RESUMEN
The occurrence of classical pancreatic lipase (PL) and pancreatic lipase-related proteins 1 and 2 (PLRP1s and 2) in the pancreas of ten mammalian species (humans, pig, rat, guinea pig, coypu, rabbit, horse, ox, goat and sheep) and two bird species (ostrich and turkey) was investigated. The lipases were purified from delipidated pancreas and identified based on the results of Western blotting analysis with anti-human PLRP2 serum, the catalytic properties and N-terminal microsequencing data. PLRP2s were detected in the pancreas of monogastric herbivorous animals (guinea pig, coypu, rabbit and horse) but not in that of ruminant herbivorous animals (ox, goat and sheep). The pancreas of carnivorous animals (dogs and cats) does not have any detectable PLRP2, but contains high levels of PL and PLRP1. By contrast, the pancreas of omnivorous animals (humans and rats) contains PL, PLRP1 and PLRP2, with the exception of porcine pancreas, where no PLRP2 was detected. In the case of bird (ostrich and turkey) pancreases, only classical PL was detected. The substrate specificity of PLRP2s was investigated using phospholipid micelles and synthetic monomolecular galactolipid films. Like human PLRP2, rabbit and horse PLRP2s are galactolipases. In polygastric herbivorous animals (ruminants), however, galactolipids are hydrolyzed via microbial enzymatic processes (involving galactolipases). The absence of galactolipids in carnivorous animals' diet may explain why no PLRP2s were detected here in the pancreas of these species.
Asunto(s)
Dieta , Conducta Alimentaria/fisiología , Lipasa/metabolismo , Secuencia de Aminoácidos , Animales , Aves/metabolismo , Hidrolasas de Éster Carboxílico/metabolismo , Humanos , Lipasa/química , Lipasa/aislamiento & purificación , Datos de Secuencia Molecular , Páncreas/enzimología , Fosfatidilcolinas/metabolismo , Fosfolipasas/metabolismo , Proteínas Recombinantes/metabolismo , Rumiantes/metabolismo , Especificidad de la Especie , Especificidad por SustratoRESUMEN
Recombinant human pancreatic lipase-related protein 2 (rHPLRP2) was produced in the protease A-deficient yeast Pichia pastoris. A major protein with a molecular mass of 50 kDa was purified from the culture medium using SP-Sepharose and Mono Q chromatography. The protein was found to be highly sensitive to the proteolytic cleavage of a peptide bond in the lid domain. The proteolytic cleavage process occurring in the lid affected both the lipase and phospholipase activities of rHPLRP2. The substrate specificity of the nonproteolyzed rHPLRP2 was investigated using pH-stat and monomolecular film techniques and various substrates (glycerides, phospholipids, and galactolipids). All of the enzyme activities were maximum at alkaline pH values and decreased in the pH 5-7 range corresponding to the physiological conditions occurring in the duodenum. rHPLRP2 was found to act preferentially on substrates forming small aggregates in solution (monoglycerides, egg phosphatidylcholine, and galactolipids) rather than on emulsified substrates such as triolein and diolein. The activity of rHPLRP2 on monogalactosyldiglyceride and digalactosyldiglyceride monomolecular films was determined and compared with that of guinea pig pancreatic lipase-related protein 2, which shows a large deletion in the lid domain. The presence of a full-length lid domain in rHPLRP2 makes it possible for enzyme activity to occur at higher surface pressures. The finding that the inhibition of nonproteolyzed rHPLRP2 by tetrahydrolipstatin and diethyl-p-nitrophenyl phosphate does not involve any bile salt requirements suggests that the rHPLRP2 lid adopts an open conformation in aqueous media.
Asunto(s)
Lipasa/fisiología , Pichia/metabolismo , Hidrolasas de Éster Carboxílico/metabolismo , Colipasas/farmacología , Glicéridos/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Lactonas/farmacología , Lipasa/antagonistas & inhibidores , Lipasa/aislamiento & purificación , Orlistat , Paraoxon/farmacología , Fosfolipasas/metabolismo , Presión , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Propiedades de Superficie , Ácido Taurodesoxicólico/farmacologíaRESUMEN
Human pancreatic lipase-related protein 2 (HPLRP2) was previously found to be secreted by the exocrine pancreas. HPLRP2 shows a high level of activity on galactolipids, and might be involved in the digestion of these common vegetable lipids. Specific antibodies were raised in rabbits using a synthetic HPLRP2 peptide selected for its weak amino acid homology with the corresponding peptides of classical human pancreatic lipase (HPL) and human pancreatic lipase-related protein 1 (HPLRP1). ELISA and Western blotting data showed that these antibodies did not react with HPL or HPLRP1. Various tissues from the digestive tract were subjected to Western blotting analysis with the specific anti-peptide HPLRP2 antibody and the expression of HPLRP2 was detected in the pancreas and colon. An ELISA was developed for specifically measuring the HPLRP2 levels in pure pancreatic juice. This procedure was performed using the anti-peptide HPLRP2 antibody as the captor antibody and a biotinylated anti-HPLRP2 polyclonal antibody as the detector antibody. The lowest HPLRP2 quantification limit was found to be 50 microg/L and the reference range for the present assay was 50 microg-500 microg/L. HPL and HPLRP2 levels were measured using specific ELISAs in pancreatic juice from patients with and without pancreatic disorders. Patients with chronic calcifying pancreatitis (CCP) had significantly lower levels of both HPL and HPLRP2 than the controls subjects. The mean HPLRP2 to HPL ratio was estimated to be 28.30% (w/w) and 23.96% (w/w) in controls subjects and CCP patients, respectively, and the difference was not significant. The levels of HPL and HPLRP2 are therefore similarly reduced in both healthy patients and CCP patients.
Asunto(s)
Tracto Gastrointestinal/química , Lipasa/biosíntesis , Jugo Pancreático/química , Secuencia de Aminoácidos , Animales , Calcinosis/metabolismo , Colon/metabolismo , Ensayo de Inmunoadsorción Enzimática/métodos , Humanos , Lipasa/análisis , Lipasa/inmunología , Datos de Secuencia Molecular , Páncreas/metabolismo , Pancreatitis Crónica/metabolismo , Conejos , Sensibilidad y EspecificidadRESUMEN
High-level constitutive expression of the human pancreatic lipase-related protein 1 (HPLRP1) was achieved using the methylotrophic yeast Pichia pastoris. The HPLRP1 cDNA, including its original leader sequence, was subcloned into the pGAPZB vector and further integrated into the genome of P. pastoris X-33 under the control of the glyceraldehyde 3-phosphate dehydrogenase (GAP) constitutive promoter. A major protein with a molecular mass of 50 kDa was found to be secreted into the culture medium and was identified using anti-HPLRP1 polyclonal antibodies as HPLRP1 recombinant protein. The level of expression reached 100-120 mg of HPLRP1 per liter of culture medium after 40 h, as attested by specific and quantitative enzyme-linked immunosorbent assay. A single cation-exchange chromatography sufficed to obtain a highly purified recombinant HPLRP1 after direct batch adsorption onto S-Sepharose of the HPLRP1 present in the culture medium, at pH 5.5. N-terminal sequencing and mass spectrometry analysis were carried out to monitor the production of the mature protein and to confirm that its signal peptide was properly processed.
Asunto(s)
Lipasa/biosíntesis , Pichia , Proteínas Recombinantes/biosíntesis , Cromatografía por Intercambio Iónico , Proteínas Fúngicas/genética , Expresión Génica/genética , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Humanos , Concentración de Iones de Hidrógeno , Lipasa/genética , Lipasa/aislamiento & purificación , Pichia/genética , Señales de Clasificación de Proteína/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Análisis de Secuencia de ProteínaRESUMEN
Epitope mapping was performed on human pancreatic lipase (HPL) using the SPOTscan method. A set of 146 short (12 amino acid residues) synthetic overlapping peptides covering the entire amino acid sequence of HPL were used to systematically assess the immunoreactivity of antisera raised in rabbits against native HPL, HPL without a lid (HPL(-lid)) and HPL covalently inhibited by diethyl p-nitrophenyl phosphate (DP-HPL). In the latter form of HPL, the lid domain controlling the access to the active site was assumed to exist in the open conformation. All the anti-lipase sera were tested in a direct ELISA, anti-HPL serum showing the greatest antibody titer. Although from the structural point of view, the differences between the various forms of HPL were restricted to the lid domain, differences in the antigenic properties of HPL were observed with the SPOTscan method, and the anti-DP-HPL antibodies showed the strongest reactivity. Most of the peptide stretches recognized included amino acid residues which are accessible at the surface of the lipase, except for those located near the active site. Two small peptides (T173-P180, V199-A207) were identified in the vicinity of the active site, their antipeptide antibodies were produced and their reactivity towards the various forms of HPL was tested in a double sandwich ELISA. No reactivity was observed under these conditions. Two antipeptide antibodies directed against two other selected peptides, P208-V221 (belonging to the beta9 loop) and I245-F258 (belonging to the lid domain) were prepared and found to react much more strongly with DP-HPL than with HPL or HPL(-lid) in a double sandwich ELISA. These antibodies should provide useful tools for monitoring the conformational changes taking place during the opening of the HPL lid domain.
Asunto(s)
Mapeo Epitopo , Epítopos/química , Lipasa/química , Oligopéptidos/química , Páncreas/enzimología , Animales , Sitios de Unión/inmunología , Ensayo de Inmunoadsorción Enzimática , Epítopos/inmunología , Humanos , Lipasa/inmunología , Oligopéptidos/inmunología , Páncreas/inmunología , Estructura Secundaria de Proteína , Conejos , Relación Estructura-ActividadRESUMEN
Human pancreatic lipase-related protein 2 (HPLRP2) was identified for the first time in pancreatic juice using specific anti-peptide antibodies and purified to homogeneity. Antibodies were raised in the rabbit using a synthetic peptide from the HPLRP2 protein sequence deduced from cDNA. Western blotting analysis showed that these antibodies did not react with classical human pancreatic lipase (HPL) or human pancreatic lipase-related protein 1 (HPLRP1) but cross-reacted with native rat PLRP2 (RPLRP2), as well as with recombinant rat and guinea-pig PLRP2 (GPLRP2). Immunoaffinity chromatography was performed on immobilized anti-recombinant HPLRP2 polyclonal antibodies to purify native HPLRP2 after conventional chromatographic steps including gel filtration and chromatrography on an anion-exchanger. The substrate specificity of HPLRP2 was investigated using various triglycerides, phospholipids and galactolipids as substrates. The lipase activity on triglycerides was inhibited by bile salts and weakly restored by colipase. The phospholipase activity of HPLRP2 on phospholipid micelles was very low. A significant level of galactolipase activity was measured using monogalactosyldiglyceride monomolecular films. These data suggest that the main physiological function of HPLRP2 is the hydrolysis of galactolipids, which are the main lipids present in vegetable food.
Asunto(s)
Lipasa/química , Anticuerpos/inmunología , Ácidos y Sales Biliares/metabolismo , Hidrolasas de Éster Carboxílico/química , Hidrolasas de Éster Carboxílico/inmunología , Hidrolasas de Éster Carboxílico/aislamiento & purificación , Hidrolasas de Éster Carboxílico/metabolismo , Colipasas/metabolismo , Humanos , Lipasa/inmunología , Lipasa/aislamiento & purificación , Lipasa/metabolismo , Jugo Pancreático/química , Jugo Pancreático/inmunología , Jugo Pancreático/metabolismo , Fosfolipasas/química , Fosfolipasas/inmunología , Fosfolipasas/aislamiento & purificación , Fosfolipasas/metabolismoRESUMEN
BACKGROUND AND AIMS: Human pancreatic lipases (HPL) include the classical HPL, and two related proteins known as pancreatic lipase-related proteins 1 and 2 (HPLRP1 and 2). The aim of this study was to develop an ELISA for specifically quantifying the classical-HPL level in sera of patients with and without pancreatic disorders. METHODS: The specific activity of various human (including classical-HPL) and microbial lipases was measured using Lipa Vitros and potentiometric (pH-stat) assays. A double sandwich ELISA was also set up, using an anti-classical-HPL polyclonal antibody and a biotinylated monoclonal antibody (mAb 146-40) specific to the classical-HPL. Sera (n = 53) were collected from patients with and without pancreatic disorders. The lipase concentration was deduced from the measured lipolytic activity and compared with the corresponding classical-HPL concentration, measured with the ELISA. RESULTS: Both the purified HPLRP2 and 3 lipases of microbial origin were found to have a significant and unexpected lipolytic activity under the standard Lipa Vitros assay, whereas the ELISA test developed in the present study was found to be specific for the classical-HPL, due to the absence of cross-reactivity between mAb 146-40, HPLRP1 and HPLRP2. The efficiency of the ELISA was assessed in terms of its reproducibility and accuracy. The lower detection limit of classical-HPL was found to be 0.03 microg/l. A good correlation was found to exist between the lipase concentrations obtained in the ELISA, pH-stat and Lipa Vitros tests, in both the control and pathological groups. CONCLUSION: This is the first time a specific method of measuring classical-HPL in human serum has been proposed. Using this ELISA, we established with the 53 sera selected in the present study, that the Lipa Vitros assay as well as the pH-stat assay were mostly detecting classical pancreatic lipase. However, it is possible that other lipases such as HPLRP2 or lipases of microbial origin, present in some pathological sera, may well interfere with the Lipa Vitros assay.