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Structure-function analysis of the EGF-CFC family member Cripto identifies residues essential for nodal signalling

Gabriella Minchiotti^1,, Giuseppe Manco², Silvia Parisi¹, Carmine T. Lago¹, Frederic Rosa^3,* and M. Graziella Persico^1,*

¹ International Institute of Genetics and Biophysics, CNR, Via G. Marconi 12, 80125 Naples, Italy
² Institute of Protein Biochemistry and Enzymology, CNR, Via G. Marconi 12, 80125 Naples, Italy
³ Ecole Normale Superieure, 46 rue d’Ulm, 75230 Paris CEDEX 05, France
^* These two authors contributed equally to this work

View larger version (31K): [in a new window]   Fig. 1. (A) Western blot analysis (using anti-Cripto antibody) of the conditioned medium of 293T cells transfected with cripto-His WT-, cripto-His Glu91Ala-expressing vectors, or vector alone. (B) Immunochemical analysis of Cripto-Fc chimera. Metabolically labelled conditioned medium from mock or cripto-Fc 293T transfectants was immunoprecipitated with protein A Sepharose and subjected to SDS-PAGE. The immunoreactive species were visualised after fluorography. (C) MZoep mutant embryos at sphere stage co-injected with recombinant Cripto-His protein and 10K dextran rhodamin. Fluorescence (a’,b’) and light (a,b) images of frontal (a,a’) and lateral (b,b’) views of injected MZoep mutant embryos. The molecular mass of protein standards is indicated (kDa).

View larger version (118K): [in a new window]   Fig. 2. Phenotypes of MZoep embryos rescued by mutant cripto RNA injections. Live embryos at 32 hours post-fertilization, lateral view anterior to the left. (A) MZoep mutant embryo, uninjected. (B) Grade 1: rescue of somites. (C) Grade 2: rescue of somites and notochord. (D) Grade 3: partial rescue of cyclopia. (E) Grade 4: full rescue. (F) Wild-type embryo. Note the notochord (arrowhead in C-E) and somites (arrow in B-D).

View larger version (56K): [in a new window]   Fig. 3. (A) Sequence alignment of the EGF-CFC proteins: mouse Cripto (mCripto), human Cripto (hCripto), zebrafish Oep (Oep), human Cryptic (hCryptic), mouse Cryptic (mCryptic), frog FRL1 (FRL1) and chicken Cripto (cCripto). The positions of the missense mutations (red residues) are shown above the alignment. Red and blue lines indicate amino acids deleted in cripto-His and in the EGF-CFC-His constructs, respectively. Blue and green shaded areas indicate the EGF and the CFC domains, respectively. Conserved residues in the EGF-CFC domain are indicated by asterisks. The white arrow indicates the predicted cleavage site of signal peptide in mouse Cripto. Black arrows indicate processing sites of recombinant Cripto-His expressed in 293T cells. (B) Western blot analysis of total lysates from 293T cells transfected with Cripto wild-type and mutant derivatives. Cells were cotransfected with Jun-Ha expression vector as internal control. The molecular mass of protein standards is indicated (kDa).

View larger version (27K): [in a new window]   Fig. 4. (A) View of Cripto model superimposed onto the basic FGF structure (2bfh). The C traces are shown. The Cripto and 2bfh traces are rendered in magenta and cyan, respectively. The r.m.s.d. of the backbone atoms (C, C-alfa and N atoms) for the two superimposed structures was 0.91Å on a total of 468 corresponding atoms. (B) Cripto model (C trace) with highlighted EGF (red) and CFC (blue) regions. The disulphide bonds (in yellow) are also shown. (C) Ribbon rendering of the Cripto model with all mutated residues drawn with a CPK representation. Mutations with a strong and less severe effect are indicated in blue and cyan, respectively while ineffective mutations are indicated in red. ß-strands are shown as green arrows and loops as yellow ropes. Orientation is different from A and B.