2-O-phosphorylation of xylose has been detected in the glycosaminoglycan-protein linkage region, GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser, of proteoglycans. Recent mutant analyses in zebrafish suggest that xylosyltransferase I and FAM20B, a protein of unknown function that shows weak similarity to a Golgi kinase encoded by four-jointed, operate in a linear pathway for proteoglycan production. In the present study, we identified FAM20B as a kinase that phosphorylates the xylose residue in the linkage region. Overexpression of FAM20B increased the amount of both chondroitin sulfate and heparan sulfate in HeLa cells, whereas the RNA interference of FAM20B resulted in a reduction of their amount in the cells. Gel-filtration analysis of the glycosaminoglycan chains synthesized in the overexpressing cells revealed that the glycosaminoglycan chains had a similar length to those in mock-transfected cells. These results suggest that FAM20B regulates the number of glycosaminoglycan chains by phosphorylating the xylose residue in the glycosaminoglycan-protein linkage region of proteoglycans.
We recently cloned human chondroitin synthase (ChSy) exhibiting the glucuronyltransferase-II (GlcAT-II) and N-acetylgalactosaminyltransferase-II (GalNAcT-II) activities responsible for the biosynthesis of repeating disaccharide units of chondroitin sulfate, but chondroitin polymerization was not demonstrated in vitro using the recombinant ChSy. We report here that the chondroitin polymerizing activity requires concomitant expression of a novel protein designated chondroitin polymerizing factor (ChPF) with ChSy. The human ChPF consists of 775 amino acids with a type II transmembrane protein topology. The amino acid sequence displayed 23% identity to that of human ChSy. The expression of a soluble recombinant form of the protein in COS-1 cells produced a protein with little GlcAT-II or GalNAcT-II activity. In contrast, coexpression of the ChPF and ChSy yielded markedly augmented glycosyltransferase activities, whereas simple mixing of the two separately expressed proteins did not. Moreover, using both UDP-glucuronic acid (GlcUA) and UDP-N-acetylgalactosamine (GalNAc) as sugar donors, chondroitin polymerization was demonstrated on the so-called glycosaminoglycan-protein linkage region tetrasaccharide sequence of ␣-thrombomodulin. These results suggested that the ChPF acts as a specific activating factor for ChSy in chondroitin polymerization. The coding region of the ChPF was divided into four discrete exons and localized to chromosome 2q35-q36. Northern blot analysis revealed that the ChPF gene exhibited a markedly different expression pattern among various human tissues, which was similar to that of ChSy. Thus, the ChPF is required for chondroitin polymerizing activity of mammalian ChSy.
Previously, we have demonstrated that co-expression of ChSy-1 (chondroitin synthase-1), with ChPF (chondroitin-polymerizing factor) resulted in a marked augmentation of glycosyltransferase activities and the expression of the chondroitin polymerase activity of ChSy-1. These results prompted us to evaluate the effects of co-expression of the recently cloned CSS3 (chondroitin sulfate synthase-3) with ChPF, because ChSy-1 and CSS3 have similar properties, i.e. they possess GalNAcT-II (N-acetylgalactosaminyltransferase-II) and GlcAT-II (glucuronyltransferase-II) activities responsible for the elongation of CS (chondroitin sulfate) chains but cannot polymerize chondroitin chains by themselves. Co-expressed CSS3 and ChPF showed not only substantial GalNAcT-II and GlcAT-II activities but also chondroitin polymerase activity. Interestingly, co-expressed ChSy-1 and CSS3 also exhibited polymerase activity. The chain length of chondroitin formed by the co-expressed proteins in various combinations was different. In addition, interactions between any two of ChSy-1, CSS3 and ChPF were demonstrated by pull-down assays. Moreover, overexpression of CSS3 increased the amount of CS in HeLa cells, while the RNA interference of CSS3 resulted in a reduction in the amount of CS in the cells. Altogether these results suggest that chondroitin polymerization is achieved by multiple combinations of ChSy-1, CSS3 and ChPF. Based on these characteristics, we have renamed CSS3 ChSy-2 (chondroitin synthase-2).
Cancer stem cells (CSCs) represent a small subpopulation of self-renewing oncogenic cells. As in many other stem cells, metabolic reprogramming has been implicated to be a key characteristic of CSCs. However, little is known about how the metabolic features of cancer cells are controlled to orchestrate their CSC-like properties. We recently demonstrated that hyaluronan (HA) overproduction allowed plastic cancer cells to revert to stem cell states. Here, we adopted stable isotope-assisted tracing and mass spectrometry profiling to elucidate the metabolic features of HA-overproducing breast cancer cells. These integrated approaches disclosed an acceleration of metabolic flux in the hexosamine biosynthetic pathway (HBP). A metabolic shift toward glycolysis was also evident by quantitative targeted metabolomics, which was validated by the expression profiles of key glycolytic enzymes. Forced expression of glutamine:fructose-6-phosphate amidotransferase 1 (GFAT1), an HBP rate-limiting enzyme, resembled the results of HA overproduction with regard to HIF-1α accumulation and glycolytic program, whereas GFAT1 inhibition significantly decreased HIF-1α protein level in HA-overproducing cancer cells. Moreover, inhibition of the HBP-HIF-1 axis abrogated HA-driven glycolytic enhancement and reduced the CSC-like subpopulation. Taken together, our results provide compelling evidence that HA production regulates the metabolic and CSC-like properties of breast cancer cells via HBP-coupled HIF-1 signaling.
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