Molecular Mapping of the Active Site of an Aging Antigen: Senescent Cell Antigen Requires Lysine(s) for Antigenicity and Is Located on an Anion-Binding Segment of Band 3 Membrane Transport Protein

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Abstract
An aging antigen, senescent cell antigen, resides on the 911 amino acid membrane protein band 3. It marks cells for removal by initiating specific IgG binding. The active antigenic sites of the aging antigen have been localized to residues 538–554 and 778–827. Two peptides within these regions interact synergistically to generate a synthetic aging antigen that is an effective inhibitor of senescent cell IgG binding to old cells. We synthesized peptides corresponding to these residues (pep-ANION 1: SKLIKIFQDHPLQKTYN, and pep-COOH: LFKPPKYHPDVPYVKR). These are extracellular regions of band 3 containing lysines which are implicated in anion transport. The contribution of lysine to the antigenicity of the aging antigen and to anion transport was examined by chemically modifying the lysines on both synthetic peptides and whole cells, and by synthesizing peptides in which glycines or arginines were substituted for lysines. Anion transport sites were localized using 16- to 18-mer peptides followed by 6- to 8-mer peptides. Functional studies with the peptide pep-COOH indicate that it contains sulfate-binding sites and inhibits sulfate transport in addition to carrying aging antigenic determinants. Substitution of arginines or glycines for lysines in pep-COOH reduces the sulfate-binding properties of the peptide although significant inhibition still occurs. Residues 812–827 (pep-COOH) and 813–818 (N6, the six amino acids on the amino side of pep-COOH) and 822–839 are inhibitors of anion transport when used in equimolar amounts with sulfate suggesting that these regions may be transport regions in situ. Results of this study indicate that: (a) lysines are required for the integrity of the aging antigenic site; (b) pep-COOH (residues 812–827) is part of senescent cell antigen and an anion-binding site; (c) pep-ANION 1 (538–554), which has been reported to be a transport segment of band 3, does not bind sulfate; (d) residues 588–602 are part of an anion binding/transport segment; (e) band 3 residues 822–839 are part of an anion binding/transport site, and (f) lysines contribute to anion binding but are not the only amino acid(s) required for anion binding and, thus, anion transport.

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