Characterization by electrospray mass spectrometry of human Ca(2+)-sensitive cytosolic phospholipase A2 produced in baculovirus-infected insect cells.
The 85-kD cytosolic phospholipase A2 (cPLA2) is a novel receptor-regulated phospholipase that is thought to initiate the production of inflammatory lipid mediators. Since cPLA2 is present only in minute amounts (less than 0.01% of total cellular protein) in various cells and tissues, we have used the baculovirus expression system to produce sufficient quantities of cPLA2 for structural and functional analysis. The cDNA for cPLA2 was cloned into a baculovirus expression vector and, upon 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 that the recombinant protein has properties indistinguishable from cPLA2 present in human monocytic U937 cells. Structural analysis of recombinant cPLA2, using electrospray mass spectrometry in conjunction with automated sequence analysis, confirmed the expected sequence and revealed two post-translational modifications of the protein, phosphorylation on at least one site, and acetylation of the N-terminal serine residue after removal of the initiating methionine. In spite of the presence of six potential N-glycosylation sites, there is no evidence that any of them is glycosylated. The baculovirus expression system should prove useful for production of cPLA2, and electrospray mass spectrometry is a rapid and accurate method for the analysis of post-translational modifications.
Becker
G W
GW
Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN 46285.
Miller
J R
JR
Kovacevic
S
S
Ellis
R M
RM
Louis
A I
AI
Small
J S
JS
Stark
D H
DH
Roberts
E F
EF
Wyrick
T K
TK
Hoskins
J
J
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002118:Calcium / Q000494:pharmacology*; D002460:Cell Line; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology*; D015870:Gene Expression; D018014:Gene Transfer Techniques; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009036:Moths; D010741:Phospholipases A / Q000737:chemistry / Q000235:genetics*; D054467:Phospholipases A2; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins
D011994:Recombinant Proteins; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium