Type II Secretory Phospholipase A2 Associated with Cell Surfaces via C-terminal Heparin-binding Lysine Residues Augments Stimulus-initiated Delayed Prostaglandin Generation

Abstract

Type II secretory phospholipase A 2 (sPLA 2 ) has been shown to be induced by a variety of proinflammatory stimuli and, therefore, has been implicated in the inflammatory process. In order to determine whether association of sPLA 2 with cell surfaces via heparan sulfate proteoglycan is important for its effects on cellular functions, we have identified the critical domain in sPLA 2 for heparin and cell surface binding and examined its role in cellular prostaglandin (PG) biosynthesis. Replacement of several conserved Lys residues in the Cterminal region of mouse and rat sPLA 2 s by Glu resulted in a marked reduction of their capacities to bind to heparin and mammalian cell surfaces without affecting their enzymatic activities toward dispersed phospholipid as a substrate. CHO cells stably transfected with wild-type sPLA 2 released about twice as much arachidonic acid (AA) during culture for 10 h with fetal calf serum and interleukin-1␤ than cells transfected with vector alone, whereas the ability to enhance AA release was impaired in sPLA 2 mutants incapable of binding to cell surfaces. AA released by wild-type sPLA 2 -transfected CHO cells was metabolized to prostaglandin E 2 via prostaglandin endoperoxide H synthase (PGHS)-2 after IL-1␤ stimulation, revealing a particular functional linkage of sPLA 2 to PGHS-2. In contrast, A23187-initiated immediate AA release over 30 min was not affected by sPLA 2 overexpression. Taken together, these results suggest that sPLA 2 expressed endogenously and anchored on cell surfaces via its C-terminal heparinbinding domain is involved in the PGHS-2-dependent delayed PG biosynthesis initiated by growth factors and cytokines during long term culture.

The Functions of Five Distinct Mammalian Phospholipase A2s in Regulating Arachidonic Acid Release

“…The separated proteins were electroblotted onto nitrocellulose membranes (Schleicher and Schuell) using a semi-dry blotter (MilliBlot-SDE system; Millipore), according to the manufacturer's instructions. The membranes were probed with antibodies against the respective PLA 2 s and visualized using the ECL Western blot analysis system (Amersham Pharmacia Biotech) as described previously (18).…”

“…(52), and the PGE 2 enzyme immunoassay kit were purchased from Cayman Chemical. Mouse sPLA 2 -IIA cDNA (18), rabbit polyclonal antibody against rat sPLA 2 -IIA (53), rat sPLA 2 -V cDNA (54), and rat sPLA 2 -IIC cDNA (16) were prepared as described previously. The sPLA 2 -IIA inhibitor LY311727 (55) was donated by Dr. R. M. Kramer (Lilly).…”

Enhancing activity and phospholipase A2 activity: two independent activities present in the enhancing factor molecule

“…Cationic residues involved in heparin binding are highly conserved in the rat, human and mouse PLA # sequences. Murakami et al [13] demonstrated by site-directed mutagenesis that the Cterminal residues Lys""* and Lys"#" are important for association of the mouse sPLA # with heparin and cell surfaces, although they are not essential for the catalytic activity. Their results showed that mutations in the two residues resulted in a reduced capacity of the molecule to bind to heparin and cell-surface heparan sulphate proteoglycans compared with wild-type sPLA # [13].…”

“…Murakami et al [13] demonstrated by site-directed mutagenesis that the Cterminal residues Lys""* and Lys"#" are important for association of the mouse sPLA # with heparin and cell surfaces, although they are not essential for the catalytic activity. Their results showed that mutations in the two residues resulted in a reduced capacity of the molecule to bind to heparin and cell-surface heparan sulphate proteoglycans compared with wild-type sPLA # [13]. In our studies, mutant 293\SK-3.5m, carrying similar mutations in the C-terminal Lys residues (Lys""* Glu and Lys"#" Glu), could bind heparin (although the affinity for heparin was not checked), and demonstrated both enhancing and PLA # activities.…”

“…PLA # s have also been shown to bind heparin, and the catalytic activity of porcine pancreatic PLA # has been shown to be inhibited by heparin [12]. However, Murakami et al [13] have reported that neither mouse nor rat sPLA # activities were inhibited by heparin in their assay system. We have reported that heparin inhibits enhancing activity as well as PLA # activities [14].…”