title: Increased N-glycosylation efficiency by generation of an aromatic sequon on N135 of antithrombin.
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.
authors.affiliation: Centro Regional de Hemodonación, Hospital Morales Meseguer, Universidad de Murcia, IMIB, Murcia, Spain.
authors.forename: Sonia
authors.initials: S
authors.lastname: Aguila
authors.affiliation: Centro Regional de Hemodonación, Hospital Morales Meseguer, Universidad de Murcia, IMIB, Murcia, Spain.
authors.forename: Irene
authors.initials: I
authors.lastname: Martínez-Martínez
authors.affiliation: Division of Cardiovascular Sciences, Laboratory of Thrombosis and Haemostasis, Centre for Applied Medical Research, University of Navarra, Pamplona, Spain.
authors.forename: Gilda
authors.initials: G
authors.lastname: Dichiara
authors.affiliation: Bioanalysis Group, IMIM-Hospital del Mar, Department of Experimental and Health Sciences, University Pompeu Fabra (UPF), Barcelona, Spain; Protein and Peptide Chemistry, Anapharm Biotech, Barcelona, Spain.
authors.forename: Ricardo
authors.initials: R
authors.lastname: Gutiérrez-Gallego
authors.affiliation: Centro Regional de Hemodonación, Hospital Morales Meseguer, Universidad de Murcia, IMIB, Murcia, Spain.
authors.forename: José
authors.initials: J
authors.lastname: Navarro-Fernández
authors.affiliation: Centro Regional de Hemodonación, Hospital Morales Meseguer, Universidad de Murcia, IMIB, Murcia, Spain.
authors.forename: Vicente
authors.initials: V
authors.lastname: Vicente
authors.affiliation: Centro Regional de Hemodonación, Hospital Morales Meseguer, Universidad de Murcia, IMIB, Murcia, Spain.
authors.forename: Javier
authors.initials: J
authors.lastname: Corral
mesh_terms: D000990:Antithrombin III / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D011994:Recombinant Proteins; D000990:Antithrombin III; D006493:Heparin