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Cadherin-mediated cell sorting not determined by binding or adhesion specificity.

Niessen CM, Gumbiner BM - J. Cell Biol. (2002)

Bottom Line: None of the different cadherin-expressing cells exhibited any adhesive specificity toward either of the two purified cadherin substrates, even though these cadherins differ considerably in their primary sequence.However, this lack of adhesive specificity did not determine whether different cadherin-expressing cells would sort from each other, and the tendency to sort was not predictable by the extent of sequence diversity in their extracellular domains.These results show that cadherins are far more promiscuous in their adhesive-binding capacity than had been expected and that the ability to sort out must be determined by mechanisms other than simple adhesive-binding specificity.

View Article: PubMed Central - PubMed

Affiliation: Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.

ABSTRACT
Cadherin adhesion molecules play important roles in the establishment of tissue boundaries. Cells expressing different cadherins sort out from each other in cell aggregation assays. To determine the contribution of cadherin binding and adhesion specificity to the sorting process, we examined the adhesion of cells to different purified cadherin proteins. Chinese hamster ovary cell lines expressing one of four different cadherins were allowed to bind to the purified cadherin extracellular domains of either human E-cadherin or Xenopus C-cadherin, and the specificity of adhesion was compared with cell-sorting assays. None of the different cadherin-expressing cells exhibited any adhesive specificity toward either of the two purified cadherin substrates, even though these cadherins differ considerably in their primary sequence. In addition, all cells exhibited similar strengthening of adhesion on both substrates. However, this lack of adhesive specificity did not determine whether different cadherin-expressing cells would sort from each other, and the tendency to sort was not predictable by the extent of sequence diversity in their extracellular domains. These results show that cadherins are far more promiscuous in their adhesive-binding capacity than had been expected and that the ability to sort out must be determined by mechanisms other than simple adhesive-binding specificity.

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Purification and characterization of recombinant extracellular cadherin proteins. The extracellular domains of HE-cadherin and C-cadherin were both expressed in CHO cells as a COOH-terminal fusion proteins with the Fc part of human IgG. Recombinant proteins were purified from the media on a Protein A column. (A) Coomassie staining of HEEC1–5Fc and CEC1–5Fc run under reducing and nonreducing conditions. (B) Immunoblot analysis of the recombinant HEEC1–5Fc and CEC1–5Fc proteins using anti–human Fc, anti-HE cadherin, or anti–C-cadherin antibodies. (C) Bead aggregation assay: protein-A–coated beads coupled to HEEC1–5Fc or CEC1–5Fc were allowed to aggregate in the presence of Ca2+ or EDTA for the indicated time period.
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fig1: Purification and characterization of recombinant extracellular cadherin proteins. The extracellular domains of HE-cadherin and C-cadherin were both expressed in CHO cells as a COOH-terminal fusion proteins with the Fc part of human IgG. Recombinant proteins were purified from the media on a Protein A column. (A) Coomassie staining of HEEC1–5Fc and CEC1–5Fc run under reducing and nonreducing conditions. (B) Immunoblot analysis of the recombinant HEEC1–5Fc and CEC1–5Fc proteins using anti–human Fc, anti-HE cadherin, or anti–C-cadherin antibodies. (C) Bead aggregation assay: protein-A–coated beads coupled to HEEC1–5Fc or CEC1–5Fc were allowed to aggregate in the presence of Ca2+ or EDTA for the indicated time period.

Mentions: To make purified cadherin substrates we expressed the extracellular domains of both the human E-cadherin (HEEC1–5Fc) and Xenopus C-cadherin (CEC1–5Fc) as fusion proteins with the Fc part of human IgG in CHO cells. This insures that the protein is secreted as a dimer, which is important because functional cadherins require dimerization of the protein (Brieher et al., 1996; Tamura et al., 1998). The adhesive properties of CEC1–5Fc already have been well characterized (Chappuis-Flament et al., 2001). The HEEC1–5Fc and CEC1–5Fc proteins were isolated from conditioned medium on protein A columns. Both proteins were isolated to near purity since the size of the most abundant bands as observed by Coomassie brilliant blue staining (Fig. 1 A) corresponded to the correct molecular weights of either HEEC1–5Fc or CEC1–5Fc. Western blot analysis confirmed the identity of both proteins (Fig. 1 B). In the following experiments, we tested proteins produced by at least two different clones of secreting cells for each cadherin EC1–5Fc chimera and several protein preparations from each clone.


Cadherin-mediated cell sorting not determined by binding or adhesion specificity.

Niessen CM, Gumbiner BM - J. Cell Biol. (2002)

Purification and characterization of recombinant extracellular cadherin proteins. The extracellular domains of HE-cadherin and C-cadherin were both expressed in CHO cells as a COOH-terminal fusion proteins with the Fc part of human IgG. Recombinant proteins were purified from the media on a Protein A column. (A) Coomassie staining of HEEC1–5Fc and CEC1–5Fc run under reducing and nonreducing conditions. (B) Immunoblot analysis of the recombinant HEEC1–5Fc and CEC1–5Fc proteins using anti–human Fc, anti-HE cadherin, or anti–C-cadherin antibodies. (C) Bead aggregation assay: protein-A–coated beads coupled to HEEC1–5Fc or CEC1–5Fc were allowed to aggregate in the presence of Ca2+ or EDTA for the indicated time period.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199232&req=5

fig1: Purification and characterization of recombinant extracellular cadherin proteins. The extracellular domains of HE-cadherin and C-cadherin were both expressed in CHO cells as a COOH-terminal fusion proteins with the Fc part of human IgG. Recombinant proteins were purified from the media on a Protein A column. (A) Coomassie staining of HEEC1–5Fc and CEC1–5Fc run under reducing and nonreducing conditions. (B) Immunoblot analysis of the recombinant HEEC1–5Fc and CEC1–5Fc proteins using anti–human Fc, anti-HE cadherin, or anti–C-cadherin antibodies. (C) Bead aggregation assay: protein-A–coated beads coupled to HEEC1–5Fc or CEC1–5Fc were allowed to aggregate in the presence of Ca2+ or EDTA for the indicated time period.
Mentions: To make purified cadherin substrates we expressed the extracellular domains of both the human E-cadherin (HEEC1–5Fc) and Xenopus C-cadherin (CEC1–5Fc) as fusion proteins with the Fc part of human IgG in CHO cells. This insures that the protein is secreted as a dimer, which is important because functional cadherins require dimerization of the protein (Brieher et al., 1996; Tamura et al., 1998). The adhesive properties of CEC1–5Fc already have been well characterized (Chappuis-Flament et al., 2001). The HEEC1–5Fc and CEC1–5Fc proteins were isolated from conditioned medium on protein A columns. Both proteins were isolated to near purity since the size of the most abundant bands as observed by Coomassie brilliant blue staining (Fig. 1 A) corresponded to the correct molecular weights of either HEEC1–5Fc or CEC1–5Fc. Western blot analysis confirmed the identity of both proteins (Fig. 1 B). In the following experiments, we tested proteins produced by at least two different clones of secreting cells for each cadherin EC1–5Fc chimera and several protein preparations from each clone.

Bottom Line: None of the different cadherin-expressing cells exhibited any adhesive specificity toward either of the two purified cadherin substrates, even though these cadherins differ considerably in their primary sequence.However, this lack of adhesive specificity did not determine whether different cadherin-expressing cells would sort from each other, and the tendency to sort was not predictable by the extent of sequence diversity in their extracellular domains.These results show that cadherins are far more promiscuous in their adhesive-binding capacity than had been expected and that the ability to sort out must be determined by mechanisms other than simple adhesive-binding specificity.

View Article: PubMed Central - PubMed

Affiliation: Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.

ABSTRACT
Cadherin adhesion molecules play important roles in the establishment of tissue boundaries. Cells expressing different cadherins sort out from each other in cell aggregation assays. To determine the contribution of cadherin binding and adhesion specificity to the sorting process, we examined the adhesion of cells to different purified cadherin proteins. Chinese hamster ovary cell lines expressing one of four different cadherins were allowed to bind to the purified cadherin extracellular domains of either human E-cadherin or Xenopus C-cadherin, and the specificity of adhesion was compared with cell-sorting assays. None of the different cadherin-expressing cells exhibited any adhesive specificity toward either of the two purified cadherin substrates, even though these cadherins differ considerably in their primary sequence. In addition, all cells exhibited similar strengthening of adhesion on both substrates. However, this lack of adhesive specificity did not determine whether different cadherin-expressing cells would sort from each other, and the tendency to sort was not predictable by the extent of sequence diversity in their extracellular domains. These results show that cadherins are far more promiscuous in their adhesive-binding capacity than had been expected and that the ability to sort out must be determined by mechanisms other than simple adhesive-binding specificity.

Show MeSH
Related in: MedlinePlus