Purified group X secretory phospholipase A2 induced prominent release of arachidonic acid from human myeloid leukemia cells

Abstract

Group X secretory phospholipase A2 (sPLA2-X) possesses several structural features characteristic of both group IB and IIA sPLA2s (sPLA2- 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 sPLA2-X, the preparation of its antibody, and the purification of native sPLA2-X. The affinity-purified sPLA2-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. NH2-terminal amino acid sequencing analysis revealed that the 13-kDa form is a putative mature sPLA2-X and the 14-kDa protein possesses a propeptide of 11 amino acid residues attached at the NH2 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-sPLA2-X has a relatively weak potency compared with the mature protein. The mature sPLA2-X induced the release of arachidonic acid from phosphatidylcholine more efficiently than other human sPLA2 groups (IB, IIA, IID, and V) and elicited a prompt and marked release of arachidonic acid from human monocytic THP-1 cells compared with sPLA2-IB and -IIA with concomitant production of prostaglandin E2. A prominent release of arachidonic acid was also observed in sPLA2-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 sPLA2-X is a unique N-glycosylated sPLA2 that releases arachidonic acid from human myeloid leukemia cells more efficiently than sPLA2-IB and -IIA.