N-Glycosylation as a Tool to Study Antithrombin Secretion, Conformation, and Function

Abstract

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 N-glycosylation 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.

Keywords: Glycosylation; antithrombin; bioengineering; coagulation; folding; serpin; thrombosis.

Figure 1

Structural representation of the mutations generated in the beta-strands of antithrombin. The consensus sequences generated at each strand are colored depending on the sheet to which they belong (red for the A-sheet, green for the B-sheet, and cyan for the C-sheet). Images were rendered with Pymol using Protein Data Bank (PDB): 1t1fA as a template.

Figure 2

Secretion of mutant antithrombins. (A) SDS-PAGE under reducing and non-reducing conditions and Western blot of the medium after 48 h of transfection. (B) The rate of secretion of mutant antithrombins. The quantification of secretion was carried out by gel densitometry 48 h after transfection. Blots cropped from different gels are delineated with white space. Full-length gels are included in the Supplementary File.

Figure 3

Inhibitory activity of antithrombin mutants. SDS-PAGE under the reduction and Western blot of the medium after 48 h of transfection and later incubation in the presence of low molecular weight heparin or unfractionated heparin with FXa (A) or thrombin (B). Full-length gels are included in the Supplementary File.

Figure 4

Secondary structures of the antithrombin mutants. Circular dichroisms (CD) spectra of wild type beta-antithrombin (S137A) (A) and mutants M315N/V317T (B), L146T (C), and F77N (D) for antithrombin monomers (solid curves) and polymers (dashed curves). Insets: CD signal at 221 nm on a thermal ramp (light curves); data are fitted here with sigmoidal curves (bold solid curves); the mid-point temperatures, shown as squares, are 54, 55, 50, and 52 °C for the wild type, M315N/V317T, L146T, and F77N, respectively; the onset of a second sigmoidal curve at higher temperatures indicates the onset of unfolding (dashed curves).