CHARACTERIZATION BY ELECTROSPRAY MASS-SPECTROMETRY OF HUMAN CA2-SENSITIVE CYTOSOLIC PHOSPHOLIPASE-A(2) PRODUCED IN BACULOVIRUS-INFECTED INSECT CELLS()

The 85-kD cytosolic phospholipase A2 (cPLA2) is a novel receptor-regul ated phospholipase that is thought to initiate the production of infla mmatory lipid mediators. Since cPLA2 is present only in minute amounts (less than 0.01 % of total cellular protein) in various cells and tis sues, we have used the baculovirus expression system to produce suffic ient quantities of cPLA2 for structural and functional analysis. The c DNA for cPLA2 was cloned into a baculovirus expression vector and, upo n infection of Spodoptera frugiperda Sf-21 cells with the recombinant virus, cPLA2 was produced at high levels (9% of total cellular soluble protein). Gel electrophoresis and immunoblot analysis demonstrated th at the recombinant protein has properties indistinguishable from cPLA2 present in human monocytic U937 cells. Structural analysis of recombi nant cPLA2, using electrospray mass spectrometry in conjunction with a utomated sequence analysis, confirmed the expected sequence and reveal ed two post-translational modifications of the protein, phosphorylatio n on at least one site, and acetylation of the N-terminal serine resid ue after removal of the initiating methionine. In spite of the presenc e of six potential N-glycosylation sites, there is no evidence that an y of them is glycosylated. The baculovirus expression system should pr ove useful for production of cPLA2, and electrospray mass spectrometry is a rapid and accurate method for the analysis of post-translational modifications.