Human Pancreatic Lipase:  An Exposed Hydrophobic Loop from the C-terminal Domain May Contribute to Interfacial Binding

Bezzine, Sofiane; Carrière, Frédéric; De, Josiane; Verger, and Robert; De, Alain

doi:10.1021/bi973136r

Cited by 34 publications

(36 citation statements)

References 35 publications

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“…These data strongly indicate that the C-domain mediates the initial substrate binding step before lipid hydrolysis. These data are in agreement with previous reports of C-domain involvement in lipid binding (5,(23)(24)(25)(26)(27)(28)(29). Indeed, treatment with antibodies to LPL or HL indicated that the C-domain influences the hydrolysis of triolein (23,24,27).…”

Section: Discussionsupporting

confidence: 92%

“…Similar studies of domain exchange and antibody inhibition on human pancreatic lipase revealed that the interfacial stability of pancreatic lipase depends on the structure of the Cdomain (28). Bezzine et al (29) showed that a hydrophobic surface loop from the C-domain (b59) may be involved in the interaction of human pancreatic lipase with the lipid/water interface. The C-terminal region 415-438 of LPL was shown to play a role in the interaction of LPL with lipid substrates, as mutations in this region were shown to alter the enzyme lipolytic activity toward triolein or both triolein and tributyrin (26).…”

Section: Discussionmentioning

confidence: 99%

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Substrate specificity of lipoprotein lipase and endothelial lipase: studies of lid chimeras

Griffon

Budreck

Long

et al. 2006

Journal of Lipid Research

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The triglyceride (TG) lipase gene subfamily, consisting of LPL, HL, and endothelial lipase (EL), plays a central role in plasma lipoprotein metabolism. Compared with LPL and HL, EL is relatively more active as a phospholipase than as a TG lipase. The amino acid loop or ''lid'' covering the catalytic site has been implicated as the basis for the difference in substrate specificity between HL and LPL. To determine the role of the lid in the substrate specificity of EL, we studied EL in comparison with LPL by mutating specific residues of the EL lid and exchanging their lids. Mutation studies showed that amphipathic properties of the lid contribute to substrate specificity. Exchanging lids between LPL and EL only partially shifted the substrate specificity of the enzymes. Studies of a double chimera possessing both the lid and the C-terminal domain (C-domain) of EL in the LPL backbone showed that the role of the lid in determining substrate specificity does not depend on the nature of the C-domain of the lipase. Using a kinetic assay, we showed an additive effect of the EL lid on the apparent affinity for HDL 3 in the presence of the EL C-domain. Endothelial lipase (EL) is a member of the triglyceride (TG) lipase gene family (1) that is synthesized by endothelial cells and by other cell types such as macrophages and hepatocytes (1-3). EL has a distinct substrate specificity profile and is relatively more active as a phospholipase than as a TG lipase (4). Thus, the ratio of TG lipase to phospholipase activity of EL is markedly less than those of LPL and HL. Relative to phospholipase activity, LPL displays the highest TG lipase activity and EL the lowest TG lipase activity of the three enzymes. EL also has greater preference for HDL, and LPL has the greater preference for VLDL (4, 5). Thus, LPL, HL, and EL seem to have evolved with distinct substrate specificities to accommodate the full spectrum of circulating lipoproteins.The lid covering the active site has been implicated as a major source of substrate specificity for LPL and HL. Dugi et al. (6) studied the hypothesis that the surface lid covering the catalytic pocket may modulate access of the substrate to the active site of LPL. Characterization of a number of mutants with altered amphipathic properties of the LPL lid showed that the disruption of the lid decreased its ability to hydrolyze an emulsified lipid substrate without affecting the ability to hydrolyze a water-soluble substrate. They proposed that the interaction between the lipoprotein substrates and the lid may in part determine substrate specificity. Chimeric lipases were also generated by exchanging the lid region between LPL and HL (7). The lid of LPL conferred preferential TG hydrolysis, as opposed to augmenting phospholipase activity in the case of the lid of HL. Preferential in vivo hydrolysis of phospholipids (PLs) was demonstrated in HL-deficient mice injected with adenovirus-expressing lipases containing the HL lid (HL or LPL with the lid of HL) compared with lipases containing the ...

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Section: Discussionsupporting

confidence: 92%

Section: Discussionmentioning

confidence: 99%

Substrate specificity of lipoprotein lipase and endothelial lipase: studies of lid chimeras

Griffon

Budreck

Long

et al. 2006

Journal of Lipid Research

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“…described in the literature result from different proteolytic processing and originate from the same gene. Epitope mapping studies using monoclonal antibodies directed against human pancreatic lipase (HPL) and various mutant lipases suggest that the beta 5 0 loop from C-terminal domain may be involved in the interaction of HPL with a lipid/water interface (Bezzine et al, 1998).…”

Section: Sequencing and Cloning Of Lipase Genementioning

confidence: 99%

Production, purification, characterization, and applications of lipases

Sharma

Chisti

Banerjee

2001

Biotechnology Advances

1,286

712

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Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) catalyze the hydrolysis and the synthesis of esters formed from glycerol and long-chain fatty acids. Lipases occur widely in nature, but only microbial lipases are commercially significant. The many applications of lipases include speciality organic syntheses, hydrolysis of fats and oils, modification of fats, flavor enhancement in food processing, resolution of racemic mixtures, and chemical analyses. This article discusses the production, recovery, and use of microbial lipases. Issues of enzyme kinetics, thermostability, and bioactivity are addressed. Production of recombinant lipases is detailed. Immobilized preparations of lipases are discussed. In view of the increasing understanding of lipases and their many applications in high-value syntheses and as bulk enzymes, these enzymes are having an increasing impact on bioprocessing. D

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“…Examination of the colipase-PTL crystal structure reveals an exposed hydrophobic loop, the ␤5Ј-loop, including residues 405-414, of the C-terminal domain that orients in same plane as the hydrophobic plateau of colipase and the lid (5). This orientation positions the loop to interact with the oil-water surface of the lipid substrate (Fig.…”

mentioning

confidence: 99%

Val-407 and Ile-408 in the β5′-Loop of Pancreatic Lipase Mediate Lipase-Colipase Interactions in the Presence of Bile Salt Micelles

Freie

Ferrato

Carrière

et al. 2006

Journal of Biological Chemistry

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In the present study, we characterized the contribution of three residues in the ␤5-loop, Val-407, Ile-408, and Leu-412, to the function of hPTL. By substituting charged residues, aspartate or lysine, in these positions, we altered the hydrophilic to lipophilic ratio of the ␤5-loop. Each of the mutants was expressed, purified, and characterized for activity and binding with both monolayers and emulsions and for binding to colipase. Experiments with monolayers and with emulsions suggested that the interaction of hPTL with a phospholipid monolayer differs from the interaction of the hPTL-colipase complex with a dicaprin monolayer or a triglyceride emulsion (i.e. neutral lipids). Val-407, Ile-408, and Leu-412 make major contributions to interactions with monolayers, whereas only Val-407 and Ile-408 appear essential for activity on triglyceride emulsions in the presence of bile salt micelles. In solutions of taurodeoxycholate at micellar concentrations, a major effect of the ␤5-loop mutations is to change the interaction between hPTL and colipase. These observations support a major contribution of residues in the ␤5-loop in the function of hPTL and suggest that a third partner, bile salt micelles or the lipid interface or both, influence the binding of colipase and hPTL through interactions with the ␤5-loop.The three-dimensional structure of human pancreatic triglyceride lipase (hPTL) 2 revealed two functional domains, a globular N-terminal domain and a ␤-sandwich C-terminal domain (residues 336 -449) (1). Since the determination of the structure, functional roles have been described for each domain. The N-terminal domain consisting of an ␣/␤-hydrolase fold contains the catalytic site. A surface loop, the lid, delineated by a disulfide bond between Cys-237 and Cys-261, covers the active site and controls the activity of hPTL (2, 3). In the inactive conformation, the lid sterically hinders access of substrate to the active site. In the active conformation, the lid adopts a new position that opens and configures the active site. The C-terminal domain, which has structural similarity to the C2 domain of other lipid-binding proteins, contributes the majority of the binding surface for colipase, a necessary cofactor for hPTL function in the duodenum (4). Until recently, colipase binding was the best documented function for the C-terminal domain.Over the past several years, several lines of evidence suggested an additional role for the C-terminal domain. Examination of the colipase-PTL crystal structure reveals an exposed hydrophobic loop, the ␤5Ј-loop, including residues 405-414, of the C-terminal domain that orients in same plane as the hydrophobic plateau of colipase and the lid (5). This orientation positions the loop to interact with the oil-water surface of the lipid substrate (Fig. 1). Next, a number of studies indicate that a homologous loop in lipoprotein lipase, a protein with structural similarity to hPTL, contributes to the lipid-binding properties of lipoprotein lipase. For instance, mutagenesis of three tryp...

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Human Pancreatic Lipase: An Exposed Hydrophobic Loop from the C-terminal Domain May Contribute to Interfacial Binding

Cited by 34 publications

References 35 publications

Substrate specificity of lipoprotein lipase and endothelial lipase: studies of lid chimeras

Substrate specificity of lipoprotein lipase and endothelial lipase: studies of lid chimeras

Production, purification, characterization, and applications of lipases

Val-407 and Ile-408 in the β5′-Loop of Pancreatic Lipase Mediate Lipase-Colipase Interactions in the Presence of Bile Salt Micelles

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