Antithrombin is the most important natural anticoagulant, as its deficiency is associated with the highest risk of thrombosis [1
]. Antithrombin deficiency is a rare congenital disease (1 in 3.000–5.000) [2
], mainly caused by mutations in SERPINC1
, the gene encoding this anticoagulant. Antithrombin deficiencies are classified as type I, when the mutation causes the absence of the protein in circulation, or type II, when the mutation allows for the presence of antithrombin variants in the plasma with null or impaired inhibitory activity [3
]. Type I deficiency can be explained by mutations destabilizing the RNA or protein. Among the last group of defects, frameshifting, splicing, and nonsense mutations usually result in truncated proteins. Additionally, 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. However, this flexibility also 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
]. Additionally, intracellular polymers are retained inside the endoplasmic reticulum, establishing aggregates that may cause toxicity (serpinopathy). In some serpins, secreted polymers have been detected [8
]. In the case of antithrombin, disulfide-linked dimers have been observed in plasma from patients with missense mutations, thereby inducing intracellular polymerization [9
Antithrombin has three beta-sheets (A-C), nine alpha-helices (A-I), and a reactive center loop. A feature that distinguishes antithrombin from other serpins is the presence of three disulfide bonds (using the numbering of the mature protein: Cys8-Cys128, Cys21-Cys95, and Cys247-Cys430). Additionally, the amino acid sequence presents 4 N-glycosylation sites (UniProtKB-P01008 (ANT3_HUMAN)). However, the Asn135 is not efficiently glycosylated, resulting in two detected glycoforms of antithrombin in plasma: Alpha, with 4 N-glycans, and beta, with 3 N-glycans [10
]. N-glycosylation requires a consensus sequon Asn-X-Ser/Thr/Cys (X can be any amino acid except Pro) [11
], and, in the case of antithrombin, the N-glycans incorporated are complex, biantennary, and feature terminal sialic acids. Typically, N-glycosylation takes place within flexible protein structures such as loops or helices, allowing interactions between the Asn in the consensus sequence and the oligosaccharide transferase complex (OST) [12
]. Furthermore, it is well known that protein glycosylation occurs during folding [14
]; therefore, we hypothesized that the folding of the strand results in an inaccessible consensus sequence for OST to transfer the oligosaccharide precursor to the Asn. Hence, the introduction of a new N-glycosylation site in each strand of this serpin could help elucidate the folding order of antithrombin. Moreover, the presence of new N-glycosylation in antithrombin might provide new clues related to the secretion, half-life, and function of this serpin, which could also be explored therapeutically.
Serpin polymerization has been a popular topic for many years [7
]. Currently, there is still debate about the mechanism of polymerization. Moreover, it is not clear if such a mechanism may be different among serpins and if polymerization occurs through different mechanisms within the same serpin [32
]. In the case of antithrombin, polymerization causes antithrombin deficiency and leads to increased thrombotic risk [9
]. The characterization of antithrombin polymerization is very challenging. For example, proteins expressed in Escherichia coli
result in the large production needed for these studies, but N-glycosylation is missing, thereby impacting antithrombin folding and secretion [33
]. Interestingly, the presence of three disulfide bridges in the tertiary structure of antithrombin allowed this serpin to form disulfide linked dimers through monomeric disulfide bridges when expressed in eukaryotic cells. Theses bridges are easily detected using SDS gels without reducing agents. Indeed, these oligomers have also been described to allow polymerization in the plasma of patients with mutations [9
]. In this study, we used N-glycosylation, a post-translational modification, to study the folding, secretion, and function of antithrombin, which may help us understand serpin misfolding. However, the creation of new N-glycosylation sequons in each strand of antithrombin had heterogeneous consequences, likely explainable not only by the presence of an additional N-glycan but also by the associated missense change(s) (Supplemental Figure S2
Our study provided intriguing results. First, although the mutations generated new N-glycosylation sequons, the glycosylation was not efficient in all cases (Table 1
). Indeed, in eight of the sixteen secreted mutants, the consensus sequence was not glycosylated. This may be explained by two mechanisms: A) Other residues surrounding the N-glycosylation signal (N-X-S/T) modulated the efficiency of glycosylation. Thus, an aromatic residue preceding the asparagine increased the efficiency [34
]. Recently, our group demonstrated that the presence of lysine residues close to the sequon impairs glycosylation efficiency (de la Morena Barrio et al., manuscript in preparation). Interestingly, in our present study, lysine residues were found in five out of the eight variants that were secreted and not glycosylated (Table 3
), confirming the relevance of these electropositive residues in modulating the efficacy of N-glycosylation. The two non-glycosylated mutants without lysines at positions up to +4 or −4 from the asparagines did not have surrounding lysines in their 3D structures. B) Alternatively, the sequon may be present within a region that is immediately folded and does not allow the enzymatic action of the OST. Notably, the remaining two variants that were not glycosylated were located within the C-terminal hairpin (strands 4B and 5B). This is one of the first folded regions [35
], supporting our hypothesis that the rapid folding of this region evades the action of the OST.
Secondly, the introduction of new N-glycosylation sequences may have influenced the folding of the protein. Antithrombin is folded into a metastable native conformation, which may transform into a hyperstable latent or polymer conformation via different environmental factors, such as increased temperatures or pH modifications [36
]. Indeed, like other serpins, even missense mutations may increase the rate of latent or polymer formation [37
]. The identification of disulfide linked dimers in most variants with an additional N-glycan (particularly among those located within the A and B sheets) demonstrated that the presence of an extra N-glycan did not interfere with this polymerization process. Interestingly, this observation suggests that this additional post-translational modification may also affect the folding of antithrombin, thus favoring the transition to a hyperstable conformation. The gel filtration profiles showed significant differences for the L146T and F77N mutants compared to the wild type. The CD spectra clearly showed that the variants M315N/V317T, L146T, and F77N were able to polymerize. However, an early sign of unfolding was observed for the F77N variant, suggesting a different structural organization. Further studies will be required to clarify whether this effect might be caused by a missense change or by the new N-glycan in the case of the F77N variant.
Third, we explored the functional consequences of additional N-glycans in this key anticoagulant serpin. Glycoforms lacking one of the N-glycans increased heparin affinity, but the inhibitory function was not affected [10
]. Our study shows for first time that the presence of an extra glycan does not significantly interfere with antithrombin anticoagulant activity, except for I219T. This variant is located at strand 3A in a region that we have already shown to be critical for protease inhibition by affecting the rate of reactive loop insertion [15
], possibly explaining the loss of functionality in this case.
Finally, our study identified the mutant M315N/V317T displaying almost a 3-fold increased secretion compared to the WT. The single mutant already improved protein secretion (Supplemental Figure S2
). Therefore, the potentially increased half-life of the hyperglycosylated protein remains to be determined, which might be a useful strategy to develop recombinant antithrombin with improved therapeutic usefulness.
In conclusion, our study identified several locations in the A- and B-sheets of antithrombin as important for the proper folding of this protein. The introduction of N-glycosylation consensus sequences was used as a strategy to detect key strands for antithrombin folding by following the secretion and functionality of the protein. Such a strategy could be useful for studying the folding of other serpins or proteins.