{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,11,21]],"date-time":"2025-11-21T05:25:28Z","timestamp":1763702728319,"version":"3.45.0"},"reference-count":47,"publisher":"MDPI AG","issue":"11","license":[{"start":{"date-parts":[[2025,11,19]],"date-time":"2025-11-19T00:00:00Z","timestamp":1763510400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"crossref","award":["32371279","32100972","32200992"],"award-info":[{"award-number":["32371279","32100972","32200992"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"crossref"}]},{"name":"Science and Technology Innovation Program of Hunan Province","award":["2025RC1034"],"award-info":[{"award-number":["2025RC1034"]}]},{"DOI":"10.13039\/501100004735","name":"Natural Science Foundation of Hunan Province","doi-asserted-by":"crossref","award":["2023JJ20007","2022JJ40052"],"award-info":[{"award-number":["2023JJ20007","2022JJ40052"]}],"id":[{"id":"10.13039\/501100004735","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100021171","name":"Guangdong Basic and Applied Basic Research Foundation","doi-asserted-by":"crossref","award":["2025A1515010385"],"award-info":[{"award-number":["2025A1515010385"]}],"id":[{"id":"10.13039\/501100021171","id-type":"DOI","asserted-by":"crossref"}]},{"name":"Third Hospital of Changsha","award":["2023TP1013"],"award-info":[{"award-number":["2023TP1013"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["JoF"],"abstract":"<jats:p>The global rise in resistance to first-line antifungal agents highlights the urgent need for new therapeutic strategies. Glycosylphosphatidylinositol (GPI)-anchored protein biosynthesis is an attractive target. The GPI mannosyltransferase I (GPI-MT-I), composed of Gpi14 and Pbn1, catalyzes the essential first mannose transfer from dolichol-phosphomannose (Dol-P-Man) to the GPI precursor. This initial mannosylation is critical for fungal cell wall integrity, yet the molecular basis of GPI-MT-I assembly and substrate recognition remains poorly understood. Here, we present the cryo-EM structure of Candida glabrata GPI-MT-I in complex with Dol-P-Man, revealing how Gpi14 and Pbn1 form a stable complex and engage the mannose donor. An AlphaFold3-predicted acceptor-bound model further defines the structural basis of acceptor substrate recognition and suggests a plausible catalytic mechanism. Comparison with structural homologs highlights a distinct mode of substrate engagement by GPI-MT-I. Together, these findings establish a mechanistic framework for GPI-MT-I function with broader implications for the GPI-MT family.<\/jats:p>","DOI":"10.3390\/jof11110819","type":"journal-article","created":{"date-parts":[[2025,11,19]],"date-time":"2025-11-19T12:55:31Z","timestamp":1763556931000},"page":"819","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Structural Insights into the Glycosylphosphatidylinositol Mannosyltransferase I Complex from Candida glabrata"],"prefix":"10.3390","volume":"11","author":[{"ORCID":"https:\/\/orcid.org\/0009-0006-7850-7655","authenticated-orcid":false,"given":"Hui","family":"Sun","sequence":"first","affiliation":[{"name":"School of Biomedical Sciences, Hunan University, Changsha 410082, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Weihong","family":"Wu","sequence":"additional","affiliation":[{"name":"School of Biomedical Sciences, Hunan University, Changsha 410082, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xiaomei","family":"Li","sequence":"additional","affiliation":[{"name":"Shanxi Academy of Advanced Research and Innovation, Taiyuan 030032, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yang","family":"Deng","sequence":"additional","affiliation":[{"name":"Hunan Provincial Key Laboratory of Anti-Resistance Microbial Drugs, The Third Hospital of Changsha, Changsha 410118, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jiarong","family":"Huang","sequence":"additional","affiliation":[{"name":"School of Biomedical Sciences, Hunan University, Changsha 410082, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Meng","family":"Yin","sequence":"additional","affiliation":[{"name":"School of Biomedical Sciences, Hunan University, Changsha 410082, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9054-3313","authenticated-orcid":false,"given":"Zhaofeng","family":"Yan","sequence":"additional","affiliation":[{"name":"School of Biomedical Sciences, Hunan University, Changsha 410082, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,11,19]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Bongomin, F., Gago, S., Oladele, R., and Denning, D. (2017). Global and Multi-National Prevalence of Fungal Diseases\u2014Estimate Precision. J. Fungi, 3.","DOI":"10.3390\/jof3040057"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"e428","DOI":"10.1016\/S1473-3099(23)00692-8","article-title":"Global incidence and mortality of severe fungal disease","volume":"24","author":"Denning","year":"2024","journal-title":"Lancet Infect. Dis."},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Katsipoulaki, M., Stappers, M.H.T., Malavia-Jones, D., Brunke, S., Hube, B., Gow, N.A.R., and Heitman, J. (2024). Candida albicans and Candida glabrata: Global priority pathogens. Microbiol. Mol. Biol. Rev., 88.","DOI":"10.1128\/mmbr.00021-23"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"42","DOI":"10.1007\/s11908-016-0549-2","article-title":"Echinocandin Resistance in Candida Species: A Review of Recent Developments","volume":"18","author":"Wiederhold","year":"2016","journal-title":"Curr. Infect. Dis. Rep."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"237","DOI":"10.2147\/IDR.S118892","article-title":"Fluconazole resistance in Candida species: A current perspective","volume":"10","author":"Berkow","year":"2017","journal-title":"Infect. Drug Resist."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"115","DOI":"10.1080\/20477724.2018.1442764","article-title":"Targeting the GPI biosynthetic pathway","volume":"112","author":"Yadav","year":"2018","journal-title":"Pathog. Glob. Health"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"355","DOI":"10.1002\/iub.1734","article-title":"Generating anchors only to lose them: The unusual story of glycosylphosphatidylinositol anchor biosynthesis and remodeling in yeast and fungi","volume":"70","author":"Komath","year":"2018","journal-title":"IUBMB Life"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"119","DOI":"10.1128\/EC.00297-06","article-title":"Comprehensive Analysis of Glycosylphosphatidylinositol-Anchored Proteins in Candida albicans","volume":"6","author":"Richard","year":"2007","journal-title":"Eukaryot. Cell"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1404","DOI":"10.1016\/j.fgb.2008.08.003","article-title":"Functional analysis of Candida albicans GPI-anchored proteins: Roles in cell wall integrity and caspofungin sensitivity","volume":"45","author":"Plaine","year":"2008","journal-title":"Fungal Genet. Biol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1520","DOI":"10.1021\/cb300235m","article-title":"Inhibiting GPI Anchor Biosynthesis in Fungi Stresses the Endoplasmic Reticulum and Enhances Immunogenicity","volume":"7","author":"McLellan","year":"2012","journal-title":"ACS Chem. Biol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"59","DOI":"10.1021\/id5000212","article-title":"Chemical Genomics-Based Antifungal Drug Discovery: Targeting Glycosylphosphatidylinositol (GPI) Precursor Biosynthesis","volume":"1","author":"Mann","year":"2014","journal-title":"ACS Infect. Dis."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"9194","DOI":"10.1038\/s41467-024-53512-x","article-title":"Structural insights into the inhibition mechanism of fungal GWT1 by manogepix","volume":"15","author":"Dai","year":"2024","journal-title":"Nat. Commun."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"e0113223","DOI":"10.1128\/aac.01132-23","article-title":"In vitro activity of manogepix and comparators against infrequently encountered yeast and mold isolates from the SENTRY Surveillance Program (2017\u20132022)","volume":"68","author":"Pfaller","year":"2024","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"993","DOI":"10.1194\/jlr.R700002-JLR200","article-title":"Thematic review series: Lipid Posttranslational Modifications. GPI anchoring of protein in yeast and mammalian cells, or: How we learned to stop worrying and love glycophospholipids","volume":"48","author":"Peter","year":"2007","journal-title":"J. Lipid Res."},{"key":"ref_15","first-page":"405","article-title":"Biosynthesis and function of GPI proteins in the yeast Saccharomyces cerevisiae","volume":"1771","author":"Pittet","year":"2007","journal-title":"Biochim. Biophys. Acta (BBA)\u2014Mol. Cell Biol. Lipids"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"775","DOI":"10.1534\/genetics.112.144485","article-title":"Architecture and Biosynthesis of the Saccharomyces cerevisiae Cell Wall","volume":"192","author":"Orlean","year":"2012","journal-title":"Genetics"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"250","DOI":"10.1093\/emboj\/20.1.250","article-title":"PIG-M transfers the first mannose to glycosylphosphatidylinositol on the lumenal side of the ER","volume":"20","author":"Maeda","year":"2001","journal-title":"EMBO J."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"939","DOI":"10.1073\/pnas.0505570103","article-title":"Pbn1p: An essential endoplasmic reticulum membrane protein required for protein processing in the endoplasmic reticulum of budding yeast","volume":"103","author":"Subramanian","year":"2006","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"9489","DOI":"10.1074\/jbc.M413867200","article-title":"PIG-V Involved in Transferring the Second Mannose in Glycosylphosphatidylinositol","volume":"280","author":"Kang","year":"2005","journal-title":"J. Biol. Chem."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"3472","DOI":"10.1091\/mbc.e07-03-0258","article-title":"Pga1 Is an Essential Component of Glycosylphosphatidylinositol-Mannosyltransferase II of Saccharomyces cerevisiae","volume":"18","author":"Sato","year":"2007","journal-title":"Mol. Biol. Cell"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"153","DOI":"10.1042\/bj3320153","article-title":"Saccharomyces cerevisiae GPI10, the functional homologue of human PIG-B, is required for glycosylphosphatidylinositol-anchor synthesis","volume":"332","author":"Escribano","year":"1998","journal-title":"Biochem. J."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"27731","DOI":"10.1074\/jbc.M101986200","article-title":"The Essential Smp3 Protein Is Required for Addition of the Side-branching Fourth Mannose during Assembly of Yeast Glycosylphosphatidylinositols","volume":"276","author":"Grimme","year":"2001","journal-title":"J. Biol. Chem."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"123","DOI":"10.1093\/jb\/mvm113","article-title":"Both Mammalian PIG-M and PIG-X are Required for Growth of GPI14-Disrupted Yeast","volume":"142","author":"Kim","year":"2007","journal-title":"J. Biochem."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"993","DOI":"10.1002\/yea.1286","article-title":"Characterization of GPI14\/YJR013w mutation that induces the cell wall integrity signalling pathway and results in increased protein production in Saccharomyces cerevisiae","volume":"22","author":"Davydenko","year":"2005","journal-title":"Yeast"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"365","DOI":"10.1002\/yea.3179","article-title":"The catalytic subunit of the first mannosyltransferase in the GPI biosynthetic pathway affects growth, cell wall integrity and hyphal morphogenesis in Candida albicans","volume":"33","author":"Singh","year":"2016","journal-title":"Yeast"},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Daniel Gietz, R., and Woods, R.A. (2002). Transformation of yeast by lithium acetate\/single-stranded carrier DNA\/polyethylene glycol method. Guide to Yeast Genetics and Molecular and Cell Biology\u2014Part B, Academic Press. Methods in Enzymology.","DOI":"10.1016\/S0076-6879(02)50957-5"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"331","DOI":"10.1038\/nmeth.4193","article-title":"MotionCor2: Anisotropic correction of beam-induced motion for improved cryo-electron microscopy","volume":"14","author":"Zheng","year":"2017","journal-title":"Nat. Methods"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"290","DOI":"10.1038\/nmeth.4169","article-title":"cryoSPARC: Algorithms for rapid unsupervised cryo-EM structure determination","volume":"14","author":"Punjani","year":"2017","journal-title":"Nat. Methods"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"493","DOI":"10.1038\/s41586-024-07487-w","article-title":"Accurate structure prediction of biomolecular interactions with AlphaFold 3","volume":"630","author":"Abramson","year":"2024","journal-title":"Nature"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1605","DOI":"10.1002\/jcc.20084","article-title":"UCSF Chimera\u2014A visualization system for exploratory research and analysis","volume":"25","author":"Pettersen","year":"2004","journal-title":"J. Comput. Chem."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"486","DOI":"10.1107\/S0907444910007493","article-title":"Features and development of Coot","volume":"66","author":"Emsley","year":"2010","journal-title":"Acta Crystallogr. Sect. D Biol. Crystallogr."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"213","DOI":"10.1107\/S0907444909052925","article-title":"PHENIX: A comprehensive Python-based system for macromolecular structure solution","volume":"66","author":"Adams","year":"2010","journal-title":"Acta Crystallogr. Sect. D Biol. Crystallogr."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1074","DOI":"10.1107\/S0907444909029436","article-title":"electronic Ligand Builder and Optimization Workbench(eLBOW): A tool for ligand coordinate and restraint generation","volume":"65","author":"Moriarty","year":"2009","journal-title":"Acta Crystallogr. Sect. D Biol. Crystallogr."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"12","DOI":"10.1107\/S0907444909042073","article-title":"MolProbity: All-atom structure validation for macromolecular crystallography","volume":"66","author":"Chen","year":"2009","journal-title":"Acta Crystallogr. Sect. D Biol. Crystallogr."},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Torres-Valdetano, \u00c1., Vallejo-Ruiz, V., Milflores-Flores, L., and Mart\u00ednez-Morales, P. (2024). Role of PIGM and PIGX in glycosylphosphatidylinositol biosynthesis and human health (Review). Biomed. Rep., 20.","DOI":"10.3892\/br.2024.1746"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"450","DOI":"10.1111\/mmi.14859","article-title":"Identification of TbPBN1 in Trypanosoma brucei reveals a conserved heterodimeric architecture for glycosylphosphatidylinositol-mannosyltransferase-I","volume":"117","author":"Cowton","year":"2021","journal-title":"Mol. Microbiol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"e00433-22","DOI":"10.1128\/mbio.00433-22","article-title":"The Leishmania donovani Ortholog of the Glycosylphosphatidylinositol Anchor Biosynthesis Cofactor PBN1 Is Essential for Host Infection","volume":"13","author":"Roberts","year":"2022","journal-title":"mBio"},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"Bohl, H., Bai, L., and Li, H. (2021). Recent Progress in Structural Studies on the GT-C Superfamily of Protein Glycosyltransferases. Macromolecular Protein Complexes III: Structure and Function, Springer. Subcellular Biochemistry.","DOI":"10.1007\/978-3-030-58971-4_6"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"1277","DOI":"10.1093\/genetics\/149.3.1277","article-title":"The PBN1 Gene of Saccharomyces cerevisiae: An Essential Gene That Is Required for the Post-translational Processing of the Protease B Precursor","volume":"149","author":"Naik","year":"1998","journal-title":"Genetics"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"575","DOI":"10.1038\/s41589-022-01219-9","article-title":"Structure, sequon recognition and mechanism of tryptophan C-mannosyltransferase","volume":"19","author":"Bloch","year":"2023","journal-title":"Nat. Chem. Biol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"1100","DOI":"10.1038\/nsmb.3491","article-title":"Molecular basis of lipid-linked oligosaccharide recognition and processing by bacterial oligosaccharyltransferase","volume":"24","author":"Boilevin","year":"2017","journal-title":"Nat. Struct. Mol. Biol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"443","DOI":"10.1038\/s41586-020-2044-z","article-title":"Structure and mechanism of the ER-based glucosyltransferase ALG6","volume":"579","author":"Bloch","year":"2020","journal-title":"Nature"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"e4582","DOI":"10.1002\/pro.4582","article-title":"Using evolutionary data to make sense of macromolecules with a \u201cface-lifted\u201d ConSurf","volume":"32","author":"Yariv","year":"2023","journal-title":"Protein Sci."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"e4519","DOI":"10.1002\/pro.4519","article-title":"DALI shines a light on remote homologs: One hundred discoveries","volume":"32","author":"Holm","year":"2022","journal-title":"Protein Sci."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"2617","DOI":"10.1038\/s41467-022-30250-6","article-title":"Molecular insights into biogenesis of glycosylphosphatidylinositol anchor proteins","volume":"13","author":"Xu","year":"2022","journal-title":"Nat. Commun."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"1439","DOI":"10.1091\/mbc.e04-09-0802","article-title":"Mammalian PIG-X and Yeast Pbn1p Are the Essential Components of Glycosylphosphatidylinositol-Mannosyltransferase I","volume":"16","author":"Ashida","year":"2005","journal-title":"Mol. Biol. Cell"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"5520","DOI":"10.1038\/s41467-023-41281-y","article-title":"Structures of liganded glycosylphosphatidylinositol transamidase illuminate GPI-AP biogenesis","volume":"14","author":"Xu","year":"2023","journal-title":"Nat. Commun."}],"container-title":["Journal of Fungi"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2309-608X\/11\/11\/819\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,11,21]],"date-time":"2025-11-21T05:21:13Z","timestamp":1763702473000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2309-608X\/11\/11\/819"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,11,19]]},"references-count":47,"journal-issue":{"issue":"11","published-online":{"date-parts":[[2025,11]]}},"alternative-id":["jof11110819"],"URL":"https:\/\/doi.org\/10.3390\/jof11110819","relation":{},"ISSN":["2309-608X"],"issn-type":[{"type":"electronic","value":"2309-608X"}],"subject":[],"published":{"date-parts":[[2025,11,19]]}}}