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Mapping the binding regions of low-density lipoprotein receptor for coagulation factor VIII

Monday, October 24, 2011 — Poster Session I

Noon – 2:00 p.m.

Natcher Conference Center




  • J Kurasawa
  • S Shestopal
  • T Lee
  • A Sarafanov


Low-density lipoprotein receptor (LDLR) is member of a family of endocytic receptors that internalize various extracellular ligands for their further intracellular degradation. It has been previously found that LDLR regulates plasma levels of coagulation factor VIII (FVIII) in vivo (Bovenschen et al, 2005). In order to map the binding region of LDLR for FVIII, we expressed the extracellular ligand-binding domain of LDLR (a cluster of seven complement-type repeats (CRs)), and its overlapping fragments, using the baculovirus expression system. These fragments were then purified using diafiltration, affinity and size exclusion chromatography and tested for the binding using surface plasmon resonance (SPR). Initially, we tested the binding of the LDLR cluster with plasma-derived FVIII and receptor-associated protein (RAP), a folding chaperone for LDLR. Results showed that the binding curves of the expressed LDLR cluster and the commercial full-length LDLR were indeed comparable indicating a relevant structure of expressed fragments. Next, both RAP and FVIII were tested for binding to the overlapping LDLR fragments and found to bind to the same pattern of the fragments. The results indicate that both RAP and FVIII bind to multiple sites on LDLR constituting the same core region within LDLR exodomain.

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