin are correlated in adult pancreas. Accordingly, reg protein could be a beta-cell-specific growth factor implicated in the maintenance of beta-cell mass, especially in adult pancreas.

Human pancreatic reg protein immunoenzymatic assay and molecular form in serum.

A direct sandwich immunoassay was developed to quantify the human reg protein, a non enzymatic pancreatic acinar protein the biological function of which remains elusive. Polystyrene balls were coated with specific IgG fraction as the first antibody and horseradish peroxidase labelled IgG was used as a second antibody. The linearity of the assay was good over a concentration range of 1.25-100 ng/ml and the good parallelism obtained between the standard and the assay dilution curves in serum and pancreatic juice indicates the absence of non-specific interfering reactions. Gel filtration of serum showed that the reg protein was eluted in the fractions corresponding to the proteins of around 25 kDa and that the chromatographic behaviour of the serum protein was identical to that of the purified pancreatic protein when added to serum. This assay was simple, specific, sensitive and reproducible and may permit the determination of low levels of reg protein in different biological fluids.

Correlation between the cystic fibrosis protein detected by isoelectric focusing and a protein of 12 kDa detected by SDS-polyacrylamide gel electrophoresis.

We have studied both by isoelectric focusing and polyacrylamide gel electrophoresis in the presence of SDS, sera of individuals homozygous (25) and heterozygous (26) for cystic fibrosis and compared them to controls (13). As in our first study [1], the protein with a pI value of 8.4 called 'cystic fibrosis protein' or CFP, was found in about 70% of homozygous and carriers and in 15% of controls. When the same sera were analysed by polyacrylamide gel electrophoresis in the presence of SDS and after staining with Coomassie Blue, an additional protein with a molecular weight close to 12,000 (P12) was present in most of the sera containing CFP, suggesting a close relationship between the two proteins. By increasing the sensitivity of staining by using silver nitrate, P12 was detected with variable intensity in almost all sera of homozygotes and heterozygotes and at the level of traces in all normal sera. These data suggest that P12, like CFP, would be a normal serum protein quantitatively increased in affected subjects.

Direct double antibody sandwich immunoassay of mucin M1 epitopes in human mucus secreting pancreatic cell lines.

A direct sandwich enzyme immunoassay using two monoclonal antibodies was developed in order to quantify mucin M1 antigens produced by two pancreatic adenocarcinoma cell lines: CAPAN-1 and CFPAC-1. As a solid phase, the wells of a microtiter plate were coated with a first monoclonal antibody, 1-13 M1 and the biotinylated monoclonal conjugate 9-13 M1 was used as the second antibody. The assay was optimized with streptavidin-peroxidase. The detection limit of the assay is 1.6 ng/ml. This ELISA is highly specific, sensitive, reproducible and quickly performed. It will permit the comparison of mucin exocytosis by the two cell lines in response to secretagogue agents and may help in the study of the pathogenesis of mucus hypersecretion such as cystic fibrosis.

Plasma and serum lactoferrin levels in cystic fibrosis. Relationship with the presence of cystic fibrosis protein.

Plasma lactoferrin concentrations were measured in blood of cystic fibrosis patients, heterozygotes and controls using a specific and sensitive enzyme immunoassay. 67 plasmas were studied (26 controls, 23 heterozygotes, 18 cystic fibrosis patients) and the results showed a statistically significant increase (p less than 0.05) of the level of plasma lactoferrin in cystic fibrosis patients (265 +/- 224 micrograms/l) compared to controls (168 +/- 100 micrograms/l) and heterozygotes (150 +/- 72 micrograms/l). Since it is well established that plasma lactoferrin level could be influenced by the number of neutrophils, a second set of experiments was performed on 20 cystic fibrosis patients on whom leukocyte counts were also made. When the 15 plasmas with normal neutrophils (in the range 2 to 6 giga/l) were considered, the mean lactoferrin level was 318 +/- 116 micrograms/l, still far above the normal values. For serum, a similar significant increase of lactoferrin concentration was observed in 33 cystic fibrosis patients (610 +/- 551 micrograms/l) compared to the values observed for 25 controls (237 +/- 155 micrograms/l) and 37 heterozygotes (272 +/- 231 micrograms/l). Cystic fibrosis protein (CFP) was identified in the same sera by isoelectric focusing and the intensity of the band was closely related to the increase of lactoferrin concentration in cystic fibrosis patients. In contrast, no difference in serum lactoferrin concentrations was observed between heterozygotes with or without CFP, indicating that the increased CFP concentration cannot be due only to altered granulocyte function.

A specific immunoradiometric assay of cationic trypsin(ogen) that does not recognize trypsin-alpha-1-proteinase inhibitor complex.

An immunoradiometric assay using two monoclonal antibodies directed to human trypsin 1 was developed for measuring trypsin(ogen) in biological fluids. The assay is different from other assays in that it is specific for cationic trypsinogen and does not recognize the alpha-1-proteinase inhibitor-trypsin complex. It can be used as a complement to classical immunoassays to characterize trypsinogen activation in pathological cases. The evaluation and the specificity of the assay are presented.

Exocrine pancreatic function in cystic fibrosis.

Pancreatic dysfunction in cystic fibrosis (CF) begins in utero and, at birth, in most cases, cystic fibrosis is characterized by an elevated level of serum immunoreactive trypsin (IRT). If most patients with CF typically present insufficient pancreatic exocrine function, 10-15% of CF patients have pancreatic sufficiency and this status is genetically determined by one or two 'mild' mutations in CF transmembrane conductance regulator (CFTR). However, with age, these patients can develop pancreatic insufficiency.

Production and characterization of two monoclonal antibodies to human pancreatic trypsin 1.

Two monoclonal antibodies (MAb) G6 and A8 directed against human trypsin 1 have been produced by hybridization of myeloma cells with spleen cells of OF1 immunized mice. Antibodies were screened by radioimmunoassay. Monoclonal antibodies were purified by affinity chromatography on Protein A-Sepharose, and we found that MAb G6 was of the IgG2b class and MAb A8 of the IgG2a class. Both MAbs had a high affinity for trypsin 1 with the respective affinity constants equal to 1.3 x 10(8) l/mol for G6 and 3.2 x 10(7) l/mol for A8. Epitope specificity was studied by western blotting, using human trypsinogens 1 and 2. Both MAbs gave a positive reaction with native trypsinogen 1 and no reaction with the same protein after reduction. Only MAb G6 reacted with trypsinogen 2 in the native form. Its affinity for trypsin 2 was found similar to that for trypsin 1 with a constant equal to 2.7 x 10(7) l/mol. Both antibodies appeared directed against conformational and not sequential epitopes.