Role of the C-sheet in the maturation of N-glycans on antithrombin: functional relevance of pleiotropic mutations.
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.


OBJECTIVE
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.


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.
Centro Regional de Hemodonación, Servicio de Hematología y Oncología Médica HU Morales Meseguer, University of Murcia, IMIB, Murcia, Spain.
S
S
Aguila
J
J
Navarro-Fernández
N
N
Bohdan
R
R
Gutiérrez-Gallego
M E
ME
de la Morena-Barrio
V
V
Vicente
J
J
Corral
I
I
Martínez-Martínez
D000293:Adolescent; D000328:Adult; D020152:Antithrombin III Deficiency / Q000235:genetics* / Q000378:metabolism; D000991:Antithrombins / Q000737:chemistry*; D004586:Electrophoresis; D006031:Glycosylation; D006493:Heparin / Q000737:chemistry; D006579:Heterozygote; D006801:Humans; D019272:Leukocyte Elastase / Q000378:metabolism; D008875:Middle Aged; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011134:Polysaccharides / Q000737:chemistry*; D011379:Prognosis; D017434:Protein Structure, Tertiary; D040901:Proteomics; D011994:Recombinant Proteins / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013917:Thrombin / Q000737:chemistry; D013927:Thrombosis / Q000235:genetics
D000991:Antithrombins; D011134:Polysaccharides; D011994:Recombinant Proteins; D006493:Heparin; D019272:Leukocyte Elastase; D013917:Thrombin
antithrombin III; antithrombin III deficiency; glycosylation; isolation & purification; recombinant proteins