LMAN1 and MCFD2 Form a Cargo Receptor Complex and Interact withCoagulation Factor VIII in the Early SecretoryPathway

Bin Zhang,Randal J Kaufman,David Ginsburg

doi:10.1074/jbc.m502160200

Bin Zhang, Randal J Kaufman + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m502160200

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Abstract

Mutations in LMAN1 (ERGIC-53) and MCFD2 are the causes of a human genetic disorder, combined deficiency of coagulation factor V and factor VIII. LMAN1 is a type 1 transmembrane protein with homology to mannose-binding lectins. MCFD2 is a soluble EF-hand-containing protein that is retained in the endoplasmic reticulum through its interaction with LMAN1. We showed that endogenous LMAN1 and MCFD2 are present primarily in complex with each other with a 1:1 stoichiometry, although MCFD2 is not required for oligomerization of LMAN1. Using a cross-linking-immunoprecipitation assay, we detected a specific interaction of both LMAN1 and MCFD2 with factor VIII, with the B domain as the most likely site of interaction. We also present evidence that this interaction is independent of the glycosylation state of factor VIII but requires native calcium concentration in the endoplasmic reticulum. The interaction of MCFD2 with factor VIII appeared to be independent of LMAN1-MCFD2 complex formation. These results suggest that LMAN1 and MCFD2 form a cargo receptor complex and that the primary sorting signals residing in the B domain direct the binding of factor VIII to LMAN1-MCFD2 through calcium-dependent protein-protein interactions. MCFD2 may function to specifically recruit factor V and factor VIII to sites of transport vesicle budding within the endoplasmic reticulum lumen.

Highlights

Folded proteins destined for secretion by anterograde transport toward the Golgi are packaged in the ER1 into COPII-coated vesicles [1]
These results suggest that LMAN1 and MCFD2 form a cargo receptor complex and that the primary sorting signals residing in the B domain direct the binding of factor VIII to LMAN1-MCFD2 through calcium-dependent protein-protein interactions
Taken together with the apparently similar half-lives for both proteins by pulse-chase analysis (Fig. 1a), these results suggest that LMAN1 and MCFD2 are associated in a 1:1 stoichiometry

Summary

Introduction

Folded proteins destined for secretion by anterograde transport toward the Golgi are packaged in the ER1 into COPII-coated vesicles [1]. These results suggest that LMAN1 and MCFD2 form a cargo receptor complex and that the primary sorting signals residing in the B domain direct the binding of factor VIII to LMAN1-MCFD2 through calcium-dependent protein-protein interactions. The involvement of LMAN1 and MCFD2 in F5F8D suggests that they function as a cargo receptor for the ER-to-Golgi transport of FV and FVIII.

Results

Conclusion