MUTUALLY EXCLUSIVE BINDING OF PEPTIDE AND INVARIANT CHAIN TO MAJOR HISTOCOMPATIBILITY COMPLEX CLASS-II ANTIGENS

The invariant chain is a membrane protein associated with the major hi stocompatibility complex class II antigens both intra- and extracellul arly. The extracellular portion of the human invariant chain (Ii) was expressed in Escherichia coli as a fusion protein with a polyhistidine tail and purified by metal affinity chromatography. The recombinant I i was used as a ligand to probe binding to the cell surface of Chinese hamster ovary cells stably transfected with human class II alpha and beta genes of the DR4 isotype. We show that recombinant Ii inhibits pe ptide loading on class II polypeptides and also the converse; the pres ence of peptide in the antigen groove prevents binding of fluorescein conjugated Ii. Moreover, blocking of Ii binding by peptide did not req uire a transition of the class II dimers to an SDS stable state. A mon oclonal antibody, L243, known to bind to (or close to) the peptide poc ket of the class II molecule likewise blocked Ii-fluorescein binding. Further, we investigated whether or not the Ii, a variety of bacterial superantigens or the CD4 molecule, have overlapping binding sites on the class II heterodimer. Of the class II ligands tested, reduced bind ing was detected for the Staphylococcus superantigen type SEB on cells precincubated with soluble Ii while the binding of the other ligands was either unchanged or marginally changed. These data clarify by a di rect biochemical approach the binding characteristics of Ii in compari son with other class II Ligands.