Purified Group X Secretory Phospholipase A2 Induced Prominent Release of Arachidonic Acid from Human Myeloid Leukemia Cells

Group X secretory phospholipase A 2 (sPLA 2 -X) possesses several structural features characteristic of both group IB and IIA sPLA 2 s (sPLA 2 -IB and -IIA) and is postulated to be involved in inflammatory responses owing to its restricted expression in the spleen and thymus. Here, we report the purification of human recombinant COOH-terminal His-tagged sPLA 2 -X, the preparation of its antibody, and the purification of native sPLA 2 -X. The affinity-purified sPLA 2 -X protein migrated as various molecular species of 13–18 kDa on SDS-polyacrylamide gels, and N -glycosidase F treatment caused shifts to the 13- and 14-kDa bands. NH 2 -terminal amino acid sequencing analysis revealed that the 13-kDa form is a putative mature sPLA 2 -X and the 14-kDa protein possesses a propeptide of 11 amino acid residues attached at the NH 2 termini of the mature protein. Separation with reverse-phase high performance liquid chromatography revealed that N -linked carbohydrates are not required for the enzymatic activity and pro-sPLA 2 -X has a relatively weak potency compared with the mature protein. The mature sPLA 2 -X induced the release of arachidonic acid from phosphatidylcholine more efficiently than other human sPLA 2 groups (IB, IIA, IID, and V) and elicited a prompt and marked release of arachidonic acid from human monocytic THP-1 cells compared with sPLA 2 -IB and -IIA with concomitant production of prostaglandin E 2 . A prominent release of arachidonic acid was also observed in sPLA 2 -X-treated human U937 and HL60 cells. Immunohistochemical analysis of human lung preparations revealed its expression in alveolar epithelial cells. These results indicate that human sPLA 2 -X is a unique N -glycosylated sPLA 2 that releases arachidonic acid from human myeloid leukemia cells more efficiently than sPLA 2 -IB and -IIA.