Jun Ishizaki

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

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, andN-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 sPLA2groups (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.

Resistance to Endotoxic Shock in Phospholipase A2 Receptor-deficient Mice

Mammals possess various types of secretory phospholipase A2, which differ in the primary structure and tissue distribution. The phosholipase A2receptor (PLA2R) recognizes group IB phospholipase A2 (PLA2-IB) and mediates the PLA2-IB-induced biological responses in non-digestive organs, including eicosanoid production and contraction of airway smooth muscles. In this study, we generated PLA2R-deficient mice to define its biological roles further. These mice are viable, fertile, and without evident histopathological abnormalities. There was no difference in the clearance of circulating PLA2-IB between wild-type and mutant mice. After challenge with bacterial lipopolysaccharide (LPS), PLA2R-deficient mice exhibited longer survival than wild-type mice. The mutant mice were also resistant to lethal effects of exogenous PLA2-IB after sensitization with sublethal dose of LPS. The plasma levels of tumor necrosis factor-α and interleukin-1β elevated after LPS treatment were significantly reduced in mutant mice compared with wild-type mice. These findings suggest a potential role of PLA2R in the progression of endotoxic shock.

Cloning and Characterization of Novel Mouse and Human Secretory Phospholipase A2s

Mammalian secretory phospholipase A2s (sPLA2s) are classified into several groups according to molecular structure and the localization of intramolecular disulfide bridges. Among them, group IIA sPLA2 has been thought to be one of the key enzymes in the pathogenesis of inflammatory diseases owing to its augmented expression under various inflammatory conditions. However, in a number of inbred mouse strains, the group IIA sPLA2 gene is naturally disrupted by a frameshift mutation. Here, we report the cloning of a cDNA encoding a novel sPLA2 expressed in the spleen of group IIA sPLA2-deficient mouse. We also cloned its human homolog and mapped its gene location on chromosome 1p36.12 near the loci of group IIA and V sPLA2 genes. The human mature sPLA2 protein consists of 125 amino acids (M r = 14,500) preceded by a 20-residue prepeptide and is most similar to group IIA sPLA2 with respect to the number and positions of cysteine residues as well as overall identity (48%). Based on these structural properties, the novel sPLA2 should be categorized into group II, called group IID to follow the already identified IIA to IIC sPLA2s. When the cDNA was expressed in COS-7 cells, PLA2 activity preferentially accumulated in the culture medium. It is maximally active at neutral to alkaline pH and with 2 mm Ca2+. In assays with individual substrates,l-α-1-palmitoyl-2-linoleoyl phosphatidylethanolamine was more efficiently hydrolyzed than the other phospholipids examined. An RNA blot hybridized with the cDNA exhibited two transcripts (2.0 and 1.0 kb) in human spleen, thymus, and colon. The expression of a novel sPLA2 mRNA was elevated in the thymus after treatment with endotoxin in rats as well as in group IIA sPLA2-deficient mice, suggesting its functional role in the progression of the inflammatory process.

cDNA cloning and sequence determination of pig gastric (H+ + K+)-ATPase

Complementary DNA to pig gastric mRNA encoding (H+ + K+)-ATPase was cloned, and its amino acid sequence was deduced from the nucleotide sequence. The enzyme contained 1034 amino acid residues (Mr. 114,285) including the initiation methionine. The sequence of pig (H+ + K+)-ATPase was highly homologous with that of the corresponding enzyme from rat, but had high degree of synonymous codon changes. Potential sites of phosphorylation by cAMP-dependent protein kinase and N-linked glycosylation sites were identified. The amino terminal region contained a lysine-rich sequence similar to that of the alpha subunit of (Na+ + K+)-ATPase, although a cluster of glycine residues was inserted into the sequence of the (H+ + K+)-ATPase. As the pig enzyme is advantageous for biochemical studies, the information of the primary structure is useful for further detailed studies.

cDNA cloning and sequence determination of rat membrane-associated phospholipase A2

Based on the partial amino acid sequences of membrane-associated phospholipase A2 (PLA2M), belonging to group II, purified from rat spleen, the cDNA encoding PLA2M was cloned by a new cloning strategy utilizing enzymatic cDNA amplification. At the N-terminus of the coded 146 residues, which were deduced from the cDNA sequence, the putative signal peptide was found despite the tight adherence of this enzyme to the membrane. The sequence of rat PLA2M exhibits 75% homology with that of human group II PLA2 in the protein-coding region. The result of RNA blot analysis showed that rat ileal mucosa contains the largest amount of the PLA2 transcript among the tissues examined.

Structure and function of phospholipase A2 receptor

Phospholipase A2 (EC 3.1.1.4: PLA2) is the group name of enzymes that cleave an acyl ester bond at the sn-2 position of glycerophospholipid (1). Mammalian PLA2s are classified into two types, secretory 14 kDa PLA2 and cytosolic higher molecular weight of PLA2 (2). Secretory PLA2 are further classified into two subgroups, group I and group II, on the basis of their characteristics in primary structure (3). Group II PLA2 (PLA2-II) is abundant at inflammatory loci and its expression is modulated by various inflammatory cytokines, and thus this type of PLA2 is thought to play some roles in the pathogenesis of inflammation (4,5). On the other hand, group I PLA2 (PLA2-I) is mainly secreted from the pancreas and the major physiological function of PLA2-I has long been thought to be digestion of phospholipids in nutrients (6). However, recent studies have revealed the presence of this type of PLA2 in several non-digestive organs such as lung and spleen (7). What is the biological role of PLA2-I in non-digestive organs? This question led us to initiate the series of studies on the PLA2 receptor.

Production of recombinant human glucagon in the form of a fusion protein in Escherichia coli; recovery of glucagon by sequence-specific digestion

SummaryRecombinant human glucagon was succesfully produced with a high level of expression in Escherichia coli as a fusion protein with human interferon γ. The synthetic gene was designed to release glucagon, which does not contain glutamic acid residues, from fusion protein with the Staphylococcus aureus strain V8 protease that specifically cleaves the peptide bond on the carboxyl side of the glutamic acid residue. The resulting glucagon was purified to homogeneity by a combination of C18 reverse-phase HPLC and ion-exchange HPLC. The yield of intact glucagon obtained from 11 of culture was approximately 12 mg. The structure of recombinant human glucagon was confirmed by HPLC and amino acid composition/sequence analyses.

Transcriptomic Analysis of Nephrotoxicity Induced by Cephaloridine, a Representative Cephalosporin Antibiotic

Cephaloridine (CER) is a classical beta-lactam antibiotic that has long served as a model drug for the study of cephalosporin antibiotic-induced acute tubular necrosis. In the present study, we analyzed gene expression profiles in the kidney of rats given subtoxic and toxic doses of CER to identify gene expression alterations closely associated with CER-induced nephrotoxicity. Male Fischer 344 rats were intravenously injected with CER at three different dose levels (150, 300, and 600 mg/kg) and sacrificed after 24 h. Only the high dose (600 mg/kg) caused mild proximal tubular necrosis and slight renal dysfunction. Microarray analysis identified hundreds of genes differentially expressed in the renal cortex following CER exposure, which could be classified into two main groups that were deregulated in dose-dependent and high dose-specific manners. The genes upregulated dose dependently mainly included those involved in detoxification and antioxidant defense, which was considered to be associated with CER-induced oxidative stress. In contrast, the genes showing high dose-specific (lesion-specific) induction included a number of genes related to cell proliferation, which appeared to reflect a compensatory response to CER injury. Of the genes modulated in both manners, we found many genes reported to be associated with renal toxicity by other nephrotoxicants. We could also predict potential transcription regulators responsible for the observed gene expression alterations, such as Nrf2 and the E2F family. Among the candidate gene biomarkers, kidney injury molecule 1 was markedly upregulated at the mildly toxic dose, suggesting that this gene can be used as an early and sensitive indicator for cephalosporin nephrotoxicity. In conclusion, our transcriptomic data revealed several characteristic expression patterns of genes associated with specific cellular processes, including oxidative stress response and proliferative response, upon exposure to CER, which may enhance our understanding of the molecular mechanisms behind cephalosporin antibiotic-induced nephrotoxicity.

Receptor‐binding capability of pancreatic phospholipase A2 is separable from its enzymatic activity

Mammalian pancreatic phospholipase A2 (PLA2‐I) has its specific receptor through which PLA2‐I induces a variety of biological responses. In this study, a fundamental relationship between the enzymatic and the receptor‐binding activities of PLA2‐I was investigated. The specific binding of PLA2‐I to the receptor was found to be independent of Ca2+ which is requisite for the PLA2 activity. On the basis of this observation, we designed and produced mutant PLA2‐Is without Ca2+‐binding abilities in order to demonstrate that the structural requirement for the enzymatic activity of PLA2‐I is not identical with that for its receptor‐binding reaction. These mutant PLA2‐Is lost almost all enzymatic activity through a disturbance at the Ca2+‐binding site, as expected, but still retained a substantial affinity to the receptor, allowing us to conclude that the receptor‐binding reaction of PLA2‐I is separable from its catalytic action.

Characterization of novel non-peptide thrombopoietin mimetics, their species specificity and the activation mechanism of the thrombopoietin receptor

A series of non-peptide small compounds discovered to be thrombopoietin receptor agonists showed species specificity to humans. Compound I could induce megakaryocyte lineage from human bone marrow cells, but not from mouse, guinea pig or cynomolgus monkey bone marrow cells. To elucidate the mechanism, we identified the pivotal amino acid residue for the receptor activation by compound I by taking advantage of its species specificity. The response of compound I to three human/mouse chimeric receptors indicated the importance of the transmembrane domain. Comparison of amino acid sequences of the transmembrane domain of the thrombopoietin receptor between human and three animal species led us to hypothesize that histidine 499 is necessary for the reactivity to the thrombopoietin mimetics. We verified the hypothesis using two mutant receptors: the human thrombopoietin receptor mutant His499Leu and the mouse thrombopoietin receptor mutant Leu490His. These results should be helpful for structure-activity relationship studies and conducting in vivo studies of thrombopoietin mimetics.