Distinct arachidonate-releasing functions of mammalian secreted phospholipase A2s in human embryonic kidney 293 and rat mastocytoma RBL-2H3 cells through heparan sulfate shuttling and external plasma membrane mechanisms.
We analyzed the ability of a diverse set of mammalian secreted phospholipase A(2) (sPLA(2)) to release arachidonate for lipid mediator generation in two transfected cell lines. In human embryonic kidney 293 cells, the heparin-binding enzymes sPLA(2)-IIA, -IID, and -V promote stimulus-dependent arachidonic acid release and prostaglandin E(2) production in a manner dependent on the heparan sulfate proteoglycan glypican. In contrast, sPLA(2)-IB, -IIC, and -IIE, which bind weakly or not at all to heparanoids, fail to elicit arachidonate release, and addition of a heparin binding site to sPLA(2)-IIC allows it to release arachidonate. Heparin nonbinding sPLA(2)-X liberates arachidonic acid most likely from the phosphatidylcholine-rich outer plasma membrane in a glypican-independent manner. In rat mastocytoma RBL-2H3 cells that lack glypican, sPLA(2)-V and -X, which are unique among sPLA(2)s in being able to hydrolyze phosphatidylcholine-rich membranes, act most likely on the extracellular face of the plasma membrane to markedly augment IgE-dependent immediate production of leukotriene C(4) and platelet-activating factor. sPLA(2)-IB, -IIA, -IIC, -IID, and -IIE exert minimal effects in RBL-2H3 cells. These results are also supported by studies with sPLA(2) mutants and immunocytostaining and reveal that sPLA(2)-dependent lipid mediator generation occur by distinct (heparanoid-dependent and -independent) mechanisms in HEK293 and RBL-2H3 cells.
Murakami
M
M
Department of Health Chemistry, School of Pharmaceutical Sciences, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan.
Koduri
R S
RS
Enomoto
A
A
Shimbara
S
S
Seki
M
M
Yoshihara
K
K
Singer
A
A
Valentin
E
E
Ghomashchi
F
F
Lambeau
G
G
Gelb
M H
MH
Kudo
I
I
D000595:Amino Acid Sequence; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D015232:Dinoprostone / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D054501:Group II Phospholipases A2; D054505:Group V Phospholipases A2; D019812:Heparan Sulfate Proteoglycans / Q000378:metabolism*; D006493:Heparin / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007150:Immunohistochemistry; D017997:Leukotriene C4 / Q000378:metabolism; D008407:Mast Cells / Q000378:metabolism; D012515:Mast-Cell Sarcoma / Q000378:metabolism*; D051379:Mice; D018613:Microscopy, Confocal; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010741:Phospholipases A / Q000378:metabolism*; D010766:Phosphorylation; D010972:Platelet Activating Factor / Q000378:metabolism; D011485:Protein Binding; D012313:RNA / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured
D019812:Heparan Sulfate Proteoglycans; D010972:Platelet Activating Factor; D011994:Recombinant Proteins; D016718:Arachidonic Acid; D017997:Leukotriene C4; D012313:RNA; D006493:Heparin; D010741:Phospholipases A; D054501:Group II Phospholipases A2; D054505:Group V Phospholipases A2; C516299:PLA2G5 protein, human; C516300:Pla2g5 protein, mouse; C516301:Pla2g5 protein, rat; D015232:Dinoprostone