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

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. The primary structure found in LCAT is conserved in LPLA₂, including three amino acid residues potentially required for catalytic activity and four cysteine residues. LPLA₂ activity was measured in COS-7 cells transfected with c-myc-conjugated mouse LPLA₂ (mLPLA₂) or mutated LPLA₂. Single alanine substitutions in the catalytic triad resulted in the elimination of LPLA₂ activity. Four cysteine residues (C65, C89, C330, and C371), conserved between LPLA₂ 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 LPLA₂ 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 mLPLA₂ activity.