Characterization of functional residues in the interfacial recognition domain of lecithin cholesterol acyltransferase (LCAT)

Peelman, Frank; Vanloo, Berlinda; Pérez-Méndez, Óscar; Decout, Anne; Verschelde, Jean-Luc; Labeur, Christine; Vinaimont, N; Verhee, Annick; Duverger, Nicolas; Brasseur, Robert; Vandekerckhove, Joël; Tavernier, Jan; Rosseneu, Maryvonne

doi:10.1093/protein/12.1.71

Cited by 34 publications

(27 citation statements)

References 32 publications

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“…In both LPLA 2 and LCAT, the region between the cysteine residues forming the disulfide bridge in the proximity of the N-terminal region is predicted to be the most hydrophobic region that is spanned as a loop containing an amphipathic helix (8,9). The loop formed in LCAT is essential for the interaction between LCAT and lipoprotein surfaces and is thought to be indispensable for the interfacial activation of LCAT (17). By analogy, the loop in LPLA 2 may also play a crucial role in the interaction of LPLA 2 with hydrophobic structures such as phospholipid membranes.…”

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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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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“…These lids have highly variable sequences in lipases and are able to form an amphipatic helix interacting with the lipid interface. It has been suggested that they destabilize the bilayer and facilitate both the binding of the hydrophobic substrate and its diffusion into the active site cavity of the enzyme (Peelman et al, 1999). A Trp residue in the lid is thought to bind the cleaved fatty acid in the active site of the enzyme (Martinelle et al, 1996).…”

Section: Identification Of the Arabidopsis Pdat Genementioning

confidence: 99%

Cloning and Functional Characterization of a Phospholipid:Diacylglycerol Acyltransferase from Arabidopsis

et al. 2004

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A new pathway for triacylglycerol biosynthesis involving a phospholipid:diacylglycerol acyltransferase (PDAT) was recently described (Dahlqvist A, Stahl U, Lenman M, Banas A, Lee M, Sandager L, Ronne H, Stymne S, [2000] Proc Natl Acad Sci USA 97: 6487–6492). The LRO1 gene that encodes the PDAT was identified in yeast (Saccharomyces cerevisiae) and shown to have homology with animal lecithin:cholesterol acyltransferase. A search of the Arabidopsis genome database identified the protein encoded by the At5g13640 gene as the closest homolog to the yeast PDAT (28% amino acid identity). The cDNA of At5g13640 (AtPDAT gene) was overexpressed in Arabidopsis behind the cauliflower mosaic virus promoter. Microsomal preparations of roots and leaves from overexpressers had PDAT activities that correlated with expression levels of the gene, thus demonstrating that this gene encoded PDAT (AtPDAT). The AtPDAT utilized different phospholipids as acyl donor and accepted acyl groups ranging from C10 to C22. The rate of activity was highly dependent on acyl composition with highest activities for acyl groups containing several double bonds, epoxy, or hydroxy groups. The enzyme utilized both sn-positions of phosphatidylcholine but had a 3-fold preference for the sn-2 position. The fatty acid and lipid composition as well as the amounts of lipids per fresh weight in Arabidopsis plants overexpressing AtPDAT were not significantly different from the wild type. Microsomal preparations of roots from a T-DNA insertion mutant in the AtPDAT gene had barely detectable capacity to transfer acyl groups from phospholipids to added diacylglycerols. However, these microsomes were still able to carry out triacylglycerol synthesis by a diacylglycerol:diacylglycerol acyltransferase reaction at the same rate as microsomal preparations from wild type.

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“…The LCAT reaction occurs primarily on the surface of high-density lipoproteins (HDL), which contains phospholipids, cholesterol, and apolipoprotein A-I (apoA-I). Although no crystal structure exists for LCAT, the study of its structure-function relationships has been facilitated by the construction of a computer model of the catalytic core of LCAT (2)(3)(4)(5)(6). Based on its amino acid composition alone, LCAT has an intermediate hydrophobicity between integral membrane proteins and apolipoproteins (7).…”

Section: Lecithin-cholesterol Acyltransferase (Lcat)mentioning

confidence: 99%

Deletion of Specific Glycan Chains Affects Differentially the Stability, Local Structures, and Activity of Lecithin-cholesterol Acyltransferase

Kosman

Jonas

2001

Journal of Biological Chemistry

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The enzymatic and interfacial binding activity of lecithin-cholesterol acyltransferase (LCAT) is affected differentially by the location and extent of its glycosylation. Two LCAT glycosylation-deficient mutants, N84Q and N384Q, were constructed, permanently expressed in Chinese hamster ovary cells, and purified to determine the effects of deleting individual glycan chains on its stability, structure, and function. These purified mutants were studied by spectroscopic structural methods and enzymatic and binding assays to develop a molecular rationale for the relationship between LCAT glycosylation and activity. The N84Q LCAT mutant did not possess measurable enzymatic activity or interfacial binding affinity for reconstituted high-density lipoproteins. In addition, in thermal and chemical denaturation studies, N84Q LCAT was found to be significantly less stable than wild-type LCAT. The N384Q variant was initially more enzymatically active than wild-type LCAT, but gradually lost activity within months; however, it retained full interfacial binding activity. Significant changes were detected over time by circular dichroism in the ␣-helical content of N384Q LCAT and in the ␤-sheet content of N84Q LCAT, compared with wild-type LCAT. Fluorescence measurements with the probe 1-anilinonapthalene-8-sulfonate suggested an alteration of the active site cavity in both mutants. In conclusion, both mutants lost catalytic activity, N84Q shortly after purification and N384Q more gradually, and were destabilized, probably because the deletion of the glycan chains altered local structural elements near the active site cavity and/or the interfacial binding regions. Lecithin-cholesterol acyltransferase (LCAT)1 is the enzyme responsible for the generation of the majority of the cholesterol esters present in human plasma. The conversion of cholesterol to cholesterol esters is necessary to facilitate reverse cholesterol transport, the process which removes excess cholesterol and phospholipids from peripheral cells and delivers them to the liver for disposal (1). The LCAT reaction occurs primarily on the surface of high-density lipoproteins (HDL), which contains phospholipids, cholesterol, and apolipoprotein A-I (apoA-I). Although no crystal structure exists for LCAT, the study of its structure-function relationships has been facilitated by the construction of a computer model of the catalytic core of LCAT (2-6). Based on its amino acid composition alone, LCAT has an intermediate hydrophobicity between integral membrane proteins and apolipoproteins (7). One modification commonly found in plasma proteins, including LCAT, that increases their solubility and hydrophilicity is glycosylation.Four N-linked complex type glycans and two O-linked glycans are found in LCAT (8, 9) and constitute around 20% of the total enzyme mass of human plasma LCAT (10). Different cell types transfected with the human LCAT gene express the glycan structure of LCAT differently (11, 12), which leads to disparities between recombinant preparations of LCAT. Chines...

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Characterization of functional residues in the interfacial recognition domain of lecithin cholesterol acyltransferase (LCAT)

Cited by 34 publications

References 32 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

Cloning and Functional Characterization of a Phospholipid:Diacylglycerol Acyltransferase from Arabidopsis

Deletion of Specific Glycan Chains Affects Differentially the Stability, Local Structures, and Activity of Lecithin-cholesterol Acyltransferase

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