Mapping the Binding Region on the Low Density Lipoprotein Receptor for Blood Coagulation Factor VIII

Autor: James H. Kurasawa, Svetlana A Shestopal, Andrey G Sarafanov, Elena Karnaukhova, Timothy K Lee, Evi Struble
Rok vydání: 2013
Předmět:
congenital
hereditary
and neonatal diseases and abnormalities
animal diseases
Molecular Sequence Data
Plasma protein binding
Immunoglobulin light chain
Binding
Competitive
Biochemistry
LDL-receptor-related protein-associated protein
law.invention
law
hemic and lymphatic diseases
Protein Interaction Mapping
Humans
Amino Acid Sequence
LDL-Receptor Related Protein-Associated Protein
Binding site
Receptor
Molecular Biology
Peptide sequence
Binding Sites
Factor VIII
Sequence Homology
Amino Acid
biology
Chemistry
Circular Dichroism
food and beverages
nutritional and metabolic diseases
Cell Biology
Surface Plasmon Resonance
Molecular biology
Peptide Fragments
Recombinant Proteins
Kinetics
Receptors
LDL
Mutation
Protein Structure and Folding
LDL receptor
Mutagenesis
Site-Directed
biology.protein
Recombinant DNA
lipids (amino acids
peptides
and proteins)
Protein Binding
Zdroj: Journal of Biological Chemistry. 288:22033-22041
ISSN: 0021-9258
Popis: 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.
Databáze: OpenAIRE
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