{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,10]],"date-time":"2026-03-10T07:14:31Z","timestamp":1773126871459,"version":"3.50.1"},"reference-count":49,"publisher":"MDPI AG","issue":"17","license":[{"start":{"date-parts":[[2025,9,4]],"date-time":"2025-09-04T00:00:00Z","timestamp":1756944000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"NSERC","award":["2022-05389"],"award-info":[{"award-number":["2022-05389"]}]},{"name":"GlycoNet","award":["2022-05389"],"award-info":[{"award-number":["2022-05389"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Molecules"],"abstract":"<jats:p>Gangliosides are essential for membrane functions, cell recognition, and maintenance of the nervous system. GM2 gangliosidosis is a group of rare genetic lysosomal storage diseases that includes Tay-Sachs disease (TSD), Sandhoff disease (SD), and AB variant. TSD and SD are characterized by deficient \u03b2-N-acetyl-hexosaminidase activity. This leads to decreased catabolism of \u03b2-N-acetyl-hexosamine-containing ganglioside GM2 in the lysosomes, damage to cells and tissues, and severe neurological symptoms. GM2 is a major ganglioside accumulating in TSD and SD, and is synthesized from GM3 by \u03b21,4-N-acetylgalactosaminyltransferase 1 (B4GALNT1, GM2 synthase). Therapies under development for GM2 gangliosidosis include adeno-associated virus gene therapy, enzyme replacement, and substrate reduction therapy (SRT). The goal of this work was to express and purify human B4GALNT1, characterize its activity, and explore its structural features by protein modeling and substrate docking. We used a panel of synthetic compounds to study their potential inhibition of B4GALNT1 activity. This work can serve to develop SRT for GM2 gangliosidosis.<\/jats:p>","DOI":"10.3390\/molecules30173615","type":"journal-article","created":{"date-parts":[[2025,9,4]],"date-time":"2025-09-04T10:55:37Z","timestamp":1756983337000},"page":"3615","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Characterization of Human Recombinant \u03b21,4-GalNAc-Transferase B4GALNT1 and Inhibition by Selected Compounds"],"prefix":"10.3390","volume":"30","author":[{"given":"Iram","family":"Abidi","sequence":"first","affiliation":[{"name":"Department of Biomedical and Molecular Sciences, Queen\u2019s University, Kingston, ON K7L 3N6, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Alexander N.","family":"Kocev","sequence":"additional","affiliation":[{"name":"Department of Biomedical and Molecular Sciences, Queen\u2019s University, Kingston, ON K7L 3N6, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3271-9947","authenticated-orcid":false,"given":"Jonathan L.","family":"Babulic","sequence":"additional","affiliation":[{"name":"Department of Biomedical and Molecular Sciences, Queen\u2019s University, Kingston, ON K7L 3N6, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5977-8453","authenticated-orcid":false,"given":"Chantelle J.","family":"Capicciotti","sequence":"additional","affiliation":[{"name":"Department of Biomedical and Molecular Sciences, Queen\u2019s University, Kingston, ON K7L 3N6, Canada"},{"name":"Department of Chemistry, Queen\u2019s University, Kingston, ON K7L 3N6, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jagdeep","family":"Walia","sequence":"additional","affiliation":[{"name":"Department of Biomedical and Molecular Sciences, Queen\u2019s University, Kingston, ON K7L 3N6, Canada"},{"name":"Department of Pathology, Queen\u2019s University, Kingston, ON K7L 3N6, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Inka","family":"Brockhausen","sequence":"additional","affiliation":[{"name":"Department of Biomedical and Molecular Sciences, Queen\u2019s University, Kingston, ON K7L 3N6, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,9,4]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"19","DOI":"10.1007\/978-1-4939-8552-4_2","article-title":"Gangliosides of the nervous system","volume":"1804","author":"Ledeen","year":"2018","journal-title":"Methods Mol. Biol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"926","DOI":"10.1111\/j.1471-4159.2010.07067.x","article-title":"Gangliosides are essential in the protection of inflammation and neurodegeneration via maintenance of lipid rafts: Elucidation by a series of ganglioside-deficient mutant mice","volume":"116","author":"Ohmi","year":"2011","journal-title":"J. Neurochem."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"46","DOI":"10.1126\/science.1174621","article-title":"Lipid rafts as a membrane-organizing principle","volume":"327","author":"Lingwood","year":"2010","journal-title":"Science"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"356","DOI":"10.1016\/S0304-4165(02)00403-8","article-title":"Beta1,4-N-acetylgalactosaminyltransferase-GM2\/GD2 synthase: A key enzyme to control the synthesis of brain-enriched complex gangliosides","volume":"1573","author":"Furukawa","year":"2002","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"14","DOI":"10.1021\/acschemneuro.9b00532","article-title":"Spatial Lipidomics Reveals Region and Long Chain Base Specific Accumulations of Monosialogangliosides in Amyloid Plaques in Familial Alzheimer\u2019s Disease Mice (5xFAD) Brain","volume":"11","author":"Kaya","year":"2020","journal-title":"ACS Chem. Neurosci."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Manich, M., Knapp, O., Gibert, M., Maier, E., Jolivet-Reynaud, C., Geny, B., Benz, R., and Popoff, M.R. (2008). Clostridium perfringens delta toxin is sequence related to beta toxin, NetB, and Staphylococcus pore-forming toxins, but shows functional differences. PLoS ONE, 3.","DOI":"10.1371\/journal.pone.0003764"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"47454","DOI":"10.18632\/oncotarget.17665","article-title":"Interaction of glycosphingolipids GD3 and GD2 with growth factor receptors maintains breast cancer stem cell phenotype","volume":"8","author":"Liang","year":"2017","journal-title":"Oncotarget"},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Itokazu, Y., Ariga, T., Fuchigami, T., and Li, D. (2024). Gangliosides in neural stem cell fate determination and nerve cell specification--preparation and administration. bioRxiv.","DOI":"10.1101\/2024.06.09.598109"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"4245","DOI":"10.1182\/blood-2006-08-039347","article-title":"Human bone marrow mesenchymal stromal cells express the neural ganglioside GD2: A novel surface marker for the identification of MSCs","volume":"109","author":"Martinez","year":"2007","journal-title":"Blood"},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Ryckman, A.E., Brockhausen, I., and Walia, J.S. (2020). Metabolism of Glycosphingolipids and Their Role in the Pathophysiology of Lysosomal Storage Disorders. Int. J. Mol. Sci., 21.","DOI":"10.3390\/ijms21186881"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"15057","DOI":"10.1038\/mtm.2015.57","article-title":"Construction of a hybrid \u03b2-hexosaminidase subunit capable of forming stable homodimers that hydrolyze GM2 ganglioside in vivo","volume":"3","author":"Tropak","year":"2016","journal-title":"Mol. Ther.\u2014Methods Clin. Dev."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"263","DOI":"10.1038\/sj.bjc.6604179","article-title":"Detection of GD2-positive cells in bone marrow samples and survival of patients with localised neuroblastoma","volume":"98","author":"Corrias","year":"2008","journal-title":"Br. J. Cancer"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"4355890","DOI":"10.1155\/2022\/4355890","article-title":"Pan-Cancer Analysis of B4GALNT1 as a Potential Prognostic and Immunological Biomarker","volume":"2022","author":"Yi","year":"2022","journal-title":"J. Immunol. Res."},{"key":"ref_14","first-page":"5395","article-title":"Genetic and enzymatic basis for the differential expression of GM2 and GD2 gangliosides in human cancer cell lines","volume":"53","author":"Yamashiro","year":"1993","journal-title":"Cancer Res."},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Chiricozzi, E., Mauri, L., Lunghi, G., Di Biase, E., Fazzari, M., Maggioni, M., Valsecchi, M., Prioni, S., Loberto, N., and Pom\u00e8, D.Y. (2019). Parkinson\u2019s disease recovery by GM1 oligosaccharide treatment in the B4galnt1+\/\u2212 mouse model. Sci. Rep., 9.","DOI":"10.1038\/s41598-019-55885-2"},{"key":"ref_16","first-page":"100513","article-title":"Human GLB1 knock-out cerebral organoids: A model system for testing AAV9-mediated GLB1 gene therapy for reducing GM1 ganglioside storage in GM1 gangliosidosis","volume":"21","author":"Latour","year":"2019","journal-title":"Mol. Genet. Metab. Rep."},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Deschenes, N.M., Cheng, C., Ryckman, A.E., Quinville, B.M., Khanal, P., Mitchell, M., Chen, Z., Sangrar, W., Gray, S.J., and Walia, J.S. (2023). Biochemical Correction of GM2 Ganglioside Accumulation in AB-Variant GM2 Gangliosidosis. Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms24119217"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"14611","DOI":"10.3390\/ijms241914611","article-title":"Efficacy of Adeno-Associated Virus Serotype 9-Mediated Gene Therapy for AB-Variant GM2 Gangliosidosis","volume":"24","author":"Vyas","year":"2023","journal-title":"Int. J. Mol. Sci."},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Coutinho, M.F., Santos, J.I., and Alves, S. (2016). Less Is More: Substrate Reduction Therapy for Lysosomal Storage Disorders. Int. J. Mol. Sci., 17.","DOI":"10.3390\/ijms17071065"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1495","DOI":"10.1016\/j.ymthe.2019.05.018","article-title":"Substrate Reduction Therapy for Sandhoff Disease through Inhibition of Glucosylceramide Synthase Activity","volume":"27","author":"Marshall","year":"2019","journal-title":"Mol. Ther."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1481","DOI":"10.1016\/S0140-6736(00)02161-9","article-title":"Novel oral treatment of Gaucher\u2019s disease with N-butyldeoxynojirimycin (OGT 918) to decrease substrate biosynthesis","volume":"355","author":"Cox","year":"2000","journal-title":"Lancet"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"26395","DOI":"10.1074\/jbc.271.42.26395","article-title":"Beta1,4-N-acetylgalactosaminyltransferase (GM2 synthase) is released from Golgi membranes as a neuraminidase-sensitive, disulfide-bonded dimer by a cathepsin D-like protease","volume":"271","author":"Jaskiewicz","year":"1996","journal-title":"J. Biol. Chem."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"540","DOI":"10.1016\/j.sbi.2012.06.007","article-title":"Recent structures, evolution and mechanisms of glycosyltransferases","volume":"22","author":"Breton","year":"2012","journal-title":"Curr. Opin. Struct. Biol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"118","DOI":"10.1016\/j.ajhg.2013.05.006","article-title":"Alteration of ganglioside biosynthesis responsible for complex hereditary spastic paraplegia","volume":"93","author":"Boukhris","year":"2013","journal-title":"Am. J. Hum. Genet."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"3618","DOI":"10.1093\/brain\/awt270","article-title":"Mutations in B4GALNT1 (GM2 synthase) underlie a new disorder of ganglioside biosynthesis","volume":"136","author":"Harlalka","year":"2013","journal-title":"Brain"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"2590","DOI":"10.1002\/ajmg.a.62880","article-title":"Functional validation of novel variants in B4GALNT1 associated with early-onset complex hereditary spastic paraplegia with impaired ganglioside synthesis","volume":"188","author":"Alecu","year":"2022","journal-title":"Am. J. Med. Genet."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"265","DOI":"10.1016\/j.expneurol.2018.10.014","article-title":"Parkinsonian GM2 synthase knock-out mice lacking mature gangliosides develop urinary dysfunction and neurogenic bladder","volume":"311","author":"Herrera","year":"2019","journal-title":"Exp. Neurol."},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Yamaguchi, T., Yamauchi, Y., Furukawa, K., Ohmi, Y., Ohkawa, Y., Zhang, Q., Okajima, T., and Furukawa, K. (2016). Expression of B4GALNT1, an essential glycosyltransferase for the synthesis of complex gangliosides, suppresses BACE1 degradation and modulates APP processing. Sci. Rep., 6.","DOI":"10.1038\/srep34505"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"19","DOI":"10.1186\/s13195-022-00966-0","article-title":"Glucosylceramide synthase inhibition reduces ganglioside GM3 accumulation, alleviates amyloid neuropathology, and stabilizes remote contextual memory in a mouse model of Alzheimer\u2019s disease","volume":"14","author":"Dodge","year":"2022","journal-title":"Alzheimer\u2019s Res. Ther."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"692","DOI":"10.1111\/jnc.15136","article-title":"Substrate reduction therapy using Genz-667161 reduces levels of pathogenic components in a mouse model of neuronopathic forms of Gaucher disease","volume":"156","author":"Blumenreich","year":"2021","journal-title":"J. Neurochem."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"5839","DOI":"10.1016\/S0021-9258(17)30345-9","article-title":"Biosynthesis of bovine thyroid gangliosides","volume":"253","author":"Pacuszka","year":"1978","journal-title":"J. Biol. Chem."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"59","DOI":"10.1111\/j.1432-1033.1981.tb05670.x","article-title":"Ganglioside biosynthesis in rat liver. Characterization of UDP-N-acetylgalactosamine\u2014GM3 acetylgalactosaminyltransferase","volume":"120","author":"Senn","year":"1981","journal-title":"Eur. J. Biochem."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"6149","DOI":"10.1074\/jbc.270.11.6149","article-title":"Substrate specificity of \u03b21,4-N-Acetylgalactosaminyltransferase in Vitro and in cDNA-transfected Cells","volume":"270","author":"Yamashiro","year":"1995","journal-title":"J. Biol. Chem."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"957","DOI":"10.1042\/bj3030957","article-title":"Beta 1-4N-acetylgalactosaminyltransferase can synthesize both asialoglycosphingolipid GM2 and glycosphingolipid GM2 in vitro and in vivo: Isolation and characterization of a beta 1-4N-acetylgalactosaminyltransferase cDNA clone from rat ascites hepatoma cell line AH7974F","volume":"303","author":"Hidari","year":"1994","journal-title":"Biochem. J."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"10662","DOI":"10.1073\/pnas.93.20.10662","article-title":"Mice with disrupted GM2\/GD2 synthase gene lack complex gangliosides but exhibit only subtle defects in their nervous system","volume":"93","author":"Takamiya","year":"1996","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"41476","DOI":"10.1074\/jbc.M007480200","article-title":"Disulfide bonds of GM2 synthase homodimers. Antiparallel orientation of the catalytic domains","volume":"275","author":"Li","year":"2000","journal-title":"J. Biol. Chem."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"5442","DOI":"10.1038\/s41467-025-60593-9","article-title":"Conformational dynamics and membrane insertion mechanism of B4GALNT1 in ganglioside synthesis","volume":"16","author":"Welland","year":"2025","journal-title":"Nat. Commun."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"217","DOI":"10.1093\/glycob\/11.3.217","article-title":"The DXD motif is required for GM2 synthase activity but is not critical for nucleotide binding","volume":"11","author":"Li","year":"2001","journal-title":"Glycobiology"},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Inamori, K.I., Nakamura, K., Shishido, F., Hsu, J.C., Nagafuku, M., Nitta, T., Ikeda, J., Yoshimura, H., Kodaira, M., and Tsuchida, N. (2024). Functional evaluation of novel variants of B4GALNT1 in a patient with hereditary spastic paraplegia and the general population. Front. Neurosci., 18.","DOI":"10.3389\/fnins.2024.1437668"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"273","DOI":"10.1042\/bj3120273","article-title":"The effects of the site-directed removal of N-glycosylation sites from beta-1,4-N-acetylgalactosaminyltransferase on its function","volume":"312","author":"Haraguchi","year":"1995","journal-title":"Biochem. J."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"1244","DOI":"10.1016\/j.bbagen.2009.05.020","article-title":"Site directed processing: Role of amino acid sequences and glycosylation of acceptor glycopeptides in the assembly of extended mucin type O-glycan core 2. Biochim","volume":"1790","author":"Brockhausen","year":"2009","journal-title":"Biophys. Acta"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"47","DOI":"10.1016\/j.carres.2012.05.027","article-title":"Structures and biosynthesis of the N- and O-glycans of recombinant human oviduct-specific glycoprotein expressed in human embryonic kidney cells","volume":"358","author":"Yang","year":"2012","journal-title":"Carbohydr. Res."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"1305","DOI":"10.1016\/j.bmc.2012.12.034","article-title":"Selective inhibition of glycosyltransferases by bivalent imidazolium salts","volume":"21","author":"Gao","year":"2013","journal-title":"Bioorg. Med. Chem."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"525","DOI":"10.1007\/s10719-006-7153-x","article-title":"UDP-Gal: GlcNAc-R beta1, 4-galactosyltransferase\u2013a target enzyme for drug design. Acceptor specificity and inhibition of the enzyme","volume":"23","author":"Brockhausen","year":"2006","journal-title":"Glycoconj. J."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"673","DOI":"10.1007\/s10719-010-9312-3","article-title":"Specificity of \u03b21,4-galactosyltransferase inhibition by 2-naphthyl 2-butanamido-2-deoxy-1-thio-\u03b2-D-glucopyranoside","volume":"27","author":"Gao","year":"2010","journal-title":"Glycoconj. J."},{"key":"ref_46","doi-asserted-by":"crossref","unstructured":"Kocev, A., Melamed, J., Wang, S., Kong, X., Vlahakis, J.Z., Xu, Y., Szarek, W.A., and Brockhausen, I. (2020). Inhibition of bacterial growth and galactosyltransferase activity of WbwC by \u03b1, \u03c9-bis(3-alkyl-1H-imidazolium)alkane salts: Effect of varying carbon content. Bioorg. Med. Chem., 28.","DOI":"10.1016\/j.bmc.2020.115494"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"465430","DOI":"10.1016\/j.chroma.2024.465430","article-title":"Highly selective split intein method for efficient separation and purification of recombinant therapeutic proteins from mammalian cell culture fluid","volume":"1736","author":"Prabhala","year":"2024","journal-title":"J. Chromatogr."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"156","DOI":"10.1038\/nchembio.2539","article-title":"Expression system for structural and functional studies of human glycosylation enzymes","volume":"14","author":"Moremen","year":"2018","journal-title":"Nat. Chem. Biol."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"773","DOI":"10.1021\/acs.bioconjchem.2c00043","article-title":"Exo-Enzymatic Cell-Surface Glycan Labeling for Capturing Glycan-Protein Interactions through Photo-Cross-Linking","volume":"33","author":"Babulic","year":"2022","journal-title":"Bioconjugate Chem."}],"container-title":["Molecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1420-3049\/30\/17\/3615\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T18:39:17Z","timestamp":1760035157000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1420-3049\/30\/17\/3615"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,9,4]]},"references-count":49,"journal-issue":{"issue":"17","published-online":{"date-parts":[[2025,9]]}},"alternative-id":["molecules30173615"],"URL":"https:\/\/doi.org\/10.3390\/molecules30173615","relation":{},"ISSN":["1420-3049"],"issn-type":[{"value":"1420-3049","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,9,4]]}}}