SYNTHESIS AND CHARACTERIZATION OF INSULIN-LIKE GROWTH FACTOR-BINDING PROTEIN (IGFBP)-7 - RECOMBINANT HUMAN MAC25 PROTEIN SPECIFICALLY BINDSIGF-I AND IGF-II

The mac25 cDNA was originally cloned from leptomeningial cells and sub sequently reisolated through differential display as a sequence prefer entially expressed in senescent human mammary epithelial cells. The de duced amino acid sequence of the human mac25 propeptide shares a 20-25 % identity to human insulin-like growth factor-binding proteins (IGFBP s), suggesting that mac25 could be another member of the IGFBP family. In the present study, we have generated recombinant human mac25 (rh-m ac25) in a baculovirus expression system and assessed its affinity for IGFs and have evaluated the pattern of expression of the mac25 gene i n human tissues. Binding of I-125-IGF-I and I-125-IGF-II to rh-mac25 w as demonstrated by Western ligand blotting after nondenaturing polyacr ylamide gel electrophoresis and by affinity cross-linking with as litt le as 2 nM rh-mac25. Specificity of rh-mac25 binding to I-125-IGFs was demonstrated by competition for rh-mac25 binding with unlabeled IGFs, but not with [QAYLL]IGF-II analog, which has 100-fold less affinity f or IGFBPs. In comparison with IGFBP-3, rh-mac25 has at least a 5-6-fol d lower affinity for IGF-I and 20-25-fold lower affinity for IGF-II. m ac25 mRNA was detectable in a wide range of normal human tissues, with decreased expression in breast, prostate, colon, and lung cancer cell lines, In conclusion, mac25 specifically binds IGFs and constitutes a new member of the IGFBP family, IGFBP-7. Its wider distribution in no rmal tissue and lower expression in several cancer cells indicate that IGFBP-7 may function as a growth-suppressing factor, as well as an IG F-binding protein.