Walker-Warburg syndrome, caused by mutations in protein O-mannosyltransferase-1 (POMT1), is an autosomal recessive disorder characterized by severe brain malformation, muscular dystrophy, and structural eye abnormalities. As humans have a second POMT, POMT2, we cloned each Drosophila ortholog of the human POMT genes and carried out RNA interference (RNAi) knockdown to investigate the function of these proteins in vivo. Drosophila POMT2 (dPOMT2) RNAi mutant flies showed a "twisted abdomen phenotype," in which the abdomen is twisted 30 -60°, similar to the dPOMT1 mutant. Moreover, dPOMT2 interacted genetically with dPOMT1, suggesting that the dPOMTs function in collaboration with each other in vivo. We expressed dPOMTs in Sf21 cells and measured POMT activity. dPOMT2 transferred a mannose to the dystroglycan protein only when it was coexpressed with dPOMT1. Likewise, dPOMT1 showed POMT activity only when coexpressed with dPOMT2, and neither dPOMT showed any activity by itself. Each dPOMT RNAi fly totally reduced POMT activity, despite the specific reduction in the level of each dPOMT mRNA. The expression pattern of dPOMT2 mRNA was found to be similar to that of dPOMT1 mRNA using whole mount in situ hybridization. These results demonstrate that the two dPOMTs function as a protein O-mannosyltransferase in association with each other, in vitro and in vivo, to generate and maintain normal muscle development.

Embryonic stem (ES) cell self-renewal and pluripotency are maintained by several signaling cascades and by expression of intrinsic factors, such as Oct3/4 and Nanog. The signaling cascades are activated by extrinsic factors, such as leukemia inhibitory factor, bone morphogenic protein, and Wnt. However, the mechanism that regulates extrinsic signaling in ES cells is unknown. Heparan sulfate (HS) chains are ubiquitously present as the cell surface proteoglycans and are known to play crucial roles in regulating several signaling pathways. Here we investigated whether HS chains on ES cells are involved in regulating signaling pathways that are important for the maintenance of ES cells. RNA interference-mediated knockdown of HS chain elongation inhibited mouse ES cell self-renewal and induced spontaneous differentiation of the cells into extraembryonic endoderm. Furthermore, autocrine/paracrine Wnt/␤-catenin signaling through HS chains was found to be required for the regulation of Nanog expression. We propose that HS chains are important for the extrinsic signaling required for mouse ES cell self-renewal and pluripotency.

The sialyl Lewis a antigen is a well known tumor marker, CA19-9, which is frequently elevated in the serum in gastrointestinal and pancreatic cancers. UDPgalactose:N-acetylglucosamine ␤1,3-galactosyltransferase(s) (␤3Gal-Ts) are required for the synthesis of the sialyl Lewis a epitope. In the present study, a novel ␤3Gal-T, named ␤3Gal-T5, was isolated from a Colo205 cDNA library using a degenerate primer strategy based on the amino acid sequences of the four human ␤3Gal-T genes cloned to date. Transfection experiments demonstrated that HCT-15 cells transfected with the ␤3Gal-T5 gene expressed all the type 1 Lewis antigens. In gastrointestinal and pancreatic cancer cell lines, the amounts of ␤3Gal-T5 transcripts were quite well correlated with the amounts of the sialyl Lewis a antigens. The ␤1,3Gal-T activity toward agalacto-lacto-N-neotetraose was also well correlated with the amounts of ␤3Gal-T5 transcripts in a series of cultured cancer cells, and in Namalwa and HCT-15 cells transfected with the ␤3Gal-T5 gene. Thus, the ␤3Gal-T5 gene is the most probable candidate responsible for the synthesis of the type 1 Lewis antigens in gastrointestinal and pancreatic epithelia and tumor cells derived therefrom. In addition, ␤3Gal-T5 is a key enzyme that determines the amounts of the type 1 Lewis antigens including the sialyl Lewis a antigen.