title: Purified group X secretory phospholipase A(2) induced prominent release of arachidonic acid from human myeloid leukemia cells.
abstract: 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.
authors.affiliation: Shionogi Research Laboratories, Shionogi and Co., Ltd., Sagisu 5-12-4, Fukushima-ku, Osaka 553-0002, Japan. kohji.hanasaki@shionogi.co.jp
authors.forename: K
authors.initials: K
authors.lastname: Hanasaki
authors.forename: T
authors.initials: T
authors.lastname: Ono
authors.forename: A
authors.initials: A
authors.lastname: Saiga
authors.forename: Y
authors.initials: Y
authors.lastname: Morioka
authors.forename: M
authors.initials: M
authors.lastname: Ikeda
authors.forename: K
authors.initials: K
authors.lastname: Kawamoto
authors.forename: K
authors.initials: K
authors.lastname: Higashino
authors.forename: K
authors.initials: K
authors.lastname: Nakano
authors.forename: K
authors.initials: K
authors.lastname: Yamada
authors.forename: J
authors.initials: J
authors.lastname: Ishizaki
authors.forename: H
authors.initials: H
authors.lastname: Arita
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000302:isolation & purification / Q000494:pharmacology; D016718:Arachidonic Acid / Q000378:metabolism*; D001095:Arachidonic Acids / Q000494:pharmacology; D016466:CHO Cells; D019556:COS Cells; D002460:Cell Line; D006224:Cricetinae; D015232:Dinoprostone / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005227:Fatty Acids / Q000378:metabolism; D054501:Group II Phospholipases A2; D018922:HL-60 Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D007951:Leukemia, Myeloid / Q000378:metabolism / Q000473:pathology; D008168:Lung / Q000201:enzymology; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology / Q000494:pharmacology; D010741:Phospholipases A / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D014316:Tritium; D014407:Tumor Cells, Cultured
chemical_list: D000906:Antibodies; D001095:Arachidonic Acids; D004791:Enzyme Inhibitors; D005227:Fatty Acids; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C081565:arachidonyltrifluoromethane; D014316:Tritium; D016718:Arachidonic Acid; D010741:Phospholipases A; D054501:Group II Phospholipases A2; D015232:Dinoprostone