Molecular cloning of the bile salt-dependent lipase of ferret lactating mammary gland: an overview of functional residues

Sbarra, Véronique; Bruneau, Nadine; Mas, Eric; Hamosh, Margit; Lagadic‐Gossmann, Dominique; Hamosh, Paul

doi:10.1016/s0005-2760(98)00067-8

Cited by 20 publications

(27 citation statements)

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“…The mRNA (2.0 kb) encoding BSDL detected in clone 3B expressing the wild-type enzyme and that detected in clone 3M2 and 5M5 expressing K61I/K62I/R63A and K32I/K56I/K61I/K62I/R63A mutagenized variants of BSDL, respectively, was of the expected size (15). Also, the cDNA probe for ␤-actin hybridized with a transcript of the right size (13), present in all selected clones. The amount of mRNA encoding BSDL, equivalent in all selected clones, correlated quite well with the amount of BSDL expressed by the corresponding clone.…”

Section: Expression Of Recombinant Mutagenized Bsdl-mentioning

confidence: 89%

“…These two plasmids were referred to as 3M (for three mutations, K61I/K62I/R63A) and 5M (for five mutations, K32I/K56I/K61I/K62I/R63A), respectively. Plasmids were completely sequenced using specific primers for BSDL sequence (13) to detect any misincorporation of bases by the polymerase within the BSDL cDNA. Mutagenized cDNA of BSDL was then digested using EcoRI restriction enzyme and ligated into pECE-1 vector from which the wild-type cDNA encoding BSDL has been excised previously following the same restriction procedure.…”

Section: Methodsmentioning

confidence: 99%

“…After migration on 1% agarose gel, RNAs (about 20 g) were transferred onto a nitrocellulose membrane (22). cDNA probes specific for pancreatic BSDL (500 bp) and for ␤-actin (300 bp) were obtained and used as described previously (13). Probes were radiolabeled by using [␣-…”

Section: Methodsmentioning

confidence: 99%

“…13) 1 is a lipolytic enzyme secreted by the acinar pancreatic cell into the duodenum, where it plays a significant role in dietary lipid digestion. BSDL has been shown to display wide substrate reactivities ranging from the hydrolysis of both long-chain and short-chain fatty acid esters of glycerol, as well as phospholipids, lysophospholipids, and esters of cholesterol and of the fat-soluble vitamin A, E, and D (1).…”

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confidence: 99%

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Site-directed Mutagenesis of the Basic N-terminal Cluster of Pancreatic Bile Salt-dependent Lipase

Aubert¹,

Sbarra

Petit-Thévenin

et al. 2002

Journal of Biological Chemistry

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Previous studies have postulated the presence of a heparin-binding site on the bile salt-dependent lipase (BSDL), whereas two bile salt-binding sites regulate the enzyme activity. One of these sites may overlap with the tentative heparin-binding site at the level of an N-terminal basic cluster consisting of positive residues Lys 32 , Lys 56 , Lys 61 , Lys 62 , and Arg 63 . The present study uses specific site-directed mutagenesis to determine the functional significance of this basic cluster. Mutations in this sequence resulted in recombinant enzymes that were able to bind to immobilized and to cell-associated heparin before moving throughout intestinal cells. Recombinant BSDL was fully active on soluble substrate, but mutants were less active on micellar cholesteryl oleate in comparison with the wild-type enzyme. Activation studies by primary (sodium taurocholate) and by secondary (sodium taurodeoxycholate) bile salts revealed that the activation of BSDL by sodium taurocholate at concentrations below the critical micellar concentration, and not that evoked by micellar bile salts, was affected by substitutions, suggesting that this Nterminal basic cluster likely represents the specific bile salt-binding site of BSDL. Substitutions also affected the activation of the enzyme promoted by anionic phospholipids, extending the function of this site to that of a cationic regulatory site susceptible to accommodate anionic ligands.

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Section: Expression Of Recombinant Mutagenized Bsdl-mentioning

confidence: 89%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Site-directed Mutagenesis of the Basic N-terminal Cluster of Pancreatic Bile Salt-dependent Lipase

Aubert¹,

Sbarra

Petit-Thévenin

et al. 2002

Journal of Biological Chemistry

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“…14, al., 1997), which differs between species both in the amount of repeated identical sequences (Sbarra et al, 1998) and in glycosylation (Wang and Hartsuck, 1993), could also be involved in the transcytosis of the enzyme. The use of heterologous systems, e.g., binding of BSDL to human CaCo-2 cells (Huang and Hui, 1990) and transcytosis of rat BSDL through human Int407 cells (Bruneau et al, 2001) and the lack of effect of PNA allowed us to rule out in first instance this assumption.…”

Section: Lox-1 Implication In Bsdl Transcytosismentioning

confidence: 99%

Lectin-like Ox-LDL Receptor Is Expressed in Human INT-407 Intestinal Cells: Involvement in the Transcytosis of Pancreatic Bile Salt–dependent Lipase

Bruneau

Richard

Silvy

et al. 2003

MBoC

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We have recently shown that the pancreatic bile salt-dependent lipase (BSDL) can be taken up by intestinal cells and transported to the blood circulation. This mechanism likely involves (specific) receptor(s) able to bind BSDL and located at the apical intestinal cell membrane. In this study, using Int407 human intestinal cells cultured to form a tight epithelium, we attempted to characterize (the) BSDL receptor(s). We found that an apical 50-kDa protein was able to bind BSDL. Further, we have demonstrated that Int407 cells expressed the lectin-like oxidized-LDL receptor (LOX-1), the upregulation of which by oxidized-LDL potentiates the transcytosis of BSDL, whereas carrageenan and to a lesser extent polyinosinic acid and fucoidan decrease the enzyme transcytosis. The mAb JTX92, which blocks the LOX-1 receptor function, also impaired the BSDL transcytosis. To confirm these results, the cDNA encoding the human intestinal receptor LOX-1 has been cloned, inserted into vectors, and transfected into Int407 cells. Overexpression of LOX-1 by these cells leads to a substantial increase in the BSDL transcytosis. Globally, these data support the view that LOX-1 could be an intestinal receptor for BSDL, which is implicated in the transcytosis of this enzyme throughout Int407 cells. INTRODUCTIONThe bile salt-dependent lipase (BSDL, EC 3.1.1.13), also referred to as carboxyl ester lipase or bile salt-stimulated lipase, is a lipolytic enzyme, synthesized and secreted by the exocrine pancreas (Lombardo, 2001). The secretion of this digestive enzyme requires the participation of the Grp94 molecular chaperone (Bruneau and Lombardo, 1995;Bruneau et al., 1998). Once in the lumen of the duodenum, BSDL, in concert with other pancreatic lipolytic enzymes and preduodenal lipase, acts to complete the digestion of dietary lipids. Indeed, upon activation by primary bile salts, BSDL catalyzes the duodenal hydrolysis of cholesterol esters into free cholesterol and fatty acids before their absorption by intestinal cells (Howles et al., 1996;Weng et al., 1999). Although controversial (Howles et al., 1996), BSDL could also participate in the intestinal free cholesterol absorption (LopezCandales et al., 1993), acting as a cholesterol transfer protein (Myers-Payne et al., 1995), or as a cholesterol reesterifying enzyme after cholesterol absorption by intestinal cells (Bhat and Brockman, 1982).A fraction of BSDL, possibly associated with Grp94, reaches the vicinity of microvilli and upon binding to the surface of intestinal cells is internalized and transported up to the blood compartment (Bruneau et al., , 2000a(Bruneau et al., , 2000b(Bruneau et al., , 2003. Consequently, BSDL has been reported in the plasma of normolipidemic patients where it associates with apolipoprotein B-containing lipoproteins such as low-density lipoprotein (LDL), chylomicron and very-low-density lipoprotein (VLDL; Bruneau et al., 2003).The pathway of BSDL throughout intestinal cells has been recently delineated using Int407 cells (Bruneau et al., 2001), which do not e...

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Lipolytic Enzymes and Hydrolytic Rancidity

Deeth

Fitz-Gerald

Advanced Dairy Chemistry Volume 2 Lipids

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Molecular cloning of the bile salt-dependent lipase of ferret lactating mammary gland: an overview of functional residues

Cited by 20 publications

References 43 publications

Site-directed Mutagenesis of the Basic N-terminal Cluster of Pancreatic Bile Salt-dependent Lipase

Site-directed Mutagenesis of the Basic N-terminal Cluster of Pancreatic Bile Salt-dependent Lipase

Lectin-like Ox-LDL Receptor Is Expressed in Human INT-407 Intestinal Cells: Involvement in the Transcytosis of Pancreatic Bile Salt–dependent Lipase

Lipolytic Enzymes and Hydrolytic Rancidity

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