Epitope mapping and immunoinactivation of human gastric lipase using five monoclonal antibodies

Abstract

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 IgGl class with a K 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.In humans, the hydrolysis of dietary triacylglycerols begins in the stomach and is catalyzed by one major enzyme; human gastric lipase (HGL) [l-31. HGL is a glycoprotein with a molecular mass of around 50 kDa, which is located in the chief cells of fundic mucosa [4]. In-vitro studies have shown that HGL hydrolyses both short-chain and long-chain triacylglycerols at comparable rates [5]. The enzyme is highly stable and active under acidic pH conditions. The optimal pH for HGL activity is 5.4 in the case of long-chain and 6 in the case of short-chain triacylglycerols.The amino acid sequence of HGL, deduced from the cDNA, consists of 379 residues [6] and shows a conservative pentapeptide (Gly-Xaa-Ser-Xaa-Gly) common to all the known lipases. . In order to further characterize these functional domains of HGL, an immunological approach was adopted here using mAb against HGL as probes for the spatial identification of the domains.In the present paper, we describe the production and functional characterization of five mAb against HGL. MATERIALS AND METHODS ProteinsHGL was purified in the laboratory from human gastric juice, using procedures described previously [15]. The protein had a specific activity of 1000 U . mg-' with tributyrin as substrate and showed a single band upon SDS/gel electrophoresis [16]. The bovine serum albumin and anti-mouseIgG -peroxidase conjugate used were from Sigma. Preparation of mAbTwo young female BALB/c mice were immunized with HGL as follows. The first and the second immunizations were carried out with 100 pg HGL emulsified in complete Freund's adjuvant, injected subcutaneously with a 10-day interval. Two weeks after the second injection, the mice were bled and the serum tested in a direct binding ELISA test. The mouse with the highest titre was selected and 100 pg pure antigen were again injected. 10 days later, three fusion-priming intraperitoneal injections of 50 pg HGL, each in 10 mM sodium phosphate, 150 mM NaCl, pH 7.4 (buffer A) were given on three subsequent days. Spleen cells were obtained 1 day after the last injection and fused with non-secreting mouse myeloma

The Importance of Non‐Charged Amino Acids in Antibody Binding to Humicola lanuginosa Lipase

“…The fact that D242N/D254N/E210Q reacts extremely heteroclitically indicates that the charge removing effect is additional and that this variant is more antigenic than the remaining one. This is in agreement with what has been found for gastric lipase, where four out of five MoAb recognized the same hydrophobic surface epitope [5]. However, the result contradicts that found for hydrophihc proteins.…”

“…Epitope mapping in a number of proteins revealed that these six amino acids are the most frequent in the analysed epitopes [4], and that antigenicity appears to be determined both by the local mobility of the region containing the epitope, and the frequency of antigenic residues. A hydrophobic epitope was identified in gastric lipase, on the surface of the folded protein to which four of the five tested monoclonal antibodies (MoAb) bound [5], thus neither the antigenicity ofthe epitope nor its individual residues reflected the properties described for the hydrophilic proteins mentioned above.…”

Human Pancreatic Lipase

“…Two ELISA methods were developed for this study. First, a simple sandwich ELISA was carried out by coating the plates with 500 ng of pure HPUwell using an identical proc edure to that previously described by Aoubala et al (13). Second, a double sandwich ELISA was performed by coating the plates with 500 ng of pure anti-HPL pAb/well (captor antibody).…”

“…Bri efly, microtiter plates were coated with 2.5 ng of native HPU well, and mAbs were cotitrated in pairs at a molar ratio of 1:1. The ELISA procedure was performed as described previously by Aoubala et al (13 ).…”

“…Monoclonal antibodies provide useful tools for investigating the structure-function relationships of proteins. This approach was previously found to be particularly fruitful in the case of colipase and human gastric lipase (11)(12)(13)(14). We have now produced five mAbs against HPL and examined the effects of these antibodies on the catalytic activity in the presence and absence of bile salts and colipase.…”