Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P11021 (BiP)
 2,049 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Factor VIII is the coagulation factor deficient in the X-chromosome-linked bleeding disorder hemophilia A. Factor VIII is homologous to blood coagulation factor V, both having a domain structure of A1-A2-B-A3-C1-C2. Previous transfection studies demonstrated that factor VIII is 10-fold less efficiently expressed than the homologous coagulation factor, factor V. The inefficient expression correlated with interaction of the factor VIII primary translation product with the protein chaperonin BiP in the lumen of the endoplasmic reticulum. In contrast, factor V was not detected in association with BiP and was secreted efficiently. To determine whether specific amino acid sequences within factor VIII inhibit secretion, we have studied the secretion of factor VIII deletion and factor VIII/factor V chimeric proteins upon transient transfection of COS-1 monkey cells. A chimeric factor VIII protein that contained the A1- and A2-domains of factor V was secreted with a similar efficiency as wild-type factor V, whereas the complementary chimera having the A1- and A2-domains of factor VIII was secreted with low efficiency, similar to wild-type factor VIII. These results suggested that sequences within the A1- and A2-domains were responsible for the low secretion efficiency of factor VIII. Secretion of A1-domain-deleted factor VIII was increased approximately 10-fold compared to wild-type factor VIII or A2-domain-deleted factor VIII. Expression of the factor VIII A1-domain alone did not yield secreted protein, whereas expression of the factor VIII A2-domain alone or the factor V A1-domain or A2-domain alone directed synthesis of secreted protein. Secretion of a hybrid in which the carboxyl-terminal 110 amino acids of the A1-domain were replaced by homologous sequences from the factor V A1-domain was also increased 10-fold compared to wild-type factor VIII, however, the secreted protein was not functional and the heavy and light chains were not associated. These results localize a 110-amino acid region within the A1-domain that inhibits factor VIII secretion. This region is clustered with multiple short peptide sequences that have potential to bind BiP.
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PMID:A 110-amino acid region within the A1-domain of coagulation factor VIII inhibits secretion from mammalian cells. 773 Mar 35

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.
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PMID:Missense mutations near the N-glycosylation site of the A2 domain lead to various intracellular trafficking defects in coagulation factor VIII. 2832 46

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.
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PMID:Molecular mechanisms of missense mutations that generate ectopic N-glycosylation sites in coagulation factor VIII. 2944 15