scholarly journals Missense mutations near the N-glycosylation site of the A2 domain lead to various intracellular trafficking defects in coagulation factor VIII

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Wei Wei ◽  
Chunlei Zheng ◽  
Min Zhu ◽  
Xiaofan Zhu ◽  
Renchi Yang ◽  
...  
Keyword(s):  
Factor Viii ◽  
Intracellular Trafficking ◽  
Coagulation Factor ◽  
Glycosylation Site ◽  
Coagulation Factor Viii ◽  
Missense Mutations ◽  
Trafficking Defects ◽  
A2 Domain

In silicoanalyses of missense mutations in coagulation factor VIII: identification of severity determinants of haemophilia A

Haemophilia ◽  
2015 ◽  
Vol 21 (5) ◽  
pp. 662-669 ◽  
Author(s):  
M. Sengupta ◽  
D. Sarkar ◽  
K. Ganguly ◽  
D. Sengupta ◽  
S. Bhaskar ◽  
...  
Keyword(s):  
Factor Viii ◽  
Coagulation Factor ◽  
Coagulation Factor Viii ◽  
Haemophilia A ◽  
Missense Mutations

Modified expression of coagulation factor VIII by addition of a glycosylation site at the N terminus of the protein

2007 ◽  
Vol 87 (2) ◽  
pp. 107-112 ◽  
Author(s):  
M. A. Srour ◽  
J. Grupp ◽  
Z. Aburubaiha ◽  
T. Albert ◽  
H. Brondke ◽  
...  
Keyword(s):  
Factor Viii ◽  
Coagulation Factor ◽  
Glycosylation Site ◽  
Coagulation Factor Viii ◽  
N Terminus

scholarly journals Molecular mechanisms of missense mutations that generate ectopic N-glycosylation sites in coagulation factor VIII

2018 ◽  
Vol 475 (5) ◽  
pp. 873-886 ◽  
Author(s):  
Wei Wei ◽  
Saurav Misra ◽  
Matthew V. Cannon ◽  
Renchi Yang ◽  
Xiaofan Zhu ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Factor Viii ◽  
Molecular Mechanisms ◽  
Complex Disease ◽  
Severe Disease ◽  
Coagulation Factor ◽  
Relative Activity ◽  
Coagulation Factor Viii ◽  
Missense Mutations ◽  
Glycosylation Sites

N-glycosylation is a common posttranslational modification of secreted and membrane proteins, catalyzed by the two enzymatic isoforms of the oligosaccharyltransferase, STT3A and STT3B. Missense mutations are the most common mutations in inherited diseases; however, missense mutations that generate extra, non-native N-glycosylation sites have not been well characterized. Coagulation factor VIII (FVIII) contains five consensus N-glycosylation sites outside its functionally dispensable B domain. We developed a computer program that identified hemophilia A mutations in FVIII that can potentially create ectopic glycosylation sites. We determined that 18 of these ectopic sites indeed become N-glycosylated. These sites span the domains of FVIII and are primarily associated with a severe disease phenotype. Using STT3A and STT3B knockout cells, we determined that ectopic glycosylation exhibited different degrees of dependence on STT3A and STT3B. By separating the effects of ectopic N-glycosylation from those due to underlying amino acid changes, we showed that ectopic glycans promote the secretion of some mutants, but impair the secretion of others. However, ectopic glycans that enhanced secretion could not functionally replace a native N-glycan in the same domain. Secretion-deficient mutants, but not mutants with elevated secretion levels, show increased association with the endoplasmic reticulum chaperones BiP (immunoglobulin heavy chain-binding protein) and calreticulin. Though secreted to different extents, all studied mutants exhibited lower relative activity than wild-type FVIII. Our results reveal differential impacts of ectopic N-glycosylation on FVIII folding, trafficking and activity, which highlight complex disease-causing mechanisms of FVIII missense mutations. Our findings are relevant to other secreted and membrane proteins with mutations that generate ectopic N-glycans.

Modified secretion of coagulation factor VIII by addition of a glycosylation site at the N terminus

2008 ◽  
Vol 28 (S 01) ◽  
pp. S99-S100
Author(s):  
M. A. Srour ◽  
J. Grupp ◽  
Z. Aburubaiha ◽  
T. Albert ◽  
H. Brondke ◽  
...  
Keyword(s):  
Factor Viii ◽  
Coagulation Factor ◽  
Glycosylation Site ◽  
Coagulation Factor Viii ◽  
N Terminus

Molecular simulation studies of human coagulation factor VIII C domain-mediated membrane binding

2015 ◽  
Vol 113 (02) ◽  
pp. 373-384 ◽  
Author(s):  
Jiangfeng Du ◽  
Kanin Wichapong ◽  
Tilman M. Hackeng ◽  
Gerry A. F Nicolaes
Keyword(s):  
Factor Viii ◽  
Membrane Binding ◽  
Coagulation Factor ◽  
Coagulation Factor Viii ◽  
Haemophilia A ◽  
C2 Domain ◽  
Missense Mutations ◽  
C2 Domains ◽  
C1 Domain ◽  
Dynamics Simulations

SummaryThe C-terminal C domains of activated coagulation factor VIII (FVIIIa) are essential to membrane binding of this crucial coagulation cofactor protein. To provide an overall membrane binding mechanism for FVIII, we performed simulations of membrane binding through coarsegrained molecular dynamics simulations of the C1 and C2 domain, and the combined C-domains (C1+C2). We found that the C1 and C2 domain have different membrane binding properties. The C1 domain uses hydrophobic spikes 3 and 4, of its total of four spikes, as major loops to bind the membrane, whereas all four of its hydrophobic loops of the C2 domain appear essential for membrane binding. Interestingly, in the C1+C2 system, we observed cooperative binding of the C1 and C2 domains such that all four C2 domain spikes bound first, after which all four loops of the C1 domain inserted into the membrane, while the net binding energy was higher than that of the sum of the isolated C domains. Several residues, mutations of which are known to cause haemophilia A, were identified as key residues for membrane binding. In addition to these known residues, we identified residues from the C1 and C2 domains, which are involved in the membrane binding process, that have not been reported before as a cause for haemophilia A, but which contribute to overall membrane binding and which are likely candidates for novel causative missense mutations in haemophilia A.

scholarly journals Inhibition of human coagulation factor VIII by monoclonal antibodies. Mapping of functional epitopes with the use of recombinant factor VIII fragments

1989 ◽  
Vol 263 (1) ◽  
pp. 187-194 ◽  
Author(s):  
A Leyte ◽  
K Mertens ◽  
B Distel ◽  
R F Evers ◽  
M J M De Keyzer-Nellen ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Factor Viii ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Procoagulant Activity ◽  
Coagulation Factor Viii ◽  
Coagulation Cascade ◽  
Restriction Enzyme Digestion ◽  
Proteolytic Activation

The epitopes of four monoclonal antibodies against coagulation Factor VIII were mapped with the use of recombinant DNA techniques. Full-length Factor VIII cDNA and parts thereof were inserted into the vector pSP64, permitting transcription in vitro with the use of a promoter specific for SP6 RNA polymerase. Factor VIII DNA inserts were truncated from their 3′-ends by selective restriction-enzyme digestion and used as templates for ‘run-off’ mRNA synthesis. Translation in vitro with rabbit reticulocyte lysate provided defined radiolabelled Factor VIII fragments for immunoprecipitation studies. Two antibodies are shown to be directed against epitopes on the 90 kDa chain of Factor VIII, between residues 712 and 741. The 80 kDa chain appeared to contain the epitopes of the other two antibodies, within the sequences 1649-1778 and 1779-1840 respectively. The effect of antibody binding to these sequences was evaluated at two distinct levels within the coagulation cascade. Both Factor VIII procoagulant activity and Factor VIII cofactor function in Factor Xa generation were neutralized upon binding to the region 1779-1840. The antibodies recognizing the region 713-740 or 1649-1778, though interfering with Factor VIII procoagulant activity, did not inhibit in Factor Xa generation. These findings demonstrate that antibodies that virtually inhibit Factor VIII in coagulation in vitro are not necessarily directed against epitopes involved in Factor VIII cofactor function. Inhibition of procoagulant activity rather than of cofactor function itself may be explained by interference in proteolytic activation of Factor VIII. This hypothesis is in agreement with the localization of the epitopes in the proximity of thrombin-cleavage or Factor Xa-cleavage sites.

Sign in / Sign up

Export Citation Format

Share Document