Expression and characterization of a truncated, soluble, low-density lipoprotein receptor.
The low-density lipoprotein (LDL) receptor mediates the clearance of apolipoprotein B- and E-containing lipoproteins from the bloodstream. In the current study, we characterized the binding properties of the amino terminus of the LDL receptor. We produced a recombinant baculovirus that encoded the first 354 amino acids, including the endogenous signal sequence, of the human LDL receptor. This truncated receptor protein (LDL-R354) was secreted from recombinant baculovirus-infected Spodoptera frugiperda (Sf-21) cells. Upon electrophoresis, LDL-R354 migrated with a mobility of 55,000. Treatment of cells with tunicamycin decreased the size of the truncated receptor, suggesting the presence of asparagine-linked carbohydrates. Nonreducing SDS-PAGE resulted in at least three discernible bands with M(r)s consistent with the truncated receptor existing as monomers and multimers, suggesting the possibility of intermolecular disulfide cross-linking. All forms of the truncated receptor bound LDL on a ligand blot in a calcium-dependent manner. The purified truncated receptor bound 125I-LDL with high affinity in a competitive binding assay (IC50 = 0.8 microgram/ml). LDL-R354 also bound calcium. This interaction was sensitive to the conformation of the ligand binding domain, as reduction of the disulfide bonds greatly decreased the affinity of the receptor for calcium. The ligand and calcium binding activities of this truncated receptor protein demonstrate that the ligand binding domain of the LDL receptor can fold into a functionally active protein.
Dirlam
K A
KA
Department of Biochemistry, University of Wisconsin-Madison 53706, USA.
Gretch
D G
DG
LaCount
D J
DJ
Sturley
S L
SL
Attie
A D
AD
D000818:Animals; D002118:Calcium / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007526:Isoelectric Point; D008077:Lipoproteins, LDL / Q000378:metabolism; D008970:Molecular Weight; D011973:Receptors, LDL / Q000737:chemistry*; D015183:Restriction Mapping; D012995:Solubility; D018411:Spodoptera
D008077:Lipoproteins, LDL; D011973:Receptors, LDL; D002118:Calcium