Chemical synthesis, structural modeling, and biological activity of the epidermal growth factor-like domain of human cripto.
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.
Lohmeyer
M
M
ICRF Molecular Oncology Laboratory, Hammersmith Hospital, Royal Postgraduate Medical School, London, U.K.
Harrison
P M
PM
Kannan
S
S
DeSantis
M
M
O'Reilly
N J
NJ
Sternberg
M J
MJ
Salomon
D S
DS
Gullick
W J
WJ
D000595:Amino Acid Sequence; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002364:Caseins / Q000378:metabolism; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003198:Computer Simulation; D004220:Disulfides / Q000737:chemistry; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000737:chemistry*; D058851:GPI-Linked Proteins; D006133:Growth Substances / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D013058:Mass Spectrometry; D008562:Membrane Glycoproteins; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D004676:Myelin Basic Protein / Q000378:metabolism; D009363:Neoplasm Proteins / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D010446:Peptide Fragments / Q000138:chemical synthesis* / Q000378:metabolism* / Q000494:pharmacology; D010766:Phosphorylation; D011487:Protein Conformation; D017510:Protein Folding; D017386:Sequence Homology, Amino Acid
D002364:Caseins; D004220:Disulfides; D058851:GPI-Linked Proteins; D006133:Growth Substances; D036341:Intercellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; D004676:Myelin Basic Protein; D009363:Neoplasm Proteins; D010446:Peptide Fragments; C070636:TDGF1 protein, human; C489450:Tdgf1 protein, mouse; D004815:Epidermal Growth Factor; D017871:Calcium-Calmodulin-Dependent Protein Kinases