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.

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

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 Mutations in Coagulation Factor VIII Are Associated with More Favorable Outcome in Patients with Cutaneous Melanoma

TH Open ◽  
2017 ◽  
Vol 01 (02) ◽  
pp. e113-e121 ◽  
Author(s):  
Zheng Ping ◽  
Abha Soni ◽  
Lance Williams ◽  
Huy Pham ◽  
Malay Basu ◽  
...  
Keyword(s):  
Factor Viii ◽  
Cutaneous Melanoma ◽  
Molecular Mechanisms ◽  
Coagulation Factor ◽  
B Cell Lymphoma ◽  
The Cancer Genome Atlas ◽  
Coagulation Factor Viii ◽  
Mutation Rates ◽  
Sequencing Data ◽  
Genomic Studies

AbstractCoagulation factor VIII (FVIII), von Willebrand factor (VWF), and ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats 13) play an important role in the regulation of normal hemostasis. However, little is known about their roles in patients with malignancy, particularly with cutaneous melanoma. Whole genome sequencing data are available for 25,719 cases in 126 cancer genomic studies for analysis. All sequencing data and corresponding pathology findings were obtained from The Cancer Genome Atlas. The cBioPortal bioinformatics tools were used for the data analysis. Our results demonstrated that mutations in genes encoding FVIII, VWF, and ADAMTS13 were reported in 92 of 126 cancer genomic studies, and high mutation rates in these three genes were observed in patients with cutaneous melanoma from three independent studies. Moreover, high mutation rates in FVIII, VWF, and ADAMTS13 were also found in patients with diffuse large B cell lymphoma (22.9%), lung small cell carcinoma (20.7%), and colon adenocarcinoma (19.4%). Among 366 melanoma cases from TCGA provisional, the somatic mutation rates of FVIII, VWF, and ADAMTS13 in tumor cells were 15, 14, and 5%, respectively. There was a strong tendency for coexisting mutations of FVIII, VWF, and ADAMTS13. Kaplan–Meier survival analysis demonstrated that melanoma patients with FVIII mutations had a more favorable overall survival rate than those without FVIII mutations (p = 0.02). These findings suggest, for the first time, that the FVIII mutation burden may have a prognostic value for patients with cutaneous melanoma. Further studies are warranted to delineate the molecular mechanisms underlying the favorable prognosis associated with FVIII mutations.

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.

scholarly journals Characterization and visualization of murine coagulation factor VIII-producing cells in vivo

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Morisada Hayakawa ◽  
Asuka Sakata ◽  
Hiroko Hayakawa ◽  
Hikari Matsumoto ◽  
Takafumi Hiramoto ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Factor Viii ◽  
Fluorescent Protein ◽  
Coagulation Factor ◽  
Coagulation Factors ◽  
Genetically Engineered ◽  
Coagulation Factor Viii ◽  
Liver Sinusoidal Endothelial Cells ◽  
Genetically Engineered Mice

AbstractCoagulation factors are produced from hepatocytes, whereas production of coagulation factor VIII (FVIII) from primary tissues and cell species is still controversial. Here, we tried to characterize primary FVIII-producing organ and cell species using genetically engineered mice, in which enhanced green fluorescent protein (EGFP) was expressed instead of the F8 gene. EGFP-positive FVIII-producing cells existed only in thin sinusoidal layer of the liver and characterized as CD31high, CD146high, and lymphatic vascular endothelial hyaluronan receptor 1 (Lyve1)+. EGFP-positive cells can be clearly distinguished from lymphatic endothelial cells in the expression profile of the podoplanin− and C-type lectin-like receptor-2 (CLEC-2)+. In embryogenesis, EGFP-positive cells began to emerge at E14.5 and subsequently increased according to liver maturation. Furthermore, plasma FVIII could be abolished by crossing F8 conditional deficient mice with Lyve1-Cre mice. In conclusion, in mice, FVIII is only produced from endothelial cells exhibiting CD31high, CD146high, Lyve1+, CLEC-2+, and podoplanin− in liver sinusoidal endothelial cells.

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