Epitope Mapping of the Transforming Growth Factor-β Superfamily Protein, Macrophage Inhibitory Cytokine-1 (MIC-1):  Identification of at Least Five Distinct Epitope Specificities

• Published in: Biochemistry (Easton) Vol. 40; no. 1; pp. 65 - 73
• Main Authors: Fairlie, W. Douglas, Russell, Patricia K, Wu, Wan M, Moore, Anthony G, Zhang, Hong-Ping, Brown, Peter K, Bauskin, Asne R, Breit, Samuel N
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 09-01-2001
• Subjects: Amino Acid Sequence; Animals; Antibodies, Monoclonal - biosynthesis; Antibodies, Monoclonal - chemistry; Antibodies, Monoclonal - genetics; Antibodies, Monoclonal - metabolism; Antibody Specificity - genetics; CHO Cells; Cricetinae; Cross Reactions - genetics; Cytokines - chemistry; Cytokines - genetics; Cytokines - immunology; Cytokines - metabolism; Epitope Mapping; Epitopes - chemistry; Epitopes - genetics; Epitopes - immunology; Epitopes - metabolism; Genetic Vectors - chemical synthesis; Genetic Vectors - immunology; Growth Differentiation Factor 15; Humans; Immune Sera - biosynthesis; Immune Sera - chemistry; Immune Sera - genetics; Immune Sera - metabolism; macrophage inhibitory cytokine 1; Mice; Molecular Sequence Data; Multigene Family - immunology; Recombinant Fusion Proteins - chemical synthesis; Recombinant Fusion Proteins - immunology; Recombinant Fusion Proteins - metabolism; Sequence Homology, Amino Acid; Transforming Growth Factor beta - chemistry; Transforming Growth Factor beta - genetics; Transforming Growth Factor beta - immunology; Transforming Growth Factor beta - metabolism; transforming growth factor-^b
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi001064p

Macrophage inhibitory cytokine-1 (MIC-1) is a divergent member of the transforming growth factor-β (TGF-β) superfamily whose increased expression is associated with macrophage activation and which is expressed highly in placenta as compared to other tissues. There are two known allelic forms of human MIC-1 due an amino acid substitution at position 6 of the mature protein. We have raised four monoclonal antibodies (MAbs) and one polyclonal antiserum to the mature protein region of human MIC-1 and have used an extensive 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 MIC-1 or with hTGF-β1, and all of the MAb epitopes were conformation-dependent. A distinct cross-reactivity pattern with the various antigens was observed for each of the monoclonal and polyclonal antibodies suggesting the presence of at least five immunogenic regions on the MIC-1 surface. One 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 other three MAbs were located near the tips of the so-called “fingers” of the protein and appeared to be partially overlapping as each involved amino acids 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 another mutant in which Cys 77 was replaced by serine enabled confirmation of the location of the MIC-1 interchain disulfide bond.