Construction and in vitro functional evaluation of a low-density lipoprotein receptor/transferrin fusion protein as a therapeutic tool for familial hypercholesterolemia

A cDNA sequence encoding a soluble form of the human low-density lipoprotei n receptor (LDL-R) was produced by RT-PCR amplification. This form of the r eceptor contains the N-terminal cysteine-rich domain, the EGF homology doma in, and the serine/threonine-rich domain, but lacks the membrane anchor as well as the cytoplasmic domain. By the same technical approach a cDNA seque nce encoding rabbit transferrin was generated. In-frame fusion of the two c DNAs produced a sequence encoding a chimeric protein potentially capable of binding LDL on the N-terminal side and the transferrin receptor on the C-t erminal side. It was expected that LDL bound to the chimeric protein could be internalized, targeted to an acidic compartment, and processed through t he pathway of the transferrin receptor. Cells transfected with the LDL-R/tr ansferrin cDNA translate, glycosylate, and secrete the corresponding protei n in the culture medium. The secreted protein binds LDL in a ligand-blottin g experiment. Finally, the chimeric protein mediates the binding and intern alization of LDL in mutant cells lacking the LDL receptor. In fact, Watanab e rabbit fibroblasts, incubated with the chimeric protein show a fourfold i ncrease in LDL binding, a fivefold increase in LDL internalization, and a s ixfold increase in LDL degradation, with respect to unincubated fibroblasts .