All three LDL receptor homology regions of the LDL receptor-related protein bind multiple ligands.
The three complete human LDL receptor homology regions of the LDL receptor-related protein (sLRP2, sLRP3, and sLRP4) have been expressed in Pichia pastoris SMD1168 with constitutive coexpression of the receptor-associated protein (RAP). Each sLRP was purified to homogeneity after deglycosylation using a combination of anion-exchange and size exclusion chromatography. Mass spectrometry and N-terminal sequencing confirmed the identity of each fragment at purified yields of several milligrams per liter. Despite the large number of disulfide linkages and glycosylation sites in each LDL receptor homology region (sLRP), all were shown to be competent for binding to several LRP1 ligands. Each sLRP also bound human RAP, which is thought to be a generalized receptor antagonist, in solution-binding experiments. As expected, sLRP2 bound the receptor-binding domain of alpha(2)-macroglobulin (residues 1304-1451). All three sLRPs bound human apolipoprotein-enriched beta very low density lipoprotein, the canonical ligand for this receptor. All three sLRPs also bound lactoferrin and thrombin-protease nexin 1 complexes. Only sLRP4 bound thrombin-antithrombin III complexes. The results show that binding-competent LDL receptor homology regions (sLRPs) can be produced in high yield in P. pastoris and readily purified. Each sLRP has binding sites for multiple ligands, but not all ligand binding could be competed by RAP.
Croy
Johnny E
JE
Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0378, USA.
Shin
William D
WD
Knauer
Mary F
MF
Knauer
Daniel J
DJ
Komives
Elizabeth A
EA
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000378:metabolism; D001667:Binding, Competitive / Q000235:genetics; D006801:Humans; D026502:LDL-Receptor Related Proteins / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D026503:Low Density Lipoprotein Receptor-Related Protein-1 / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D026561:Low Density Lipoprotein Receptor-Related Protein-2 / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary / Q000235:genetics; D051381:Rats; D011973:Receptors, LDL / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D012995:Solubility; D014162:Transfection; D000511:alpha-Macroglobulins / Q000378:metabolism
D000906:Antibodies; D026502:LDL-Receptor Related Proteins; C419827:LRP3 protein, human; D008024:Ligands; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D026561:Low Density Lipoprotein Receptor-Related Protein-2; D011973:Receptors, LDL; D000511:alpha-Macroglobulins