Structural and Mechanistic Insights into the Catalytic-Domain-Mediated Short-Range Glycosylation Preferences of GalNAc-T4

Matilde De Las Rivas, Earnest James Paul Daniel, Helena Coelho, Erandi Lira-Navarrete, Lluis Raich, Ismael Compañón, Ana Diniz, Laura Lagartera, Jesús Jiménez-Barbero, Henrik Clausen, Carme Rovira, Filipa Marcelo, Francisco Corzana, Thomas A. Gerken, Ramon Hurtado-Guerrero

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

Mucin-type O-glycosylation is initiated by a family of polypeptide GalNAc-transferases (GalNAc-Ts) which are type-II transmembrane proteins that contain Golgi luminal catalytic and lectin domains that are connected by a flexible linker. Several GalNAc-Ts, including GalNAc-T4, show both long-range and short-range prior glycosylation specificity, governed by their lectin and catalytic domains, respectively. While the mechanism of the lectin-domain-dependent glycosylation is well-known, the molecular basis for the catalytic-domain-dependent glycosylation of glycopeptides is unclear. Herein, we report the crystal structure of GalNAc-T4 bound to the diglycopeptide GAT GAGAGAGT TPGPG (containing two α-GalNAc glycosylated Thr (T ), the PXP motif and a "naked" Thr acceptor site) that describes its catalytic domain glycopeptide GalNAc binding site. Kinetic studies of wild-type and GalNAc binding site mutant enzymes show the lectin domain GalNAc binding activity dominates over the catalytic domain GalNAc binding activity and that these activities can be independently eliminated. Surprisingly, a flexible loop protruding from the lectin domain was found essential for the optimal activity of the catalytic domain. This work provides the first structural basis for the short-range glycosylation preferences of a GalNAc-T.

Original languageEnglish
Pages (from-to)1274-1290
Number of pages17
JournalACS Central Science
Volume4
Issue number9
DOIs
Publication statusPublished - 26 Sep 2018

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    De Las Rivas, M., Paul Daniel, E. J., Coelho, H., Lira-Navarrete, E., Raich, L., Compañón, I., ... Hurtado-Guerrero, R. (2018). Structural and Mechanistic Insights into the Catalytic-Domain-Mediated Short-Range Glycosylation Preferences of GalNAc-T4. ACS Central Science, 4(9), 1274-1290. https://doi.org/10.1021/acscentsci.8b00488