Molecular mapping of functional antibody binding sites of α4 integrin

Abstract

Integrin α4β1 is a leukocyte receptor for fibronectin and vascular cell adhesion molecule-1 (VCAM-1). It is important in inflammatory recruitment of leukocytes, lymphopoiesis, and a number of developmental events. Here we have mapped a panel of functional monoclonal antibodies (mAbs) recognizing the integrin α4 chain, using murine/human chimeric constructs expressed in COS7 cells. We find that: 1) mAbs that induce homotypic aggregation (epitope A mAbs) map to the most N-terminal 100 amino acids of the human α4 chain; 2) mAbs that block adhesion of α4β1 to VCAM-1 and fibronectin (epitope B mAbs) map to a 52-amino-acid region between residues 152 and 203 of human α4; 3) epitope B mAbs that do or do not induce aggregation (epitope B2 and B1 mAbs, respectively) map to the same regions and are therefore indistinguishable by this analysis; 4) mAbs that neither induce homotypic aggregation nor block adhesion (epitope C mAbs) map to a distinct region of the molecule comprising amino acids 422-606. The N-terminal region of the α4 chain identified by functional A and B epitope mAbs does not correspond to ligand binding sites identified in other α subunits, such as cation binding sites or the 'I- domain,' which α4 lacks, and thus represents a novel site for epitope functionality among the integrins.