OC 49.2 - Identification of structural elements in factor V and factor VIII that determine their routes in the early secretory pathway

Background: Secretion of FV and FVIII is assisted by a cargo receptor complex of LMAN1 and MCFD2. Mutations in either LMAN1 or MCFD2 lead to the combined deficiency of FV and FVIII. The mechanism of how FV and FVIII are transported by this cargo receptor complex is unclear.

Aims: We aim to identify domains and N-glycans on FV and FVIII that mediate LMAN1-MCFD2-dependent secretion.

Methods: We generated various mutants of FV and FVIII and tested their secretion in wild-type (WT), LMAN1 knockout (KO), and MCFD2 KO HepG2 cells. In vitro results are validated in a novel mouse model expressing B domain-deleted (BDD) FVIII.

Results: In LMAN1 and MCFD2 deficient cell lines, full-length (FL)-FV and FVIII secretion is reduced to less than 10% of WT cells, but BDD-FV and BDD-FVIII secretion has no statistically significant reduction, suggesting that the B domain is involved in directing FVIII into the LMAN1-MCFD2 secretion pathway. FVIII with the elimination of all N-glycans in the B domain has an intermediate decrease in secretion in LMAN1 and MCFD2 KO cells, suggesting that B-domain glycans play a limited role in the entry into the LMAN1-MCFD2 secretion pathway. Secretion analysis of a series of partial B deleted mutants of FVIII showed that dependence on the LMAN1-MCFD2 pathway strengthens with the increase of B domain size and glycan content. Moreover, expression of the B-A3 domains of FVIII in LMAN1 and MCFD2 KO cells showed decreased secretion compared with WT cells. Next, we crossed a mouse with B-domain deletion with LMAN1 or MCFD2 KO mice. Results show that, unlike FL-FVIII, BDD-FVIII secretion is not deceased in LMAN1 or MCFD2 KO mice.

Conclusion(s): The B domain governs the entry of FV and FVIII into the LMAN1-MCFD2-dependent secretion pathway. LMAN1-MCFD2-mediated secretion is regulated by the size and glycan content of the B domain and the adjacent A3 domain.