The carbohydrate chains of glycolipids and glycoproteins at the cell surface are known to undergo many rapid changes during embryogenesis. These developmental changes include (a) alteration of branching structure in lactoseries carbohydrate chains, identified as I/i interconversion (1), (b) modification of terminal chains, responsible for the transition from Ii to ABH or SSEA-1 (reviewed in 2), (c) transition of globoseries antigen expression through pk, p, and Forssman (3), and (d) switching of core structure synthesis from one series to another, such as SSEA-3 ÷ globoseries to SSEA-1 + lactoseries (4). Although these structural changes have been defined clearly using various monoclonal antibodies, their physiological significance is unclear.The appearance of SSEA-1 on the embryo surface after the third cleavage division correlates approximately in time with the onset of compaction (5). During compaction, blastomeres maximize their intercellular contacts and generate a polar distribution of microvilli (6). Identification of SSEA-1 as the X hapten (Gall31 ---* 4[Fucal --0 3]GIcNAcI31 ~ R) (7-9) suggested the possibility of a 3-fucosyl-N-acetyllactosamine recognition system in the early mouse embryo. To test this hypothesis, we have studied the effect of lacto-N-fucopentaose III, its analogues, and their multivalent lysyllysine conjugates on compaction.
Materials and Methods
Purification of Oligosaccharides.A mixture of lacto-N-fucopentaose (LNFP) I, II, and III was obtained from human milk by the method of Kobata (10). LNFP I, II, and III (unreduced) were separated after acetylation by preparative thin-layer chromatography on HPTLC plates (J. T. Baker Chemical Co., Phiilipsburg, NJ) using a solvent system of butylacetate-acetone-water (25:8:1). After deacetylation, purified pentasaccharides were lyophilized and stored at -20 ° C. Chitotriose (GlcNAcI31 --~ 3GlcNA031 ---, 3GIcNAc) was a gift from Professor Toshiaki Osawa, University of Tokyo.Covalent Attachment of Oligosaccharides to LysyUysine.
We previously proposed specific interaction of Le(x) (Gal beta 1-->4 [Fuc alpha 1-->3]-GlcNAc beta 1-->3Gal) with Le(x) as a basis of cell adhesion in pre-implantation embryos and in aggregation of F9 teratocarcinoma cells, based on several lines of evidence (Eggens et al., J. Biol Chem (1989) 264:9476-9484). We now present additional evidence for this concept, based on autoaggregation studies of plastic beads coated with glycosphingolipids (GSLs) bearing Le(x) or other epitopes, and affinity chromatography on Le(x)-columns of multivalent lactofucopentaose III (Le(x) oligosaccharide) conjugated with lysyllysine. Comparative adhesion studies of Le(x)-expressing tumour cells vs their Le(x)-non-expressing variants showed that only Le(x)-expressing cells adhere to Le(x)-coated plates and are involved in tumour cell aggregation, in analogy to F9 cell aggregation. The major carrier of Le(x) determinant in F9 cells is not GSL but rather polylactosaminoglycan ('embryoglycan'), and we demonstrated autoaggregation of purified embryoglycan in the presence of Ca2+, and reversible dissociation in the absence of Ca2+ (addition of EDTA). Defucosylated embryoglycan did not show autoaggregation under the same conditions. Thus, Le(x)-Le(x) interaction has been demonstrated on a lactosaminoglycan basis as well as a GSL basis. A molecular model of Le(x)-Le(x) interaction based on minimum energy conformation with involvement of Ca2+ is presented.
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