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Structural and mechanistic insights into the catalytic-domain-mediated short-range glycosylation preferences of galnac-t4

DOI: 10.1021/acscentsci.8b00488 DOI Help

Authors: Matilde De Las Rivas (BIFI, University of Zaragoza, BIFI-IQFR (CSIC)) , Earnest James Paul Daniel (Case Western Reserve University) , Helena Coelho (Universidade de Nova de Lisboa; CIC bioGUNE; University of the Basque Country) , Erandi Lira-navarrete (University of Copenhagen) , Lluis Raich (Universitat de Barcelona) , Ismael Compañón (Universidad de La Rioja) , Ana Diniz (Universidade de Nova de Lisboa) , Laura Lagartera (IQM-CSIC) , Jesús Jiménez-barbero (CIC bioGUNE; University of the Basque Country; Ikerbasque, Basque Foundation for Science) , Henrik Clausen (University of Copenhagen) , Carme Rovira (Universitat de Barcelona) , Filipa Marcelo (Universidade de Nova de Lisboa) , Francisco Corzana (Universidad de La Rioja) , Thomas A. Gerken (Case Western Reserve University) , Ramon Hurtado-guerrero (University of Zaragoza; Fundación ARAID)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Acs Central Science

State: Published (Approved)
Published: September 2018
Diamond Proposal Number(s): 10121

Open Access Open Access

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.

Subject Areas: Biology and Bio-materials, Chemistry


Instruments: I03-Macromolecular Crystallography

Documents:
acscentsci.8b00488.pdf

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