Morena Barrio, María Eugenia de la

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Morena Barrio, María Eugenia de la

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Morena Barrio, María Eugenia de la

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Universidad de Murcia. Departamento de Medicina

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Role of the C-sheet in the maturation of N-glycans on antithrombin: functional relevance of pleiotropic mutations
(2014-04-15) Águila Martínez, Sonia; Navarro Fernández, José Luis; Bohdan, N.; Gutiérrez Gallego, R.; Morena Barrio, María Eugenia de la; Vicente García, Vicente; Corral de la Calle, Javier; Martínez-Martínez, I.; Medicina Interna; Facultad de Medicina
Open Access
Neutrophil extracellular traps and von Willebrand factor are allies that negatively influence COVID-19 outcomes
(Wiley, 2021-01) Águila Martínez, Sonia; Fernández-Pérez, M. P.; Reguilón-Gallego, L.; de Los Reyes-García, A. M.; Miñano, A.; Bravo-Pérez, C.; García-Barberá, N.; Gómez-Verdú, J. M.; Martínez, C.; Morena Barrio, María Eugenia de la; Corral de la Calle, Javier; Bernal Morell, Enrique; Herranz Marín, María Teresa; Vicente García, Vicente; González-Conejero Hilla, Rocío; Lozano Almela, María Luisa; Medicina Interna
Open Access
N-Glycosylation as a Tool to Study Antithrombin Secretion, Conformation, and Function.
(MDPI, 2021-06-06) Águila Martínez, Sonia; Noto, Rosina; Luengo-Gil, Ginés; Espín, Salvador; Bohdan, Nataliya; Morena Barrio, María Eugenia de la; Peñas, Julia; Rodenas, Maria Carmen; Vicente García, Vicente; Corral de la Calle, Javier; Manno, Mauro; Martínez-Martínez, Irene; Medicina Interna
N-linked glycosylation is a crucial post-translational modification involved in protein folding, function, and clearance. N-linked glycosylation is also used therapeutically to enhance the half-lives of many proteins. Antithrombin, a serpin with four potential N-glycosylation sites, plays a pivotal role in hemostasis, wherein its deficiency significantly increases thrombotic risk. In this study, we used the introduction of N-glycosylation sites as a tool to explore what effect this glycosylation has on the protein folding, secretion, and function of this key anticoagulant. To accomplish this task, we introduced an additional N-glycosylation sequence in each strand. Interestingly, all regions that likely fold rapidly or were surrounded by lysines were not glycosylated even though an Nglycosylation sequon was present. The new sequon in the strands of the A- and B-sheets reduced secretion, and the B-sheet was more sensitive to these changes. However, the mutations in the strands of the C-sheet allowed correct folding and secretion, which resulted in functional variants. Therefore, our study revealed crucial regions for antithrombin secretion and could potentially apply to all serpins. These results could also help us understand the functional effects of natural variants causing type-I deficiencies.
Open Access
Amelioration of the severity of heparin-binding antithrombin mutations by posttranslational mosaicism
(American Society of Hematology, 2012-04-12) Martínez-Martínez, Irene; Navarro-Fernández, José; Ostergaad, Alice; Gutierrez-Gallego, Ricardo; Padilla, José; Miñano, Antonia; Pascual, Cristina; Martínez, Constantino; Morena-Barrio, María Eugenia de la; Pedersen, Shona; Kristensen, Soren Risom; Corral, Javier; Bohdan, Nataliya; Morena Barrio, María Eugenia de la; Águila Martínez, Sonia; Vicente García, Vicente; Corral de la Calle, Javier; Medicina
The balance between actions of procoagulant and anticoagulant factors protects organisms from bleeding and thrombosis. Thus, antithrombin deficiency increases the risk of thrombosis, and complete quantitative deficiency results in intrauterine lethality. However, patients homozygous for L99F or R47C antithrombin mutations are viable. These mutations do not modify the folding or secretion of the protein, but abolish the glycosaminoglycan-induced activation of antithrombin by affecting the heparin-binding domain. We speculated that the natural β-glycoform of antithrombin might compensate for the effect of heparin-binding mutations. We purified α- and β-antithrombin glycoforms from plasma of 2 homozygous L99F patients. Heparin affinity chromatography and intrinsic fluorescence kinetic analyses demonstrated that the reduced heparin affinity of the α-L99F glycoform (K(D), 107.9 ± 3nM) was restored in the β-L99F glycoform (K(D), 53.9 ± 5nM) to values close to the activity of α-wild type (K(D), 43.9 ± 0.4nM). Accordingly, the β-L99F glycoform was fully activated by heparin. Similar results were observed for recombinant R47C and P41L, other heparin-binding antithrombin mutants. In conclusion, we identified a new type of mosaicism associated with mutations causing heparin-binding defects in antithrombin. The presence of a fully functional β-glycoform together with the activity retained by these variants helps to explain the viability of homozygous and the milder thrombotic risk of heterozygous patients with these specific antithrombin
Open Access
Compound heterozygosity involving Antithrombin Cambridge II (p.Ala416Ser) in antithrombin deficiency.
(Elsevier, 2013-03-10) Águila Martínez, Sonia; Martínez-Martínez, Irene; Collado, Miriam; Llamas, Pilar; Antón, Ana I.; Martínez-Redondo, Consuelo; Padilla, José; Miñano, Antonia; Morena Barrio, María Eugenia de la; García-Avello, Ángel; Vicente García, Vicente; Corral de la Calle, Javier; Medicina Interna
Background: The characterization of natural mutants identified in patients with antithrombin deficiency has helped to identify functional domains or regions of this key anticoagulant and the mechanisms involved in the deficiency, as well as to define the clinical prognosis. Recently, we described an abnormal glycosylation in a pleiotropic mutant (K241E) that explained the impaired heparin affinity and the mild risk of thrombosis in carriers. Objectives: To evaluate the effects of different natural pleiotropic mutations on the glycosylation of antithrombin and their functional effects. Methods: Five pleiotropic mutations identified in patients with antithrombin deficiency and located at each one of the strands of the C-sheet were selected (K241E, M251I, M315K, F402L, and P429L). Recombinant mutants were generated and purified. Glycoform heterogeneity and conformational sensitivity were studied with electrophoresis, proteomic analysis, and glycomic analysis. Heparin affinity was evaluated from intrinsic fluorescence. Reactivity assays with factor Xa, thrombin and neutrophil elastase in the presence or absence of heparin were also performed. Results and Conclusions: Pleiotropic mutants, except for that with the M315K mutation, which affects a non-exposed residue, showed two glycoforms. Variant 1, with abnormal glycosylation, had reduced heparin affinity and severely affected reactivity with the target proteases. In contrast, variant 2, with similar electrophoretic mobility and heparin affinity to wild-type antithrombin, had impaired inhibitory activity that was partially compensated for by activation with heparin. Our results suggest the C-sheet of antithrombin as a new region that is relevant for proper maturation of the N-glycans. Therefore, pleiotropic mutations lead to glycosylation defects that are responsible for the reduced heparin affinity