Le<sup>x</sup> glycan and E-cadherin (Ecad) are co-expressed at embryonal stem (ES) cells and embryonal carcinoma (EC) cells. While the structure and function of Ecad mediating homotypic adhesion of these cells have been well established, evidence that Le<sup>x</sup> glycan also mediates such adhesion is weak, despite the fact that Le<sup>x</sup> oligosaccharide inhibits the compaction process. To provide stronger evidence, we knocked out Ecad gene in EC and ES cells to establish F9 Ecad (-/-) and D3M Ecad (-/-) cells, which highly express Le<sup>x</sup> glycan but do not express Ecad at all. Both F9 Ecad (-/-) and D3M Ecad (-/-) cells displayed strong autoaggregation in the presence of Ca<sup>2+</sup>, while PYS-2 cells, which express trace amount of Ecad and undetectable level of Le<sup>x</sup> glycan, did not display autoaggregation. In addition, F9 Ecad -/- and D3M Ecad -/- cells displayed strong adhesion to plates coated with Le<sup>x</sup> glycosphingolipid (III(3)FucnLc4Cer), in dose-dependent manner, in the presence of Ca<sup>2+</sup>. Thus, ES or EC cells display autoaggregation and strong adhesion to Le<sup>x</sup>-coated plates in the absence of Ecad, further supporting the notion of Le<sup>x</sup> self-recognition (i.e., Le<sup>x</sup>-to-Le<sup>x</sup> interaction) in cell adhesion
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