Abstract
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.
Highlights
ApoE, a 34-kDa protein composed of 299 amino acids, is an important functional component of chylomicrons and very low, intermediate, and high density lipoproteins
In a lipid-free form, the 22-kDa amino-terminal domain is folded into an elongated four-helix bundle; basic residues that have been implicated in low density lipoprotein receptor (LDLR) binding are situated on helix 4, where they form a region with strong electropositive potential [3]
The LDLR, an integral membrane protein composed of 831 amino acids, mediates the uptake of lipoproteins through its ability to bind to apoE and apoB
Summary
Vol 275, No 10, Issue of March 10, pp. 7109 –7116, 2000 Printed in U.S.A. (Received for publication, September 1, 1999, and in revised form, December 14, 1999). Monoclonal antibody 2E8 is specific for an epitope that coincides with the binding site of the low density lipoprotein receptor (LDLR) on human apoE. Electrostatic Forces in Antibody Mimicry in coordinating the divalent ion These acidic residues were thought to be available for ionic interactions with the basic residues in the receptor-binding sites of apoE and apoB; these new structural observations have necessitated a reassessment of the existing models for the binding of the LDLR to its ligands [9]. The antigen-binding site, or paratope of an antibody, is composed mainly of amino acids residing within the three heavy and light chain complementarity-determining regions [12], which exist as looped structures extending from the surface of the folded variable domains [13]. We propose a model of the 2E81⁄7apoE immune complex that is consistent with the tertiary structures of the 2E8 paratope and its epitope on apoE and with the apoE-binding characteristics of the 2E8 variants
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