title: Mapping the binding region on the low density lipoprotein receptor for blood coagulation factor VIII.
abstract: Low density lipoprotein receptor (LDLR) was shown to mediate clearance of blood coagulation factor VIII (FVIII) from the circulation. To elucidate the mechanism of interaction of LDLR and FVIII, our objective was to identify the region of the receptor necessary for binding FVIII. Using surface plasmon resonance, we found that LDLR exodomain and its cluster of complement-type repeats (CRs) bind FVIII in the same mode. This indicated that the LDLR site for FVIII is located within the LDLR cluster. Similar results were obtained for another ligand of LDLR, α-2-macroglobulin receptor-associated protein (RAP), a common ligand of receptors from the LDLR family. We further generated a set of recombinant fragments of the LDLR cluster and assessed their structural integrity by binding to RAP and by circular dichroism. A number of fragments overlapping CR.2-5 of the cluster were positive for binding RAP and FVIII. The specificity of these interactions was tested by site-directed mutagenesis of conserved tryptophans within the LDLR fragments. For FVIII, the specificity was also tested using a single-chain variable antibody fragment directed against the FVIII light chain as a competitor. Both cases resulted in decreased binding, thus confirming its specificity. The mutagenic study also showed an importance of the conserved tryptophans in LDLR for both ligands, and the competitive binding results showed an involvement of the light chain of FVIII in its interaction with LDLR. In conclusion, the region of CR.2-5 of LDLR was defined as the binding site for FVIII and RAP.
authors.affiliation: Center for Biologics Evaluation and Research, Food and Drug Administration, Rockville, Maryland 20852, USA.
authors.forename: James H
authors.initials: JH
authors.lastname: Kurasawa
authors.forename: Svetlana A
authors.initials: SA
authors.lastname: Shestopal
authors.forename: Elena
authors.initials: E
authors.lastname: Karnaukhova
authors.forename: Evi B
authors.initials: EB
authors.lastname: Struble
authors.forename: Timothy K
authors.initials: TK
authors.lastname: Lee
authors.forename: Andrey G
authors.initials: AG
authors.lastname: Sarafanov
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D002942:Circular Dichroism; D005169:Factor VIII / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D026563:LDL-Receptor Related Protein-Associated Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D011973:Receptors, LDL / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance
chemical_list: D026563:LDL-Receptor Related Protein-Associated Protein; D010446:Peptide Fragments; D011973:Receptors, LDL; D011994:Recombinant Proteins; D005169:Factor VIII
keywords: Blood Coagulation Factors; Factor VIII; Hemophilia A; Lipoprotein Receptor; Receptor Structure-Function; Recombinant Protein Expression