Molecular basis of LMAN1 in coordinating LMAN1-MCFD2 cargo receptor formation and ER-to-Golgi transport of FV/FVIII

Abstract

The LMAN1-MCFD2 (lectin, mannose binding 1/multiple coagulation factor deficiency protein 2) cargo receptor complex transports coagulation factors V (FV) and VIII (FVIII) from the endoplasmic reticulum (ER) to the ER-Golgi intermediate compartment (ERGIC). LMAN1 (ERGIC-53) is a hexameric transmembrane protein with a carbohydrate recognition domain (CRD) on the ER luminal side. Here, we show that mutations in the first beta sheet of the CRD abolish MCFD2 binding without affecting the mannose binding, suggesting that LMAN1 interacts with MCFD2 through its N-terminal beta sheet, consistent with recently reported crystal structures of the CRD-MCFD2 complex. Mutations in the Ca(2+)- and sugar-binding sites of the CRD disrupt FV and FVIII interactions, without affecting MCFD2 binding. This interaction is independent of MCFD2, as LMAN1 mutants defective in MCFD2 binding can still interact with FVIII. Thus, the CRD of LMAN1 contains distinct, separable binding sites for both its partner protein (MCFD2) and the cargo proteins (FV/FVIII). Monomeric LMAN1 mutants are defective in ER exit and unable to interact with MCFD2, suggesting that the oligomerization of LMAN1 is necessary for its cargo receptor function. These results point to a central role of LMAN1 in regulating the binding in the ER and the subsequent release in the ERGIC of FV and FVIII.

Figure 1

The CRD domain of LMAN1 is responsible for MCFD2 binding in vivo. (A) Diagram of LMAN1 mutants used in the experiments. SS, signal peptide; F, Flag; TM, transmembrane. KKFF represents the last 4 amino acids of wild-type LMAN1 important for ER exit and retrieval, while KKAA represents mutations of the last 2 amino acids. (B) Co-IP of LMAN1 mutants with MCFD2. COS1 cells were cotransfected with Flag-tagged LMAN1 mutants and myc-tagged wild-type MCFD2. Cell lysates were immunoprecipitated with anti-myc for MCFD2 and anti-Flag for LMAN1.

Figure 2

The first β-sheet of the CRD is the binding motif for MCFD2. (A) Alignment of the first 56 amino acids of the mature human LMAN1 with the orthologs from chimp, monkey, bovine, rat, and mouse. Amino acid residues that differ from the consensus are boxed. The secondary structures are indicated on top of the sequences. The locations of deletions (arrows) and point mutations (arrow heads) used in the study are denoted under the sequences. (B) Mannose binding activities of different LMAN1 mutants. COS1 cells were transfected with the wild-type and the indicated LMAN1 mutants. LMAN1 proteins that are in the cell lysate and that are eluted from the mannose agarose beads are detected by Western blot analysis. Lysate lanes represent 20% of the input added to the mannose beads. (C) Co-IP of LMAN1 mutants with MCFD2. COS1 cells were cotransfected with Flag-tagged LMAN1 mutants and myc-tagged wild-type MCFD2. Cell lysates were immunoprecipitated with anti-myc for MCFD2 and anti-Flag for LMAN1.

Figure 3

Oligomerization of LMAN1 is required for MCFD2 binding. (A) Mannose binding activities of different LMAN1 mutants. COS1 cells were transfected with the wild-type and the indicated LMAN1 mutants. LMAN1 proteins that are in the cell lysate and that are eluted from the mannose agarose beads are detected by Western blot analysis. Lysate lanes represent 20% of the input added to the mannose beads. (B) Co-IP of LMAN1 mutants with MCFD2. COS1 cells were cotransfected with Flag-tagged ΔHelix or ΔHM mutant and myc-tagged wild-type MCFD2. Cell lysates were immunoprecipitated with anti-myc for MCFD2 and anti-Flag for LMAN1.

Figure 4

LMAN1 monomer is defective in ER exit. (A) Immunofluorescence staining of the wild-type LMAN1 (WT), ΔHelix, and ΔHM mutants. LMAN1 proteins were detected with a monoclonal anti-Flag antibody. TRAP-α was used as an ER marker. (B) ER exit of wild-type LMAN1, ΔHelix, ΔHM, KKAA, and C466A/C475A mutants. HeLa cells were transfected with the indicated LMAN1 constructs. Membrane from transfected cells were incubated with rat liver cytosol. COPII vesicles were isolated and analyzed for the presence of a resident ER protein, ribophorin I, the endogenous LMAN1, the transfected LMAN1 mutants, and a control cargo protein, SEC22B.

Figure 5

BiFC analysis of the interactions of LMAN1 mutants with MCFD2 in living cells. (A) Fusion protein levels in cotransfection experiments were detected by immunoblotting (IB) using the indicated antibodies. Cells were lysed immediately after microscopic observations. (B) BiFC signals relative to the wild-type LMAN1 and MCFD2 fusion proteins. Fifty cotransfected cells from each set of transfection were counted, with the background subtracted for each image. Bars represent means ± SD from 3 replicates. Statistical analysis was performed using the Student t test, and asterisks indicate significant differences (P < .05).

Figure 6

The Ca²⁺- and sugar-binding sites in the CRD interacts with FV and FVIII. COS-1 cells were cotransfected with the wild-type or indicated LMAN1 mutants, along with either FV (A) or FVIII (A-B). Cells were incubated with or without DSP before lysis. Cell lysates were immunoprecipitated with preimmune serum (pre) as a negative control, anti-Flag (Flag) for LMAN1, and anti-FV (F5) or -FVIII (F8) for the indicated coagulation factor.