Mapping the CD4 binding domain of gp17, a glycoprotein secreted from seminal vesicles and breast carcinomas

gp17, a secretory CD4-binding factor isolated from the human seminal plasma , is identical to the gross cystic disease fluid protein-15, a specific mar ker for primary and metastatic breast tumors. We previously demonstrated th at gp17 binds to CD4 with high affinity and strongly inhibits T lymphocyte apoptosis induced by sequential cross-linking of CD4 and T cell receptor (T CR). To further characterize the,gp17/CD4 interaction and map the gp17 bind ing site, we produced a secreted form of recombinant gp17 fused to human Ig G1 Fc, gp17-Ig. We showed that gp17-Ig exhibits a binding affinity for CD4 similar to that of native gp17. As no information about gp17 structure is p resently available, 99 overlapping gp17 peptides were synthesized by the Sp ot method, which allowed the mapping of two CD4 binding regions. Alanine sc anning of CD4-reactive peptides identified critical residues, selected for site-directed mutagenesis. Nine gp17-Ig mutants were generated and characte rized. Three residues within the carboxyterminal region were identified as the major binding domain to CD4. The Spot method combined with mutagenesis represents a refined approach to distinguish the contact residues from the ones contributing to the conformation of the CD4-binding domain.