Dependence on vitamin K-dependent protein S for eukaryotic cell secretion of the beta-chain of C4b-binding protein.

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 ß-chain (C4BPß(+)), the chains being linked by disulfide bridges. PS binds to the ß-chain with high affinity. In plasma, PS is in molar excess over C4BPß(+) and due to the high affinity, all C4BPß(+) molecules contain a bound PS. Taken together with the observation that PS-deficient patients have decreased levels of C4BPß(+), this raises the question of whether PS is important for secretion of the ß-chain from the cell. To test this hypothesis, HEK293 cells were stably and transiently transfected with ß-chain cDNA in combinations with cDNAs for PS and/or the α-chain. The concentration of ß-chains in the medium increased after co-transfection with PS cDNA, but not by α-chain cDNA, suggesting secretion of the ß-chains from the cells to be dependent on concomitant synthesis of PS, but not of the α-chains. Thus, ß-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 ß-chain were formed intracellularly, in the endoplasmic reticulum. In conclusion, our results demonstrate that successful secretion of ß-chains depends on intracellular complex formation with PS, but not on the α-chains. This provides an explanation for the decreased ß-chain levels observed in PS-deficient patients.