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 lipoprotein receptor (LDL-R) was produced by RT-PCR amplification. This form of the receptor contains the N-terminal cysteine-rich domain, the EGF homology domain, and the serine/threonine-rich domain, but lacks the membrane anchor as well as the cytoplasmic domain. By the same technical approach a cDNA sequence encoding rabbit transferrin was generated. In-frame fusion of the two cDNAs produced a sequence encoding a chimeric protein potentially capable of binding LDL on the N-terminal side and the transferrin receptor on the C-terminal side. It was expected that LDL bound to the chimeric protein could be internalized, targeted to an acidic compartment, and processed through the pathway of the transferrin receptor. Cells transfected with the LDL-R/transferrin cDNA translate, glycosylate, and secrete the corresponding protein in the culture medium. The secreted protein binds LDL in a ligand-blotting experiment. Finally, the chimeric protein mediates the binding and internalization of LDL in mutant cells lacking the LDL receptor. In fact, Watanabe rabbit fibroblasts, incubated with the chimeric protein show a fourfold increase in LDL binding, a fivefold increase in LDL internalization, and a sixfold increase in LDL degradation, with respect to unincubated fibroblasts.
Parise
F
F
Dipartimento di Scienze Biomediche, Università di Modena e Reggio Emilia, Italy.
Simone
L
L
Croce
M A
MA
Ghisellini
M
M
Battini
R
R
Borghi
S
S
Tiozzo
R
R
Ferrari
S
S
Calandra
S
S
Ferrari
S
S
Calandra
S
S
Ferrari
S
S
D000818:Animals; D019556:COS Cells; D018076:DNA, Complementary; D005347:Fibroblasts / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006801:Humans; D006938:Hyperlipoproteinemia Type II / Q000628:therapy*; D008077:Lipoproteins, LDL / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011233:Precipitin Tests; D011817:Rabbits; D011973:Receptors, LDL / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D014168:Transferrin / Q000235:genetics* / Q000378:metabolism
D018076:DNA, Complementary; D008077:Lipoproteins, LDL; D011973:Receptors, LDL; D011993:Recombinant Fusion Proteins; D014168:Transferrin