Increased N-Glycosylation Efficiency by Generation of an Aromatic Sequon on N135 of Antithrombin

The inefficient glycosylation of consensus sequence on N135 in antithrombin explains the two glycoforms of this key anticoagulant serpin found in plasma: α and β, with four and three N-glycans, respectively. The lack of this N-glycan increases the heparin affinity of the β-glycoform. Recent studies...

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Main Authors:	Águila, Sonia, Martínez-Martínez, Irene, Dichiara, Gilda, Gutiérrez-Gallego, Ricardo, Navarro-Fernández, José, Vicente, Vicente, Corral, Javier
Format:	Online
Language:	English
Published:	Public Library of Science 2014
Online Access:	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4259341/

id	pubmed-4259341
recordtype	oai_dc
spelling	pubmed-42593412014-12-15 Increased N-Glycosylation Efficiency by Generation of an Aromatic Sequon on N135 of Antithrombin Águila, Sonia Martínez-Martínez, Irene Dichiara, Gilda Gutiérrez-Gallego, Ricardo Navarro-Fernández, José Vicente, Vicente Corral, Javier Research Article The inefficient glycosylation of consensus sequence on N135 in antithrombin explains the two glycoforms of this key anticoagulant serpin found in plasma: α and β, with four and three N-glycans, respectively. The lack of this N-glycan increases the heparin affinity of the β-glycoform. Recent studies have demonstrated that an aromatic sequon (Phe-Y-Asn-X-Thr) in reverse β-turns enhances N-glycosylation efficiency and stability of different proteins. We evaluated the effect of the aromatic sequon in this defective glycosylation site of antithrombin, despite of being located in a loop between the helix D and the strand 2A. We analyzed the biochemical and functional features of variants generated in a recombinant cell system (HEK-EBNA). Cells transfected with wild-type plasmid (K133-Y-N135-X-S137) generated 50% of α and β-antithrombin. The S137T, as previously reported, K133F, and the double mutant (K133F/S137T) had improved glycosylation efficiency, leading to the secretion of α-antithrombin, as shown by electrophoretic and mass analysis. The presence of the aromatic sequon did not significantly affect the stability of this conformationally sensitive serpin, as revealed by thermal denaturation assay. Moreover, the aromatic sequon hindered the activation induced by heparin, in which is involved the helix D. Accordingly, K133F and particularly K133F/S137T mutants had a reduced anticoagulant activity. Our data support that aromatic sequons in a different structural context from reverse turns might also improve the efficiency of N-glycosylation. Public Library of Science 2014-12-08 /pmc/articles/PMC4259341/ /pubmed/25485983 http://dx.doi.org/10.1371/journal.pone.0114454 Text en © 2014 Águila et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
repository_type	Open Access Journal
institution_category	Foreign Institution
institution	US National Center for Biotechnology Information
building	NCBI PubMed
collection	Online Access
language	English
format	Online
author	Águila, Sonia Martínez-Martínez, Irene Dichiara, Gilda Gutiérrez-Gallego, Ricardo Navarro-Fernández, José Vicente, Vicente Corral, Javier
spellingShingle	Águila, Sonia Martínez-Martínez, Irene Dichiara, Gilda Gutiérrez-Gallego, Ricardo Navarro-Fernández, José Vicente, Vicente Corral, Javier Increased N-Glycosylation Efficiency by Generation of an Aromatic Sequon on N135 of Antithrombin
author_facet	Águila, Sonia Martínez-Martínez, Irene Dichiara, Gilda Gutiérrez-Gallego, Ricardo Navarro-Fernández, José Vicente, Vicente Corral, Javier
author_sort	Águila, Sonia
title	Increased N-Glycosylation Efficiency by Generation of an Aromatic Sequon on N135 of Antithrombin
title_short	Increased N-Glycosylation Efficiency by Generation of an Aromatic Sequon on N135 of Antithrombin
title_full	Increased N-Glycosylation Efficiency by Generation of an Aromatic Sequon on N135 of Antithrombin
title_fullStr	Increased N-Glycosylation Efficiency by Generation of an Aromatic Sequon on N135 of Antithrombin
title_full_unstemmed	Increased N-Glycosylation Efficiency by Generation of an Aromatic Sequon on N135 of Antithrombin
title_sort	increased n-glycosylation efficiency by generation of an aromatic sequon on n135 of antithrombin
description	The inefficient glycosylation of consensus sequence on N135 in antithrombin explains the two glycoforms of this key anticoagulant serpin found in plasma: α and β, with four and three N-glycans, respectively. The lack of this N-glycan increases the heparin affinity of the β-glycoform. Recent studies have demonstrated that an aromatic sequon (Phe-Y-Asn-X-Thr) in reverse β-turns enhances N-glycosylation efficiency and stability of different proteins. We evaluated the effect of the aromatic sequon in this defective glycosylation site of antithrombin, despite of being located in a loop between the helix D and the strand 2A. We analyzed the biochemical and functional features of variants generated in a recombinant cell system (HEK-EBNA). Cells transfected with wild-type plasmid (K133-Y-N135-X-S137) generated 50% of α and β-antithrombin. The S137T, as previously reported, K133F, and the double mutant (K133F/S137T) had improved glycosylation efficiency, leading to the secretion of α-antithrombin, as shown by electrophoretic and mass analysis. The presence of the aromatic sequon did not significantly affect the stability of this conformationally sensitive serpin, as revealed by thermal denaturation assay. Moreover, the aromatic sequon hindered the activation induced by heparin, in which is involved the helix D. Accordingly, K133F and particularly K133F/S137T mutants had a reduced anticoagulant activity. Our data support that aromatic sequons in a different structural context from reverse turns might also improve the efficiency of N-glycosylation.
publisher	Public Library of Science
publishDate	2014
url	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4259341/
_version_	1613165181024600064

Increased N-Glycosylation Efficiency by Generation of an Aromatic Sequon on N135 of Antithrombin

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