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.
Hiraoka
Miki
M
Nephrology Division, Department of Internal Medicine, University of Michigan, Ann Arbor, MI, USA.
Abe
Akira
A
Shayman
James A
JA
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry; D001921:Brain / Q000378:metabolism; D019556:COS Cells; D002384:Catalysis; D020134:Catalytic Domain; D002417:Cattle; D002522:Chlorocebus aethiops; D002845:Chromatography; D003545:Cysteine / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006639:Histidine / Q000737:chemistry; D006801:Humans; D015151:Immunoblotting; D007668:Kidney / Q000378:metabolism; D008247:Lysosomes / Q000201:enzymology*; D008264:Macrophages / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D016366:Open Reading Frames; D010713:Phosphatidylcholines / Q000737:chemistry; D010714:Phosphatidylethanolamines / Q000737:chemistry; D010741:Phospholipases A / Q000737:chemistry*; D054467:Phospholipases A2; D010743:Phospholipids / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011663:Pulmonary Surfactants / Q000737:chemistry; D051381:Rats; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry; D013501:Surface-Active Agents / Q000737:chemistry; D014162:Transfection
D004220:Disulfides; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D010743:Phospholipids; D011663:Pulmonary Surfactants; D013501:Surface-Active Agents; D001224:Aspartic Acid; C483858:phosphatidylethanolamine; D012694:Serine; D006639:Histidine; D010741:Phospholipases A; D054467:Phospholipases A2; D003545:Cysteine; D000409:Alanine