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.

Figure 1. Expression of antithrombin variants.

SDS-PAGE under reducing conditions and western blot of recombinant antithrombin variants from intracellular lysates or conditioned medium of HEK-EBNA cells 24 h after transfection. As loading control in cellular lysates we showed the expression of β-actin. As control of electrophoretic mobility we used a mixture of α and β-antithrombin purified from plasma of healthy subjects.

Figure 2. MALDI-TOF mass spectra of purified antithrombin variants.

Figure 3. Function of antithrombin variants.

Anti-FXa activity of antithrombin proteins secreted to the conditioned medium in presence of heparin. Results are expressed as a percentage of the activity of the S137T variant. Each bar represents the mean ± standard deviation (SD) of two independent experiments performed in duplicate. The differences between mutants were tested by paired t-test (p-value). The “*” indicated differences statistically significant with p<0.05.

Figure 4. Scheme of binding of antithrombin and heparin.

Initial rapid equilibrium, K₁, between antithrombin, AT, and pentasaccharide, H, leads to complex, AT.H, followed by rapid conformational change via k₂ to a high heparin affinity, highly fluorescence complex, AT*.H.

Figure 5. Ribbon diagram of antithrombin.

A) Structural representation of the localization of N135 (red spheres) in native antithrombin (PDB code 1T1FA). Strand 2A and helix D are displayed in cyan. B) Stick representation of the WT glycosylation consensus sequence (K133/S137 or (i) and (i+4) position) on native and activated antithrombin (AT) (PDB code 1T1F and 2GD4, respectively). All structures were rendered in Pymol (www.pymol.org).