title: Generation of a Potent Low Density Lipoprotein Receptor-related Protein 1 (LRP1) Antagonist by Engineering a Stable Form of the Receptor-associated Protein (RAP) D3 Domain.
abstract: The low density lipoprotein receptor-related protein 1 (LRP1) is a member of the low density lipoprotein receptor family and plays important roles in a number of physiological and pathological processes. Expression of LRP1 requires the receptor-associated protein (RAP), a molecular chaperone that binds LRP1 and other low density lipoprotein receptor family members in the endoplasmic reticulum and traffics with them to the Golgi where the acidic environment causes its dissociation. Exogenously added RAP is a potent LRP1 antagonist and binds to LRP1 on the cell surface, preventing ligands from binding. Following endocytosis, RAP dissociates in the acidic endosome, allowing LRP1 to recycle back to the cell surface. The acid-induced dissociation of RAP is mediated by its D3 domain, a relatively unstable three-helical bundle that denatures at pH <6.2 due to protonation of key histidine residues on helices 2 and 3. To develop an LRP1 inhibitor that does not dissociate at low pH, we introduced a disulfide bond between the second and third helices in the RAP D3 domain. By combining this disulfide bond with elimination of key histidine residues, we generated a stable RAP molecule that is resistant to both pH- and heat-induced denaturation. This molecule bound to LRP1 with high affinity at both neutral and acidic pH and proved to be a potent inhibitor of LRP1 function both in vitro and in vivo, suggesting that our stable RAP molecule may be useful in multiple pathological settings where LRP1 blockade has been shown to be effective.
authors.affiliation: From the Center for Vascular and Inflammatory Disease and the Department of Surgery, University of Maryland School of Medicine, Baltimore, Maryland 21201.
authors.forename: Joni M
authors.initials: JM
authors.lastname: Prasad
authors.affiliation: From the Center for Vascular and Inflammatory Disease and the Department of Surgery, University of Maryland School of Medicine, Baltimore, Maryland 21201.
authors.forename: Mary
authors.initials: M
authors.lastname: Migliorini
authors.affiliation: From the Center for Vascular and Inflammatory Disease and the Department of Surgery, University of Maryland School of Medicine, Baltimore, Maryland 21201.
authors.forename: Rebeca
authors.initials: R
authors.lastname: Galisteo
authors.affiliation: From the Center for Vascular and Inflammatory Disease and the Department of Surgery, University of Maryland School of Medicine, Baltimore, Maryland 21201.
authors.forename: Dudley K
authors.initials: DK
authors.lastname: Strickland
mesh_terms: D000818:Animals; D002460:Cell Line; D006801:Humans; D006863:Hydrogen-Ion Concentration; D026563:LDL-Receptor Related Protein-Associated Protein / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology*; D026503:Low Density Lipoprotein Receptor-Related Protein-1 / Q000037:antagonists & inhibitors*; D051379:Mice; D008810:Mice, Inbred C57BL; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D011485:Protein Binding; D011489:Protein Denaturation; D015202:Protein Engineering; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011973:Receptors, LDL / Q000037:antagonists & inhibitors*; D025521:Tumor Suppressor Proteins / Q000037:antagonists & inhibitors*
chemical_list: D026563:LDL-Receptor Related Protein-Associated Protein; C475476:LRP1 protein, human; D026503:Low Density Lipoprotein Receptor-Related Protein-1; C486645:Lrp1 protein, mouse; D010446:Peptide Fragments; D011973:Receptors, LDL; D025521:Tumor Suppressor Proteins
keywords: LRP1; RAP; histidine switch; protein stability; protein structure; receptor endocytosis; site-directed mutagenesis; surface plasmon resonance (SPR)