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.
Highlights
Antithrombin is the most important natural anticoagulant, as its deficiency is associated with the highest risk of thrombosis [1]
We generated 18 different mutants, each one containing an additional N-glycosylation consensus sequence located in different strands of antithrombin
Serpin polymerization has been a popular topic for many years [7,23,24,25,26,27,28,29,30,31]
Summary
Antithrombin is the most important natural anticoagulant, as its deficiency is associated with the highest risk of thrombosis [1]. Certain missense mutations can cause type I deficiencies through their effects on protein folding [4] since misfolded proteins can degrade in lysosomes or accumulate inside the endoplasmic reticulum [5]. As a member of serpins (serine protease inhibitors), antithrombin is characterized by great structural flexibility facilitating its efficient inhibitory capacity. This flexibility favors abnormal folding into hyperstable conformations (latent or polymer), with a central sheet formed by six beta strands and a consequent loss of function [6,7]. Intracellular polymers are retained inside the endoplasmic reticulum, establishing aggregates that may cause toxicity (serpinopathy). In the case of antithrombin, disulfide-linked dimers have been observed in plasma from patients with missense mutations, thereby inducing intracellular polymerization [9]
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