A proposed architecture for lecithin cholesterol acyl transferase (LCAT): Identification of the catalytic triad and molecular modeling

Peelman, Frank; Vinaimont, N; Verhee, Annick; Vanloo, Berlinda; Verschelde, Jl; Labeur, Christine; S, Séguret-Macé; Duverger, Nicolas; Hutchinson, Gillian; Vandekerckhove, Joël; Tavernier, Jan; Rosseneu, Maryvonne

doi:10.1002/pro.5560070307

Cited by 88 publications

(98 citation statements)

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“…The phenylalanine at residue 103 in LCAT is one of two amino acid residues involved in the formation of the oxyanion hole (14) and is conserved in mLPLA 2 and other LPLA 2 s (Fig. 1).…”

Section: Discussionmentioning

confidence: 99%

Structure and function of lysosomal phospholipase A2: identification of the catalytic triad and the role of cysteine residues

Hiraoka¹,

Abe²,

Shayman

2005

Journal of Lipid Research

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Lysosomal phospholipase A 2 (LPLA 2 ) is an acidic phospholipase that is highly expressed in alveolar macrophages and that may play a role in the catabolism of pulmonary surfactant. The primary structure found in LCAT is conserved in LPLA 2 , including three amino acid residues potentially required for catalytic activity and four cysteine residues. LPLA 2 activity was measured in COS-7 cells transfected with c-myc -conjugated mouse LPLA 2 (mLPLA 2 ) or mutated LPLA 2 . Single alanine substitutions in the catalytic triad resulted in the elimination of LPLA 2 activity. Four cysteine residues (C65, C89, C330, and C371), conserved between LPLA 2 and LCAT, were replaced with alanine. Quadruple mutations at C65, C89, C330, and C371, double mutations at C65 and C89, and a single mutation at C65 or C89 resulted in the elimination of activity. Double mutations at C330 and C371 and a single mutation at C330 or C371 resulted in a partial reduction of activity. Thus, the presence of a disulfide bond between C330 and C371 is not required for LPLA 2 activity. We propose that one disulfide bond between C65 and C89 and free cysteine residues at C330 and C371 and the triad, serine-198, aspartic acid-360, and histidine-392, are required for the full expression of mLPLA 2 activity. Recently, a novel lysosomal phospholipase A 2 (LPLA 2 ) was purified from bovine brain (1), and the bovine, mouse, and human genes encoding LPLA 2 were cloned (2). LPLA 2 is highly expressed in the alveolar macrophages of rats and mice (3). Granulocyte-macrophage colony-stimulating factor-deficient mice, a model of pulmonary alveolar proteinosis (4), exhibited significantly lower LPLA 2 activity in alveolar macrophages compared with wild-type mice, consistent with a role for LPLA 2 in the phospholipid catabolism of pulmonary surfactant (3).LPLA 2 has both transacylase and phospholipase A 2 activities under acidic conditions (1, 5). Divalent cations are not required for LPLA 2 activity, although calcium slightly enhances enzyme activity (1). The purified LPLA 2 is a water-soluble glycoprotein consisting of a single peptide chain with a molecular mass of 45 kDa (1). The primary structure of LPLA 2 , deduced from DNA sequences encoding LPLA 2 , is highly conserved between mammals, including mouse, rat, human, and bovine. The amino acid sequence of LPLA 2 is 49% identical to that of LCAT ( Fig. 1 ). Based on their primary structures, both enzymes are members of the ␣␤ -hydrolase superfamily and have the amino acid residues forming a catalytic triad (2, 6). In general, the triad consists of three amino acid residues, serine, aspartic acid/glutamic acid, and histidine, a structure that is essential for the hydrolysis reaction. We previously demonstrated that the serine-198 (S198) residue within a putative lipase motif sequence and within the catalytic triad is required for phospholipase A 2 activity (2).In addition, there are four cysteine residues that are conserved between LPLA 2 and LCAT (2, 6). Two of the cysteines are proximate to the N terminu...

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“…The phenylalanine at residue 103 in LCAT is one of two amino acid residues involved in the formation of the oxyanion hole (14) and is conserved in mLPLA 2 and other LPLA 2 s (Fig. 1).…”

Section: Discussionmentioning

confidence: 99%

Structure and function of lysosomal phospholipase A2: identification of the catalytic triad and the role of cysteine residues

Hiraoka¹,

Abe²,

Shayman

2005

Journal of Lipid Research

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“…The discovery of lipid-interacting helical peptides at the entrance of the lipid binding cavities of the lipases (28), along with the predicted cavities of CETP (29) and lecithin cholesterol acyl transferase (30), suggests that the mechanism of lipid acquisition described here for MTP might apply to other lipid binding proteins.…”

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confidence: 97%

A Mechanism of Membrane Neutral Lipid Acquisition by the Microsomal Triglyceride Transfer Protein

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Anderson

Ritchie

et al. 2000

Journal of Biological Chemistry

121

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Chylomicrons (CM)1 and very low density lipoproteins (VLDL) are among the largest macromolecular complexes secreted from eukaryotic cells. The assembly of neutral lipids and phospholipids into CM and VLDL is nucleated around a single molecule of apoB and requires a microsomal triglyceride transfer protein (MTP) complexed to the endoplasmic reticulumresident protein, protein disulfide isomerase (PDI). The function of the MTP-PDI complex is to supply apoB with sufficient lipid to form a soluble lipoprotein. Defects of apoB and MTP cause hypobetalipoproteinemia and abetalipoproteinemia, respectively (1, 2).ApoB and MTP have structural homology with lamprey lipovitellin (LV) (3). LV contains an N-terminal ␤-barrel (amino acids 17-296), an ␣-helical structure (amino acids 297-614), and a C-terminal lipid binding cavity (4). The structural relationship between MTP, apoB, and LV is supported by conservation of the gene and protein structure (3,5). Important features of the quaternary structure of the lamprey LV homodimer are adapted in MTP to form a heterodimer with PDI and to associate with apoB during lipoprotein production (3, 5). The defining difference between MTP, apoB, and LV is related to their C-terminal lipid binding structures, which associate with different amounts of lipid (3). LV binds principally phospholipid with a stoichiometry of ϳ35 molecules/subunit (6). MTP binds 1-5 molecules of lipid (7). ApoB has a long C-terminal extension (ϳ3500 amino acids), which incorporates a large neutral lipid core (8).Here, we have addressed the mechanism by which MTP-PDI acquires neutral lipid from phospholipid bilayers for the assembly of VLDL and CMs. In the absence of a crystal structure for MTP, we derived a homology model to guide mutagenesis and biophysical studies. The experimental data substantiate the overall predictions of the model and provide insights into the mechanism of lipid acquisition and binding. EXPERIMENTAL PROCEDURESModeling-Models were developed on the alignment shown in Fig. 1 and the x-ray crystal structure of lamprey LV (Protein Data Bank accession number 1LLV), refined to an R-value of 0.19 at 2.8 Å resolution (4). The C-sheet was modeled using INSIGHT interactive graphics software and the Homology computer program (Biosym Technologies, San Diego) and the A-sheet with the general purpose modeling program O (9). Models were energy minimized and the quality of the coordinates assessed as described (3).Mutagenesis and Expression Studies-Mutagenesis was performed by a polymerase chain reaction-based strategy (3). All constructs were sequenced before use. Transfections, preparation of cell extracts, and triglyceride transfer activities were performed as described (2, 3).Triglyceride Binding and Fusogenic Activity-Wild-type (WT) and mutant MTP-PDI complexes were purified as described (10). Donor small unilamellar vesicles (SUVs) were prepared as described (2), purified to homogeneity (11), and incubated with MTP (w/w 70:1) for 2 h at 37°C. Lipid-protein complexes were separated on a Sepharose CL-4B...

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“…The primary sequence of LCAT is well conserved between mammalian species (6). A structural model for LCAT predicts the conformation of a catalytic triad formed by SerAsp-His residues involved in the phospholipase reaction (7). Mammalian LCATs are primarily expressed in the liver and secreted to the plasma where they circulate in association with HDL (8).…”

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confidence: 99%

A Lipolytic Lecithin:Cholesterol Acyltransferase Secreted by Toxoplasma Facilitates Parasite Replication and Egress

Pszenny

Ehrenman

Romano

et al. 2016

Journal of Biological Chemistry

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The protozoan parasite Toxoplasma gondii develops within a parasitophorous vacuole (PV) in mammalian cells, where it scavenges cholesterol. When cholesterol is present in excess in its environment, the parasite expulses this lipid into the PV or esterifies it for storage in lipid bodies. Here, we characterized a unique T. gondii homologue of mammalian lecithin:cholesterol acyltransferase (LCAT), a key enzyme that produces cholesteryl esters via transfer of acyl groups from phospholipids to the 3-OH of free cholesterol, leading to the removal of excess cholesterol from tissues. TgLCAT contains a motif characteristic of serine lipases "AHSLG" and the catalytic triad consisting of serine, aspartate, and histidine (SDH) from LCAT enzymes. TgL-CAT is secreted by the parasite, but unlike other LCAT enzymes it is cleaved into two proteolytic fragments that share the residues of the catalytic triad and need to be reassembled to reconstitute enzymatic activity. TgLCAT uses phosphatidylcholine as substrate to form lysophosphatidylcholine that has the potential to disrupt membranes. The released fatty acid is transferred to cholesterol, but with a lower transesterification activity than mammalian LCAT. TgLCAT is stored in a subpopulation of dense granule secretory organelles, and following secretion, it localizes to the PV and parasite plasma membrane. LCAT-null parasites have impaired growth in vitro, reduced virulence in animals, and exhibit delays in egress from host cells. Parasites overexpressing LCAT show increased virulence and faster egress. These observations demonstrate that TgLCAT influences the outcome of an infection, presumably by facilitating replication and egress depending on the developmental stage of the parasite.The phospholipase A 2 (PLA 2 ) 2 family of serine lipases comprises more than 30 enzymes in mammals. The PLA 2 members hydrolyze the sn-2 ester of phospholipids, yielding lysophospholipid and releasing a fatty acid (1). Approximately one-third of the PLA 2 family members are secreted from cells and display various localizations post-secretion, reflecting their specialized biological functions (2). Among the secretory PLA 2 , lecithin: cholesterol acyltransferase (LCAT; EC 2.3.1.43) is characterized by dual activity, PLA 2 and acyltransferase. In mammals, this enzyme catalyzes the transacylation of the sn-2 fatty acid liberated from various phospholipids (e.g. phosphatidylcholine or phosphatidylethanolamine) to the 3-␤-hydroxyl group on the A-ring of cholesterol, thereby forming cholesteryl esters (3-5). The primary sequence of LCAT is well conserved between mammalian species (6). A structural model for LCAT predicts the conformation of a catalytic triad formed by SerAsp-His residues involved in the phospholipase reaction (7). Mammalian LCATs are primarily expressed in the liver and secreted to the plasma where they circulate in association with HDL (8). These enzymes are components of the reverse cholesterol transport pathway by which cholesterol from peripheral cells is delivered to the liver f...

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A proposed architecture for lecithin cholesterol acyl transferase (LCAT): Identification of the catalytic triad and molecular modeling

Cited by 88 publications

References 50 publications

Structure and function of lysosomal phospholipase A2: identification of the catalytic triad and the role of cysteine residues

Structure and function of lysosomal phospholipase A2: identification of the catalytic triad and the role of cysteine residues

A Mechanism of Membrane Neutral Lipid Acquisition by the Microsomal Triglyceride Transfer Protein

A Lipolytic Lecithin:Cholesterol Acyltransferase Secreted by Toxoplasma Facilitates Parasite Replication and Egress

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