Redundant and segregated functions of granule-associated heparin-binding group II subfamily of secretory phospholipases A2 in the regulation of degranulation and prostaglandin D2 synthesis in mast cells.
We herein demonstrate that mast cells express all known members of the group II subfamily of secretory phospholipase A2 (sPLA2) isozymes, and those having heparin affinity markedly enhance the exocytotic response. Rat mastocytoma RBL-2H3 cells transfected with heparin-binding (sPLA2-IIA, -V, and -IID), but not heparin-nonbinding (sPLA2-IIC), enzymes released more granule-associated markers (beta-hexosaminidase and histamine) than mock- or cytosolic PLA2alpha (cPLA2alpha)-transfected cells after stimulation with IgE and Ag. Site-directed mutagenesis of sPLA2-IIA and -V revealed that both the catalytic and heparin-binding domains are essential for this function. Confocal laser and electron microscopic analyses revealed that sPLA2-IIA, which was stored in secretory granules in unstimulated cells, accumulated on the membranous sites where fusion between the plasma membrane and granule membranes occurred in activated cells. These results suggest that the heparin-binding sPLA2s bind to the perigranular membranes through their heparin-binding domain, and lysophospholipids produced in situ by their enzymatic action may facilitate the ongoing membrane fusion. In contrast to the redundant role of sPLA2-IIA, -IID, and -V in the regulation of degranulation, only sPLA2-V had the ability to markedly augment IgE/Ag-stimulated immediate PGD2 production, which reached a level comparable to that elicited by cPLA2alpha. The latter observation reveals an unexplored functional segregation among the three related isozymes expressed in the same cell population.
Enomoto
A
A
Department of Health Chemistry, Showa University School of Pharmaceutical Sciences, Tokyo, Japan.
Murakami
M
M
Valentin
E
E
Lambeau
G
G
Gelb
M H
MH
Kudo
I
I
D000818:Animals; D015550:Cell Degranulation / Q000235:genetics; D002478:Cells, Cultured; D003594:Cytoplasmic Granules / Q000201:enzymology* / Q000378:metabolism; D054501:Group II Phospholipases A2; D006493:Heparin / Q000378:metabolism*; D007527:Isoenzymes / Q000096:biosynthesis / Q000145:classification / Q000378:metabolism / Q000502:physiology; D008297:Male; D008407:Mast Cells / Q000201:enzymology* / Q000378:metabolism / Q000502:physiology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D010741:Phospholipases A / Q000096:biosynthesis / Q000145:classification / Q000378:metabolism / Q000502:physiology*; D054467:Phospholipases A2; D015230:Prostaglandin D2 / Q000096:biosynthesis*; D011485:Protein Binding / Q000235:genetics; D051381:Rats; D013347:Subcellular Fractions / Q000201:enzymology / Q000502:physiology; D014162:Transfection; D014407:Tumor Cells, Cultured
D007527:Isoenzymes; D006493:Heparin; D010741:Phospholipases A; D054501:Group II Phospholipases A2; D054467:Phospholipases A2; D015230:Prostaglandin D2