In preimplantation embryos, stage-specific embryonic antigen (SSEA)-1, SSEA-3, and SSEA-4 are well-known carbohydrate marker antigens that show a stage-specific expression. SSEA-1 is usually carried by glycoproteins and/or glycolipids, while SSEA-3 and SSEA-4 are mostly carried by glycosphingolipids, which are the major constituents of raft microdomains. Biochemical analysis and confocal laser-scanning microscopic study of raft microdomains have been conducted in cultured cell lines. Here, we show the dynamic movement of SSEA-4 in the raft microdomains of preimplantation mouse embryos by immunostaining with 6E2, a novel anti-SSEA-4 monoclonal antibody, and subsequently discuss the role of raft microdomains in early embryogenesis.
The most important consideration for the synthesis of oligosaccharides is the stereocontrol of glycosylation reactions. Recently, we developed new methods for efficient α-sialylation and α-galactosylation by exploiting the effects of a remote substituent on the reactivity and/or stereoselectivity of glycosyl donors. These methods have enabled the efficient synthesis of various natural biologically important glycans.
Notch signaling regulates numerous cell fate decisions during development. In a genetic screen designed to isolate new components of this pathway, mutations in a novel gene, rumi, were identified to cause a temperature-sensitive phenotype. At 28-30°C, rumi clones exhibited a full-blown Notch phenotype in all the examined tissues. However, at 18°C, only a mild Notch phenotype was evident. Notch accumulated intracellularly and in the cell membrane of rumi cells, but was not properly processed despite normal binding to Delta. Rumi is an endoplasmic reticulum (ER)-retained protein with a highly conserved CAP10 domain. Our studies showed that Rumi is a protein O-glucosyltransferase (Poglut), capable of adding glucose in an O-linkage to serine residues in the consensus C1-X-S-X-P-C2 sequence within the EGF repeats of the Notch extracellular domain. These data indicate that Rumi regulates Notch folding and/or trafficking and allows signaling at the cell membrane by O-glucosylation of Notch in the ER. Here, we, in collaboration with Dr. Hugo Bellen's laboratory, describe our method of discovery of Rumi (1). We also highlight the functional importance of O-linked sugar modifications on Notch.