Definition of a physiologic aging autoantigen by using synthetic peptides of membrane protein band 3: localization of the active antigenic sites.(1)
Senescent cell antigen (SCA) appears to be involved in the removal of erythrocytes in clinical hemolytic anemias and the removal of malaria infected erythrocytes. Band 3, the membrane protein from which SCA is derived, is a ubiquitous protein. It has been found in diverse cell types and tissues besides erythrocytes. Band 3 is a major transmembrane structural protein that attaches the plasma membrane to the internal cell cytoskeleton by binding to band 2.1 (ankyrin).
The first peptide (anion 1, residues 538-554) includes two important amino acids. Lys-539 is a covalent binding site for the anion transport inhibitor, diisothiocyanatostilbene disulfonate (DIDS), and Tyr-553 is radioiodinated by extracellular lactoperoxidase. The second peptide (anion 2, residues 588-602) is situated toward the end of the region and is probably intracellular because the potential N-glycosylation site at ASN593 is not glycosylated. The last peptide from the carboxyl-terminal end (“COOH”, residues 812-827) contains both hydrophobic and hydrophilic regions. The lysines found in this region may comprise another binding site for DIDS. SCIgG recognizes antigenic determinants that lie within a putative anion-transport region, residues 538-554 (anion 1), and a transport site containing a cluster of lysines toward the carboxyl terminus, residues 812-827 (peptide COOH). Anion 1 and COOH interact together to form a three-dimensional structure that functions as an aging antigen.
Residues 814-829 contain a DIDS binding site or the availability of Tyr-553 for external radioiodination. Close steric association must be maintained by external loops 02 and 04. When these regions are associated on the same band 3 monomer, then band 3 loops back upon itself so that these regions are contiguous. Alternatively, the functional assembly may be dimers in which close associations of loops 02 and 04 form between separate molecules. Development of a model incorporating band 3 was designated loop 04 that contains the peptide COOH and its component N6, which are potent inhibitors of the binding of autoantibodies to the aging antigen to erythrocytes.
30 um for this “single concentration” assay because our two synergistic peptides, anion 1 and COOH, give >95% inhibition at this concentration, whereas “noninhibitory” peptides give <20% inhibition. Peptides that were negative by immunoblotting were also negative in the inhibition assay. The competitive inhibition assay shows that significant inhibition of senescent cell IgG binding is obtained only with the peptide comprising residues 822-839, which is on the carboxyl-terminal side of peptide COOH and contains six amino acids of COOH. The competitive inhibition assay shows that inhibition of 52 +/- 4% was obtained with the peptide formed of residues 597-614. Residues 526-543 and 549-566 could not be tested because of low solubility of the peptides.
Active antigenic determinants of SCA reside on peptides anion 1 and COOH within residues 538-554 and 788-827. These peptides reside in highly conserved regions.