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

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dc.contributor.authorÁguila Martínez, Sonia
dc.contributor.authorMartínez-Martínez, Irene
dc.contributor.authorDichiara, Gilda
dc.contributor.authorGutiérrez-Gallego, Ricardo
dc.contributor.authorNavarro Fernández, José Luis
dc.contributor.authorVicente García, Vicente
dc.contributor.authorCorral de la Calle, Javier
dc.contributor.departmentMedicina Interna
dc.date.accessioned2026-02-16T09:54:17Z
dc.date.available2026-02-16T09:54:17Z
dc.date.copyright©2014 Aguila et al.
dc.date.issued2014-12-08
dc.description.abstractThe inefficient glycosylation of consensus sequence on N135 in antithrombin explains the two glycoforms of this key anticoagulant serpin found in plasma: a and b, with four and three N-glycans, respectively. The lack of this N-glycan increases the heparin affinity of the b-glycoform. Recent studies have demonstrated that an aromatic sequon (Phe-Y-Asn-X-Thr) in reverse b-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 (HEKEBNA). Cells transfected with wild-type plasmid (K133-Y-N135-X-S137) generated 50% of a and b-antithrombin. The S137T, as previously reported, K133F, and the double mutant (K133F/S137T) had improved glycosylation efficiency, leading to the secretion of a-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 Nglycosylation.
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dc.identifier.citationÁguila S, Martínez-Martínez I, Dichiara G, Gutiérrez-Gallego R, Navarro-Fernández J, Vicente V, et al. (2014) Increased N-Glycosylation Efficiency by Generation of an Aromatic Sequon on N135 of Antithrombin. PLoS ONE 9(12): e114454.
dc.identifier.doihttps://doi.org/10.1371/journal.pone.0114454
dc.identifier.eissn1932-6203
dc.identifier.urihttp://hdl.handle.net/10201/205343
dc.languageeng
dc.publisherPublic Library of Science(PLOS)
dc.relationThis study was supported by PI12/00657 (ISCIII and FEDER), and Redes RIC (RD12/0042/0050) (ISCIII and FEDER), S. Aguila holds a FPI grant from Ministerio de Ciencia e Innovacion,
dc.relation.publisherversionhttps://journals.plos.org/plosone/article?id=10.1371/journal.pone.0114454
dc.rightsAttribution 4.0 International*
dc.rights.accessRightsinfo:eu-repo/semantics/openAccess
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/*
dc.subject.odsObjetivo 3: Salud
dc.titleIncreased N-glycosylation efficiency by generation of an aromatic sequon on N135 of antithrombin.
dc.typeinfo:eu-repo/semantics/article
dc.type.versioninfo:eu-repo/semantics/publishedVersion
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