Kyushu University Academic Staff Educational and Research Activities Database
List of Papers
NOZOMU OKINO Last modified date:2023.06.27

Associate Professor / Molecular Bioscience / Department of Bioscience and Biotechnology / Faculty of Agriculture

1. Nyunoya, Hayato; Ishibashi, Yohei; Ito, Makoto; Okino, Nozomu, Significance of mitochondrial fatty acid β-oxidation for the survivability of Aurantiochytrium limacinum ATCC MYA-1381 during sugar starvation., Bioscience, Biotechnology, and Biochemistry, 10.1093/bbb/zbac141, 86, 11, 1524-1535, 2022.10.
2. Nyunoya, Hayato; Noda, Tatsuki; Kawamoto, You; Hayashi, Yasuhiro; Ishibashi, Yohei; Ito, Makoto; Okino, Nozomu, Lack of increment 5 Desaturase Activity Impairs EPA and DHA Synthesis in Fish Cells from Red Sea Bream and Japanese Flounder, MARINE BIOTECHNOLOGY, 10.1007/s10126-021-10040-9, 23, 3, 472-481, 2021.06.
3. Nozomu Okino, Mengbai Li, Qingjun Qu, Tomoko Nakagawa, Yasuhiro Hayashi, Mitsufumi Matsumoto, Yohei Ishibashi, Makoto Ito, Two bacterial glycosphingolipid synthases responsible for the synthesis of glucuronosylceramide and alpha-galactosylceramide, Journal of Biological Chemistry, 10.1074/jbc.RA120.013796, 295, 31, 10709-10725, 2020.07, Bacterial glycosphingolipids such as glucuronosylceramide and galactosylceramide have been identified as ligands for invariant natural killer T cells and play important roles in host defense. However, the glycosphingolipid synthases required for production of these ceramides have not been well-characterized. Here, we report the identification and characterization of glucuronosylceramide synthase (ceramide UDP-glucuronosyltransferase [Cer-GlcAT]) in Zymomonas mobilis, a Gram-negative bacterium whose cellular membranes contain glucuronosylceramide. On comparing the gene sequences that encode the diacylglycerol GlcAT in bacteria and plants, we found a homologous gene that is widely distributed in the order Sphingomonadales in the Z. mobilis genome. We first cloned the gene and expressed it in Escherichia coli, followed by protein purification using nickel-Sepharose affinity and gel filtration chromatography. Using the highly enriched enzyme, we observed that it has high glycosyltransferase activity with UDP-glucuronic acid and ceramide as sugar donor and acceptor substrate, respectively. Cer-GlcAT deletion resulted in a loss of glucuronosylceramide and increased the levels of ceramide phosphoglycerol, which was expressed in WT cells only at very low levels. Furthermore, we found sequences homologous to Cer-GlcAT in Sphingobium yanoikuyae and Bacteroides fragilis, which have been reported to produce glucuronosylceramide and α-galactosylceramide, respectively. We expressed the two homologs of the cer-glcat gene in E. coli and found that each gene encodes Cer-GlcAT and Cer-galactosyltransferase, respectively. These results contribute to the understanding of the roles of bacterial glycosphingolipids in host-bacteria interactions and the function of bacterial glycosphingolipids in bacterial physiology..
4. Satoshi Ishii, Atsumi Taguchi, Nozomu Okino, Makoto Ito, Hiroki Maruyama, Determination of globotriaosylceramide analogs in the organs of a mouse model of Fabry disease, Journal of Biological Chemistry, 10.1074/jbc.RA120.012665, 295, 17, 5577-5587, 2020.04, Fabry disease is a heritable lipid disorder caused by the low activity of α-galactosidase A and characterized by the systemic accumulation of globotriaosylceramide (Gb3). Recent studies have reported a structural heterogeneity of Gb3 in Fabry disease, including Gb3 isoforms with different fatty acids and Gb3 analogs with modifications on the sphingosine moiety. However, Gb3 assays are often performed only on the selected Gb3 isoforms. To precisely determine the total Gb3 concentration, here we established two methods for determining both Gb3 isoforms and analogs. One was the deacylation method, involving Gb3 treatment with sphingolipid ceramide N-deacylase, followed by an assay of the deacylated products, globotriaosylsphingosine (lyso-Gb3) and its analogs, by ultra-performance LC coupled to tandem MS (UPLC-MS/MS). The other method was a direct assay established in the present study for 37 Gb3 isoforms and analogs/isoforms by UPLC-MS/MS. Gb3s from the organs of symptomatic animals of a Fabry disease mouse model were mainly Gb3 isoforms and two Gb3 analogs, such as Gb3(+18) containing the lyso-Gb3(+18) moiety and Gb3(-2) containing the lyso- Gb3(-2) moiety. The total concentrations and Gb3 analog distributions determined by the two methods were comparable. Gb3(+18) levels were high in the kidneys (24% of total Gb3) and the liver (13%), and we observed Gb3(-2) in the heart (10%) and the kidneys (5%). These results indicate organ-specific expression ofGb3analogs, insights that may lead to a deeper understanding of the pathophysiology of Fabry disease..
5. Yohei Ishibashi, Keisuke Aoki, Nozomu Okino, Masahiro Hayashi, Makoto Ito, A thraustochytrid-specific lipase/phospholipase with unique positional specificity contributes to microbial competition and fatty acid acquisition from the environment, Scientific reports, 10.1038/s41598-019-52854-7, 9, 1, 2019.12, Thraustochytrids are heterotrophic marine protists that are considered as important decomposers in the marine ecosystem; however, how they digest and uptake lipid nutrients from the environment is largely unknown. Genomic clustering analysis using thraustochytrid draft genome databases revealed that novel proteins with a Lipase_3 domain are commonly present in thraustochytrids, including Aurantiochytrium limacinum. After heterologous expression and His tag-based purification, protein ID: 145138 was identified as lipase/phospholipase capable of hydrolyzing triacylglycerol (TG) and phosphatidylcholine (PC). 145138 was secreted into the medium, and deletion of the 145138 gene in A. limacinum reduced the degradation of extracellular lipids. Fatty acids generated by 145138 were reused for the biosynthesis of PC and TG, and 145138 allowed A. limacinum to survive in the medium containing TG as a sole carbon source. 145138 hydrolyzed all the acyl-ester linkages of TG; however, the enzyme showed strict positional specificity toward phospholipids, generating 2-acyl lysophospholipids. The 2-acyl lysophospholipids showed stronger antimicrobial activity compared with 1-acyl lysophospholipids. These results suggested that 145138 is a bifunctional enzyme that contributes to the acquisition of lipid nutrients from the environment, as well as to generate antimicrobial lysophospholipids that are beneficial for competition with bacteria over lipid nutrients in the marine environment..
6. Akihiro Morio, Jian Xu, Akitsu Masuda, Yurie Kinoshita, Masato Hino, Daisuke Morokuma, Hatsumi M. Goda, Nozomu Okino, Makoto Ito, Hiroaki Mon, Ryosuke Fujita, Takahiro Kusakabe, Man Lee, Expression, purification, and characterization of highly active endo-α-N-acetylgalactosaminidases expressed by silkworm-baculovirus expression system, Journal of Asia-Pacific Entomology, 10.1016/j.aspen.2019.01.009, 22, 2, 404-408, 2019.06, The O-glycosidase, endo-α-N-acetylgalactosaminidase from Enterococcus faecalis (endoEF) catalyzes the cleavage of core 1 and core 3 type O-linked disaccharides between GalNAc and serine or threonine residues from glycoproteins. The endoEF has broad substrate specificity and thus is extensively utilized for the structural and functional analysis of the O-linked glycans. In this study, we expressed and purified the recombinant endoEF (rEndoEF) by using the silkworm-baculovirus expression vector system (Silkworm-BEVS) and confirmed the deglycosylation activity of rEndoEF targeting reporter glycoproteins, which was equivalent to the commercial O-glycosidase. Thus, our study provides important clues to produce highly active rEndoEF O-glycosidases employing silkworm-BEVS as an alternative..
7. Eri Nutahara, Eriko Abe, Shinya Uno, Yohei Ishibashi, Takashi Watanabe, Masahiro Hayashi, Nozomu Okino, Makoto Ito, The glycerol-3-phosphate acyltransferase PLAT2 functions in the generation of DHA-rich glycerolipids in Aurantiochytrium limacinum F26-b, PloS one, 10.1371/journal.pone.0211164, 14, 1, 2019.01, Thraustochytrids possess docosahexaenoic acid (DHA, 22:6n-3) as acyl chain(s) of triacylglycerol (TG) and phosphatidylcholine (PC), some of which contain multiple DHAs. However, little is known about how these DHA-rich glycerolipids are produced in thraustochytrids. In this study, we identified PLAT2 in Aurantiochytrium limacinum F26-b as a glycerol-3-phosphate (G3P) acyltransferase (GPAT) by heterologous expression of the gene in budding yeast. Subsequently, we found that GPAT activity was reduced by disruption of the PLAT2 gene in A. limacinum, resulting in a decrease in DHA-containing lysophosphatidic acid (LPA 22:6). Conversely, overexpression of PLAT2 increased both GPAT activity and LPA 22:6. These results indicate that PLAT2 is a GPAT that transfers DHA to G3P in vivo as well as in vitro. Overexpression of the PLAT2 gene increased the production of a two DHA-containing diacylglycerol (DG 44:12), followed by an increase in the three DHA-containing TG (TG 66:18), two-DHA-containing TG (TG 60:12), and two DHA-containing PC (PC 44:12). However, overexpression of PLAT2 did not increase DHA-free DG (DG32:0), which was preferentially converted to three 16:0-containing TG (TG 48:0) but not two 16:0-contain-ing PC (PC 32:0). Collectively, we revealed that DHA-rich glycerolipids are produced from a precursor, LPA 22:6, which is generated by incorporating DHA to G3P by PLAT2 in the A. limacinum..
8. Yoshihara, Toru; Satake, Hiroyuki; Nishie, Toshikazu; Okino, Nozomu; Hatta, Toshihisa; Otani, Hiroki; Naruse, Chie; Suzuki, Hiroshi; Sugihara, Kazushi; Kamimura, Eikichi; Tokuda, Noriyo; Furukawa, Keiko; Fururkawa, Koichi; Ito, Makoto; Asano, Masahide, Lactosylceramide synthases encoded by B4galt5 and 6 genes are pivotal for neuronal generation and myelin formation in mice, PLOS GENETICS, 10.1371/journal.pgen.1007545, 14, 8, 2018.08.
9. Yibo Huang, Yujiro Higuchi, Kazuki Mori, Ryoko Yamashita, Nozomu Okino, Kosuke Tashiro, Kaoru Takegawa, Draft genome sequence of Sphingobacterium sp. strain HMA12, which encodes endo-β-Nacetylglucosaminidases and can specifically hydrolyze fucose-containing oligosaccharides, Genome Announcements, 10.1128/genomeA.01525-17, 6, 8, 2018.02, The genome sequence of the soil bacterium Sphingobacterium sp. strain HMA12, the culture supernatant of which exhibited endo-β-N-acetylglucosaminidase (ENGase) activity, was examined for ENGase-encoding genes. Here, we report the characterization of new genes of ENGases, obtained by whole-genome shotgun sequencing, that are capable of specifically hydrolyzing fucose-containing oligosaccharides..
10. Nozomu Okino, Hiroyoshi Wakisaka, Yohei Ishibashi, Makoto Ito, Visualization of Endoplasmic Reticulum and Mitochondria in Aurantiochytrium limacinum by the Expression of EGFP with Cell Organelle-Specific Targeting/Retaining Signals, Marine Biotechnology, 10.1007/s10126-018-9795-7, 1-11, 2018.01.
11. Chan Yeong Kwak, Seung Yeol Park, Chung Geun Lee, Nozomu Okino, Makoto Ito, Jung Hoe Kim, Enhancing the sialylation of recombinant EPO produced in CHO cells via the inhibition of glycosphingolipid biosynthesis, Scientific Reports, 10.1038/s41598-017-13609-4, 7, 1, 2017.12, Sialylation regulates the in vivo half-life of recombinant therapeutic glycoproteins, affecting their therapeutic efficacy. Levels of the precursor molecule cytidine monophospho-N-acetylneuraminic acid (CMP-Neu5Ac) are considered a limiting factor in the sialylation of glycoproteins. Here, we show that by reducing the amount of intracellular CMP-Neu5Ac consumed for glycosphingolipid (GSL) biosynthesis, we can increase the sialylation of recombinant human erythropoietin (rhEPO) produced in CHO cells. Initially, we found that treating CHO cells with a potent inhibitor of GSL biosynthesis increases the sialylation of the rhEPO they produce. Then, we established a stable CHO cell line that produces rhEPO in the context of repression of the key GSL biosynthetic enzyme UDP-glucose ceramide glucosyltransferase (UGCG). These UGCG-depleted cells show reduced levels of gangliosides and significantly elevated levels of rhEPO sialylation. Upon further analysis of the resulting N-glycosylation pattern, we discovered that the enhanced rhEPO sialylation could be attributed to a decrease in neutral and mono-sialylated N-glycans and an increase in di-sialylated N-glycans. Our results suggest that the therapeutic efficacy of rhEPO produced in CHO cells can be improved by shunting intracellular CMP-Neu5Ac away from GSL biosynthesis and toward glycoprotein sialylation..
12. Takashi Watanabe, Ryo Sakiyama, Yuya Iimi, Satomi Sekine, Eriko Abe, Kazuko H. Nomura, Kazuya Nomura, Yohei Ishibashi, Nozomu Okino, Masahiro Hayashi, Makoto Ito, Regulation of TG accumulation and lipid droplet morphology by the novel TLDP1 in Aurantiochytrium limacinum F26-b, Journal of Lipid Research, 10.1194/jlr.M079897, 58, 12, 2334-2347, 2017.12, Thraustochytrids are marine single-cell protists that produce large amounts of PUFAs, such as DHA. They accumulate PUFAs in lipid droplets (LDs), mainly as constituent(s) of triacylglycerol (TG). We identified a novel protein in the LD fraction of Aurantiochytrium limacinum F26-b using 2D-difference gel electrophoresis. The protein clustered with orthologs of thraustochytrids; however, the cluster was evolutionally different from known PAT family proteins or plant LD protein; thus, we named it thraustochytrid-specific LD protein 1 (TLDP1). TLDP1 surrounded LDs when expressed as a GFP-tagged form. Disruption of the tldp1 gene decreased the content of TG and number of LDs per cell; however, irregular and unusually large LDs were generated in tldp1-deficient mutants. Although the level of TG synthesis was unchanged by the disruption of tldp1, the level of TG degradation was higher in tldp1-deficient mutants than in the WT. These phenotypic abnormalities in tldp1-deficient mutants were restored by the expression of tldp1. These results indicate that TLDP1 is a thraustochytrid-specific LD protein and regulates the TG accumulation and LD morphology in A. limacinum F26-b.—Watanabe, T., R. Sakiyama, Y. Iimi, S. Sekine, E. Abe, K. H. Nomura, K. Nomura, Y. Ishibashi, N. Okino, M. Hayashi, and M. Ito. Regulation of TG accumulation and lipid droplet morphology by the novel TLDP1 in Aurantiochytrium limacinum F26-b..
13. Nozomu Okino, Makoto Ito, Molecular mechanism for sphingosine-induced Pseudomonas ceramidase expression through the transcriptional regulator SphR, Scientific Reports, 6, 38797, 2016.12.
14. 深野 泰史, Nozomu Okino, Shigeki Furuya, Makoto Ito, A label-free impedance-based whole cell assay revealed a new G protein-coupled receptor ligand for mouse microglial cell migration, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 10.1016/j.bbrc.2016.07.119, 478, 2, 624-630, 2016.09.
15. Takashi Watanabe, 谷 元洋, Yohei Ishibashi, 遠藤郁実, Nozomu Okino, Makoto Ito, Ergosteryl-β-glucosidase (Egh1) involved in sterylglucoside catabolism and vacuole formation in Saccharomyces cerevisiae. , Glycobiology, 25, 10, 1079-1089, 2015.06.
16. Takashi Watanabe, Tomoharu Ito, Hatsumi Goda, Yohei Ishibashi, Tomofumi Miyamoto, Kazutaka Ikeda, Ryo Taguchi, Nozomu Okino, Makoto Ito, Sterylglucoside Catabolism in Cryptococcus neoformans with Endoglycoceramidase-related Protein 2 (EGCrP2), the First Steryl-beta-glucosidase Identified in Fungi, JOURNAL OF BIOLOGICAL CHEMISTRY, 10.1074/jbc.M114.616300, 290, 2, 1005-1019, 2015.01.
17. Hyun Lee, Jong Kil Lee, Min Hee Park, Yu Ri Hong, Hugo H. Marti, Hyongbum Kim, Yohei Okada, Makoto Otsu, Eul-Ju Seo, Jae-Hyung Park, Jae-Hoon Bae, Nozomu Okino, Xingxuan He, Edward H. Schuchman, Jae-sung Bae, Hee Kyung Jin, Pathological roles of the VEGF/SphK pathway in Niemann-Pick type C neurons, NATURE COMMUNICATIONS, 10.1038/ncomms6514, 5, 2014.11.
18. Yohei Ishibashi, Yusuke Nagamatsu, Tomofumi Miyamoto, Naoyuki Matsunaga, Nozomu Okino, Kuniko Yakaguchi, Makoto Ito, A novel ether-linked phytol-containing digalactosylglycerolipid in the marine green alga, Ulva pertusa, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 10.1016/j.bbrc.2014.08.056, 452, 4, 873-880, 2014.10.
19. Eriko Abe, Kazutaka Ikeda, Eri Nutahara, Masahiro Hayashi, Atsushi Yamashita, Ryo Taguchi, Kosaku Doi, Daiske Honda, Nozomu Okino, Makoto Ito, Novel Lysophospholipid Acyltransferase PLAT1 of Aurantiochytrium limacinum F26-b Responsible for Generation of Palmitate-Docosahexaenoate-Phosphatidylcholine and Phosphatidylethanolamine, PLOS ONE, 10.1371/journal.pone.0102377, 9, 8, 2014.08.
20. Masato Honda, Akemi Muta, Taiki Akasaka, Yoshiyuki Inoue, Yohei Shimasaki, Kurunthachalam Kannan, Nozomu Okino, Yuji Oshima, Identification of perfluorooctane sulfonate binding protein in the plasma of tiger pufferfish Takifugu rubripes, ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY, 10.1016/j.ecoenv.2013.11.010, 104, 409-413, 2014.06.
21. Ami Oizumi, Hitoshi Nakayama, Nozomu Okino, Chihiro Iwahara, Katsunari Kina, Ryo Matsumoto, Hideoki Ogawa, Kenji Takamori, Makoto Ito, Yasushi Suga, Kazuhisa Iwabuchi, Pseudomonas-Derived Ceramidase Induces Production of Inflammatory Mediators from Human Keratinocytes via Sphingosine-1-Phosphate, PLOS ONE, 10.1371/journal.pone.0089402, 9, 2, e89402, 2014.02.
22. Hyun Lee, Jong Kil Lee, Yong Chul Bae, Song Hyun Yang, Nozomu Okino, Edward H. Schuchman, Tadashi Yamashita, Jae-sung Bae, Hee Kyung Jin, Inhibition of GM3 Synthase Attenuates Neuropathology of Niemann-Pick Disease Type C by Affecting Sphingolipid Metabolism, Molecules and Cells, 37, 2, 161-171, 2014.02.
23. Ami Oizumi, Hitoshi Nakayama, Nozomu Okino, Chihiro Iwahara, Katsunari Kina, Ryo Matsumoto, Hideoki Ogawa, Kenji Takamori, Makoto Ito, Yasushi Suga, Kazuhisa Iwabuchi, Pseudomonas-Derived Ceramidase Induces Production of Inflammatory Mediators from Human Keratinocytes via Sphingosine-1-Phosphate, PLOS ONE, 10.1371/journal.pone.0089402, 9, 2, 2014.02.
24. Rumana Tasmin, Yohei Shimasaki, Michito Tsuyama, Xuchun Qiu, Fatma Khalil, Nozomu Okino, Naotaka Yamada, Shinji Fukuda, Ik-Joon Kang, Yuji Oshima, Elevated water temperature reduces the acute toxicity of the widely used herbicide diuron to a green alga, Pseudokirchneriella subcapitata, ENVIRONMENTAL SCIENCE AND POLLUTION RESEARCH, 10.1007/s11356-013-1989-y, 21, 2, 1064-1070, 2014.01.
25. Junichiro Ohara, Keishi Sakaguchi, Yuji Okita, Nozomu Okino, Makoto Ito, Two Fatty Acid Elongases Possessing C18-Delta 6/C18-Delta 9/C20-Delta 5 or C16-Delta 9 Elongase Activity in Thraustochytrium sp ATCC 26185, MARINE BIOTECHNOLOGY, 10.1007/s10126-013-9496-1, 15, 4, 476-486, 2013.08.
26. Shin-ichi Chisada, Kohei Shimizu, Haruna Kamada, Naoyuki Matsunaga, NOZOMU OKINO, Makoto Ito, Vibrios adhere to epithelial cells in the intestinal tract of red sea bream, Pagrus major, utilizing GM4 as an attachment site, FEMS MICROBIOLOGY LETTERS, 10.1111/1574-6968.12082, 341, 1, 18-26, 2013.04.
27. Yohei Ishibashi, Utaro Kobayashi, Atsushi Hijikata, Keishi Sakaguchi, Hatsumi M. Goda, Tomohiro Tamura, NOZOMU OKINO, Makoto Ito, Preparation and characterization of EGCase I, applicable to the comprehensive analysis of GSLs, using a rhodococcal expression system, JOURNAL OF LIPID RESEARCH, 10.1194/jlr.D028951, 53, 10, 2242-2251, 2012.10.
28. Hitomi Kajiwara, Sakurako Katayama, Yoshimitsu Kakuta, NOZOMU OKINO, Makoto Ito, Toshiki Mine, Takeshi Yamamoto, Loss-of-Function Mutation in Bi-Functional Marine Bacterial Sialyltransferase, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 10.1271/bbb.120133, 76, 9, 1639-1644, 2012.09.
29. Takanori Matsuda, Keishi Sakaguchi, Rie Hamaguchi, Takumi Kobayashi, Eriko Abe, Yoichiro Hama, Masahiro Hayashi, Daiske Honda, Yuji Okita, Shinichi Sugimoto, NOZOMU OKINO, Makoto Ito, Analysis of Delta 12-fatty acid desaturase function revealed that two distinct pathways are active for the synthesis of PUFAs in T. aureum ATCC 34304, JOURNAL OF LIPID RESEARCH, 10.1194/jlr.M024935, 53, 6, 1210-1222, 2012.06.
30. Ishibashi Y, Ikeda K, Sakaguchi K, Okino N, Taguchi R, Ito M., Quality control of fungi-specific glucosylceramide in Cryptococcus neoformans by a novel glucocerebrosidase EGCrP1., J. Biol. Chem., 287, 1, 368-381, 2012.01.
31. Matsuda T, Sakaguchi K, Kobayashi T, Abe E, Kurano N, Sato A, Okita Y, Sugimoto S, Hama Y, Hayashi M, Okino N, Ito M., Molecular cloning of a Pinguiochrysis pyriformis oleate-specific microsomal {Delta}12-fatty acid desaturase and functional analysis in yeasts and thraustochytrids., J. Biochem., 150, 4, 375-383, 2011.10.
32. Matsunaga N, Chisada S, Fujioka H, Takashima K, Okino N, Ito M., Glycosphingolipid receptor of mariculture fish intestines for phathogenic vibrios., Fish. Sci. , 77, 4, 583-590, 2011.07.
33. Nabetani T, Makino A, Hullin-Matsuda F, Hirakawa TA, Takeoka S, Okino N, Ito M, Kobayashi T, Hirabayashi Y., Multiplex analysis of sphingolipids using amine-reactive tags (iTRAQ)., J. Lipid. Res., 52, 6, 1294-1302, 2011.06.
34. Sato B, Katagiri YU, Miyado K, Okino N, Ito M, Akutsu H, Okita H, Umezawa A, Fujimoto J, Toshimori K, Kiyokawa N., Lipid rafts enriched in monosialylGb5Cer carrying the stage-specific embryonic antigen-4 epitope are involved in development of mouse preimplantation embryos at cleavage stage., BMC Dev. Biol. , 11, 22, 2011.06.
35. Takara T, Nakagawa T, Isobe M, Okino N, Ichinose S, Omori A, Ito M., Purification, molecular cloning, and application of a novel sphingomyelin-binding protein (clamlysin) from the brackishwater clam, Corbicula japonica., Biochim. Biophys. Acta. , 1811, 5, 323-32, 2011.05.
36. Kobayashi T, Sakaguchi K, Matsuda T, Abe E, Hama Y, Hayashi M, Honda D, Okita Y, Sugimoto S, Okino N, Ito M., Increase of EPA in Thraustochytrids through Expression of a Fatty Acid Δ5 Desaturase Gene Driven by the Thraustochytrid Ubiquitin Promoter., Appl. Environ. Microbiol., 77, 11, 3870-3876, 2011.05.
37. Okino, N., Ikeda, R. and Ito, M., Expression, Purification, and Characterization of a Recombinant Neutral Ceramidase from Mycobacterium tuberculosis., Bioscience, Biotechnology, and Biochemistry, 74, 2, 316-321, 2010.02.
38. X. Xu, Y. Horibata, M. Inagaki, Y. Hama, K. Sakaguchi, N. Okino, and M. Ito, A novel fucosyl glycosphingolipid of brine shrimp that is highly sensitive to endoglycoceramidase., Glycobiology, 19, 12, 1446-1451, 2009.12.
39. Chisada SI, Yoshimura Y, Sakaguchi K, Uemura S, Go S, Ikeda K, Uchima H, Matsunaga N, Ogura K, Tai T, Okino N, Taguchi R, Inokuchi J, Ito M., Zebrafish and mouse alpha2,3-sialyltranserases responsible for synthesizing GM4 ganglioside., J Biol Chem, 284, 44, 30534-30546, 2009.10.
40. Iwatani T, Okino N, Sakakura M, Kajiwara H, Takakura Y, Kimura M, Ito M, Yamamoto T, Kakuta Y., Crystal structure of alpha/beta-galactoside alpha2,3-sialyltransferase from a luminous marine bacterium, Photobacterium phosphoreum., FEBS Lett. , 583(12):2083-2087, 2009.06.
41. Zama K, Hayashi Y, Ito S, Hirabayashi Y, Inoue T, Ohno K, Okino N, Ito M., Simultaneous quantification of glucosylceramide and galactosylceramide by normal phase HPLC using O-phtalaldehyde derivatives prepared with sphingolipid ceramide N-deacylase., Glycobiology, 19, 7, 767-775, 2009.05.
42. Ishibashi Y, Nagamatsu Y, Meyer S, Imamura A, Ishida H, Kiso M, Okino N, Geyer R, Ito M., Transglycosylation-based Fluorescent Labeling of 6-Gala Series Glycolipids by EGALC., Glycobiology, 19, 7, 797-807, 2009.05.
43. Inoue T, Okino N, Kakuta Y, Hijikata A, Okano H, Goda HM, Tani M, Sueyoshi N, Kambayashi K, Matsumura H, Kai Y, Ito M., Mechanistic insights into the hydrolysis and synthesis of ceramide by neutral ceramidase., J Biol Chem, 284(14):9566-9577, 2009.04.
44. Takakura Y, Tsunashima M, Suzuki J, Usami S, Kakuta Y, Okino N, Ito M, Yamamoto T., Tamavidins--novel avidin-like biotin-binding proteins from the Tamogitake mushroom., FEBS J, 276(5):1383-1397., 2009.03.
45. Hayashi Y, Zama K, Abe E, Okino N, Inoue T, Ohno K, Ito M., A sensitive and reproducible fluorescent-based HPLC assay to measure the activity of acid as well as neutral beta-glucocerebrosidases., Anal Biochem, 2008.11.
46. Goda HM, Ushigusa K, Ito H, Okino N, Narimatsu H, Ito M., Molecular cloning, expression, and characterization of a novel endo-alpha-N-acetylgalactosaminidase from Enterococcus faecalis., Biochem Biophys Res Commun, 375(4):541-546, 2008.10.
47. Noguchi J, Hayashi Y, Baba Y, Okino N, Kimura M, Ito M, Kakuta Y., Crystal structure of the covalent intermediate of human cytosolic beta-glucosidase., Biochem Biophys Res Commun, 374, 549-552, 2008.09.
48. Y. Kakuta, N. Okino, H. Kajiwara, M. Ichikawa, Y. Takakura, M. Ito, and T. Yamamoto, Crystal Structure of Vibrionaceae Photobacterium sp. JT-ISH-224 a2,6-Sialyltransferase in a Ternary Complex With Donor Product CMP and Acceptor Substrate Lactose., Glycobiology, 18, 66-73, 2008.01.
49. Hayashi Y, Okino N, Kakuta Y, Shikanai T, Tani M, Narimatsu H, Ito M, Klotho-related Protein Is a Novel Cytosolic Neutral beta-Glycosylceramidase., J Biol Chem, 282(42), 30889-30900, 2007.10.
50. Okino N, Kakuta Y, Kajiwara H, Ichikawa M, Takakura Y, Ito M, Yamamoto T., Purification, crystallization and preliminary crystallographic characterization of the alpha 2,6-sialyltransferase from Photobacterium sp. JT-ISH-224., Acta Crystallogr Sect F Struct Biol Cryst Commun, 63(Pt 8):662-664, 2007.08.
51. Ishibashi Y, Kiyohara M, Okino N, Ito M, Synthesis of fluorescent glycosphingolipids and neoglycoconjugates which contain 6-gala oligosaccharides using the transglycosylation reaction of a novel endoglycoceramidase (EGALC)., J Biochem (Tokyo), 142(2), 239-246, 2007.08.
52. Ishibashi Y, Nakasone T, Kiyohara M, Horibata Y, Sakaguchi K, Hijikata A, Ichinose S, Omori A, Yasui Y, Imamura A, Ishida H, Kiso M, Okino N, Ito M., A novel endoglycoceramidase hydrolyzes oligogalactosylceramides to produce galactooligosaccharides and ceramides., J Biol Chem, 282(15), 11386-11396, 2007.04.
53. N. Okino and M. Ito, Ceramidase Enhances Phospholipase C-induced Hemolysis by Pseudomonas aeruginosa, J. Biol. Chem. , 282, 6021-6030, 2007.03.
54. Xu X, Monjusho H, Inagaki M, Hama Y, Yamaguchi K, Sakaguchi K, Iwamori M, Okino N, Ito M., Fucosyl-GM1a, an endoglycoceramidase-resistant ganglioside of porcine brain., J Biochem (Tokyo), 141(1), 1-7, 2007.01.
55. Y. Hayashi, Y. Horibata, K. Sakaguchi, N. Okino, and M. Ito, A Sensitive and Reproducible Assay to Measure the Activity of Glucosylceramide Synthase and Lactosylceramide Synthase using HPLC and Fluorescent Substrates., Anal. Biochem., 10.1016/j.ab.2005.05.029, 345, 2, 181-186, 345, 181-186 (2005), 2005.01.
56. Y. -H. Hwang, M. Tani, T. Nakagawa, N. Okino, and M. Ito, Subcellular Localization of Human Neutral Ceramidase Expressed in HEK293 cells., Biochem. Biophys. Res. Commun., 10.1016/j.bbrc.2005.03.134, 331, 1, 37-42, 331, 37-42 (2005), 2005.01.
57. S. Chisada, Y. Horibata, Y. Hama, M. Inagaki, N. Furuya, N. Okino, and M. Ito, The Glycosphingolipid Receptor for Vibrio trachuri in the Red Sea Bream Intestine Is a GM4 Ganglioside which Contains 2-hydroxy Fatty Acids., Biochem. Biophys. Res. Commun., 10.1016/j.bbrc.2005.05.110, 333, 2, 367-373, 333, 367-373 (2005), 2005.01.
58. Horibata, K. Sakaguchi, N. Okino, H. Iida, M. Inagaki, T. Fujisawa, Y. Hama, and M. Ito, Unique catabolic pathway of glycosphingolipids in a hydrozoan, Hydra magnipapillata, involving endoglycoceramidase, J. Biol. Chem., 10.1074/jbc.M401460200, 279, 32, 33379-33389, 279, pp33379-33389, 2004.08.
59. M. Tani, N. Okino, N. Sueyoshi, and M. Ito, Conserved amino acid residues in the COOH-terminal tail are indispensable for the correct folding and localization and enzyme activity of neutral ceramidase, J. Biol. Chem., 10.1074/jbc.M404012200, 279, 28, 29351-29358, 279, pp29351-29358, 2004.06.
60. Y. Yoshimura, M. Tani, N. Okino, H. Iida, and M. Ito, Molecular cloning and functional analysis of zebrafish neutral ceramidase, J. Biol. Chem., 10.1074/jbc.M405598200, 279, 42, 44012-44022, 279, pp44012-44022, 2004.01.
61. H. Monjusho, N. Okino, M. Tani, M. Maeda, M. Yoshida, and M. Ito, A Neutral Ceramidase Homologue of Dictyostelium discoideum Exhibits an Acidic pH Optimum, Biochem. J., 10.1042/BJ20030652, 376, 473-479, 376, pp473-479, 2003.12.
62. X. He, N. Okino, R. Dhami, A. Dagan, S. Gatt, H. Schulze, K. Sandhoff, and E. H. Schuchman, Purification and Characterization of Recombinant, Human Acid Ceramidase, J. Biol. Chem., 278, pp32978-32986, 2003.08.
63. N. Okino, X. He, S. Gatt, K. Sandhoff, M. Ito, and E. H. Schuchman, The Reverse Activity of Human Acid Ceramidase, J. Biol. Chem., 10.1074/jbc.M303310200, 278, 32, 29948-29953, 278, pp29948-29953, 2003.08.
64. N. Okino, K. Mori, and M. Ito, Genomic Structure and Promoter Analysis of the Mouse Neutral Ceramidase Gene, Biochem. Biophys. Res. Commun., 10.1016/S0006-291X(02)02540-8, 299, 1, 160-166, 299, pp160-166, 2002.11.
65. T. Osaki, N. Okino, F. Tokunaga, S. Iwanaga, and S. Kawabata, Proline-rich Cell Surface Antigens of Horseshoe Crab Hemocytes Are Substrates for Protein Cross-linking with a Clotting Protein Coagulin, J. Biol. Chem., 10.1074/M206773200, 277, 42, 40084-40090, 277, pp40084 - 40090, 2002.10.
66. Y. Yoshimura, N. Okino, M. Tani, and M. Ito, Molecular Cloning and Characterization of a Secretory Neutral Ceramidase of Drosophila melanogaster, J. Biochem., 132, 2, 229-236, 132, pp229-236, 2002.08.
67. Katsuhiro Kita, Noriyuki Sueyoshi, Nozomu Okino, Masanori Inagaki, Hideharu Ishida, Makoto Kiso, Shuhei Imayama, Takashi Nakamura, Makoto Ito, Activation of bacterial ceramidase by anionic glycerophospholipids
Possible involvement in ceramide hydrolysis on atopic skin by Pseudomonas ceramidase, Biochemical Journal, 10.1042/0264-6021:3620619, 362, 3, 619-626, 2002.03, We have reported previously that the ceramidase from Pseudomonas aeruginosa AN17 isolated from a patient with atopic dermatitis requires detergents for hydrolysis of ceramide (Cer) [Okino, Tani, Imayama and Ito (1998) J. Biol. Chem. 273, 14368-14373]. In the present study, we report that some glycerophospholipids strongly activated the hydrolysis of Cer by Pseudomonas ceramidase in the absence of detergents. Among the glycerophospholipids tested, cardiolipin was most effective in stimulating hydrolysis of Cer followed by phosphatidic acid, phosphatidylethanolamine and phosphatidylglycerol, whereas phosphatidylcholine, lysophosphatidic acid and diacylglycerol were less effective. Interestingly, Staphylococcus aureus-derived lipids, which contain cardiolipin and phosphatidylglycerol as major lipid components, also strongly enhanced the hydrolysis of normal Cer, as well as the human skin-specific ω-hydroxyacyl Cer, by the enzyme in the absence of detergents. It was confirmed that several strains of P. aeruginosa, including AN17, secrete a significant amount of staphylolytic proteases to lyse S. aureus cells, resulting in the release of cardiolipin and phosphatidylglycerol. Since both P. aeruginosa and S. aureus are suspected of being present in microflora of atopic skin, we speculate that S. aureus-derived glycerophospholipids stimulate the hydrolysis of Cer in atopic skin by bacterial ceramidase..
68. N. Sueyoshi, K. Kita, N. Okino, K. Sakaguchi, T. Nakamura, M. Ito, Molecular cloning and expression of Mn2+-dependent sphingomyelinase/hemolysin of an aquatic bacterium, pseudomonas sp. strain TK4, Journal of bacteriology, 10.1128/JB.184.2.540-546.2002, 184, 2, 540-546, 2002.01, We report here the molecular cloning and expression of a hemolytic sphingomyelinase from an aquatic bacterium, Pseudomonas sp. strain TK4. The sphingomyelinase gene was found to consist of 1,548 nucleotides encoding 516 amino acid residues. The recombinant 57.7-kDa enzyme hydrolyzed sphingomyelin but not phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, phosphatidic acid, or phosphatidylethanolamine, indicating that the enzyme is a sphingomyelin-specific sphingomyelinase C. The hydrolysis of sphingomyelin by the enzyme was found to be most efficient at pH 8.0 and activated by Mn2+. The enzyme shows quite a broad specificity, i.e., it hydrolyzed 4-nitrobenz-2-oxa-1,3-diazole (NBD)-sphingomyelin with short-chain fatty acids and NBD-sphingosylphosphorylcholine, the latter being completely resistant to hydrolysis by any sphingomyelinase reported so far. Significant sequence similarities were found in sphingomyelinases from Bacillus cereus, Staphylococcus aureus, Listeria ivanovii, and Leptospira interrogans, as well as a hypothetical protein encoded in Chromobacterium violaceum, although the first three lacked one-third of the sequence corresponding to that from the C terminus of the TK4 enzyme. Interestingly, the deletion mutant of strain TK4 lacking 186 amino acids at the C-terminal end hydrolyzed sphingomyelin, whereas it lost all hemolytic activity, indicating that the C-terminal region of the TK4 enzyme is indispensable for the hemolytic activity..
69. Mari Kakiuchi, Nozomu Okino, Noriyuki Sueyoshi, Sachiyo Ichinose, Akira Omori, Shun Ichiro Kawabata, Kuniko Yamaguchi, Makoto Ito, Purification, characterization, and cDNA cloning of α-N-acetylgalactosamine-specific lectin from starfish, Asterina pectinifera, Glycobiology, 10.1093/glycob/12.2.85, 12, 2, 85-94, 2002.01, We report here the purification, characterization, and cDNA cloning of a novel N-acetylgalactosamine-specific lectin from starfish, Asterina pectinifera. The purified lectin showed 19-kDa, 41-kDa, and 60-kDa protein bands on SDS-PAGE, possibly corresponding to a monomer, homodimer, and homotrimer. Interestingly, on 4-20% native PAGE the lectin showed at least nine protein bands, among which oligomers containing six to nine subunits had potent hemagglutination activity for sheep erythrocytes. The hemagglutination activity of the lectin was specifically inhibited by N-acetylgalactosamine, Tn antigen, and blood group A trisaccharide, but not by N-acetylglucosamine, galactose, galactosamine, or blood group B trisaccharide. The specificity of the lectin was further examined using various glycosphingolipids and biotin-labeled lectin. The lectin was found to bind to Gb5Cer, but not Gb4Cer, Gb3Cer, GM1a, GM2, or asialo-GM2, indicating that the lectin specifically binds to the terminal α-GalNAc at the nonreducing end. The hemagglutination activity of the lectin was completely abolished by chelation with EDTA or EGTA and completely restored by the addition of CaCl2. cDNA cloning of the lectin showed that the protein is composed of 168 amino acids, including a signal sequence of 18 residues, and possesses the typical C-type lectin motif. These findings indicate that the protein is a C-type lectin. The recombinant lectin, produced in a soluble form by Escherichia coli, showed binding activity for asialomucin in the presence of Ca2+ but no hemagglutination..
70. S. Mitsutake, M. Tani, N. Okino, K. Mori, H. Iida, S. Yoshida, A. Omori, T. Nakamura, and M. Ito, Purification, Characterization, Molecular Cloning and Subcellular Distribution of Neutral Ceramidase of Rat Kidney., J. Biol. Chem., 10.1074/jbc.M102233200, 276, 28, 26249-26259, 276, pp26249-26359, 2001.07.
71. Horibata Y, Okino N, Ichinose S, Omori A, Ito M., Purification, Characterization and cDNA Cloning of a Novel Acidic Endoglycoceramidase from the Jellyfish, Cyanea nozakii, J. Biol. Chem., 10.1074/jbc.M003575200, 275, 40, 31297-31304, 275, pp31297-31304, 2000.10.
72. Katsuhiro Kita, Nozomu Okino, Makoto Ito, Reverse hydrolysis reaction of a recombinant alkaline ceramidase of Pseudomonas aeruginosa, Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids, 10.1016/S1388-1981(00)00029-9, 1485, 2-3, 111-120, 2000.05, Recently, we purified an alkaline ceramidase (CDase) of Pseudomonas aeruginosa and found that the enzyme catalyzed a reversible reaction in which the N-acyl linkage of ceramide was hydrolyzed or synthesized [J. Biol. Chem. 273 (1998) 14368-14373]. Here, we report the characterization of the reverse hydrolysis reaction of the CDase using a recombinant enzyme. The reverse hydrolysis reaction of the CDase was clearly distinguishable from the reaction of an acyl-coenzyme A (CoA) dependent N-acyltransferase, because the CDase catalyzed the condensation of a free fatty acid to sphingosine (Sph) without cofactors but did not catalyze the transfer of a fatty acid from acyl-CoA to Sph. The reverse hydrolysis reaction proceeded most efficiently in the presence of 0.05% Triton X-100 at neutral pH, while the hydrolysis reaction tended to be favored with an increase in the concentration of the detergent at alkaline pH. The specificity of the reverse reaction for fatty acids is quite broad; saturated and unsaturated fatty acids were efficiently condensed to Sph. In contrast, the stereo-specificity of the reverse reaction for the sphingoid bases is very strict; the D-erythro form of Sph, not the L- erythro or D/L-threo one, was only acceptable for the reverse reaction. Chemical modification of the enzyme protein affected or did not affect both the hydrolysis and reverse reactions to the same extent, suggesting that the two reactions are catalyzed at the same catalytic domain..
73. M. Tani, N. Okino, K. Mori, T. Tanigawa, H. Izu, and M. Ito, Molecular Cloning of the Full-length cDNA Encoding Mouse Neutral Ceramidase, J. Biol. Chem., 10.1074/jbc.275.15.11229, 275, 15, 11229-11234, 275, pp11229-11234, 2000.04.
74. M. Tani, N. Okino, S. Mitsutake, T. Tanigawa, H. Izu, and M. Ito, Purification and Characterization of a Novel Ceramidase from Mouse Liver, J. Biol. Chem., 275, pp3462-3468, 2000.02.
75. Keishi Sakaguchi, Nozomu Okino, Noriyuki Sueyoshi, Hiroyuki Izu, Makoto Ito, Cloning and expression of gene encoding a novel endoglycoceramidase of Rhodococcus sp. strain C9, Journal of biochemistry, 10.1093/oxfordjournals.jbchem.a022725, 128, 1, 145-152, 2000.01, Endoglycoceramidase (EGCase) is an enzyme capable of cleaving the glycosidic linkage between oligosaccharides and ceramides of various glycosphingolipids. We previously reported that the Asn-Glu-Pro (NEP) sequence is part of the active site of EGCase of Rhodococcus sp. strain M- 777. This paper describes the molecular cloning of a new EGCase gene utilizing the NEP sequence from the genomic library of Rhodococcus sp. strain C9, which was clearly distinguishable from M-777 by 16S rDNA analysis. C9 EGCase possessed an open reading frame of 1,446 bp encoding 482 amino acids, and showed 78% and 76% identity to M-777 EGCase II at the nucleotide and amino acid levels, respectively. Interestingly, C9 EGCase showed the different specificity to the M-777 enzyme: it hydrolyzed b-series gangliotetraosylceramides more slowly than the M-777 enzyme, whereas both enzymes hydrolyzed a-series gangliosides and neutral glycosphingolipids to the same extent..
76. N. Okino, S. Ichinose, A. Omori, S. Imayama, T. Nakamura, and M. Ito, Molecular cloning, sequencing, and expression of the gene encoding alkaline ceramidase from Pseudomonas aeruginosa. Cloning of a ceramidase homologue from Mycobacterium tuberculosis, J Biol Chem., 10.1074/jbc.274.51.36616, 274, 51, 36616-36622, 274, pp36616-36622., 1999.12.
77. Keishi Sakaguchi, Nozomu Okino, Hiroyuki Izu, Makoto Ito, The Glu residue in the conserved Asn-Glu-Pro sequence of endoglycoceramidase is essential for enzymatic activity, Biochemical and Biophysical Research Communications, 10.1006/bbrc.1999.0855, 260, 1, 89-93, 1999.06, Endoglycoceramidase (EGCase) is an enzyme capable of cleaving the glycosidic linkage between oligosaccharides and ceramides of various glycosphingolipids. We previously cloned the gene encoding EGCase II of Rhodococcus sp. M-777 and reported that the deduced amino acid sequence contained the Asn-Glu-Pro (NEP) sequence, conserved as part of the active site of family A cellulases (endo-1,4-β-glucanases). The NEP sequence was also found in the deduced amino acid sequence of the newly cloned EGCase gene of Rhodococcus sp. C9. Replacement of the Glu residue in the NEP sequence with Gln or Asp by site directed mutagenesis caused marked loss of enzymatic activity in both the M-777 and C9 EGCases but did not affect the expression of EGCase protein. This result clearly indicated that the NEP sequence is part of the active site of EGCase, in which the Glu residue plays an important role in the catalytic reaction, possibly in the same manner as in endo-1,4-β-glucanase..
78. Motohiro Tani, Nozomu Okino, Susumu Mitsutake, Makoto Ito, Specific and sensitive assay for alkaline and neutral ceramidases involving C12-NBD-ceramide, Journal of Biochemistry, 125, 4, 746-749, 1999.04, A fluorescent analogue of ceramide, C12-NBD-ceramide, was found to be hydrolyzed much faster than 14C-labeled ceramide by alkaline ceramidase from Pseudomonas aeruginosa and neutral ceramidase from mouse liver, while this substrate was relatively resistant to acid ceramidase from plasma of the horseshoe crab. The radioactive substrate was used more preferentially by the acid ceramidase. It should be noted that C6-NBD-ceramide, which is usually used for ceramidase assays, was hardly hydrolyzed by any of the enzymes examined, compared to C12-NBD)-ceramide. For the alkaline and neutral enzymes, the V(max) and k (V(max)/K(m)) with C12-NBD-ceramide were much higher than those with 14C-ceramide. In contrast, for the acid enzyme these parameters with C12-NBD-ceramide were less than half those with the radioisotope-labeled substrate. It is noteworthy that the labeling of ceramide with NBD did not itself reduce the K(m) of the alkaline enzyme, but did that of the neutral enzyme. It was also found that C12-NBD-ceramide was preferentially hydrolyzed by the alkaline and neutral enzymes, but not the acid one, in several mammalian cell lines. This study clearly shows that the attachment of NBD, but not dansyl, increases the susceptibility of ceramide to alkaline and neutral enzyme, and decreases that to acid enzymes. Thus the use of this substrate provides a specific and sensitive assay for alkaline and neutral ceramidases..
79. Yoshinori Ohnishi, Nozomu Okino, Makoto Ito, Shuhei Imayama, Ceramidase activity in bacterial skin flora as a possible cause of ceramide deficiency in atopic dermatitis, Clinical and Diagnostic Laboratory Immunology, 6, 1, 101-104, 1999.01, A marked decrease in the content of ceramide has been reported in the horny layer of the epidermis in atopic dermatitis (AD). This decrease impairs the permeability barrier of the epidermis, resulting in the characteristic dry and easily antigen-permeable skin of AD, since ceramide serves as the major water-holding molecule in the extracellular space of the horny layer. On the other hand, the skin of such patients is frequently colonized by bacteria, most typically by Staphylococcus aureus, possessing genes such as those for sphingomyelinase, which are related to sphingolipid metabolism. We therefore tried to identify a possible correlation between the ceramide content and the bacterial flora obtained from the skin of 25 patients with AD versus that of 24 healthy subjects, using a thin-layer chromatographic assay of the sphingomyelin-associated enzyme activities secreted from the bacteria. The findings of the assay demonstrated that ceramidase, which breaks ceramide down into sphingosine and fatty acid, was secreted significantly more from the bacterial flora obtained from both the lesional and the nonlesional skin of patients with AD than from the skin of healthy subjects; sphingomyelinase, which breaks sphingomyelin down into ceramide and phosphorylcholine, was secreted from the bacterial flora obtained from all types of skin at similar levels for the patients with AD and the healthy controls. The finding that the skin of patients with AD is colonized by ceramidase-secreting bacteria thus suggests that microorganisms are related to the deficiency of ceramide in the horny layer of the epidermis, which increases the hypersensitivity of skin in AD patients by impairing the permeability barrier..
80. N. Okino, M. Tani, S. Imayama, and M. Ito, Purification and Characterization of a Novel Alkaline Ceramidase from Pseudomonas aeruginosa, J. Biol. Chem., 10.1074/jbc.273.23.14368, 273, 23, 14368-14373, 273, pp14368-14373, 1998.06.