Title : Missense mutations near the N-glycosylation
site of the A2
domain lead to various intracellular trafficking defects in
coagulation factor VIII
Abstract :
- Missense mutation is the most common mutation type in hemophilia
- However, the majority of missense mutations remain uncharacterized
- Here we characterize how hemophilia mutations near the unused N-glycosylation site of the A2 domain ( N582 ) of FVIII affect protein conformation and intracellular trafficking
- N582 is located in the middle of a short 310-helical turn ( D580-S584 ), in which most amino acids have multiple hemophilia mutations
- All 14 missense mutations found in this 310-helix reduced secretion levels of the A2 domain and full-length FVIII
- Secreted mutants have decreased activities relative to WT FVIII
- Selected mutations also lead to partial glycosylation of N582 , suggesting that rapid folding of local conformation prevents glycosylation of this site in wild-type FVIII
- Protease sensitivity, stability and degradation of the A2 domain vary among mutants, and between non-glycosylated and glycosylated species of the same mutant
- Most of the mutants interact with the ER chaperone BiP , while only mutants with aberrant glycosylation interact with calreticulin
- Our results show that the short 310-helix from D580 to S584 is critical for proper biogenesis of the A2 domain and FVIII , and reveal a range of molecular mechanisms by which FVIII missense mutations lead to moderate to severe hemophilia A