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D Lombardo

Publications and source records attributed to D Lombardo.

At least 55 records · Page 3Linked to original sources
PubMed1997

Association of bile-salt-dependent lipase with membranes of human pancreatic microsomes is under the control of ATP and phosphorylation.

Bile-salt-dependent lipase (BSDL) is secreted by the pancreas into the duodenum, where it catalyses the hydrolysis of dietary lipid esters on activation by bile salts. The secretion pathway of BSDL is comparable with that of other digestive enzymes produced by pancreatic acinar cells. However, in contrast with these other enzymes, BSDL is partly associated with endoplasmic reticulum membranes as part of a folding complex, including a Grp94-related protein to which BSDL is transiently linked. The release of BSDL from membranes occurs once its glycosylation is completed [Bruneau and Lombardo (1995) J. Biol. Chem. 270, 13524-13533]. In the present study, investigations concerning the mechanism of association/dissociation of BSDL with membranes of microsomes were performed. For this purpose the role of ATP and that of the possible phosphorylation of BSDL were examined. For the first time, it is shown that human pancreatic BSDL is phosphorylated, probably at a serine residue, during its transport within the acinar cell. The phosphorylation of BSDL is provoked by calphostin C, an inhibitor of protein kinase C. In the presence of 1-(isoquinolinesulphonyl)2-methylpiperazine, a non-specific inhibitor of serine/threonine protein kinase A, C or G, or of calcium chelator 1,2-bis(O-aminophenoxy)ethane-N,N,N', N'-tetra-acetic tetra(acetoxymethyl)ester, the phosphorylation of BSDL elicited by calphostin C is abolished. These data suggested that the phosphorylation of BSDL within human pancreatic microsomes is under the control of a cascade of protein kinases. We have also shown that the phosphorylation of BSDL appears to be involved in the release of the enzyme from microsome membranes. Nevertheless ATP, which modifies the conformation of BSDL, triggers this association, and an unhydrolysable ATP analogue was unable to promote it.

Adenosine Triphosphate↗
PubMed1997

Investigation of two glycosylated forms of bile-salt-dependent lipase in human pancreatic juice.

Pure human pancreatic bile-salt-dependent lipase, devoid of its oncofetal glycoform [Mas, E., Abouakil, N., Roudani, S., Miralles, F., Guy-Crotte., O., Figarella, C., Escribano, M. J. & Lombardo, D. (1993) Biochem. J. 289, 609-615], was analyzed on immobilized concanavalin A (ConA). Two variants were separated: an unabsorbed ConA-unreactive fraction; and an absorbed ConA-reactive fraction. Carbohydrate compositions of ConA-reactive and ConA-unreactive fractions were not significantly different, and analysis of 3H-labelled oligosaccharides liberated from these fractions on the ConA-Sepharose column indicated that the fractionation of the bile-salt-dependent lipase on this column depends upon oligosaccharide structures. The activity of the ConA-reactive fraction was however much lower, independent of the substrate (4-nitrophenyl hexanoate or cholesteryl esters), than that of the ConA-unreactive fraction. Therefore, catalytic constants for the hydrolysis of 4-nitrophenyl hexanoate were determined; both fractions had quite similar Km, while the kcat for the ConA-unreactive fraction was 3-4-fold higher than that of the ConA-reactive fraction. ConA-reactive and ConA-unreactive fractions were shown to have slightly different molecular masses and different amino acid compositions. Cleavage patterns after cyanogen bromide treatment of the ConA-reactive and ConA-unreactive fractions suggested that the ConA-reactive (high Mr form) and ConA-unreactive (low Mr form) forms could be different isoforms of the bile-salt-dependent lipase secreted by the human pancreas.

Amino Acids↗
PubMed1997

Pancreatic bile salt-dependent lipase activity in serum of normolipidemic patients.

Bile salt-dependent lipase (BSDL, E.C. 3.1.1.-) is a digestive enzyme secreted by the pancreatic acinar cell. Once in the duodenum, the enzyme, upon activation by primary bile salts, hydrolyzes dietary lipid esters such as cholesteryl esters and lipid-soluble vitamin esters. This enzyme is partially transferred from the duodenum or pancreas to the circulation where it has been postulated to exert a systemic action on atheroma-generating oxidized-low density lipoprotein (LDL). In the present study, sera from 40 healthy normolipidemic volunteers were used to investigate the possible linkage between circulating BSDL, lipids, and lipoproteins. We showed, firstly, that pancreatic-like BSDL activity can be detected in these serums. Secondly, BSDL activity increased significantly with the level of LDL-cholesterol and was also positively linked to the serum concentration of Apo B100 and Apo A-I. Thirdly, we also established that BSDL was associated with LDL, in part by a specific interaction with Apo B100, while no interaction was found with Apo A-I. No linkage with other recorded parameters (triglycerides, phospholipids, and high density lipoprotein-cholesterol) was detected. Because an increase in LDL-cholesterol represents an important risk factor for atheroma, the concomitant increase in BSDL, which can metabolize atherogenic LDL, suggests for the first time that this circulating enzyme may exert a positive effect against atherosclerosis.

Adult↗
PubMed1997

The oncofetal J28 epitope involves fucosylated O-linked oligosaccharide structures of the fetoacinar pancreatic protein.

The fetoacinar pancreatic protein (FAP), characterized by the mAb J28, is an oncofetal form of bile salt dependent lipase (BSDL), the expression of which is related to pancreatic differentiation and neoplastic processes. Because the J28 epitope, recognized by mAb J28, is suggested to be dependent upon carbohydrates, we have attempted to gain information about the structure of this epitope. Indeed, treatment of FAP with sodium periodate abolished the reactivity of the protein to mAb J28, which demonstrates the implication of oligosaccharides in the structure of the J28 epitope. FAP offers both O-linked and N-linked carbohydrate structures, of which, as we have determined, one is involved. Peptides obtained after cyanogen bromide cleavage were desialylated then separated by affinity chromatography on an immobilized peanut agglutinin agarose column. The peptide retained on this column carried out the reactivity with the mAb J28. Although some differences in amino acid analysis were observed, the N-terminal sequence of this peptide correlates with that of the C-terminal part of the enzyme. Carbohydrate analysis of the peptide bearing the J28 epitope revealed fucose, galactose, N-acetylgalactosamine, N-acetylglucosamine, and N-acetylneuraminic acid. The competition observed between mAb J28 and Ulex europaeus I lectin for binding to the J28 epitope suggested that fucose residue alpha (1-2) linked to a galactose residue was implicated in the structure of the J28 epitope. Alternatively, the loss of the mAb J28 reactivity upon treatment of FAP either with bovine kidney or bovine epididymis fucosidase was observed indicating that fucose residues linked at the alpha (1-2) and alpha (1-6) positions may be involved in the establishment of the structure of the J28 epitope. These observations suggest that mAb J28 recognized a particular fucosylated O-linked oligosaccharide structure located at the mucin-like extended C-terminal part of FAP.

Antibodies, Monoclonal↗
PubMed1996

Pancreatic carboxyl ester lipase: a circulating enzyme that modifies normal and oxidized lipoproteins in vitro.

Pancreatic carboxyl ester lipase (CEL) hydrolyzes cholesteryl esters (CE), triglycerides (TG), and lysophospholipids, with CE and TG hydrolysis stimulated by cholate. Originally thought to be confined to the gastrointestinal system, CEL has been reported in the plasma of humans and other mammals, implying its potential in vivo to modify lipids associated with LDL, HDL (CE, TG), and oxidized LDL (lysophosphatidylcholine, lysoPC). We measured the concentration of CEL in human plasma as 1.2+/-0.5 ng/ml (in the range reported for lipoprotein lipase). Human LDL and HDL3 reconstituted with radiolabeled lipids were incubated with purified porcine CEL without or with cholate (10 or 100 microM, concentrations achievable in systemic or portal plasma, respectively). Using a saturating concentration of lipoprotein-associated CE (4 microM), with increasing cholate concentration there was an increase in the hydrolysis of LDL- and HDL3-CE; at 100 microM cholate, the present hydrolysis per hour was 32+/-2 and 1.6+/-0.1, respectively, indicating that CEL interaction varied with lipoprotein class. HDL3-TG hydrolysis was also observed, but was only approximately 5-10% of that for HDL3-CE at either 10 or 100 microM cholate. Oxidized LDL (OxLDL) is enriched with lysoPC, a proatherogenic compound. After a 4-h incubation with CEL, the lysoPC content of OxLDL was depleted 57%. Colocalization of CEL in the vicinity of OxLDL formation was supported by demonstrating in human aortic homogenate a cholate-stimulated cholesteryl ester hydrolytic activity inhibited by anti-human CEL IgG. We conclude that CEL has the capability to modify normal human LDL and HDL composition and structure and to reduce the atherogenicity of OxLDL by decreasing its lysoPC content.

Animals↗
PubMed1996

Digestive lipases of the newborn ferret: compensatory role of milk bile salt-dependent lipase.

The amount of mRNA hybridizing to bile salt-dependent lipase and to colipase-dependent lipase probes as well as their translation into active proteins were quantified in the adult and newborn pancreas and lactating mammary gland from the ferret, a species whose milk, similar to that of the human, has bile salt-dependent lipase. The concentration of colipase-dependent lipase mRNA correlated with the amount of activity found in the adult and newborn pancreas, whereas neither mRNA nor activity of this enzyme was detected in the kit pancreas or in the lactating mammary gland. These data indicate that colipase-dependent lipase is actually expressed in adult pancreas and might represent the main lipolytic system in the adult. mRNA hybridizing to the bile salt-dependent lipase probe used in this study were detected in adult and in newborn ferret pancreas as well as in lactating mammary gland. However, the bile salt-dependent lipase activity expressed in the newborn pancreas was very low when compared with the activity expressed either in the mammary gland or in the adult pancreas. These data argue for a compensatory role of milk bile salt-dependent lipase in lipid digestion in the newborn. The hydrolysis of dietary fat might be initiated by preduodenal lipase, the activity of which is only two times lower in the gastric mucosa of the newborn than in the adult ferret. The high concentration of mRNA hybridizing to the bile salt-dependent lipase probe associated with a very poor bile salt-dependent lipase activity and protein suggests either that these mRNA are very unstable or that they are poorly translated into an active pancreatic bile salt-dependent lipase.

Animals↗
PubMed1995

Recombinant human dihydroorotate dehydrogenase: expression, purification, and characterization of a catalytically functional truncated enzyme.

An N-terminally truncated cDNA for human dihydroorotate dehydrogenase (DHODase) was placed under the control of the inducible T7 lac promoter in a pyrimidine auxotrophic strain of Escherichia coli lacking the endogenous enzyme. Induction of gene expression rescued growth in media lacking exogenous pyrimidines. The recombinant enzyme was purified to homogeneity from detergent extracts of bacterial membranes by two chromatographic steps. The purity of the resulting enzyme was judged to be > 95% based on SDS-PAGE with Coomassie staining. The enzyme displays an apparent molecular weight of ca. 40 kDa on SDS-PAGE and ca. 120 kDa on native size-exclusion chromatography, suggesting that the native enzyme is multimeric. Recombinant DHODase displayed a specific activity and Km for dihydroorotate that were similar to those for the enzymes from bovine and human liver tissue. The pH dependence of the activity of the recombinant enzyme was likewise similar to that of the enzyme from human liver and may indicate the involvement of a critical histidine residue in catalytic turnover; only eight histidine residues remain in the truncated version of DHODase used here. The catalytic activity of the recombinant enzyme is inhibited in a dose-dependent fashion by the histidine-selective modifying agent diethylpyrocarbonate. These results further suggest a potential role for histidine in enzyme turnover. Brequinar sodium, an experimental drug which has been shown to be a nanomolar noncompetitive inhibitor of mammalian DHODases, inhibited the activity of the purified recombinant enzyme with a Ki value similar to that for enzyme derived from human liver tissue. The recombinant DHODase thus displays enzymatic behavior similar to the 50-kDa full-length human liver enzyme, illustrating that the catalytically essential structural features of the enzyme, as well as the site of Brequinar binding, are contained within the 40-kDa truncated version of the enzyme that was expressed here.

Amino Acid Sequence↗
PubMed1995

Bile salt-dependent lipase transcripts in human fetal tissues.

In human fetal pancreas, we identified two cDNA transcripts of the bile salt-dependent lipase (BSDL) using reverse transcription followed by polymerase chain reaction (RT-PCR). The sequence of four overlapping segments obtained by RT-PCR matched the sequence of the 2.2 kb cDNA cloned from human adult pancreas (Reue et al. (1991) J. Lipid Res. 32, 267-276). A second RT-PCR product of approx. 1.1 kb was evidenced, the sequence of which corresponds to that of the BSDL-pseudogene transcript (Nilsson et al. (1993) Genomics, 17, 416-422). The short transcript is present in all tissues examined whereas the former one (2.2 kb) is either poorly (in liver and kidney) or not at all expressed in adult tissues, excepted in the pancreas. On the other hand, the 2.2 kb transcript specific of the BSDL gene was detected in all fetal tissues examined as early as the 6th week of gestation. Results also suggested that the fetal pancreas contains more 2.2 kb transcript than its adult counterpart. Therewith, BSDL was immuno-precipitated from fetal liver. The role of BSDL-gene expression during the fetal life is discussed.

Amino Acid Sequence↗
PubMed1995

Association of bile-salt-dependent lipase with membranes of human pancreatic microsomes.

Immunolocalization studies indicated that, in contrast to other enzyme markers of human pancreatic secretion, bile-salt-dependent lipase (BSDL) was partly but specifically associated with endoplasmic reticulum membranes. In microsomes, temperature-induced phase separation using Triton X-114 elucidated the partition of BSDL between the aqueous phase and the detergent-rich phase containing hydrophilic and membrane proteins, respectively. The size of the membrane-associated BSDL (approx. 100 kDa) is compatible with that of the fully processed enzyme. Fucosylated O- and N-linked oligosaccharide structures were detected by means of specific lectins. The membrane-associated BSDL might therefore be released from membranes between the trans-Golgi compartment (where terminal fucose residues were added) and the zymogen granules where BSDL was mainly found in the soluble fraction. Even though BSDL associated with membranes was enzymically active, it appeared less efficient than the soluble form. The association of BSDL with membranes was pH-dependent and optimal association occurred between pH 5-6. The membrane-associated BSDL was released by KBr which suggests that the association of BSDL with microsomal membranes involves ionic interactions. Lipid-protein interactions are probably not involved in this association as BSDL did not associate with liver microsome membranes. We attempted to characterize the putative ligand and showed that BSDL and a 94-kDa protein, immunologically related to a glucose-regulated protein of 94 kDa (Grp94), were co-immunoprecipitated by specific antibodies directed against each individual species. It is suggested that the biogenesis of the human pancreatic BSDL involves an association with intracellular membranes and that its folding may be assisted by molecular chaperones.

Adult↗
PubMed1995

Chaperone function of a Grp 94-related protein for folding and transport of the pancreatic bile salt-dependent lipase.

In its fundamental attributes, the secretion pathway of the pancreatic bile salt-dependent lipase (BSDL) followed that described for all enzymes involved in regulated secretion. This route was inhibited by drugs that affect protein synthesis and intracellular transport. In the presence of monensin, BSDL was solely detected in microsome membrane fractions. The association of BSDL with intracellular membranes involved a protein complex, formed by at least two proteins of 94 and 56 kDa. In cells experiencing the metabolic stress due to azetidine-2-carboxylic acid, BSDL was additionally associated with a protein of 46 kDa. Affinity blotting showed that BSDL bound directly to the 94-kDa protein (p94). It was suggested that p94 could be a molecular chaperone, further identified as related to the 94-kDa glucose regulated protein (Grp 94). The membrane-associated BSDL (i.e. BSDL bound to the Grp 94-related p94) was O- and N-glycosylated and consequently appeared released from membranes in the trans-Golgi compartment. Therefore and for the first time, it is suggested that a multiprotein complex including the chaperone Grp 94-related p94 protein may play an essential role in the folding and transport of BSDL. One hypothesis is that the association of BSDL with membrane via the Grp 94-related p94 along its secretion pathway is required for its complete O-glycosylation, which occurs on the extended mucin-like structures present on the C-terminal part of the protein.

Animals↗
PubMed1994

Horse pancreatic lipase. The crystal structure refined at 2.3 A resolution.

Pancreatic lipase (EC 3.1.1.3) plays a key role in dietary fat digestion by converting triacylglycerols into 2-monoacylglycerols and free fatty acids in the intestine. Although the crystallographic structures of the human pancreatic lipase and of a human lipase-porcine colipase complex have been solved, no refined structure of pancreatic lipase has yet been published. The crystal structure of the horse enzyme was solved by the molecular replacement method from the model of the human pancreatic lipase and subsequently refined to 2.3 A resolution. The final model contains two molecules of 449 amino acid residues each in the asymmetric unit, 705 well-defined water molecules and two calcium ions. The two molecules in the asymmetric unit of the orthorhombic crystals are related by a 2-fold non-crystallographic symmetry axis as in the case of the human lipase. However, the association between the two molecules in their respective crystal forms is different. The overall molecular structure of the horse lipase is very similar to that of the human enzyme. The horse lipase is made up of two well-defined domains. The N-terminal domain which bears the active centre has a typical alpha/beta hydrolase fold topology. The C-terminal domain which is devoted to colipase binding has a beta-sheet sandwich topology. Comparison of equivalent C alpha atom positions between the final model of the horse lipase and the human lipase structure shows only slight differences which are mainly located in the C-terminal domain. The horse enzyme possesses the common features of the known mammalian and microbial lipases, in particular the "flap" covering the catalytic triad. In addition to more precise information concerning these features, the elucidation of the horse lipase crystal structure allowed us to better understand the structural basis of the kinetic behaviour of pancreatic lipases towards a soluble substrate, p-nitrophenyl acetate, and the different sensitivity of these enzymes towards limited proteolysis.

Amino Acid Sequence↗
PubMed1994

Efficacy and safety evaluation of lacidipine compared with amlodipine in mild-to-moderate hypertensive patients.

Hypertension is well recognized to be an important cardiovascular risk factor and antihypertensive therapy has been shown to decrease cardiovascular mortality and morbidity as blood pressure control is achieved. At present, management of hypertension is obtained with effective compounds that exhibit a satisfactory safety profile. Among the antihypertensive drugs, calcium antagonists have been proven to possess this property. In the present study, our objective was to compare the antihypertensive effect of two new long-acting dihydropyridines, lacidipine and amlodipine, as once-daily monotherapies in mild-to-moderate hypertensive patients. Eighty hypertensive patients were recruited and after a 3-week washout period were randomized to receive lacidipine 4 mg once daily or amlodipine 10 mg once daily. After 4 weeks, hydrochlorothiazide 12.5 mg was added in patients not adequately controlled, and patients were treated for a total of 8 weeks. At this time, supine mean diastolic blood pressure decrease was 16 mm Hg in the lacidipine group and 10 mm Hg in the amlodipine group (p < = 0.01). Adverse events were reported in 28% of patients treated with lacidipine and in 48% of patients receiving amlodipine. Results of our pilot clinical experience show that lacidipine is a well-tolerated and effective compound, compared with amlodipine, in mild-to-moderate hypertensive patients.

Adult↗
PubMed1994

Expression of a 46 kDa protein in human pancreatic tumors and its possible relationship with the bile salt-dependent lipase.

Bile salt-dependent lipase (BSDL), an enzyme normally found in human pancreatic secretions is a 100 kDa glycoprotein. A BSDL-specific 477 bp cDNA probe was prepared by performing polymerase chain reaction experiments. This cDNA was used to probe mRNAs extracted from human pancreatic tissue and tumoral cell lines. Two mRNAs were detected in normal human pancreas at 2.2 and 1.3 to 1.5 kb. In human pancreatic tumoral cells, mRNAs encoding for the BSDL were detected using in situ hybridization, and proteins with an M(r) of 46,000 to 48,000 were translated into an in vitro system using mRNAs extracted from these cells. Using an immunoprecipitation procedure, we observed here that the specific BSDL polyclonal antibodies recognized three proteins of 100 +/- 5 (p100), 46 +/- 2 (p46) and 22.7 +/- 1.2 (p23) kDa, respectively in the soluble extracts of normal adult human pancreas. The p100 protein was probably the glycosylated product resulting from the translation of the 2.2 kb transcript. The p46 protein, which electrophoresed as a doublet was the main component immunoprecipitated from extract of a differentiated human pancreatic adenocarcinoma as well as from the extracts of two pancreatic cell lines, BxPC-3 and SOJ-6. In addition, the p46 immunoform of the BSDL was detected in cell-free medium from SOJ-6 cell line and its expression was found to be correlated with the secretion of an esterolytic activity on 4-nitrophenyl caproate, whereas the BxPC-3 cell line neither secreted the p46 nor showed any esterolytic activity on this substrate. The p46 may be either a short variant of BSDL resulting from the translation of the 1.3 to 1.5 kb transcript or a protein structurally related to the enzyme. The p46 doublet immunoform was detected in the human pancreatic secretion.

Base Sequence↗
PubMed1993

Bile salt-dependent lipase biosynthesis in rat pancreatic AR 4-2 J cells. Essential requirement of N-linked oligosaccharide for secretion and expression of a fully active enzyme.

This study used the rat pancreatic AR 4-2 J cell line as a model system to investigate the role of glycosylation in bile salt-dependent lipase (BSDL) secretion and esterolytic activity. Results indicated that AR 4-2 J cells synthesized BSDL, the esterolytic activity of which was the greater part of the cell activity toward 4-nitrophenylcaproate. The protein thus expressed was glycosylated and had a molecular mass approximately 74 kDa. Exposure of these cells to tunicamycin significantly decreased [3H]mannose incorporation, while [35S]methionine incorporation in trichloroacetic acid-precipitable material was not modified. Tunicamycin treatment of AR 4-2 J cells lead to a lower molecular mass form (approximately 70 kDa) of BSDL which did not incorporate [3H]mannose. The nonglycosylated low M(r) form of the enzyme was not secreted as shown by the decreasing activity in cell-free medium which paralleled the time-dependent secretion of the enzyme. Tunicamycin had no effect on BSDL synthesis. Nevertheless, the nonglycosylated BSDL was apparently not degraded in any cell compartment as shown in part by the enzyme activity accumulation within cells upon brefeldin A treatment. The BSDL expressed by AR 4-2 J cells was characterized by a Km of 68 +/- 30 microM and a kcat of 106 +/- 19 min-1. The sequestrated BSDL due to tunicamycin treatment of cells presents a significant increase of Km of over 10 times to 757 +/- 303 microM. kcat was affected by a factor of approximately 4-445 +/- 22 min-1. These data correlated with an approximately 2.5-fold decrease of the esterolytic activity following inhibition of the N-glycosylation of the protein. The nonglycosylated BSDL was less stable to temperature than the native form. Processing inhibitors (castanospermine, 1-deoxymannojirimycin, and swainsonine) had no effect either on the enzyme activity or on its secretion. Results suggested first that the transfer of the oligosaccharide precursor to the nascent BSDL is essential for the folding of the fully active BSDL. Second that the glycan structure is required for the enzyme secretion.

1-Deoxynojirimycin↗