Three-dimensional analysis of CD6 site-directed mutagenesis and monoclonalantibody binding studies using the x-ray structure of mac-2 binding protein and a molecular model of the CD6 ligand binding domain

The extracellular region of CD6 consists of three scavenger receptor cystei ne-rich (SRCR) domains and binds activated leukocyte cell adhesion molecule (ALCAM), a member of the immunoglobulin superfamily (IgSF). Residues impor tant for the CD6-ALCAM interaction have previously been identified by mutag enesis. A total of 22 CD6 residues were classified according to their impor tance for anti-CD6 monoclonal antibody (mAb) and/or ALCAM binding. The thre e-dimensional structure of the SRCR domain of Mac-2 binding protein has rec ently been determined, providing a structural prototype for the SRCR protei n superfamily. This has made a thorough three-dimensional analysis of CD6 m utagenesis and mAb binding experiments possible. Mutation of buried residue s compromised both mAb and ALCAM binding, consistent with the presence of s tructural perturbations. However, several residues whose mutation affected both mAb and ALCAM binding or, alternatively, only ligand binding were foun d to map to the surface in the same region of the domain. This suggests tha t the CD6 ligand binding site and epitopes of tested mAbs overlap and provi des an explanation for the finding that these mAbs effectively block ALCAM binding. An approximate molecular model of CD6 was used to delineate the AL CAM binding site.