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Increased N-glycosylation efficiency by generation of an aromatic sequon on N135 of antithrombin

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dc.contributor.author Águila, Sonia
dc.contributor.author Martínez-Martínez, Irene
dc.contributor.author Dichiara, Gilda
dc.contributor.author Gutiérrez Gallego, Ricardo, 1968-
dc.contributor.author Navarro-Fernández, José
dc.contributor.author Vicente, Vicente
dc.contributor.author Corral, Javier
dc.date.accessioned 2015-05-25T07:13:13Z
dc.date.available 2015-05-25T07:13:13Z
dc.date.issued 2014
dc.identifier.citation Aguila S, Martínez-Martínez I, Dichiara G, Gutiárrez-Gallego R, Navarro-Fernández J, Vicente V, Corral J. Increased N-glycosylation efficiency by generation of an aromatic sequon on N135 of antithrombin. PLoS ONE. 2014;9(12):e114454. DOI: 10.1371/journal.pone.0114454
dc.identifier.issn 1932-6203
dc.identifier.uri http://hdl.handle.net/10230/23631
dc.description.abstract 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.
dc.description.sponsorship This work has been supported by PI12/00657 (ISCIII,(www.isciii.es) & FEDER), and Redes RIC (RD12/0042/0050 & RD12/0042/0009) (ISCIII & FEDER), S.A is holder of a FPI grant from Ministerio de Ciencia e Innovación (www.idi.mineco.gob.es), I.M.M. is a Miguel Servet researcher from ISCIII and J.N.F. holders a post-doctoral contract Sara Borrell from ISCIII.
dc.format.mimetype application/pdf
dc.language.iso eng
dc.publisher Public Library of Science (PLoS)
dc.relation.ispartof PLoS ONE. 2014;9(12):e114454
dc.rights 2014 Águila et al. 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 credited
dc.rights.uri https://creativecommons.org/licenses/by/4.0/
dc.title Increased N-glycosylation efficiency by generation of an aromatic sequon on N135 of antithrombin
dc.type info:eu-repo/semantics/article
dc.identifier.doi http://dx.doi.org/10.1371/journal.pone.0114454
dc.rights.accessRights info:eu-repo/semantics/openAccess
dc.type.version info:eu-repo/semantics/publishedVersion

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