Chemical synthesis, structural modeling, and biological activity of the epidermal growth factor-like domain of human...

Cripto, also known as human teratocarcinoma-derived growth factor 1 (TDGF-1), contains a 40 amino acid region with some similarity to the epidermal growth factor (EGF) domain. However, sequence homology is largely restricted to the classical cysteine/glycine motif with only limited similarities in other regions. Significant differences to human EGF include the absence of all seven residues between the two N-terminal half-cystines and a five-residue shorter loop between the third and fourth half-cystines. We examine the hypothesis that, in spite of these differences, cripto can adopt the characteristic EGF-like 1-3, 2-4, 5-6 disulfide bond pattern. A comparative structural model of the growth factor cripto was constructed on the basis of its similarity to EGF, transforming growth factor alpha (TGF-alpha), and the EGF-like domain of human clotting factor IX. The predicted disulfide bridges and disulfide-bridged loops were analyzed and appear viable in the modeled structure. Moreover, to ascertain the importance of disulfide arrangement for cripto bioactivity, two 47-residue peptides were synthesized and then refolded using either a simple oxidative or a controlled sequential refolding protocol. The cripto peptides were tested for their ability to stimulate MAP-kinase activity, for inhibition of beta-casein induction, and for Shc phosphorylation in MDA-MB 453 human mammary carcinoma cells and HC-11 mouse mammary epithelial cells. Data suggest that cripto does adopt the 1-3, 2-4, 5-6 disulfide pattern and thus forms the classical EGF-like fold in spite of the significant deletions within the folding domain. The predicted structure of cripto shows some of the characteristics of both the ErbB1- and ErbB3/ErbB4-binding growth factors.