Epitope mapping of the transforming growth factor-beta superfamily protein, macrophage inhibitory cytokine-1 (MIC-1): Identification of at least fivedistinct epitope specificities

Macrophage inhibitory cytokine-l (MIC-1) is a divergent member of the trans forming growth factor-beta (TGF-beta) superfamily whose increased expressio n is associated with macrophage activation and which is expressed highly in placenta as compared to other tissues. There an two known allelic forms of human MIG-I due an amino acid substitution at position 6 of the mature pro tein. We have raised four monoclonal antibodies (MAbs) and one polyclonal a ntiserum to the mature protein region of human MIC-1 and have used an exten sive panel of MIC-1 relatives, mutants, and chimeras to map their epitopes. None of the MAbs were able to cross-react with either the murine homologue of MIG-I or with hTGF-beta1, and all of the MAb epitopes were conformation -dependent. A distinct cross-reactivity pattern with the various antigens w as observed for each of the monoclonal and polyclonal antibodies suggesting the presence of at least five immunogenic regions on the MIG-I surface. On e of the MAbs is directed against the amino terminus of the protein and can distinguish between the two allelic forms of MIC-1. The epitopes for the o ther three MAbs were located near the tips of the so-called "fingers" of th e protein and appeared to be partially overlapping as each involved amino a cids in the region 24-37. In one case, it was possible to mutate murine MIC -1 so that it could be recognized by one of the MAbs. Finally, the use of a nother mutant in which Cys 77 was replaced by serine enabled confirmation o f the location of the MIC-1 interchain disulfide bond.