Refolding and characterization of the functional ligand-binding domain of human lectin-like oxidized LDL receptor.
Lectin-like oxidized low-density lipoprotein receptor (LOX-1), a type II membrane protein that can recognize a variety of structurally unrelated macromolecules, plays an important role in host defense and is implicated in atherogenesis. To understand the interaction between human LOX-1 and its ligands, in this study the functional C-type lectin-like domain (CTLD) of LOX-1 was reconstituted at high efficiency from inactive aggregates in Escherichia coli using a refolding technique based on an artificial chaperone. The CD spectra of the purified domain suggested that the domain has alpha-helical structure and the blue shift of Trp residues was observed on refolding of the domain. Like wild-type hLOX-1, the refolded CTLD domain was able to bind modified LDL. Thus, even though CTLD contains six Cys residues that form disulfide bonds, it recovered its specific binding ability on refolding. This suggests that the correct disulfide bonds in CTLD were formed by the artificial chaperone technique. Although the domain lacked N-glycosylation, it showed high affinity for its ligand in surface plasmon resonance experiments. Thus, unglycosylated CTLD is sufficient for binding modified LDL.
Xie
Qiuhong
Q
National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan.
Matsunaga
Shigeru
S
Shi
Xiaohua
X
Ogawa
Setsuko
S
Niimi
Setsuko
S
Wen
Zhesheng
Z
Tokuyasu
Ken
K
Machida
Sachiko
S
D001665:Binding Sites; D002942:Circular Dichroism; D004926:Escherichia coli; D006031:Glycosylation; D006801:Humans; D008024:Ligands; D017510:Protein Folding; D020673:Protein Renaturation; D017434:Protein Structure, Tertiary; D011973:Receptors, LDL / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051131:Receptors, Oxidized LDL; D051127:Scavenger Receptors, Class E; D013050:Spectrometry, Fluorescence; D020349:Surface Plasmon Resonance; D014364:Tryptophan / Q000737:chemistry
D008024:Ligands; C495137:OLR1 protein, human; D011973:Receptors, LDL; D051131:Receptors, Oxidized LDL; D051127:Scavenger Receptors, Class E; D014364:Tryptophan