Abstract
Total glycans were isolated and purified from Lytechinus pictus embryos at early developmental stages by gel-filtration chromatography after pronase and DNase digestion, and alkali-borohydride treatment. Fractionation by Superose 6 and HPLC gel-filtration chromatography revealed three major glycan fractions of 580, 150 and 2 kDa consistently throughout development up to the stage of end gastrula. The 580-kDa and the 150-kDa glycan fractions isolated from fertilized eggs up to the stage of end gastrula are highly acidic, whereas the 2-kDa glycan fractions have no detectable uronic acid residues and charged groups. Chemical analysis of the glycan fractions showed that their content of neutral hexoses, uronic acid, GlcNAc, GalNAc and sulphate changes during development. The resistance of the 580-kDa and the 150-kDa glycan fractions to glycosaminoglycan-degrading enzymes indicates a structure which is different from the glycosaminoglycans. The incorporation of [3H]glucosamine into the 580-kDa, the 150-kDa and the 2-kDa glycan fractions showed that glycan synthesis increases in a linear fashion from the stage of early blastulation to end of gastrulation. Maximal incorporation of the radioligand occurs in the 2-kDa glycan fractions, with the highest rate observed between the stages of mesenchyme blastula and early gastrulation. Immunological studies, using a monoclonal antibody which inhibits cell aggregation, showed that the total glycans isolated from morula, early blastulation, early gastrulation and the end of gastrulation carry cell-adhesion epitopes. The number of these epitopes, as indicated by the intensity of the immunostaining, increases from morula formation to end-gastrulation stages and correlates with the increased rate of morphogenetic movements. These results suggest that controlled expression of the cell-adhesion glycan epitopes play an important role in sea urchin gastrulation.
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