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

doi:10.1021/acscentsci.8b00488

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

^*Corresponding author for this work

Glycomics Program

21 Citations (Scopus)

54 Downloads (Pure)

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 language	English
Journal	A C S Central Science
Volume	4
Issue number	9
Pages (from-to)	1274-1290
Number of pages	17
DOIs	https://doi.org/10.1021/acscentsci.8b00488
Publication status	Published - 2018

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Structural and Mechanistic Insights into the Catalytic-Domain-Mediated Short-Range Glycosylation Preferences of GalNAc-T4Final published version, 5.02 MBLicence: Unspecified

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De Las Rivas, M., Paul Daniel, E. J., Coelho, H., Lira-Navarrete, E., Raich, L., Compañón, I., Diniz, A., Lagartera, L., Jiménez-Barbero, J., Clausen, H., Rovira, C., Marcelo, F., Corzana, F., Gerken, T. A., & Hurtado-Guerrero, R. (2018). Structural and Mechanistic Insights into the Catalytic-Domain-Mediated Short-Range Glycosylation Preferences of GalNAc-T4. A C S Central Science, 4(9), 1274-1290. https://doi.org/10.1021/acscentsci.8b00488

De Las Rivas, M, Paul Daniel, EJ, Coelho, H, Lira-Navarrete, E, Raich, L, Compañón, I, Diniz, A, Lagartera, L, Jiménez-Barbero, J, Clausen, H, Rovira, C, Marcelo, F, Corzana, F, Gerken, TA & Hurtado-Guerrero, R 2018, 'Structural and Mechanistic Insights into the Catalytic-Domain-Mediated Short-Range Glycosylation Preferences of GalNAc-T4', A C S Central Science, vol. 4, no. 9, pp. 1274-1290. https://doi.org/10.1021/acscentsci.8b00488

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

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.",

author = "{De Las Rivas}, Matilde and {Paul Daniel}, {Earnest James} and Helena Coelho and Erandi Lira-Navarrete and Lluis Raich and Ismael Compa{\~n}{\'o}n and Ana Diniz and Laura Lagartera and Jes{\'u}s Jim{\'e}nez-Barbero and Henrik Clausen and Carme Rovira and Filipa Marcelo and Francisco Corzana and Gerken, {Thomas A.} and Ramon Hurtado-Guerrero",

year = "2018",

doi = "10.1021/acscentsci.8b00488",

language = "English",

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pages = "1274--1290",

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T1 - Structural and Mechanistic Insights into the Catalytic-Domain-Mediated Short-Range Glycosylation Preferences of GalNAc-T4

AU - De Las Rivas, Matilde

AU - Paul Daniel, Earnest James

AU - Coelho, Helena

AU - Lira-Navarrete, Erandi

AU - Raich, Lluis

AU - Compañón, Ismael

AU - Diniz, Ana

AU - Lagartera, Laura

AU - Jiménez-Barbero, Jesús

AU - Clausen, Henrik

AU - Rovira, Carme

AU - Marcelo, Filipa

AU - Corzana, Francisco

AU - Gerken, Thomas A.

AU - Hurtado-Guerrero, Ramon

PY - 2018

Y1 - 2018

N2 - 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.

AB - 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.

U2 - 10.1021/acscentsci.8b00488

DO - 10.1021/acscentsci.8b00488

M3 - Journal article

AN - SCOPUS:85053694658

SN - 2374-7951

VL - 4

SP - 1274

EP - 1290

JO - A C S Central Science

JF - A C S Central Science

IS - 9

ER -

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

Abstract

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