A Trypanosoma brucei β3 glycosyltransferase superfamily gene encodes a β1-6 GlcNAc-transferase mediating N-glycan and GPI anchor modification

Duncan, S. M., Nagar, R., Damerow, M., Yashunsky, D. V., Buzzi, B., Nikolaev, A. V. and Ferguson, M. A.J. (2021) A Trypanosoma brucei β3 glycosyltransferase superfamily gene encodes a β1-6 GlcNAc-transferase mediating N-glycan and GPI anchor modification. Journal of Biological Chemistry, 297(4), 101153. (doi: 10.1016/j.jbc.2021.101153) (PMID:34478712) (PMCID:PMC8477195)

Abstract

The parasite Trypanosoma brucei exists in both a bloodstream form (BSF) and a procyclic form (PCF), which exhibit large carbohydrate extensions on the N-linked glycans and glycosylphosphatidylinositol (GPI) anchors, respectively. The parasite's glycoconjugate repertoire suggests at least 38 glycosyltransferase (GT) activities, 16 of which are currently uncharacterized. Here, we probe the function(s) of the uncharacterized GT67 glycosyltransferase family and a β3 glycosyltransferase (β3GT) superfamily gene, TbGT10. A BSF-null mutant, created by applying the diCre/loxP method in T. brucei for the first time, showed a fitness cost but was viable in vitro and in vivo and could differentiate into the PCF, demonstrating nonessentiality of TbGT10. The absence of TbGT10 impaired the elaboration of N-glycans and GPI anchor side chains in BSF and PCF parasites, respectively. Glycosylation defects included reduced BSF glycoprotein binding to the lectin ricin and monoclonal antibodies mAb139 and mAbCB1. The latter bind a carbohydrate epitope present on lysosomal glycoprotein p67 that we show here consists of (-6Galβ1-4GlcNAcβ1-)≥4 poly-N-acetyllactosamine repeats. Methylation linkage analysis of Pronase-digested glycopeptides isolated from BSF wild-type and TbGT10 null parasites showed a reduction in 6-O-substituted- and 3,6-di-O-substituted-Gal residues. These data define TbGT10 as a UDP-GlcNAc:βGal β1-6 GlcNAc-transferase. The dual role of TbGT10 in BSF N-glycan and PCF GPI-glycan elaboration is notable, and the β1-6 specificity of a β3GT superfamily gene product is unprecedented. The similar activities of trypanosome TbGT10 and higher-eukaryote I-branching enzyme (EC 2.4.1.150), which belong to glycosyltransferase families GT67 and GT14, respectively, in elaborating N-linked glycans, are a novel example of convergent evolution.

• Item Type: Articles
• Additional Information: This work was supported by a Wellcome Trust Investigator Award to M. A. J. F. (101842/Z13/Z).
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Duncan, Mr Samuel

Creator Roles

Duncan, S.: Data curation, Formal analysis, Investigation, Writing – original draft, Writing – review and editing

• Authors: Duncan, S. M., Nagar, R., Damerow, M., Yashunsky, D. V., Buzzi, B., Nikolaev, A. V., and Ferguson, M. A.J.
• College/School: College of Medical Veterinary and Life Sciences > School of Biodiversity, One Health & Veterinary Medicine
• Journal Name: Journal of Biological Chemistry
• Publisher: Elsevier
• ISSN: 0021-9258
• ISSN (Online): 1083-351X
• Published Online: 01 September 2021
• Copyright Holders: Copyright © 2021 The Authors
• First Published: First published in Journal of Biological Chemistry 297(4):101153
• Publisher Policy: Reproduced under a Creative Commons license