Human Pancreatic Lipase: Colipase Dependence and Interfacial Binding of Lid Domain Mutants

Bezzine, Sofiane; Ferrato, Francine; Ivanova, М.; Lopez, Véronique; Verger, Robert; Carrière, Frédéric

doi:10.1021/bi982601x

Cited by 71 publications

(64 citation statements)

References 43 publications

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“…Binding of hPTL and the Mutants to Tributyrin and Trioctanoin Emulsions-The interfacial binding of hPTL and the mutants was assayed by mixing the enzyme with a trioctanoin or tributyrin emulsion as previously described (14,15). A 5-fold molar excess of colipase and varying amounts of taurodeoxycholate were included in the incubations as indicated.…”

Section: Methodsmentioning

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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Section: Methodsmentioning

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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“…All fragments were calculated to be ␣-helix and structures were optimized by a Simplex energy minimization procedure. The behavior of each helix at the IMPALA 6 hydrophobic/ hydrophilic interface (20) was then calculated by inserting the peptide at every Å of the model membrane from Ϫ40 Å (water medium) to 0 Å (membrane center), rotating the helix around its axis and rotating the helix axis with respect to the interface plane. At each step, the best position was selected as the lowest IMPALA restraint value.…”

Section: Methodsmentioning

confidence: 99%

“…The lipids (500 mg) were solubilized in 5 ml of chloroform/methanol (2:1, v/v). An aliquot (500 l) of the mixture was dried under nitrogen, resuspended in 3 ml of 6 The abbreviations used are: IMPALA, integral membrane protein and lipid association; TP, tilted peptide; NaTDC, sodium taurodeoxycholate; HuLip, human pancreatic lipase; E600, diethyl p-nitrophenyl phosphate; PLRP2, pancreatic lipase-related protein 2; ASA, accessible surface area. a 1 mM Tris-HCl buffer, pH 7.5, containing 150 mM NaCl and 5 mM CaCl 2 , and sonicated for 5 min at 95% power level and a frequency of 20.178 Hz (Sonoreactor Undatim, Japan) in a ice/ethanol bath.…”

Section: Methodsmentioning

confidence: 99%

“…It was first thought to account for the interfacial activation of pancreatic lipase but the presence of a full-length lid in most pancreatic lipase-related proteins that display no interfacial activation rules out this explanation. Actually, it is not the lid per se but rather its motion and stabilization in the open conformation that are implicated in the activation of the lipase (6,7). Contradictory results were reported for the lid implication in the lipase binding to an interface.…”

mentioning

confidence: 99%

“…Contradictory results were reported for the lid implication in the lipase binding to an interface. In one study (5), the deletion of the [240 -260] region impeded lipase binding whereas, in another study (6), the binding was maintained but the mini-lid lipase had very weak activity. Investigation of crystal structures supported the idea that side chains of the [251-259] lid fragment might be involved in the hydrophobic groove interacting with the substrate (8).…”

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confidence: 99%

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Role of the Lid Hydrophobicity Pattern in Pancreatic Lipase Activity

Thomas

Allouche

Basyn

et al. 2005

Journal of Biological Chemistry

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Pancreatic lipase is a soluble globular protein that must undergo structural modifications before it can hydrolyze oil droplets coated with bile salts. The binding of colipase and movement of the lipase lid open access to the active site. Mechanisms triggering lid mobility are unclear. The *KNILSQIVDIDGI* fragment of the lid of the human pancreatic lipase is predicted by molecular modeling to be a tilted peptide. Tilted peptides are hydrophobicity motifs involved in membrane fusion and more globally in perturbations of hydrophobic/hydrophilic interfaces. Analysis of this lid fragment predicts no clear consensus of secondary structure that suggests that its structure is not strongly sequence determined and could vary with environment. Point mutations were designed to modify the hydrophobicity profile of the [240 -252] fragment and their consequences on the lipase-mediated catalysis were tested. Two mutants, in which the tilted peptide motif was lost, also have poor activity on bile saltcoated oil droplets and cannot be reactivated by colipase. Conversely, one mutant in which a different tilted peptide is created retains colipase dependence. These results suggest that the tilted hydrophobicity pattern of the [240 -252] fragment is neither important for colipase binding to lipase, nor for interfacial binding but is important to trigger the maximal catalytic efficiency of lipase in the presence of bile salt.The pancreatic lipase-colipase complex plays a key role in dietary fat absorption in the intestine by converting triglycerides into more polar products able to cross the brush-border membrane of enterocytes. The lipase is fully active on water-insoluble substrates that form lipid/water interfaces, a phenomenon called interfacial activation. In vivo, oil droplets consist of a bulk substrate phase surrounded by a monolayer of amphiphilic compounds, mainly biliary lipids (phosphatidylcholine, cholesterol and bile salts) that prevent lipase adsorption. To counteract the inhibitory effect of biliary lipids, the pancreas secretes a small protein, colipase, which anchors lipase to the biliary lipid-coated water/ lipid interface. The water-soluble lipase must therefore partition between aqueous and lipid phases before lipolysis can occur.Structural studies (1-3) have shown that lipases possess a two-domain organization, the N-terminal domain bearing the active site and the C-terminal domain bearing the colipase binding site. One specific feature of lipase is the shielding of its catalytic site by a surface loop (lid) controlling the access of the substrate. Therefore, movement of the lid domain is an absolute requirement for the lipase to adopt an active conformation.The role of the lid in lipolysis has been investigated by site-directed mutagenesis, lid exchange, or lid deletion (4 -6). It was first thought to account for the interfacial activation of pancreatic lipase but the presence of a full-length lid in most pancreatic lipase-related proteins that display no interfacial activation rules out this explanation. Actua...

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The role of the surgeon in whether patients with lymph node‐positive colon cancer see a medical oncologist

Luo

Giordano

Zhang

et al. 2007

Cancer

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Background Neurofibromatosis type 1 (NF1) is characterized by low‐grade tumors of the central and peripheral nervous system. There is also an increased risk of developing malignant tumors. Glioblastoma is an uncommon, malignant tumor of children that is even less frequently observed in children with NF1. Procedure We performed a retrospective review of patients with NF1 and glioblastoma to determine specific clinical and pathologic indicators of overall prognosis. Results Five patients were identified from the CHB/DFCI database for whom pathologic and imaging studies were available. All pathologic specimens demonstrated vascular proliferation and necrosis. All samples stained positively for p53. Chromogenic in situ hybridization (CISH) for epidermal growth factor receptor (EGFR) copy numbers was increased, PTEN copy numbers were normal and the promoter of the O(6)‐methylguanine‐DNA methyltransferase (MGMT) gene was unmethylated in the one patient evaluated. In the same time period, there were 56 patients without NF1 diagnosed with glioblastoma who were treated at our institution. Although the small sample size precludes formal statistical analysis, the 2‐year survival of patients with NF1 is 60% with median overall survival of 9.25 years compared to non‐NF1 patients with a 2‐year survival of 25% and median overall survival 1.08 years. Conclusions This study provides preliminary evidence that children with NF1 may be at risk for glioblastoma, but that these patients have an increased survival compared to children without NF1. Additional molecular studies will be required to determine if the pathogenesis of these tumors differs from glioblastoma in children without NF1. Pediatr Blood Cancer 2010;54:890–896 © 2010 Wiley‐Liss, Inc.

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Human Pancreatic Lipase: Colipase Dependence and Interfacial Binding of Lid Domain Mutants

Cited by 71 publications

References 43 publications

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

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

Role of the Lid Hydrophobicity Pattern in Pancreatic Lipase Activity

The role of the surgeon in whether patients with lymph node‐positive colon cancer see a medical oncologist

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