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A novel C-type lectin regulating cell growth, cell adhesion and cell differentiation of the multipotent epithelium in budding tunicates

Jun Matsumoto*, Chiaki Nakamoto, Shigeki Fujiwara, Toshitsugu Yubisui and Kazuo Kawamura{ddagger}

Laboratory of Cellular and Molecular Biotechnology, Faculty of Science, Kochi University, Kochi 780-8520, Japan
* Present address: Biomolecular Engineering Department, National Institute of Bioscience and Human Technology, AIST, 1-1 Higashi, Tsukuba, Ibaragi 305-8566, Japan



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  		Fig. 1. Purification of novel galactose-binding proteins from P. misakiensis. (A) Anion exchange chromatography of crude extracts. The third peak (arrowhead) was further fractionated. (B) Gel filtration HPLC. The highest peak (arrowhead) has a retention time of about 20 minutes. (C) SDS-PAGE of the highest peak after gel filtration HPLC. (D) P18 and P15 after a second anion exchange chromatography step. (E) Affinity chromatography of P18 and P15, on immobilized galactose. After washing with the binding buffer containing 1 mM CaCl2, the column was eluted with 5 mM EDTA. (F) SDS-PAGE of each fraction after affinity chromatography.

 


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  		Fig. 2. Effect of P18 and P15 on cell growth and aggregation of the atrial epithelium in culture, 4 days after inoculation. (A) Control. Cells were allowed to grow in the serum-supplemented medium. Scale bar, 50 µm. (B) Experiment. The proteins (about 36 µg/ml) were added to the serum-supplemented medium. Note that cells aggregate. Scale bar, 50 µm. (C) In situ hybridization showing in vivo expression of {alpha}-integrin homolog (blue) in the atrial epithelium (arrow). Scale bar, 20 µm. (D) In vitro induction of {alpha}-integrin homolog by P18 and P15 (brown). Cell aggregates in culture were suspended by pipetting, mounted on cover slips coated with poly-L-lysine, and immunostained (arrow). Scale bar, 10 µm.

 


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  		Fig. 3. Nucleotide and deduced amino acid sequences of TC14s. (A) TC14-2. Shaded letters indicate N-terminal and internal amino acid sequences of P18, determined by peptide microsequencing. (B) TC14-3. Shaded letters indicate N-terminal amino acid sequence of P15. Letters marked by dots are Kozak’s consensus motif, those with a double underline are the signal peptide and those with a single underline are the polyadenylation signal. (C) Multiple alignment of TC14-1, TC14-2 and TC14-3. Four half cysteines (asterisks) are conserved perfectly. Carbohydrate-binding amino acids, E,N, and DD, are also conserved. A box indicates sequence similarity to E-selectin. These sequence data of TC14-1, TC14-2 and TC14-3 are available from EMBL/GenBank under accession numbers AB049563, AB049564 and AB049565, respectively.

 


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  		Fig. 4. Preparation of recombinant TC14-2 (rTC14-2) and rTC14-3. (A,B) Bacterial lysates after IPTG induction of GST fusion proteins. (C,D) Affinity purification of rTC14-2 and rTC14-3 after thrombin digestion. (E,F) Affinity chromatography of rTC14-2 (E) and rTC14-3 (F), using immobilized galactose. Elution profiles show that both rTC14-2 and rTC14-3 have calcium-dependent, galactose-binding activity.

 


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  		Fig. 5. Effects of recombinant proteins on cell growth and differentiation of the atrial epithelium in culture, 5 days after inoculation in the serum-supplemented medium. (A-C) Phase contrast microscopy. Scale bar, 50 µm. (D-G) Immunocytochemistry. Cells were suspended in the medium by pipetting and mounted on poly-L-lysine-coated cover slips. Cells, except those in F, were stained with the monoclonal antibody recognizing specifically alkaline phosphatase of the atrial epithelium. In F, the gastric epithelium-specific antibody was used. (A,D) rTC14-2 (3 nmol/ml). (B,E,F) rTC14-3 (3 nmol/ml). (C,G) rTC14-3 (3 nmol/ml) + 50 µM galactose. Scale bar, 25 µm.

 


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  		Fig. 6. Expression of TC14-3 during the asexual life span of P. misakiensis. (A) Blots of crude extracts and immunostaining with anti-TC14-3 monoclonal antibody. Lane 1, young functional animals; lane 2, adult animals of budding stage; lane 3, growing buds; lane 4, 1-day developing buds; lane 5, 2-day developing buds; lane 6, 3-day developing buds. (B) Section of the body wall of adult animal. Mesenchymal cells are stained. Scale bar, 25 µm. (C) Section of the pharynx of adult animal. Mesenchymal cells are stained heavily, and the pharyngeal epithelium is stained faintly (arrow). Scale bar, 25 µm. (D) Whole-mount of growing bud. Arrows indicate the atrial epithelium. Scale bar, 250 µm. (E) Section of growing bud. Both mesenchymal cells and atrial epithelium are stained. Scale bar, 25 µm. (F) Whole mount of 1-day-developing bud. Signal becomes weak at the proximal (boxed) area where morphogenetic events are in progress. Scale bar, 1 mm. (G) Section of boxed area in F. Scale bar, 25 µm. ae, atrial epithelium; e, epidermis (white dashed lines); m, mesenchymal cell.

 


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  		Fig. 7. Possible role of TC14-3 in asexual reproduction of P. misakiensis. (A) The atrial epithelium is composed of differentiated pigment cells. (B) During blastogenesis, dedifferentiation takes place. (C) Dedifferentiated cells become progenitors of various tissues and organs. (D) Respective progenitor cells enter the terminal differentiation pathways to become nerves, gastric epithelium, pharyngeal epithelium and others. TRAMP is a retinoic acid-inducible serine protease that promotes cell growth and, possibly, dedifferentiation of the atrial epithelium. TC14-3 is expressed constitutively by mesenchymal cells and by the atrial epithelium during budding stages. It blocks cell growth, dedifferentiation and the resultant terminal differentiation of the atrial epithelium, thus contributing to the maintenance of multipotent cells. The effect of TC14-3 is strong but reversible, as it is cancelled with ease by galactose.

 

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