Noriaki Iijima

Molecular cloning, expression, and characterization of secretory phospholipase A2 in tobacco.

Phospholipase A2 (PLA2) activity was investigated in various tissues of tobacco (Nicotiana tabacum). PLA2 activity in the flower was 15 times higher than that in the leaf, stem, and root. PLA2 activity in the flower appears to have originated from both Ca2+-dependent and-independent PLA2. A cDNA clone for protein with homology to animal secretory PLA2 (sPLA2), denoted as Nt PLA2, was isolated from the tobacco flower. The cDNA of Nt PLA2 encoded a mature protein of 127 amino acid residues with a putative signal peptide of 30 residues. The amino acid sequence for mature Nt PLA2 contains 12 cysteines, a Ca2+ binding loop, and a catalytic domain that are commonly conserved in animal sPLA2. The Nt PLA2 mRNA was mainly expressed in the root and stem of tobacco. The recombinant Nt PLA2 was expressed as a fusion protein with thioredoxin in Escherichia coli. From the bacterial cell lysate, the fusion protein was recovered in soluble form and cleaved by Factor Xa proteinase. Then the recombinant mature Nt PLA2 was purified by ion exchange chromatography. It was discovered that the purified Nt PLA2 essentially requires Ca2+, for the enzyme activity when the activity was determined using mixed-micellar phospholipid substrates with sodium cholate. The optimal activity of Nt PLA2 was at pH 8–10 when PC was used as a substrate.

Localization of group IB phospholipase A2 isoform in the gills of the red sea bream, Pagrus (Chrysophrys) major

We previously reported that PLA(2) activity in the gills is higher than that in other tissues in red sea bream and purified PLA(2) from the gills belongs to the group IB PLA(2) as well as other red sea bream PLA(2)s. In this study, we reconfirmed that the level of PLA(2) activity is extremely high in the gills compared with other tissues, and gill PLA(2) was detected only in the gills by immunoblotting and inhibition test using anti-gill PLA(2) monoclonal antibody. The level of PLA(2) activity and protein expression in the gills are well correlated. Fish can be roughly divided into high and low groups based on the level of PLA(2) activity. Gill PLA(2) was detected in the gills of the high group, but not the low group by immunoblotting. In the gills of the high group, gill PLA(2) was detected in the mucous cells and pavement cells located on the surface of gill epithelia by immunohistochemistry. On the other hand, positive signals were observed only in the mucous cells by in situ hybridization. We also isolated inactive proPLA(2), having AR propeptide, preceding the mature enzyme from the gill extract. These results suggest that gill PLA(2) is synthesized as an inactive proPLA(2) in the mucous cells and is secreted to the surface of gill epithelia.

Cloning and expression of group IB phospholipase A2 isoforms in the red sea bream, Pagrus major

Two cDNA encoding red sea bream DE-1 and DE-2 phospholipases A2 (PLA2) were cloned from the hepatopancreas of red sea bream, Pagrus (Chrysophrys) major. The cDNA of DE-1 PLA2 encoded a mature protein of 125 amino acid residues with an apparent signal peptide of 20 residues and propeptide of 5 residues, and that of DE-2 PLA2, a mature protein of 126 amino acid residues with an apparent signal peptide of 17 residues and propeptide of 6 residues. Comparison of the predicted amino acid sequences for mature DE-1 and DE-2 PLA2 showed that both proteins contain 14 cysteines including Cys 11 and 77 and a pancreatic loop, which are commonly conserved in group IB PLA2; however, the identity in amino acid sequence between DE-1 and DE-2 PLA2 was low (47%). A previous report concerning the cDNA cloning of red sea bream gill G-3 PLA2 and the present results represent the first cloning and sequencing of three distinct isoforms of group IB PLA2 in a single fish species, red sea bream. Reverse transcription-polymerase chain reaction analysis showed that DE-1 PLA2 mRNA was expressed in the hepatopancreas, pyloric ceca, intestine, spleen, gonad, stomach, and kidney, whereas gill G-3 PLA2 mRNA was expressed only in the gills and gonad. The expression of DE-2 PLA2 mRNA was detected in all of the tissues analyzed. These results indicate that three distinct isoforms of group IB PLA2, DE-1 and DE-2 PLA2 in hepatopanceas and gill G-3 PLA2, are expressed in a tissue-specific manner in red sea bream.

The prostaglandin synthesis in marine fish thrombocyte

1. Prostaglandin (PG) syntheses from labelled highly unsaturated fatty acids were investigated in washed thrombocyte suspensions of four species of marine fish, flounder (Paralichthys olivaceus), black seabream (Acanthopagrus schlegeli), black rockfish (Sebastes schlegeli), and red seabream (Pagrus major). 2. Synthesized PGs were analyzed by thin-layer radiochromatogram scanner and high-performance liquid chromatography. 3. When [1-(14)C]arachidonic acid (AA) was incubated with washer thrombocyte suspension, AA was mainly converted to PGF(2alpha), PGE2, and PGD2 in all species. 4. It was suggested that [1-(14)C]eicosapentaenoic acid was mainly converted in PGs only in black rockfish thrombocytes, and the metabolites were mainly PGE3 and PGD3. 5 However, [1-(14)C]docosahexaenoic acid was not cyclized into PGs in all the species.

Purification and characterization of glycerolipid acyl-hydrolase from the red alga Gracilaria vermiculophylla

A glycerolipid acyl-hydrolase was purified 19-fold with a yield of 11% from the prostaglandin-producing red alga Gracilaria vermiculophylla by ammonium sulfate precipitation, anion-exchange chromatoraphy and gel filtration chromatography. Sodium dodecylsulfate-polyacrylamide gel electrophoresis of the final preparation showed a single band corresponding to a molecular mass of 20 kDa, but Superdex 200 fast protein liquid chromatography exhibited a molecular mass of 40 kDa. Accordingly, it was suggested that the purified enzyme was a homodimer of a 20 kDa subunit. The optimal temperature and pH were 37°C and 7–8, respectively. The purified enzyme catalyzed hydrolysis of the acyl groups of both glycoglycerolipids and phospholipids, especially monogalacto-syldiacylglycerol and phosphatidylcholine. These results suggest that the enzyme hydrolyze the membrane lipids of the alga to release various saturated and unsaturated fatty acids, including arachidonic acid as substrate for prostaglandin synthesis.

Purification, characterization, and molecular cloning of group I phospholipases A2 from the gills of the red sea bream, Pagrus major

Phospholipase A2 (PLA2) activity was investigated in various tissues of male and female red sea bream. In both male and female fishes, the specific activity of PLA2 in the gills was 70 times higher than that in other tissues, such as the adipose tissue, intestine, and hepatopancreas. Therefore, we tried to purify PLA2 from the gill filaments of red sea bream to near homogeneity by sequential chromatography on Q-Sepharose Fast Flow, Butyl-Cellulofine, and DEAE-Sepharose Fast Flow columns, and by reversed-phase high-performance liquid chromatography. Two minor and one major PLA2, tentatively named G-1, G-2 and G-3 PLA2, were purified, and all showed a single band with an apparent molecular mass of approximately 15 kDa by sodium dodecylsulfate-polyacrylamide gel electrophoresis. The exact molecular mass values of G-1, G-2, and G-3 PLA2 were 14,040, 14,040 and 14,005 Da, respectively. G-1, G-2, and G-3 PLA2 had a Cys 11 and were all identical in N-terminal amino acid sequences from Ala-1 to Glu-56. A full-length cDNA encoding G-3 PLA2 was cloned by reverse transcriptase-polymerase chain reaction and rapid amplification of cDNA ends methods, and G-3 PLA2 was found to be classified to group IB PLA2 from the deduced amino acid sequence. G-1, G-2, and G-3 PLA2 had a pH optimum in an alkaline region at around pH 9–10 and required Ca2+ essentially for enzyme activity, using a mixed-micellar phosphatidylcholine substrate with sodium cholate. These results demonstrate that three group 1 PLA2, G-1, G-2, and G-3 PLA2, are expressed in the gill filaments of red sea bream.

Characterization of Secretory Phospholipase A2 with Phospholipase A1 Activity in Tobacco, Nicotiana tabacum (L.)

A cDNA encoding protein with homology to plant secretory phospholipase A2 (sPLA2), denoted as Nt1 PLA2, was isolated from tobacco (Nicotiana tabacum). The cDNA encodes a mature protein of 118 amino acid residues with a putative signal peptide of 29 residues. The mature form of Nt1 PLA2 has 12 cysteines, Ca2+ binding loop and catalytic site domain that are commonly conserved in plant sPLA2s. The recombinant Nt1 PLA2 was expressed as a fusion protein with thioredoxin in E. coli BL21 cells and was purified by an ion exchange chromatography after digestion of the fusion proteins by Factor Xa protease to obtain the mature form. Interestingly, Nt1 PLA2 could hydrolyze the ester bond at the sn-1 position of glycerophospholipids as well as at the sn-2 position, when the activities were determined using mixed-micellar phospholipids with sodium cholate. Both activities for the sn-1 and -2 positions of glycerophospholipids required Ca2+ essentially, and maximal activities were found in an alkaline region when phosphatidylcholine, phosphatidylglycerol or phosphatidylethanolamine was used as a substrate. The level of Nt1 PLA2 mRNA was detected at a higher level in tobacco flowers than stem, leaves and roots, and was induced by salicylic acid.

Ontogeny of gene expression of group IB phospholipase A(2) isoforms in the red sea bream, Pagrus (Chrysophrys) major

The red sea bream (Pagrus major) was previously found to express mRNAs for two group IB phospholipase A(2) (PLA(2)) isoforms, DE-1 and DE-2, in the digestive organs, including the hepatopancreas, pyloric caeca, and intestine. To characterize the ontogeny of the digestive function of these PLA(2)s, the present study investigated the localization and expression of DE-1 and DE-2 PLA(2) genes in red sea bream larvae/juveniles and immature adults, by in situ hybridization. In the adults, DE-1 PLA(2) mRNA was expressed in pancreatic acinar cells. By contrast, DE-2 PLA(2) mRNA was detected not only in digestive tissues, such as pancreatic acinar cells, gastric glands of the stomach, epithelial cells of the pyloric caeca, and intestinal epithelial cells, but also in non-digestive ones, including cardiac and lateral muscle fibers and the cytoplasm of the oocytes. In the larvae, both DE-1 and DE-2 PLA(2) mRNAs first appeared in pancreatic tissues at 3 days post-hatching (dph) and in intestinal tissue at 1 dph, and expression levels for both gradually increased after this point. In the juvenile stage at 32 dph, DE-1 PLA(2) mRNA was highly expressed in pancreatic tissue, and DE-2 PLA(2) mRNA was detected in almost all digestive tissues, including pancreatic tissue, gastric glands, pyloric caeca, and intestine, including the myomere of the lateral muscles. In conclusion, both DE-1 and DE-2 PLA(2) mRNAs are already expressed in the digestive organs of red sea bream larvae before first feeding, and larvae will synthesize both DE-1 and DE-2 PLA(2) proteins.

Molecular cloning and effect of c-fos mRNA on pharmacological stimuli in the goldfish brain.

c-fos is an immediate early gene, and is rapidly and transiently induced in neurons of the central nervous system according to their activities. To investigate neuronal activities in the brain of the goldfish (Carassius auratus), we considered that expression of c-fos mRNA would be an available marker for the neuronal activities. Therefore, we firstly isolated a cDNA clone encoding c-Fos from the goldfish brain by RT-PCR and RACE methods. A full length cDNA of the goldfish c-fos was composed of 1044 bp open reading frame. The amino acid sequence of the goldfish c-Fos was approximately 56-90% identical to those of other teleostean fish c-Fos. Northern blot analysis showed that the expression of c-fos mRNA was rapidly and transiently induced in the brain of the goldfish by the intraperitoneal administration of kainic acid. We also showed that the identification of the c-fos mRNA expression site by in situ hybridization will be able to be used as an anatomical marker for the identification of the activated neuronal region in the goldfish brain.

Monoclonal antibodies to plasma high density lipoprotein (HDL) of eel (Anguilla japonica).

Six week-old female mice (Balb/c) injected intraperitonealy with 50 micrograms of eel high density lipoprotein (HDL) emulsified with equal volume of adjuvant three times every two weeks. Three weeks after the third injection, hyperimmunized mice were boosted by injection of 100 micrograms of HDL. After 5 days, the best responding mouse to injected HDL was sacrificed, and spleen cells were fused with mouse myeloma cells (Sp2/O-Ag14), and hybridomas were cultured in a selection medium. Monoclonal antibodies specific to apolipoprotein A-I or A-II (apoA-I or apoA-II) of HDL were obtained by cloning and recloning the hybridomas. Eighteen monoclonal antibodies specific to apoA-I and/or apoApII were isolated. Antibodies in the culture medium were purified by a HiTrap Protein G or an eel-HDL column. These purified antibodies belong to the subclass IgG1. The monoclonal antibodies specific to eel apoA-I and apoA-II secreted by clone 10D12 and 2G3, respectively, interact with serum proteins of some fish species such as red-sea bream and carp. The anti-eel apoA-I antibody of 10D12 did not bind to serum proteins of rat, rabbit, and chicken, while the anti-eel apoA-II of 2G3 antibody did.