Lamb pregastric lipase (LPGL) was purified from pharyngeal tissues. The purification procedure was based on an aqueous extract containing 0.7% Tween 80 which was chromatographed on DEAE-cellulose anion-exchanger and adsorbed on HA-Ultrogel followed by gel filtration on Ultrogel AcA-54. The final enzymatic preparation, where the overall activity recovery was 3%, showed a single protein band on SDS-PAGE with a molecular mass of 50 kDa. LPGL is a glycoprotein containing approx. 14% (w/w) of carbohydrate. Extensive deglycosylation using peptide N-glycosidase F yielded a protein with an apparent molecular mass of 43 kDa. An uncontrolled proteolysis of LPGL during the purification lead to a 45 kDa form which was previously observed in human lysosomal acid lipase (HLAL) and rabbit gastric lipase (RGL). The labile bond X54-Leu55 was identified. Isoelectric focusing of LPGL reveals a major band corresponding to an isoelectric point of 4.8. The pure enzyme displayed specific activities of 950 U mg-1, 300 U mg-1 and 30 U mg-1 at pH 6.0, using tributyroylglycerol, trioctanoylglycerol and trioleoylglycerol as substrates, respectively. Using Western blot analysis, a cross-immunoreactivity of LPGL was observed with purified anti-human gastric lipase polyclonal antibodies. Determination of the amino-acid sequence of 62 residues revealed a high degree of homology with other known preduodenal lipases.

TWO sandwich enzyme linked immunosorbent assays (ELISA) were developed for evaluating the surface excess at the lipid/water interface of the human gastric lipase (HGL) and two anti-HGL monoclonal antibodies (mAbs). These assays were adapted to the monomolecular film technique used previously for measuring lipase kinetics. HGL and the two anti-HGL mAbs (4-3 and 218-13) were biotinylated without any significant loss of their biological activities occurring. They were further detected by ELISA using either anti-HGL or anti-mouse IgG polyclonal antibodies as specific captors before being revealed using a streptavidin-peroxidase conjugate as tracer. The detection limit was 25 and 85 pg in the case of HGL and mAb, respectively. By combining the above sandwich ELISA technique with the monomolecular film technique, it was possible for the first time to measure the enzymatic activity of HGL on 1,2-didecanoyl-sn-glycerol (dicaprin) monolayers as well as to determine the corresponding interfacial excess of the enzyme. The HGL turnover number increased steadily with the lipid packing. The specific activities determined on dicaprin films spread at 35 mN . m(-1) were found to be in the range of the values measured under optimal bulk assay conditions, using tributyrin emulsion as a substrate [i.e., 1000 mu mol/(min . mg of enzyme)]. At a given Lipase concentration in the water subphase, the interfacial binding of HGL to the nonhydrolyzable egg yolk phosphatidylcholine (egg PC) monolayers was found to be 10 times lower than that in the case of dicaprin monolayers. Given the low tensioactivity of the mAbs of the IgG isotype [Ivanova et al. (1993) Colloids Surf. BI, 17-22], we also investigated the effects of five anti-HGL mAbs (mAbs 4-3, 25-4, 35-2, 83-15, and 218-13) on the catalytic activity as well as on the interfacial binding of HGL to lipid/water interfaces. Four out of these five mAbs (mAbs 4-3, 25-4, 35-2, and 83-15) were found to significantly reduce the lipolytic activity of HGL. Moreover, three of the four inhibitory mAbs (mAbs 4-3, 25-4, and 35-2) were found to reduce the specific activity of HGL, while mAb 83-15 had no effect on the specific activity. These results clearly indicate that the latter mAb (83-15) complexed with HGL mainly affects the binding of the enzyme to the lipid/water interface, while the other three inhibitory mAbs (mAbs 4-3, 25-4, and 35-2) affect both the binding and the catalytic steps of HGL.

Several monoclonal antibodies (mAbs) were prepared against human pancreatic lipase (HPL). Two enzyme-linked immunosorbent assay (ELISA) procedures were set up for screening hybridomas producing specific antibodies. Four mAbs (81-23, 146-40, 315-25, and 320-24) of the IgG1 isotype were found to react with HPL in both simple sandwich and double sandwich ELISAs, while mAb 248-31, of the IgG2b isotype, reacted only with HPL in a double sandwich ELISA. The results of Western blot analysis carried out with native and SDS-denatured HPLs indicated that mAb 248-31 recognized only native HPL, while all the other mAbs recognized both forms of HPL. Since mAb 248-31 did not recognize SDS-denatured HPL, it was not possible to localize its epitope. To carry out epitope mapping along the primary sequence of HPL, four fragments (14, 26, 30, and 36 kDa) resulting from a limited chymotryptic cleavage of HPL were characterized by Western blotting as well as N-terminal amino acid sequence analysis. Of the above five anti-HPL mAbs, four (81-23, 248-31, 315-25, and 320-24) were found to inhibit the lipolytic activity of HPL (in both the presence and absence of bile salts and colipase), while mAb 146-40 had no inhibitory effects. The epitope recognized by mAb 146-40 was found to be located in the N-terminal domain (Lys1-Phe335). Combined immunoinactivation and epitope mapping studies showed that three inhibitory mAbs (81-23, 315-25, and 320-24) recognize overlapping epitopes from the hinge region between the N- and C-terminal domains of HPL, belonging to the 26-kDa fragment. In the presence of lipids, a significant decrease has been observed in the bending angle between the N- and C-terminal domains of the HPL tertiary structure (van Tilbeurgh, H., Egloff, M. P., Martinez, C., Rugani, N., Verger, R. and Cambillau, C. (1993) Nature 362, 814-820). From the present immunochemical data, we further propose that locking the hinge movement with mAbs may induce lipase immunoinactivation.

Human (HGL) and rabbit (RGL) gastric lipases were cleaved by trypsin and the resulting peptides were characterized. Exposure of HGL to trypsin led to the production of three identified fragments (H1, H2 and H3) resulting from cleavage sites at Lys-4 and Arg-229. Fragments H2 (Lys-4-Arg-229) and H3 (Glu-230-Lys-379) were derived from fragment H1 (Lys-4-Lys-379). The single disulfide bridge (Cys-236-Cys-244) of the molecule is localized in fragment H3. Out of the three cysteine residues conserved in all known gastric lipases, the free sulfhydryl group (Cys-227) was localized in fragment H2. Immunoblots, carried out with the tryptic fragments of HGL and anti-HGL mAbs, revealed that five inhibitory mAbs immunoreacted selectively with the N-terminal fragment H2, whereas two other non inhibitory mAbs immunoreacted exclusively with the C-terminal fragment H3. Trypsin also cleaved RGL at two sites (Arg-55 and Arg-229) leading to four identifiable fragments (R1, R2, R3 and R4). One cleavage site (Arg-229) was found to be identical in both RGL and HGL. We propose that this latter site is localized between the two domains of native gastric lipases.

Human gastric lipase (HGL) is the first lipolytic enzyme involved in the digestion of dietary lipids along the gastrointestinal tract. We describe an improved procedure for isolating the enzyme using immunoaffinity chromatography in combination with ion-exchange chromatography. The purified enzyme, showing a single band on SDS-PAGE, expressed a specific activity of 1000 U/mg using tributyrin as the substrate. We also describe a specific enzyme-linked immunosorbent assay (ELISA) procedure for measuring duodenal HGL levels. The ELISA was performed using an anti-HGL polyclonal antibody (pAb) as the captor antibody and a biotinylated monoclonal antibody (mAb) as the detector antibody. With the double sandwich ELISA technique, HGL in the range of 1-60 ng/ml was measured in less than 5 h. Identical HGL concentrations were obtained using the above ELISA procedure when compared to those based on the enzymatic activity using the potentiometric method (correlation coefficient: r = 0.95). No significant interference from other duodenal components was observed, as proved by the quantitative HGL determinations performed on intestinal samples.

Five monoclonal antibodies (mAb) directed against human gastric lipase (HGL) have been produced by hybridization of myeloma cells with spleen cells of BALB/c immunized mice. All these mAb belong to the IgG1 class with a kappa light chain. The effects of these mAb on the enzymic activity of HGL were studied and used to define three classes of antibodies, depending upon their immunoinactivation properties. As determined by ELISA and immunoinactivation studies, four overlapping epitopes were found to be part of the functional sites of the enzyme. The mAb appear to be suitable probes for studying the lipid binding and catalytic domains of HGL. The results of the ELISA additivity test were used to describe tentatively the epitopes of HGL in terms of a schematic spatial map.

Pancreatic juice is naturally supersaturated in calcium and bicarbonate ions. A mechanism controlling CaCO3 crystal formation and growth is therefore necessary to prevent duct clogging. The present study shows that lithostathine, a glycoprotein present in human pancreatic juice at a concentration in the range of 10 mumol/L, could be involved in such a control. Lithostathine in concentrations greater than 1.5 mumol/L significantly delayed crystal nucleation and inhibited growth of preformed CaCO3 crystals from supersaturated solutions. Adsorption of lithostathine on crystals was shown by immunodetection. Albumin also adsorbed on CaCO3 crystals, but neither albumin nor other pancreatic secretory proteins inhibited crystal nucleation or growth. Lithostathine adsorbed to sites specifically inhibiting crystal growth with a dissociation constant (Kd) = 0.9 x 10(-6) mol/L. The glycosylated amino-terminal undecapeptide generated by limited trypsin hydrolysis inhibited CaCO3 crystal growth with a Kd = 3.0 x 10(-6) mol/L, similar to that of lithostathine. On the contrary, the carboxy-terminal polypeptide was inactive. A synthetic undecapeptide identical to the N-terminal end but not glycosylated was equally active. The activity disappeared upon digestion of the undecapeptide with V8 protease. The N-terminal undecapeptide of lithostathine is therefore essential to the inhibitory activity of the protein on CaCO3 crystal growth.

Secretory forms of the pancreatic stone protein (PSP S, Mr 17, 500-22,000) have been purified from human pancreatic juice. PSP S are inhibitors of CaCO3 crystal growth. The presence of similar proteins in bovine, canine, monkey, porcine, and rat pancreatic secretion was investigated in terms of biological role and immunological relationship. Pancreatic proteins were analyzed by electrophoretic separation and by subsequent immunoblotting with a rabbit polyclonal antibody against human PSP. A single immunoreactive form was detected in dog, pig, and rat (Mr 17,000), and two distinct immunoreactive forms were observed in cow and monkey (Mr 15,000 and 17,000). Inhibition of CaCO3 crystal growth was demonstrated in dog and rat. Further kinetic studies of the inhibition process in the rat showed that PSP S binds to the crystal surface according to a Langmuir adsorption isotherm with a dissociation constant (Kd) of 1.5 x 10(-6) M. These results suggest that proteins homologous to human PSP S are present in other mammalian species and may act as stabilizers of Ca2+ -supersaturated pancreatic juice.

Chronic calcifying pancreatitis (CCP) is characterized by the presence of stones in pancreatic ducts. Calcium carbonate (CaCO3) is the main constituent of stones, to which is associated an organic matrix consisting primarily of one protein of Mr 14,000, the pancreatic stone protein or PSP. PSP is not present as such in pancreatic juice, but in polymorphic forms with higher molecular weights. These secretory forms (PSP S2-5, Mr 16-19,000) are synthesized in the acinar cells of the pancreas and secreted along the same secretory pathway as the exocrine enzymes. The heterogeneity of the forms of higher Mr (PSP S2-5) is probably due to different glycosylation patterns. PSP and PSP S1 are generated by the cleavage of an Arg-Ile bond in the N-terminal part of PSP S2-5. The N-terminal sequence of PSP (40 amino acids) is identical to that of PSP S1, whose complete sequence (133 amino acids) has been determined. Yet, the two proteins differ by their pI. Pancreatic juice is normally supersaturated in CaCO3, suggesting the presence of a stabilizer preventing CaCO3 precipitation. The PSP S could play that role, since an activity inhibiting the nucleation and growth in vitro of CaCO3 crystals was found in pancreatic juice, associated with these proteins. Moreover, PSP S concentration was significantly lower in the pancreatic juice of patients with CCP than in control patients. Proteins homologous to PSP S were also found in the dog, rat, swine, monkey and ox. They constitute a new family of pancreatic secretory proteins, whose biological role would be to maintain pancreatic juice in a stable state towards CaCO3.

In recent studies performed on pancreatic stones from patients with alcoholic pancreatitis, a novel secretory protein was identified: the pancreatic stone protein (PSP Mr 14,000). This protein suppresses CaCO3 precipitation, and could therefore stabilize normally supersaturated pancreatic juice. Crystallographic analysis of stones from patients with nutritional pancreatitis (NP), as well as alcoholic pancreatitis (AP), revealed that the main constituent was calcite (CaCO3). In the present study, we investigated the organic matrix of NP stones. In the 14 cases studied, the organic matrix was rendered soluble after mineral dissolution with EDTA + citrate. Analysis of the isolated matrix revealed the presence of one major protein (Mr 14,000), and of a minor protein (Mr 30,000), which is in fact an aggregate form of the 14,000 Mr protein. Using PSP antibodies, complete immunological identity was found between PSP, the immunoreactive form of PSP present in nonactivated pancreatic juice, and the protein matrix of NP stones. Moreover, protein matrix of NP stones also inhibited the nucleation of CaCO3 crystal, and decreased their growth rate in vitro. The presence of PSP in all AP and NP stones suggests that it plays a key role in stone formation during the course of chronic pancreatitis. These results also suggest the existence of some pathophysiological links between these two apparently different etiological forms of calcifying pancreatitis.

The primary structure of a pancreatic stone protein form has been elucidated for the first time. The protein studied was the lowest-Mr form prepared from human pancreatic juice (PSP S1). The N-terminal sequence up to residue 65 had already been determined. The five peptides obtained after staphylococcal protease digestion of the carboxymethylated reduced and succinylated PSP S1 enabled the deduction of the entire sequence. The tryptic peptides arising from the digest of cyclohexanedione--treated PSP S1 and the amino acids released by carboxypeptidase P digestion of PSP S1 confirmed the data of the sequence. The peptides were purified by Sephadex filtration and, if required, by chromatography on DEAE-cellulose or thin-layer cellulose. The amino acid sequences of the peptides were determined with a sequencer. From the sequence data it was deduced that the PSP S1 polypeptide chain contains 133 amino acid residues and has a Mr of 15,000.

Recently, in our laboratory, a protein extracted from human pancreatic stones was characterized and purified and a specific antibody was obtained. This pancreatic stone protein (PSP) was shown to have an inhibitory effect on the CaCO3 crystal growth in vitro. The cellular origin of such a protein and its repartition along the digestive tract were studied by immunolocalization (protein A-colloidal gold method) at the ultrastructural level. Surgical biopsies of pancreata from normal or chronic pancreatitis patients, needle liver biopsies, gastric mucosa, and jejunum and duodenum biopsies were minced and fixed in the Karnovsky medium or in buffered 4% paraformaldehyde. The specimens were washed in buffer, dehydrated through ethanol, and embedded in Epon 812. Ultrathin sections, collected on uncoated nickel grids, were submitted to the following reactives at room temperature: protein A 1 mg/ml, anti-PSP (1:2 to 1:100), and protein A-colloidal gold. The specificity of the localization was checked by substituting buffer or nonimmune rabbit serum to anti-PSP. The stone protein was markedly present in the zymogen granules and condensing vacuoles of the normal pancreatic acinar cells, the label was found in the acinar and ductal lumen. In chronic pancreatitis, the localization of PSP, when it occurred, was extremely weak in the acinar cells. No PSP was specifically characterized in hepatocytes, gastric mucosa, and enterocytes. However, a weak but specific reaction was found in the secretory granules of Paneth cells. These results in pancreas confirm the acinar secretory origin of the PSP and are in good agreement with its possible function in stabilizing pancreatic juice in vivo, which is normally supersaturated in calcium carbonate.(ABSTRACT TRUNCATED AT 250 WORDS)