The Impacts of G4S Mutation on N-glycosylation and conformation of the Human Coagulation Factor IX GLA domain: In silico and In vitro Analysis

Abstract

Missense mutations are the most prevalent form of mutation in hemophilia B patients. These alterations may result in the creation of novel and non-native N-glycosylation sites (Asn-X-Ser/Thr) through single amino acid substitutions. The pathogenic mechanisms of N-glycosylation mutations in hemophilia B patients have not been extensively studied yet. By survey among known missense mutations, we found only one N-glycosylation mutation in the γ-carboxyglutamic-rich (GLA) domain of the human coagulation factor IX (hFIX). This mutation that was reported in patients with mild and moderate hemophilia B, is caused by G4S amino acid substitution. To investigate the possibility of glycan attachment to the novel N-glycosylation site in G4S-mutant hFIX and the occurrence of hyperglycosylation, site-directed mutagenesis was applied to introduce the selected mutation into the coding sequence of the hFIX. The nucleotide sequences of the both native and G4S-mutant hFIX were separately cloned into the pcDNA3.1 expression plasmid and transiently expressed in HEK293T cells. Our results from gradient SDS-PAGE and western blotting analysis of the both recombinant native and mutant hFIX demonstrated no glycan attachment to the new N-glycosylation site in the G4S-mutant hFIX. Molecular dynamics (MD) simulation was also conducted to provide atomistic insights into structure and behavior of the native and G4S-mutant GLA domains in the both free and membrane-bound states. The results revealed that the mutation slightly affected the dynamic behavior of the mutant GLA domain. The conformational analysis proved that the native GLA domain had less fluctuation and more stability than the mutant GLA domain. The slight conformational changes may influence the binding capacity and interaction of the mutant GLA domain to phospholipid bilayer which is necessary for coagulation activity of the hFIX. These findings were in accordance with the nature of the G4S mutation which causes mild hemophilia B.