Submitted on June 14, 2005
Revised on July 27, 2005
Accepted on August 2, 2005
Structure and function of lysosomal phospholipase A2: identification of the catalytic triad and the role of cysteine residues
Miki Hiraoka, Akira Abe, and James A. Shayman
Internal Medicine, University of Michigan, Ann Arbor, MI 48109-0676
Corresponding Author: jshayman{at}umich.edu
Lysosomal phospholipase A2 (LPLA2) is an acidic phospholipase that is highly expressed in alveolar macrophages and that may play a role in the catabolism of pulmonary surfactant. This lysosomal enzyme has 49% homology to lecithin:cholesterol acyltransferase (LCAT) and belongs to the 
,
hydrolase superfamily. The primary structure found in LCAT is conserved in LPLA2, including three amino acid residues potentially required for catalytic activity and four cysteine residues. To evaluate the role of these conserved amino acids in LPLA2 function, LPLA2 activity was measured in the soluble fraction of COS-7 cells transiently transfected with a c-myc-conjugated mouse LPLA2 or mutated mLPLA2. Each amino acid residue in the putative catalytic triad of LPLA2 was substituted with alanine. Each single point mutation resulted in the elimination of LPLA2 activity. Four cysteine residues (C65, C89, C330 and C371) are conserved between mouse, rat, and human LPLA2s, and are present in LCAT. The corresponding cysteine residues in mLPLA2 were replaced with alanine. Quadruple mutations at C65, C89, C330 and C371, double mutations at C65 and C89 and single mutation at C65 or C89 resulted in the elimination of LPLA2 activity. Double mutations at C330 and C371 and single mutation at C330 or C371 resulted in a partial reduction of LPLA2 activity. Thus the presence of a disulfide bond between C330 and C371 is not required for LPLA2 activity. Bovine LPLA2 containing four cysteine residues was absorbed to an organomercury agarose column and eluted from the column with 2-mercaptoethanol, consistent with the presence of at least two free cysteine residues. We propose that LPLA2 contains one disulfide bond between C65 and C89 and free cysteine residues at C330 and C371 and that the triad, S198, D360 and H392, is required for the full expression of LPLA2 activity.
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