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

Int J Mol Sci. 2021 Jan 6;22(2):516. doi: 10.3390/ijms22020516.

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.

MeSH terms

  • Antithrombin III / chemistry
  • Antithrombin III / genetics
  • Antithrombin III / metabolism
  • Antithrombin Proteins / chemistry*
  • Antithrombin Proteins / genetics
  • Antithrombin Proteins / metabolism*
  • Circular Dichroism
  • Glycosylation
  • Humans
  • Models, Molecular
  • Mutant Proteins / chemistry
  • Mutant Proteins / genetics
  • Mutant Proteins / metabolism
  • Mutation
  • Protein Conformation*
  • Protein Processing, Post-Translational*
  • Thrombosis

Substances

  • Antithrombin Proteins
  • Mutant Proteins
  • Antithrombin III