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

Abstract

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...

Positional specificity of lysosomal phospholipase A2

“…We currently view Lpla2 as primarily a phospholipase A with moderate transacylase activity. As reported previously, Lpla2 is completely inhibited by DPF (9), cannot transfer free fatty acid or fatty acyl-CoA to NAS (1), and has the catalytic triad amino acid residues (serine, aspartate, and histidine) in common with some hydrolases (10). Sitedirected mutagenesis at the putative catalytic serine residue of Lpla2 abolishes both the PLA 2 and transacylase activities of Lpla2.…”

The Secretion and Uptake of Lysosomal Phospholipase A2 by Alveolar Macrophages

“…The LPLA 2 deficient cells were treated with recombinant mouse LPLA 2 with or without a site directed mutation at the catalytic site. In the mutated LPLA 2 , the catalytic serine residue was converted to alanine (7). As reported previously, the wild-type mouse LPLA 2 and the mutated mouse LPLA 2 can be transiently expressed in transfected COS-7 cells.…”

“…LPLA 2 has 49% identity to lecithin-cholesterol acyl-transferase and belongs to the ␣␤-hydrolase superfamily. The catalytic triad and four cysteine residues conserved in LPLA 2 are preserved in lecithin-cholesterol acyl-transferase (7).…”

The measurement of lysosomal phospholipase A2 activity in plasma

“…LPLA2 contains a catalytic triad structure that consists of three amino residues, serine, aspartic acid, and histidine, and is essential for the enzymatic activity ( 9 ). Such triad structures are found in many hydrolases such as proteases, lipases, phospholipases, and esterases.…”

“…The reaction product, 1-O -acyl-NAS, is easily isolated by TLC and detected by charring. To the best of our knowledge, the ceramide transacylation found in cell extracts is LPLA2 specifi c ( 3,8,9 ). Therefore, to establish a conventional assay method to detect LPLA2 activity in extracellular fl uids, we applied and developed the transacylation-based assay method to measure LPLA2 activities in plasma and serum.…”