Abstract
The anticoagulant vitamin K-dependent protein S (PS) circulates in plasma in two forms, 30% free and 70% being bound to the complement regulatory protein C4b-binding protein (C4BP). The major C4BP isoform consists of 7 α-chains and 1 β-chain (C4BPβ(+)), the chains being linked by disulfide bridges. PS binds to the β-chain with high affinity. In plasma, PS is in molar excess over C4BPβ(+) and due to the high affinity, all C4BPβ(+) molecules contain a bound PS. Taken together with the observation that PS-deficient patients have decreased levels of C4BPβ(+), this raises the question of whether PS is important for secretion of the β-chain from the cell. To test this hypothesis, HEK293 cells were stably and transiently transfected with β-chain cDNA in combinations with cDNAs for PS and/or the α-chain. The concentration of β-chains in the medium increased after co-transfection with PS cDNA, but not by α-chain cDNA, suggesting secretion of the β-chains from the cells to be dependent on concomitant synthesis of PS, but not of the α-chains. Thus, β-chains that were not disulfide-linked to the α-chains were secreted in complex with PS, either as monomers or dimers. Pulse-chase demonstrated that the complexes between PS and β-chain were formed intracellularly, in the endoplasmic reticulum. In conclusion, our results demonstrate that successful secretion of β-chains depends on intracellular complex formation with PS, but not on the α-chains. This provides an explanation for the decreased β-chain levels observed in PS-deficient patients.
Highlights
32038 JOURNAL OF BIOLOGICAL CHEMISTRY inhibitor [2, 3]
The binding between C4b-binding protein (C4BP) -chain and protein S (PS) is of very high affinity (0.1 nM) [27], and because PS circulates in molar surplus over C4BPϩ, PS occupies all -chains in human plasma
Proteins in cell medium collected from HEK293 cells stably transfected with C4BPB/PROS1 in pBudCE4.1 and C4BPA in pcDNA3 were subjected to barium citrate absorption, and the concentration of C4BP, C4BP -chain, and PS was measured in the obtained fractions
Summary
Plasma PS [29], recombinant PS [30], and plasma C4BPϩ [31, 32] were purified, as previously described. HEK293, HepG2, Hep3B, and MIHA [35] cell lines were cultured in complete medium, DMEM (Invitrogen) supplemented with 4 mM L-glutamine (Invitrogen), 50 units/ml penicillin, 50 g/ml streptomycin (Invitrogen), 10% fetal bovine serum (Invitrogen), and 10 g/ml vitamin K The medium was replaced with methionine/cysteine- and serumfree DMEM (Invitrogen) supplemented with 4 mM L-glutamine, 50 units/ml penicillin, 50 g/ml streptomycin, and 10 g/ml vitamin K, and the cells were incubated for 60 min. The individual protein concentrations were measured by using different sets of in-house antibodies, as described in the supplemental Experimental Procedures. Stepwise Purification and Characterization of Recombinant PS and C4BP—About 900 ml of OptiMEM collected from cells stably transfected with PROS1 and C4BPB in pBud, and C4BPA in pcDNA were used to analyze the different components. The purified proteins were frozen and stored at Ϫ20 °C until analysis
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