The C Domain Mediates the Export of FVIII from the Endoplasmic Reticulum By Interacting with MCFD2

Coagulation factor V (FV) and VIII (FVIII) are large plasma glycoproteins important for hemostasis and thrombosis. After undergoing folding and quality control in the endoplasmic reticulum (ER), FV and FVIII are packaged into COPII (coat protein complex-II) vesicles for transport to the Golgi. We have previously shown that packaging of FV and FVIII into COPII vesicles requires a cargo receptor. This receptor is a Ca²⁺-dependent protein complex formed by LMAN1 and MCFD2, two proteins that cycle between the ER and the cis-Golgi. Mutations in either LMAN1 or MCFD2 cause the combined deficiency of FV and FVIII, a rare bleeding disorder characterized by the reduction of both FV and FVIII to 5-30% of normal. Cargo receptors are thought to be transmembrane proteins that interact with cargo in the ER lumen and with the COPII coat on the cytoplasmic side of the ER. However, it is not clear how this cargo receptor recognizes and transports cargo. The requirement of a soluble protein (MCFD2) is a unique feature that has not been observed in other known cargo receptors.

The basic hypothesis in the receptor-mediated ER-to-Golgi transport model is that specific signals are embedded in cargo proteins that are recognized by their cognate receptors. We previously showed that FV and FVIII interact with both LMAN1 and MCFD2. To identify signals in FV and FVIII that are recognized by MCFD2, we performed immunoprecipitation (IP) of Myc-tagged MCFD2 with a series of Flag-tagged FVIII domain deletion mutants. The light chain of FVIII co-immunoprecipitates with MCFD2, using either anti-Myc or anti-Flag antibodies. The co-IP is mediated through the C domain, not the A3 domain of the light chain. When expressed individually, the C1 domain retains strong co-IP with MCFD2, while the C2 domain shows weaker co-IP with MCFD2. Similarly, only the C domain of FV can co-IP with MCFD2, consistent with the notion that the signal recognized by the LMAN1-MCFD2 complex is a common feature shared by FV and FVIII. To confirm the interaction of the C domain with MCFD2, we used the Bimolecular Fluorescence Complementation assay to detect the interaction in situ. MCFD2 and C domain mutants were separately fused to the N-terminal and the C-terminal halves of the yellow fluorescent protein. When the C1 domain and MCFD2 fusion proteins were co-transfected into HeLa cells, fluorescence signals were detected in live cells, indicating that an interaction between the C1 domain and MCFD2. Transfection of the A3 domain of FVIII produced no fluorescence signals.

We further found that the C1 domain of the light chain can significantly increase the heavy chain secretion in pulse-chase experiments. To examine the interaction of MCFD2 with FVIII in vivo, we generated transgenic mice expressing the heavy chain (HC) and light chain (LC) of canine FVIII driven by the liver-specific transthyretin promoter that have been bred into the hemophilia A background. Transgene expression is 30-40 ng/ml for HC and 150-250 ng/ml for LC. We crossed these canine FVIII transgenic mice with our MCFD2 knockout mice and obtained the Mcfd2+/+/HC, Mcfd2-/-/HC, Mcfd2+/+/LC and Mcfd2-/-/LC mouse lines, all of which are in the hemophilia A background. HC and LC levels in plasma were measured by canine FVIII-specific ELISA in 3 month-old mice. Results showed that the plasma LC level was 40% lower in Mcfd2-/- / LC mice than that in Mcfd2+/+/ LC mice (P<0.01), while plasma HC was indistinguishable between Mcfd2+/+/HC and Mcfd2-/-/HC mice. These results indicate that efficient LC secretion is dependent on MCFD2 in vivo, consistent with our cell-based results.

In conclusion, our results suggest that MCFD2 recognizes sorting signals located in the C1 and C2 domains of FV and FVIII. The identification of such signals validates the specific cargo and cargo receptor pairing and highlights a direct role of MCFD2 in ER-to-Golgi transport of FV and FVIII.