N1922S Mutation In the Factor VIII A3 Domain Produces a Rate-Limiting, Domain-Specific Folding Defect Leading to Hyposecretion of a Functional Protein

Abstract 2209

Factor VIII (fVIII) contains a domain sequence designated A1-A2-B-ap-A3-C1-C2. Mutation of Asn1922 to Ser (N1922S) in the A3 domain results in moderate to severe hemophilia A. However, it is unclear whether this mutation leads to secretion of cross-reactive material positive dysfunctional protein or decreased secretion of fVIII protein. We investigated the fVIII activity and antigen levels in a N1922S patient and found an activity level of 1.7% and an antigen of <4 ng/ml suggesting a secretion defect. To investigate this further, we constructed a B-domain deleted human fVIII cDNA encoding this mutation, designated N1922S fVIII, and compared its heterologous expression to non-mutated “wild-type” fVIII (wt-fVIII) in a baby hamster kidney-derived cell line. Levels of fVIII expression in cell culture media measured by antigen-capture ELISA were 0.011 and 0.73 mg/ml for N1922S fVIII and wt-fVIII, respectively. The corresponding media levels of fVIII activity measured by one-stage coagulation assay were 0.03 and 3.5 U/ml for N1922S fVIII and wt-fVIII, respectively. These values correspond to specific activities of 2800 and 4800 U/mg for N1922S fVIII and wt-fVIII, respectively. Consistent with this, both N1922S fVIII and wt-fVIII were over twenty-fold activatable by thrombin in the one-stage coagulation assay. These comparable coagulant activities of N1922S fVIII and wt-fVIII indicate that the N1922S mutation produces a kinetic block in the synthesis of a functionally normal fVIII protein. In contrast to media levels of fVIII, in-cell Western analysis revealed that intracellular levels of N1922S fVIII were similar to wt-fVIII. However, specific activities of N1922S fVIII and wt-fVIII in cell lysates were 290 and 6800 U/mg, respectively, indicating the presence of large amounts of a non-functional N1922S fVIII folding intermediate. Immunofluorescence microscopy demonstrated co-localization of wt-fVIII with both endoplasmic reticulum (ER)- and Golgi-resident proteins. In contrast, N1922S fVIII co-localized only with ER-resident proteins, indicating a kinetic block in intracellular trafficking between the ER and the Golgi. To investigate further whether the defect in N1922S fVIII trafficking was related to protein misfolding, we compared lysate-to-media antigenic ratios of N1922S fVIII and wt-fVIII using a panel of non-overlapping monoclonal antibodies (MAbs) consisting of one anti-A1, one anti-A2, three anti-A3, one anti-C1 and two anti-C2 MAbs. Lysate-to-media antigenic ratios for the anti-A1, anti-A2 and anti-C2 MAbs were similar between N1922S fVIII and wt-fVIII. In contrast, lysate-to-media ratios of the three anti-A3 MAbs and the anti-C1 MAb were markedly decreased for N1922S fVIII compared to wt-fVIII. This result indicates that the A1, A2 and C2 domains in N1922S fVIII fold independently into antigenically intact tertiary structures, but that folding is stalled in mutant A3 domain and its contiguous C1 domain. Because Asn1922 is buried in the interface of the two cupredoxin-like A3 subdomains in the two available X-ray structures of fVIII (Shen BW et al. Blood 2008;111:1240-1247; Ngo JC, et al. Structure 2008;16:597-606), the kinetic defect associated with this mutation may be due to slow association of intact A3 subdomains. This domain-specific defect in protein folding and intracellular trafficking is a novel mechanism for secretion defects leading to hemophilia A.