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First published online 20 January 2004
doi: 10.1242/jcs.00903

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Minimal mutations are required to effect a radical change in function in CEA family members of the Ig superfamily

Fakhraddin Naghibalhossaini^* and Clifford P. Stanners

Department of Biochemistry and McGill Cancer Centre, McGill University, Montreal, Quebec, Canada H3G 1Y6

View larger version (16K): [in a new window]   Fig. 1. Sequence comparison of the carboxy-terminal domain of CEA gene family members: (a) Nucleotides. Underlined bases are stop codons, and dashes show a naturally occurring base deletion in comparison with the CEACAM1 gene. (b) Amino acids. Underlined amino acids are the known GPI anchor attachment sites and stars show the position of stop codons. Bold letters indicate the position of amino acid mutations in various constructed, mutant CC1-4L proteins. The TM domain of mutant GPI-linked CC1-tAT protein and the positions of its mutations (bold letters) are also shown in b for comparison.

View larger version (28K): [in a new window]   Fig. 2. FACS analysis before and after PI-PLC treatment. FACS profiles giving CEA and CC1-mutant cell surface expression on LR-73 transfectant cells with and without treatment with PI-PLC to remove GPI-anchored molecules. Profiles for CEA (positive GPI-linked control), CC1-4L (negative TM-linked control) and Neo (background control) transfectants are also shown.

View larger version (64K): [in a new window]   Fig. 3. GPI-processing evaluation of various CC1-4L mutants by cold nonionic detergent solubility assay. Immunoblot analysis of cold Triton X-100 extracts of LR-73 transfectant cells. Neo transfectants and naturally occurring CEA family members, CEA, CEACAM6 (GPI-linked), and CC1-4L (TM-linked) were used as controls. a-d represent results for four separate immunoblots.

View larger version (45K): [in a new window]   Fig. 4. Nature of proteins in inefficiently GPI-processed CC1 mutant, CC1-t. (a) Immunoblot of the supernatant of LR(CC1-t) transfectants with and without PI-PLC treatment. PI-PLC treatment released only one band (lane 1), which corresponds to the TX-100-insoluble, higher Mr band (lane 3). Two other low Mr bands, which could be seen in both PI-PLC-treated and untreated cells (lane 1 and 2) but not in TX-100 extracts (lane 3 and 4), are presumably the result of other cross-reacting components in the reaction medium (see Materials and Methods). (b) Immunoblot of both TX-100-soluble and -insoluble fractions with (lanes 3 and 4) and without (lanes 1 and 2) treatment with Endo H. Only the major lower Mr, TX-100-soluble band (lane 3) was sensitive to Endo H digestion.

View larger version (15K): [in a new window]   Fig. 5. Homotypic intercellular adhesion mediated by mutant GPI-anchored CEACAM1 protein. CC1-tAT transfectants of LR-73 cells were subjected to the homotypic adhesion assay in suspension. The percentage of single cells was measured as a function of time in suspension. Neo (vector alone) and CC1-4L transfectant cells were used as negative and positive controls, respectively. The mean expression levels of CC1-4L and CC1-tAT by FACS analysis were 280 and 98 fluorescence units, respectively.

View larger version (82K): [in a new window]   Fig. 6. Effect of mutant GPI-anchored CEACAM1 protein on morphological myogenic differentiation. Photomicrographs of Hematoxylin-stained cultures of various L6 transfectants incubated in differentiation medium for 7 days. Magnification, 400x. FACS profiles show the relative cell surface expression level of CEA proteins in the cell cultures tested.

View larger version (94K): [in a new window]   Fig. 7. Effect of mutant GPI-anchored CEACAM1 protein on biochemical myogenic differentiation. Left column: phase contrast light photomicrographs; right column: corresponding anti-myosin antibody plus FITC-conjugated, anti-mouse IgG-stained fluorescent photomicrographs of cultures of various transfectant cells incubated for 4 days in differentiation medium. Cytoplasmic staining of multinucleated myotubes can be seen for L6(Neo) and L6(CC1-4L), indicating myosin synthesis and myogenic differentiation. Magnification, 600x.