Publication: Increased N-glycosylation efficiency by generation of an aromatic sequon on N135 of antithrombin.
Authors
Águila Martínez, Sonia ; Martínez-Martínez, Irene ; Dichiara, Gilda ; Gutiérrez-Gallego, Ricardo ; Navarro Fernández, José Luis ; Vicente García, Vicente ; Corral de la Calle, Javier
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Publisher
Public Library of Science(PLOS)
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DOI
https://doi.org/10.1371/journal.pone.0114454
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info:eu-repo/semantics/article
Description
Abstract
The 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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Citation
Águila 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.
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