Elucidation of the Roles of Individual Asparagine-Linked Glycans Outside of the B Domain on Factor VIII Secretion
Authors: Selvaraj Sundar Rajan , Miao Hongzhi , Pipe Steven 
Published: October 2019

Abstract
Abstract 2238
Post-translational modifications play vital roles in the secretion, function, intermolecular interactions and degradation of most secreted and transmembrane proteins. Factor VIII (FVIII) is a heavily glycosylated protein with up to 25 asparagine (Asn)-linked glycans, the bulk of which are present within its B domain. However, deletion of the B domain is not deleterious to FVIII expression and function. In addition, FVIII has several potential Asn-linked glycosylation sequons in its other domains, of which four have been experimentally deduced to be glycosylated: Asn41 and Asn239 in the A1 domain, Asn1810 in the A3 domain and Asn2118 in the C1 domain. Of these, Asn239 and Asn2118 have been determined to comprise complex oligomannose structures. Such complex oligomannose structures have been proposed to play a role in mediating interaction with immunomodulatory cells (i.e. dendritic cells). The present study was aimed at delineating the role(s) of these four Asn-linked glycans in the expression of FVIII in vitro and in vivo and to identify possible bioengineering targets to influence FVIII expression, clearance and processing by immunomodulatory cells. Individual Asn residues were mutated to glutamine (Gln) to create single and multiple glycosylation mutants in both full length (FVIII-WT) and B domain-deleted (BDD)–FVIII, by site-directed mutagenesis. A variant of BDD-FVIII completely devoid of Asn-linked glycans, designated as Degly-BDD-FVIII, was also generated. Transient transfections of the mutants were carried out in COS-1 and CHO cells and their secretion and function were analyzed and compared to that of the respective native FVIII proteins. Antigen and activity assays revealed that the secretion and function of Asn41Gln and Asn1810Gln mutants were only modestly affected (85–90% of WT) but a more significant reduction was observed in the case of Asn239Gln mutant (35–50% of WT). Interestingly, there was no significant difference in secretion or function for Asn2118Gln in either FVIII-WT or BDD-FVIII protein backbones. The double mutants, Asn41/239Gln and Asn239/2118Gln behaved similarly to that of Asn239Gln mutant (30–45% of WT). The triple mutants, Asn41/239/2118Gln and Asn239/1810/2118Gln showed a further decline in secretion (∼30-40% of WT) while Degly-BDD-FVIII demonstrated secretion of only about 15–20% of BDD-FVIII. The FVIII specific activity of each of these glycosylation mutants was similar to the native FVIII proteins. An ELISA-based Von Willebrand Factor (VWF) binding assay revealed no significant differences between immunoaffinity-purified FVIII-WT and Asn2118Gln mutant in their ability to bind VWF. Findings from in vivo expression (via hydrodynamic tail vein injection of plasmid DNA) of these glycosylation mutants in a F8−/− (exon 16 knock-out) hemophilia A mouse model were similar to the in vitro results in the cell lines. Plasma FVIII activity levels were measured 24 hrs post-injection via orbital bleed. While Asn2118Gln (5.2 – 6 U/mL) did not exhibit any difference from BDD-FVIII (4.8 – 5.9 U/ml), Asn239Gln (1.9 – 2.4 U/ml) was expressed at less than 50% of BDD-FVIII levels. The expression of Degly-BDD-FVIII (0.4 – 0.7 U/ml) was further reduced to ∼10% of BDD-FVIII levels. Taken together, these results indicate that of the four Asn-linked glycans, Asn239 was the most crucial for proper secretion of FVIII whereas, Asn2118 did not contribute to the efficiency of FVIII expression. The oligosaccharide structure on Asn239 is positioned at the A1-A2 interface and likely contributes to proper protein folding. However, the sugar moieties on Asn2118 have been shown to be positioned at the A3-C1 domain interface and postulated to participate in packing and stabilization (Shen et al, 2008). This would have suggested that disruption of this residue within the C1 domain might have a deleterious effect on protein secretion or function. Our results with Asn2118Gln in both FVIII-WT and BDD-FVIII protein backbones suggest that this Asn-linked glycosylation can be eliminated without any impact on FVIII expression or function including no impact on FVIII-VWF interaction. This Asn-linked glycan, therefore, could be targeted in bioengineering strategies to determine if eliminating this particular oligomannose structure might impact mannose-receptor mediated uptake of FVIII by dendritic cells.

Disclosures:
No relevant conflicts of interest to declare.