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Journal of Lipid Research, Vol. 46, 2441-2447, November 2005
Copyright © 2005 by American Society for Biochemistry and Molecular Biology

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

Miki Hiraoka^1,2, Akira Abe¹ and James A. Shayman³

Nephrology Division, Department of Internal Medicine, University of Michigan, Ann Arbor, MI

Published, JLR Papers in Press, August 16, 2005. DOI 10.1194/jlr.M500248-JLR200

¹ M. Hiraoka and A. Abe contributed equally to this work.

² Present address of M. Hiraoka: Ophthalmology Department, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8603, Japan.

³ To whom correspondence should be addressed. e-mail: 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. 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.

Supplementary key words surfactant • macrophage • lipase • phospholipid • phosphatidylcholine • phosphatidylethanolamine

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