Identification of Receptor Ligands in Apo B100 Reveals Potential Functional Domains

Abstract

LDL, VLDL and other members of the low-density lipoparticles (LLPs) enter cells through a large family of receptors. The actual receptor ligand(s) in apolipoprotein B100, one of the main proteins of LLP, remain(s) unknown. The objective of this study was to identify true receptor ligand(s) in apo B100, a molecule of 4,563 residues. Apo B100 contains 33 analogues of Cardin-Weintraub Arginine/Lysine-based receptor ligand motifs and shares key Lysine motifs and sequence similarity with the LDL receptor-associated protein, RAP, MESD, and heat shock proteins. Eleven FITC-labeled synthetic peptides of 21 – 42 residues, with at least one ligand, were tested for binding and internalization using HeLa cells. All peptides bind but display different binding capacities and patterns. Peptides B0013, B0582, B2366, and B2932 mediate endocytosis and appear in distinct sites in the cytoplasm. B0708 and B3181 bind and remain on the cell surface as aggregates/clusters. Peptides B3119 (Site A) and B3347 (Site B), the putative ligands showed low binding and no cell entry capacity. Apo B100 regions in this study share similarities with related proteins of known function including chaperone proteins and Apo BEC Stimulating Protein, and not directly related proteins, e.g., the DNA-binding domain of Interferon Regulatory Factors, MSX2-Interacting Protein, and snake venom Zinc metalloproteinase-disintegrin-like proteins.

The Structure of ApoB100 from Human Low-density Lipoprotein

“…Black arrows point in the N-to-C direction of the protein chains D. View of the R-face with insert 4 depicted as ribbons along with insert 0 (baseplate insert) and insert 2 depicted as ropes. E. Inserts 6 and 8 are depicted as ropes with proline residues (maroon), fragment B3181[57] (purple), residue 3276 (pink), and disulfide bond (yellow) highlighted.…”

“…Although the function of insert 6, as with insert 8, is not apparent from the structure alone, current evidence suggests that the most probable location of the LDL-receptor (LDL-r) binding site (RBS) falls within the region loosely spanning these inserts, with some of the strongest evidence pointing to regions within insert 6 [5,[56][57][58][59][60][61][62][63][64][65]. For example, of the two most widely discussed proposed locations, termed Site A and Site B, Site A is positioned partially within insert 6 (residues 3173-3185), while Site B falls within the β-belt between inserts 6 and 8 (residues 3379-3395).…”

“…For example, of the two most widely discussed proposed locations, termed Site A and Site B, Site A is positioned partially within insert 6 (residues 3173-3185), while Site B falls within the β-belt between inserts 6 and 8 (residues 3379-3395). A more recent study identified the fragment between residues 3209-3242 within the α-helix of insert 6 (Figure 3E) as the principal candidate for the RBS [57], while another study reported that an antibody whose epitope contains residue 3276, which is within the β-domain of insert 6 (Figure 3E), completely blocks LDL-r binding [56]. As a preliminary experiment, we used AF2 to predict the potential complex of LDL-r and insert 6, resulting in a model that located the binding site fully within the fragment spanning residues 3209-3242, albeit with only modest pae scores at the interface (Extended Data Figure 10A-C).…”

“…The top-ranking AF2 prediction of LDL-r (colored sequentially) in complex with insert 6 (tan). The residues corresponding to fragment B3181 [57] are colored green. Also shown (right) are the predicted contacts (within 6 Å) between the two protein chains colored by relative pae score (yellow = lower pae and higher confidence).…”

“…The top-ranking AF2 prediction of LDL-r (colored sequentially) in complex with insert 6 (tan). The residues corresponding to fragment B3181 [57]…”