Structure-function relationship and immunochemical mapping of external and intracellular antigenic sites on the lymphocyte activation inducer molecule, AIM/CD69

Authors

  • Paloma Sánchez-Mateos,

    1. Servicio de Inmunologia, Hospital de la Princesa, Universidad Autónoma de Madrid, Madrid
    Search for more papers by this author
  • Francisco Sánchez-Madrid

    Corresponding author
    1. Servicio de Inmunologia, Hospital de la Princesa, Universidad Autónoma de Madrid, Madrid
    • Serivicio de Inmunologiá, Hospital de la Princesa, Diego de León, E-28006 Madrid, Spain
    Search for more papers by this author

Abstract

Human activation inducer molecule (AIM/CD69), a dimeric glycoprotein structure of 33 and 27 kDa, is the earliest inducible cell surface antigen expressed during lymphocyte activation and is implicated in the induction of T and B cell proliferative responses. Cross-competition monoclonal antibodies (mAb) binding assays have allowed the definition of four antigenic epitopes. Three of them (antigenic sites El-3) are extracellular while the fourth (site I) is a sequential epitope localized intracellularly and highly conserved interspecies. Site E1 is shown to be an immunodominant antigenic determinant closely related to a functional domain of AIM important for triggering of T cell proliferation. Studies of peptide fragmentation of the two isolated AIM subunits with different proteases have demonstrated that both AIM chains are differentially glycosylated forms of a single 24-kDa core protein. Moreover, the two denatured and isolated AIM chains share common epitope(s) as demonstrated by their reactivity with an mAb by both Western blot analysis and immunoprecipitation of the separated AIM subunits. Biosynthesis studies revealed the rapid appearance of two intermediate precursor forms of 29 and 26 kDa which arise from the 24-kDa unglycosylated AIM polypeptide. This 24-kDa unglycosylated form could be also precipitated from iodinated cells pretreated with tunicamycin, indicating that glycosylation of the protein was neither required for AIM cell surface expression nor for acquisition of external epitopes E1-E3. Cell treatment with pronase resulted in the loss of the external epitopes E1-3 and the generation of a proteolytic peptide of 16 kDa that could be precipitated by the anti-AIM mAb specific for the internal site I. This proteolytic fragment retained the transmem-brane and cytoplasmic regions of the molecule where both epitope I and phosphorylation sites reside. These results demonstrate that AIM is an integral membrane homodimeric glycoprotein with a large cytoplasmic domain probably involved in the activation signals transduced through this molecule to lymphocytes.

Ancillary