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

Abstract

Macrophage inhibitory cytokine-1 (MIC-1) is a divergent member of the transforming growth factor-beta (TGF-beta) 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-beta1, 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.

Plasma MIC-1 correlates with systemic inflammation but is not an independent determinant of nutritional status or survival in oesophago-gastric cancer

“…In brief, the mouse MAb 26G6H6 was used for antigen capture; and the sheep PAb 233-P was used for detection (Moore et al, 2000;Fairlie et al, 2001;Brown et al, 2002). The hMIC-1 plasma concentration was determined by reference to a standard curve constructed using recombinant hMIC-1 as the standard.…”

Anoxia induces macrophage inhibitory cytokine-1 (MIC-1) in glioblastoma cells independently of p53 and HIF-1

“…Equal protein loading was confirmed by Ponceau red staining. MIC-1 and VEGF were detected using sheep polyclonal antiserum 233 anti-human MIC-1 (Bootcov et al, 1997;Fairlie et al, 2001) or mouse anti-human VEGF (clone 26503.11, dilution 1 : 125; R&D Systems), respectively. The corresponding secondary antibodies used were PODconjugated Rabbit Anti-sheep IgG (DAKO) and Horseradish POD-conjugated Anti-mouse IgG (Sigma-Aldrich).…”

Macrophage Inhibitory Cytokine-1 H6D Polymorphism, Prostate Cancer Risk, and Survival

“…9). Different amino acid properties and the close proximity of this variant to the critical stabilizing cystine residue at position 7 suggest a role in MIC-1 stability and/or function (10). There has been a recent report of a statistically weak negative association between H6D and prostate cancer risk [odds ratio (OR), 0.83; 95% confidence interval (95% CI), 0.69-0.99; ref .…”