This set of Protein Engineering Questions & Answers for Exams focuses on “Applications – Engineering Blood Clotting Factor VIII – 2”.
1. B domain of FVIII is necessary for the clotting activity.
a) True
b) False
View Answer
Explanation: The above statement is false. Although the B domain of FVIII comprises 40% of the total protein, it is not necessary for the clotting activity of FVIII. Replacement of the B domain by linker sequence has been used for several B domain ‘deleted’ or ‘truncated’ bioengineered FVIII variants.
2. Which of the following are a successful acute and prophylactic protein bypassing agent for haemophilia with inhibitors?
a) Recombinant FVIa
b) Recombinant FVa
c) Recombinant FVIIIa
d) Recombinant FVIIa
View Answer
Explanation: Recombinant FVIIa is a successful acute and prophylactic protein bypassing agent for haemophilia with inhibitors. Its major efficacy limitation is its short half-life, which is around 2 hours. Recombinant FVIa, recombinant FVa, or recombinant FVIIIa are not successful acute and prophylactic protein bypassing agents for haemophilia with inhibitors.
3. Which of the following catalyzes the conversion of prothrombin to thrombin?
a) Platelets
b) Fibrin
c) Pectinase
d) Thromboplastin
View Answer
Explanation: Thromboplastin catalyzes the conversion of prothrombin to thrombin. Platelets, fibrin, or pectinase do not catalyze the conversion of prothrombin to thrombin. Bivalent calcium ions are also required for this conversion reaction.
4. Elimination of disulfide bond between which two amino acid residues within the A3 domain increases FVIII secretion by 2-fold?
a) C1414 and C1456
b) C1123 and C1158
c) C1785 and C1798
d) C1899 and C1903
View Answer
Explanation: Elimination of disulfide bond between C1899 and C1903 (within the A3 domain) through glycine substitutions increases FVIII secretion by 2-fold from mammalian cell culture, this enhancement is speculated to be due to preventing interactions with free thiols that hinder FVIII secretion.
5. The interaction between FVIII and the Endoplasmic Reticulum (ER) chaperone BiP enhances FVIII secretion.
a) True
b) False
View Answer
Explanation: The above statement is false. The interaction between FVIII and the Endoplasmic Reticulum (ER) chaperone BiP limits the FVIII secretions. Disruption of this interaction through specific FVIII amino acid substitutions enhances FVIII secretion 2-fold from mammalian cell culture.
6. Protein-engineered bypassing agents have the potential to treat bleeding in haemophilia patients with inhibitors.
a) False
b) True
View Answer
Explanation: The above statement is true. Protein-engineered bypassing agents have the potential to treat bleeding in haemophilia patients with inhibitors. Like FVIIIa, these proteins are designed to promote hemostasis through mechanisms that bypass both FVIII and FIX activity.
7. Which of the following factors require LMAN1-facilitated transport from the ER to the Golgi?
a) FV and FVI
b) FVII and FVI
c) FVI and FVIII
d) FVIII and FV
View Answer
Explanation: FVIII and FV require LMAN1-facilitated transport from the ER to the Golgi, which is dependent on N-linked oligosaccharides that occur mostly in the B domain. FVI and FVII do not require LMAN1-facilitated transport from the ER to the Golgi.
8. Which of the following enzyme cleaves fibrinogen into fibrin which polymerizes and crosslinks (using factor VIII) into a blood clot?
a) Pepsin
b) Lipase
c) Furin
d) Thrombin
View Answer
Explanation: Thrombin cleaves fibrinogen into fibrin which polymerizes and crosslinks (using factor VIII) into a blood clot. Pepsin, lipase, or furin do not cleave fibrinogen into fibrin which polymerizes and crosslinks into a blood clot.
9. Furin Cleavage is necessary for FVIII biological functions.
a) True
b) False
View Answer
Explanation: The above statement is false. Furin Cleavage is not necessary for FVIII biological functions. In a study, where FVIII BDD (B domain deleted factor VIII) was bioengineered to avoid furin processing by deleting the cleavage sequence enhanced FVIII secretion 3-fold.
10. Which of the following can inactivate the activated FVIII?
a) Inactivated protein B
b) Activated protein B
c) Inactivated protein C
d) Activated protein C
View Answer
Explanation: Once activated, FVIIIa is inactivated by the dissociation of the A2-domain from the heterotrimer and/or by the proteolytic inactivation by activated protein C (APC). Activated FVIII cannot be activated by activated/inactivated protein B.
11. Small changes in the amino acid sequence of proteins can have a profound effect on their immunogenicity.
a) False
b) True
View Answer
Explanation: The above statement is true. Small changes in the amino acid sequence of proteins can have a profound effect on their immunogenicity. The potential advantage of bioengineered variants in gene therapy for haemophilia must be balanced against the potential risk of exacerbating the immunogenicity of the transgene.
12. Removing which of the following enzyme cleavage site impedes the A2 dissociation?
a) Rennin
b) Pepsin
c) Furin
d) Thrombin
View Answer
Explanation: Removing the thrombin cleavage site impedes the A2 dissociation. This removal of thrombin cleavage site R740 and R1649 in a B domain truncated FVIII causes the A2 domain to remain covalently bonded through the peptide chain to the A3 domain. Removal of furin cleavage site does not impede the A2 dissociation.
13. The potency of gene therapy vectors can be enhanced by using bioengineered extended half-life (EHL) FVIII or FIX fusion proteins as transgenes.
a) False
b) True
View Answer
Explanation: The above statement is true. The potency of gene therapy vectors can be enhanced by using bioengineered extended half-life (EHL) FVIII or FIX fusion proteins as transgenes. These EHL products fuse half-lifeextending proteins, such as the IgG1-Fc domain or albumin with FVIII or FIX variants.
14. How many alternatively spliced transcripts are produced by factor VIII gene?
a) Five
b) Seven
c) Three
d) Two
View Answer
Explanation: The factor VIII gene produces two alternatively spliced transcripts. Transcript variant 1 encodes a large glycoprotein, isoform a, which circulates in plasma and associates with von Willebrand factor in a non-covalent complex. This protein undergoes multiple cleavage events. Transcript variant 2 encodes a putative small protein, isoform b.
15. APC inactivates FVIIIa by proteolytic cleavage at which of the following sites?
a) R313 and/or R512
b) R454 and/or R565
c) R234 and/or R554
d) R336 and/or R562
View Answer
Explanation: APC inactivates FVIIIa by proteolytic cleavage at R336 (A1 domain) and/or R562 (A2 domain). Amino acid substitutions at these positions have resulted in FVIII variants with increased hemostatic efficacy in in vitro and in vivo assays.
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