Mechanism of antithrombin deficiency due to the novel variant C32W in the C-terminus of the signal peptide.

Introduction: We identified a novel variant of the antithrombin (AT) gene (SERPINC1), c.96 T>G, p.Cys32Trp (C32W), located at the signal peptide cleavage site in a patient with congenital AT deficiency. The impact of signal peptide variants on the intracellular trafficking and secretion of AT proteins has not been previously studied. Thus, we analyzed the intracellular dynamics and signal peptide cleavage of the C32W variant of AT (AT-C32W).

Materials and methods: Wild-type AT (AT-WT) and AT-C32W expression vectors were transfected into HEK293 cells. Functional analyses were performed using western blotting and proteasome inhibition experiments. Signal peptide cleavage was evaluated by peptide sequencing.

Results: The AT antigen levels in the cell lysates and culture supernatants of the AT-C32W were reduced to 3.8% and 4.8%, respectively. Following proteasome inhibition, the AT-C32W level increased to 71.5% of that for AT-WT. Peptide sequencing identified a fragment corresponding to the N-terminal end of the signal peptide exclusively in AT-C32W.

Discussion: These results suggest that the signal peptide of AT-C32W is not cleaved properly, which causes intracellular degradation of AT-C32W by proteasomes that results in type I AT deficiency. Further studies on the intracellular dynamics of such variants may clarify the mechanisms underlying AT deficiency.