title: Sterylglucoside catabolism in Cryptococcus neoformans with endoglycoceramidase-related protein 2 (EGCrP2), the first steryl-β-glucosidase identified in fungi.
abstract: Cryptococcosis is an infectious disease caused by pathogenic fungi, such as Cryptococcus neoformans and Cryptococcus gattii. The ceramide structure (methyl-d18:2/h18:0) of C. neoformans glucosylceramide (GlcCer) is characteristic and strongly related to its pathogenicity. We recently identified endoglycoceramidase-related protein 1 (EGCrP1) as a glucocerebrosidase in C. neoformans and showed that it was involved in the quality control of GlcCer by eliminating immature GlcCer during the synthesis of GlcCer (Ishibashi, Y., Ikeda, K., Sakaguchi, K., Okino, N., Taguchi, R., and Ito, M. (2012) Quality control of fungus-specific glucosylceramide in Cryptococcus neoformans by endoglycoceramidase-related protein 1 (EGCrP1). J. Biol. Chem. 287, 368-381). We herein identified and characterized EGCrP2, a homologue of EGCrP1, as the enzyme responsible for sterylglucoside catabolism in C. neoformans. In contrast to EGCrP1, which is specific to GlcCer, EGCrP2 hydrolyzed various β-glucosides, including GlcCer, cholesteryl-β-glucoside, ergosteryl-β-glucoside, sitosteryl-β-glucoside, and para-nitrophenyl-β-glucoside, but not α-glucosides or β-galactosides, under acidic conditions. Disruption of the EGCrP2 gene (egcrp2) resulted in the accumulation of a glycolipid, the structure of which was determined following purification to ergosteryl-3β-glucoside, a major sterylglucoside in fungi, by mass spectrometric and two-dimensional nuclear magnetic resonance analyses. This glycolipid accumulated in vacuoles and EGCrP2 was detected in vacuole-enriched fraction. These results indicated that EGCrP2 was involved in the catabolism of ergosteryl-β-glucoside in the vacuoles of C. neoformans. Distinct growth arrest, a dysfunction in cell budding, and an abnormal vacuole morphology were detected in the egcrp2-disrupted mutants, suggesting that EGCrP2 may be a promising target for anti-cryptococcal drugs. EGCrP2, classified into glycohydrolase family 5, is the first steryl-β-glucosidase identified as well as a missing link in sterylglucoside metabolism in fungi.
authors.affiliation: From the Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Watanabe
authors.affiliation: From the Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.
authors.forename: Tomoharu
authors.initials: T
authors.lastname: Ito
authors.affiliation: From the Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.
authors.forename: Hatsumi M
authors.initials: HM
authors.lastname: Goda
authors.affiliation: From the Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.
authors.forename: Yohei
authors.initials: Y
authors.lastname: Ishibashi
authors.affiliation: the Graduate School of Pharmaceutical Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan.
authors.forename: Tomofumi
authors.initials: T
authors.lastname: Miyamoto
authors.affiliation: the Institute for Advanced Biosciences, Keio University, 246-2 Mizukami, Kakuganji, Tsuruoka, Yamagata 997-0052, Japan, and.
authors.forename: Kazutaka
authors.initials: K
authors.lastname: Ikeda
authors.affiliation: the Department of Biomedical Sciences, College of Life and Health Sciences, Chubu University, 1200 Matsumoto-cho, Kasugai-shi, Aichi 487-8501, Japan.
authors.forename: Ryo
authors.initials: R
authors.lastname: Taguchi
authors.affiliation: From the Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.
authors.forename: Nozomu
authors.initials: N
authors.lastname: Okino
authors.affiliation: From the Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan, makotoi@agr.kyushu-u.ac.jp.
authors.forename: Makoto
authors.initials: M
authors.lastname: Ito
mesh_terms: D044602:Cellulases / Q000235:genetics* / Q000378:metabolism; D002518:Ceramides / Q000737:chemistry / Q000378:metabolism*; D003453:Cryptococcosis / Q000201:enzymology* / Q000473:pathology; D003455:Cryptococcus neoformans / Q000235:genetics* / Q000378:metabolism / Q000472:pathogenicity; D005656:Fungal Proteins / Q000235:genetics*; D005963:Glucosylceramides / Q000378:metabolism; D006017:Glycolipids / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans
chemical_list: D002518:Ceramides; D005656:Fungal Proteins; D005963:Glucosylceramides; D006017:Glycolipids; D044602:Cellulases; D006026:Glycoside Hydrolases; C050427:endoglycoceramidase
keywords: Fungi; Glycolipid; Glycosidase; Metabolism; Sterol