Binding of an antibody mimetic of the human low density lipoprotein receptor to apolipoprotein E is governed through electrostatic forces. Studies using site-directed mutagenesis and molecular modeling.
Monoclonal antibody 2E8 is specific for an epitope that coincides with the binding site of the low density lipoprotein receptor (LDLR) on human apoE. Its reactivity with apoE variants resembles that of the LDLR: it binds well with apoE3 and poorly with apoE2. The heavy chain complementarity-determining region (CDRH) 2 of 2E8 shows homology to the ligand-binding domain of the LDLR. To define better the structural basis of the 2E8/apoE interaction and particularly the role of electrostatic interactions, we generated and characterized a panel of 2E8 variants. Replacement of acidic residues in the 2E8 CDRHs showed that Asp(52), Glu(53), and Asp(56) are essential for high-affinity binding. Although Asp(31) (CDRH1), Glu(58) (CDRH2), and Asp(97) (CDRH3) did not appear to be critical, the Asp(97) --> Ala variant acquired reactivity with apoE2. A Thr(57) --> Glu substitution increased affinity for both apoE3 and apoE2. The affinities of wild-type 2E8 and variants for apoE varied inversely with ionic strength, suggesting that electrostatic forces contribute to both antigen binding and isoform specificity. We propose a model of the 2E8.apoE immune complex that is based on the 2E8 and apoE crystal structures and that is consistent with the apoE-binding properties of wild-type 2E8 and its variants. Given the similarity between the LDLR and 2E8 in terms of specificity, the LDLR/ligand interaction may also have an important electrostatic component.
Raffaï
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Lipoprotein and Atherosclerosis Group, University of Ottawa Heart Institute, Ottawa Civic Hospital, Ottawa, Ontario K1Y TW7, Canada.
Weisgraber
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KH
MacKenzie
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Rupp
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B
Rassart
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E
Hirama
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Innerarity
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TL
Milne
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D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism*; D000936:Antigen-Antibody Complex / Q000737:chemistry; D001057:Apolipoproteins E / Q000378:metabolism*; D003543:Cysteamine / Q000494:pharmacology; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D011973:Receptors, LDL / Q000276:immunology / Q000378:metabolism*; D055672:Static Electricity; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D001057:Apolipoproteins E; D007143:Immunoglobulin Heavy Chains; D011973:Receptors, LDL; D003543:Cysteamine