Volume 118, Issue 1, Pages 70-80 (January 2000) pS2/TFF1 interacts directly with the VWFC cysteine-rich domains of mucins  Catherine Tomasetto, Régis Masson, José–Luis Linares, Corinne Wendling, Olivier Lefebvre, Marie–Pierre Chenard, Marie–Christine Rio  Gastroenterology  Volume 118, Issue 1, Pages 70-80 (January 2000) DOI: 10.1016/S0016-5085(00)70415-X Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 Interactions between mTFF1 and clones isolated in the screen. The bait used to transform the yeast strain YRG2 was pBD-mTFF1. Clockwise from the asterisk, the interactors were pAD, pAD-mTFF1, pAD-m6, pAD-m37, pAD-m50,and pAD-m62. Yeast transformants were plated onto (A) SD/Trp− Leu− and (B) SD/Trp− Leu− His− in the presence of 1 mmol/L 3AT. Gastroenterology 2000 118, 70-80DOI: (10.1016/S0016-5085(00)70415-X) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 Nucleotide and deduced amino acid sequences of pAD-m6. Nucleotides and amino acids are numbered on the left and on the right. The 2 VWFC domains are shown by gray boxes, and the cysteine residues involved in the consensus are represented by bold characters. Encircled amino acids at positions 198 and 200 correspond to the first and last residues of pAD-m6Δ2 and pAD-m6Δ1, respectively. The EMBL/Genbank accession number for this sequence is AJ010437. Gastroenterology 2000 118, 70-80DOI: (10.1016/S0016-5085(00)70415-X) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 Nucleotide and deduced amino acid sequences of pAD-m37, pAD-m50, and pAD-m62. Nucleotides and amino acids are numbered on the left and on the right. The 2 VWFC domains are shown by gray boxes, and the cysteine residues involved in the consensus are represented by bold characters. Limits of clones m37, m50, and m62 are indicated by white, gray, and black arrowheads, respectively. Encircled amino acids at positions 616 and 702 correspond to the first and last residues of pAD-m62Δ3 and pAD-m62Δ1, respectively. The EMBL/Genbank accession number for this sequence is AJ010792. Gastroenterology 2000 118, 70-80DOI: (10.1016/S0016-5085(00)70415-X) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 Expression analysis of clones m6 and m62. The Northern blot contained approximatively 10 μg of total RNAs isolated from mouse colon (lane 1), small intestine (lane 2), and stomach (lane 3). The blot was successively hybridized using (A) m6, (B) m62, and (C) the internal loading control 36B4. rRNA size markers (S values) are indicated (right). Gastroenterology 2000 118, 70-80DOI: (10.1016/S0016-5085(00)70415-X) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 Expression analysis of clones m6 and m62 by in situ hybridization of mouse tissues. (A) Section of the small intestine hybridized with an m6-specific antisense probe. Goblet cells are labeled (blue); enterocytes are negative. (B) Section of the stomach antrum hybridized with an m62-specific antisense probe. Surface epithelial mucous cells are labeled (blue) (original magnification 20×). Gastroenterology 2000 118, 70-80DOI: (10.1016/S0016-5085(00)70415-X) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 6 Alignment of the conserved domains of the clones isolated with the human MUC2 protein. (A) Schematic representation of MUC2 containing 4 D domains (open boxes), 2 heavily glycosylated repeated regions (gray boxes), 2 VWFC domains (striped boxes), and a CTCK domain (black box). (B) Alignment of VWFC domains of the murine mucins mMuc2 and mMuc5AC with corresponding regions in MUC2. Homologous amino acids are indicated in the consensus as identical (*), conserved (:) and semi-conserved (.). The 10 cysteine residues involved in the VWFC domain signature are represented by bold characters. Gastroenterology 2000 118, 70-80DOI: (10.1016/S0016-5085(00)70415-X) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 7 Interactions between mTFF1 and mucin proteins within the yeast 2-hybrid system. (A) Bait construct pBTMN-mTFF1 was paired with interactor constructs; the relative positions of the different contructs to the MUC2 protein organization are represented. (B) Interactions were measured by quantitative β-galactosidase assays; the mean ± SE number of β-galactosidase activity units obtained for each cotransfection is presented. (C) Growth on selective medium. Gastroenterology 2000 118, 70-80DOI: (10.1016/S0016-5085(00)70415-X) Copyright © 2000 American Gastroenterological Association Terms and Conditions