Missense mutations near the N-glycosylation site of the A2 domain lead to various intracellular trafficking defects in coagulation factor VIII.
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.
Genomic Medicine Institute, Cleveland Clinic Lerner Research Institute, Cleveland, OH, USA.|State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences &Peking Union Medical College, Tianjin, China.
Wei
W
Wei
Genomic Medicine Institute, Cleveland Clinic Lerner Research Institute, Cleveland, OH, USA.
Chunlei
C
Zheng
Department of Pathology, Karamay Central Hospital, Karamay, Xinjiang, China.
Min
M
Zhu
State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences &Peking Union Medical College, Tianjin, China.
Xiaofan
X
Zhu
State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences &Peking Union Medical College, Tianjin, China.
Renchi
R
Yang
Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, KS, USA.
Saurav
S
Misra
Genomic Medicine Institute, Cleveland Clinic Lerner Research Institute, Cleveland, OH, USA.
Bin
B
Zhang
D000483:Alleles; D000818:Animals; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005169:Factor VIII / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D005838:Genotype; D006031:Glycosylation; D006467:Hemophilia A / Q000097:blood / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D020125:Mutation, Missense; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011487:Protein Conformation; D000072417:Protein Domains; D054730:Protein Interaction Domains and Motifs / Q000235:genetics*; D021381:Protein Transport; D059748:Proteolysis
D005169:Factor VIII; D046988:Proteasome Endopeptidase Complex