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Assays and Utilization of Enzymes Involved in Glycolipid Metabolism in Bacteria and Fungi

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Glycolipids

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2613))

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Abstract

Microbial glycosphingolipid (GSL)-degrading enzymes with unique specificity are useful tools for GSL research. On the other hand, some microbial glycolipids, not only GSLs but also steryl glucosides, are closely related to pathogenicity, and, thus, the metabolism of microbial glycolipids is attracting attention as a target for antibiotics. This chapter describes the assays and utilization of microbial enzymes useful for glycolipid research and those involved in pathogenicity or host immune reactions.

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References

  1. Ito M, Yamagata T (1986) A novel glycosphingolipid-degrading enzyme cleaves the linkage between the oligosaccharide and ceramide of neutral and acidic glycosphingolipids. J Biol Chem 261(30):14278–14282. https://doi.org/10.1016/S0021-9258(18)67015-2

    Article  CAS  Google Scholar 

  2. Ito M, Kurita T, Kita K (1995) A novel enzyme that cleaves the N-acyl linkage of ceramides in various glycosphingolipids as well as sphingomyelin to produce their lyso forms. J Biol Chem 270(41):24370–24374. https://doi.org/10.1074/jbc.270.41.24370

    Article  CAS  Google Scholar 

  3. Okino N, Li M, Qu Q, Nakagawa T, Hayashi Y, Matsumoto M, Ishibashi Y, Ito M (2020) Two bacterial glycosphingolipid synthases responsible for the synthesis of glucuronosylceramide and α-galactosylceramide. J Biol Chem 295(31):10709–10725. https://doi.org/10.1074/jbc.RA120.013796

    Article  CAS  Google Scholar 

  4. Normile TG, McEvoy K, Del Poeta M (2020) Steryl glycosides in fungal pathogenesis: an understudied immunomodulatory adjuvant. J Fungi 6(1):25. https://doi.org/10.3390/jof6010025

    Article  CAS  Google Scholar 

  5. Ishibashi Y, Ikeda K, Sakaguchi K, Okino N, Taguchi R, Ito M (2012) Quality control of fungus-specific glucosylceramide in Cryptococcus neoformans by Endoglycoceramidase-related protein 1 (EGCrP1). J Biol Chem 287(1):368–381. https://doi.org/10.1074/jbc.M111.311340

    Article  CAS  Google Scholar 

  6. Watanabe T, Ito T, Goda HM, Ishibashi Y, Miyamoto T, Ikeda K, Taguchi R, Okino N, Ito M (2015) Sterylglucoside catabolism in Cryptococcus neoformans with endoglycoceramidase-related protein 2 (EGCrP2), the first Steryl-beta-glucosidase identified in fungi. J Biol Chem 290(2):1005–1019. https://doi.org/10.1074/jbc.M114.616300

    Article  CAS  Google Scholar 

  7. Horibata Y, Okino N, Ichinose S, Omori A, Ito M (2000) Purification, characterization, and cDNA cloning of a novel acidic endoglycoceramidase from the jellyfish, Cyanea nozakii. J Biol Chem 275(40):31297–31304. https://doi.org/10.1074/jbc.M003575200

    Article  CAS  Google Scholar 

  8. Li Y-T, Ishikawa Y, Li S-C (1987) Occurrence of ceramide-glycanase in the earthworm, Lumbricusterrestris. Biochem Biophys Res Commun 149(1):167–172. https://doi.org/10.1016/0006-291X(87)91619-6

    Article  CAS  Google Scholar 

  9. Li S-C, Degasperi R, Muldrey JE, Li Y-T (1986) A unique glycosphingolipid-splitting enzyme (ceramide-glycanase from leech) cleaves the linkage between the oligosaccharide and the ceramide. Biochem Biophys Res Commun 141(1):346–352. https://doi.org/10.1016/S0006-291X(86)80375-8

    Article  CAS  Google Scholar 

  10. Ishibashi Y, Kobayashi U, Hijikata A, Sakaguchi K, Goda HM, Tamura T, Okino N, Ito M (2012) Preparation and characterization of EGCase I, applicable to the comprehensive analysis of GSLs, using a rhodococcal expression system. J Lipid Res 53(10):2242–2251. https://doi.org/10.1194/jlr.D028951

    Article  CAS  Google Scholar 

  11. Izu H, Izumi Y, Kurome Y, Sano M, Kondo A, Kato I, Ito M (1997) Molecular cloning, expression, and sequence analysis of the endoglycoceramidase II gene from Rhodococcus species strain M-777. J Biol Chem 272(32):19846–19850. https://doi.org/10.1074/jbc.272.32.19846

    Article  CAS  Google Scholar 

  12. 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 (2007) A novel endoglycoceramidase hydrolyzes oligogalactosylceramides to produce galactooligosaccharides and ceramides. J Biol Chem 282(15):11386–11396

    Article  CAS  Google Scholar 

  13. Ito M, Yamagata T (1989) Purification and characterization of glycosphingolipid-specific endoglycosidases (endoglycoceramidases) from a mutant strain of Rhodococcus sp. evidence for three molecular species of endoglycoceramidase with different specificities. J Biol Chem 264(16):9510–9519. https://doi.org/10.1016/S0021-9258(18)60561-7

    Article  CAS  Google Scholar 

  14. Caines MEC, Vaughan MD, Tarling CA, Hancock SM, Warren RAJ, Withers SG, Strynadka NCJ (2007) Structural and mechanistic analyses of endo-Glycoceramidase II, a membrane-associated family 5 glycosidase in the Apo and GM3 ganglioside-bound forms. J Biol Chem 282(19):14300–14308. https://doi.org/10.1074/jbc.M611455200

    Article  CAS  Google Scholar 

  15. Han YB, Chen LQ, Li Z, Tan YM, Feng Y, Yang GY (2017) Structural insights into the broad substrate specificity of a novel Endoglycoceramidase I belonging to a new subfamily of GH5 Glycosidases. J Biol Chem 292(12):4789–4800. https://doi.org/10.1074/jbc.M116.763821

    Article  CAS  Google Scholar 

  16. Higashi H, Ito M, Fukaya N, Yamagata S, Yamagata T (1990) Two-dimensional mapping by the high-performance liquid chromatography of oligosaccharides released from glycosphingolipids by endoglycoceramidase. Anal Biochem 186(2):355–362. https://doi.org/10.1016/0003-2697(90)90094-p

    Article  CAS  Google Scholar 

  17. Fujitani N, Takegawa Y, Ishibashi Y, Araki K, Furukawa JI, Mitsutake S, Igarashi Y, Ito M, Shinohara Y (2011) Qualitative and quantitative cellular glycomics of glycosphingolipids based on rhodococcal endoglycosylceramidase-assisted glycan cleavage, glycoblotting-assisted sample preparation, and matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry analysis. J Biol Chem 286(48):41669–41679. https://doi.org/10.1074/jbc.M111.301796

    Article  CAS  Google Scholar 

  18. Ito M, Ikegami Y, Yamagata T (1991) Activator proteins for glycosphingolipid hydrolysis by endoglycoceramidases. Elucidation of biological functions of cell-surface glycosphingolipids in situ by endoglycoceramidases made possible using these activator proteins. J Biol Chem 266(12):7919–7926. https://doi.org/10.1016/S0021-9258(20)89537-4

    Article  CAS  Google Scholar 

  19. Ito M, Ikegami Y, Tai T, Yamagata T (1993) Specific hydrolysis of intact erythrocyte cell-surface glycosphingolipids by endoglycoceramidase. Lack of modulation of erythrocyte glucose transporter by endogenous glycosphingolipids. Eur J Biochem 218(2):637–643. https://doi.org/10.1111/j.1432-1033.1993.tb18417.x

    Article  CAS  Google Scholar 

  20. Ito M, Ikegami Y, Yamagata T (1993) Kinetics of endoglycoceramidase action toward cell-surface glycosphingolipids of erythrocytes. Eur J Biochem 218(2):645–649. https://doi.org/10.1111/j.1432-1033.1993.tb18418.x

    Article  CAS  Google Scholar 

  21. Ito M, Ikegami Y, Omori A, Yamagata T (1991) Conversion of endoglycoceramidase-activator II by trypsin to the 27.9 kDa polypeptide possessing full activity: purification of activator for endoglycoceramidase by trypsin treatment followed by trypsin-inhibitor agarose column application. J Biochem 110(3):328–332. https://doi.org/10.1093/oxfordjournals.jbchem.a123580

    Article  CAS  Google Scholar 

  22. Iwamori M, Ohta Y, Uchida Y, Tsukada Y (1997) Arthrobacter ureafaciens sialidase isoenzymes, L, M1 and M2, cleave fucosyl GM1. Glycoconj J 14(1):67–73. https://doi.org/10.1023/A:1018513015459

    Article  CAS  Google Scholar 

  23. Xu X, Monjusho H, Inagaki M, Hama Y, Yamaguchi K, Sakaguchi K, Iwamori M, Okino N, Ito M (2007) Fucosyl-GM1a, an endoglycoceramidase-resistant ganglioside of porcine brain. J Biochem 141(1):1–7. https://doi.org/10.1093/jb/mvm014

    Article  CAS  Google Scholar 

  24. Matsubara T, Hayashi A (1981) Structural studies on glycolipid of shellfish III. Novel glycolipids from Turbo cornutus. J Biochem 89(2):645–650. https://doi.org/10.1093/oxfordjournals.jbchem.a133241

    Article  CAS  Google Scholar 

  25. Watanabe T, Tani M, Ishibashi Y, Endo I, Okino N, Ito M (2015) Ergosteryl-beta-glucosidase (Egh1) involved in sterylglucoside catabolism and vacuole formation in Saccharomyces cerevisiae. Glycobiology 25(10):1079–1089. https://doi.org/10.1093/glycob/cwv045

    Article  CAS  Google Scholar 

  26. Rella A, Mor V, Farnoud AM, Singh A, Shamseddine AA, Ivanova E, Carpino N, Montagna MT, Luberto C, Del Poeta M (2015) Role of Sterylglucosidase 1 (Sgl1) on the pathogenicity of Cryptococcus neoformans: potential applications for vaccine development. Front Microbiol 6:836. https://doi.org/10.3389/fmicb.2015.00836

    Article  Google Scholar 

  27. Kita K, Kurita T, Ito M (2001) Characterization of the reversible nature of the reaction catalyzed by sphingolipid ceramide N-deacylase. A novel form of reverse hydrolysis reaction. Eur J Biochem 268(3):592–602. https://doi.org/10.1046/j.1432-1327.2001.01907.x

    Article  CAS  Google Scholar 

  28. Kurita T, Izu H, Sano M, Ito M, Kato I (2000) Enhancement of hydrolytic activity of sphingolipid ceramide N-deacylase in the aqueous-organic biphasic system. J Lipid Res 41(5):846–851. https://doi.org/10.1016/S0022-2275(20)32394-4

    Article  CAS  Google Scholar 

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Acknowledgments

This research was supported by AMED (Grant Number 20gm0910010h0105) and the Japanese Ministry of Education, Culture, Science, and Technology (JP19H02888).

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Authors and Affiliations

  1. Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Fukuoka, Japan

    Makoto Ito, Yohei Ishibashi, Takashi Watanabe & Nozomu Okino

  2. Department of Pathophysiology and Metabolism, Kawasaki Medical School, Okayama, Japan

    Takashi Watanabe

  3. Tokyo Chemical Industry Co., Ltd., Tokyo, Japan

    Jun Iwaki

  4. TAKARA BIO INC., Shiga, Japan

    Toyohisa Kurita

Authors
  1. Makoto Ito
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  2. Yohei Ishibashi
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  3. Takashi Watanabe
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  4. Jun Iwaki
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  5. Toyohisa Kurita
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  6. Nozomu Okino
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Corresponding authors

Correspondence to Makoto Ito or Nozomu Okino .

Editor information

Editors and Affiliations

  1. Department of Chemistry, Graduate School of Science, Osaka University, Osaka, Japan

    Kazuya Kabayama

  2. Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, Sendai, Japan

    Jin-ichi Inokuchi

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© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Ito, M., Ishibashi, Y., Watanabe, T., Iwaki, J., Kurita, T., Okino, N. (2023). Assays and Utilization of Enzymes Involved in Glycolipid Metabolism in Bacteria and Fungi. In: Kabayama, K., Inokuchi, Ji. (eds) Glycolipids. Methods in Molecular Biology, vol 2613. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2910-9_18

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  • DOI: https://doi.org/10.1007/978-1-0716-2910-9_18

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2909-3

  • Online ISBN: 978-1-0716-2910-9

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