Chemical Synthesis, Structural Modeling, and Biological Activity of the Epidermal Growth Factor-like Domain of Human Cripto

• Published in: Biochemistry (Easton) 1997-04, Vol.36 (13), p.3837-3845
• Main Authors: Lohmeyer, Matthias, Harrison, Paul M, Kannan, Subha, DeSantis, Marta, O'Reilly, Nicola J, Sternberg, Michael J. E, Salomon, David S, Gullick, William J
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Calcium-Calmodulin-Dependent Protein Kinases - metabolism; Caseins - metabolism; Cell Line; Chromatography, High Pressure Liquid; Computer Simulation; Disulfides - chemistry; Enzyme Activation; Epidermal Growth Factor - chemistry; GPI-Linked Proteins; Growth Substances - chemistry; Growth Substances - metabolism; Humans; Intercellular Signaling Peptides and Proteins; Mass Spectrometry; Membrane Glycoproteins; Mice; Models, Molecular; Molecular Sequence Data; Myelin Basic Protein - metabolism; Neoplasm Proteins - chemistry; Neoplasm Proteins - metabolism; Neoplasm Proteins - pharmacology; Peptide Fragments - chemical synthesis; Peptide Fragments - metabolism; Peptide Fragments - pharmacology; Phosphorylation; Protein Conformation; Protein Folding; Sequence Homology, Amino Acid
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi961542p

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 α (TGF-α), 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 β-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.