A recombinant apoA-1--protein A hybrid reproduces the binding parameters of HDL to its receptor.
We have constructed a plasmid, pLM8, containing the coding sequence of the mature human apoA-1 fused to the coding sequence of the IgG-binding domains of protein A (PA) from Staphylococcus aureus. The hybrid gene is transcribed in Escherichia coli under the control of a heat-sensitive repressor, leading to the synthesis of large amounts of hybrid protein (apoA-1--PA). The hybrid protein was purified by denaturation with urea and alkali, renaturation and affinity chromatography on an IgG Sepharose column. ApoA-1--PA is soluble and has an Mr of 316 kd, as determined by gel filtration. This is five times the monomer size of 62 kd, predicted from the sequence and found by SDS-PAGE analysis. Cell surface binding activity of the hybrid protein was tested using two different cell types (J774 macrophages and Fao hepatocytes) and compared to human high density lipoprotein (HDL). High-affinity binding was found for both ligands in both cell lines (Kd = 3.4 X 10(-8)M in Fao cells, 4.9 X 10(-8) M in J774 cells for apoA-1--PA and 3.0 X 10(-8) M in Fao cells, 2.8 X 10(-8) M in J774 cells for HDL), with approximately 2 X 10(5) high-affinity binding sites per cell. ApoA-1--PA and HDL effectively competed with each other for binding to the cell surface. Additionally, they both bound to a 110-kd polypeptide on a ligand blot, identifying an HDL receptor. The binding parameters of HDL were very similar to those of apoA-1--PA.(ABSTRACT TRUNCATED AT 250 WORDS)
Monaco
L
L
European Molecular Biology Laboratory, Heidelberg, FRG.
Bond
H M
HM
Howell
K E
KE
Cortese
R
R
D000818:Animals; D016632:Apolipoprotein A-I; D001054:Apolipoproteins A / Q000235:genetics / Q000378:metabolism*; D001667:Binding, Competitive; D002352:Carrier Proteins; D002460:Cell Line; D005796:Genes; D006801:Humans; D007700:Kinetics; D008075:Lipoproteins, HDL / Q000378:metabolism*; D008114:Liver Neoplasms, Experimental; D010957:Plasmids; D055503:Protein Multimerization; D016601:RNA-Binding Proteins; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism*; D018110:Receptors, Lipoprotein; D011994:Recombinant Proteins / Q000378:metabolism*; D013205:Staphylococcal Protein A / Q000235:genetics / Q000378:metabolism*
D016632:Apolipoprotein A-I; D001054:Apolipoproteins A; D002352:Carrier Proteins; D008075:Lipoproteins, HDL; D016601:RNA-Binding Proteins; D011956:Receptors, Cell Surface; D018110:Receptors, Lipoprotein; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A; C031616:high density lipoprotein receptors; C075197:high density lipoprotein binding protein