UDP xylose synthase 1 is required for morphogenesis and histogenesis of the craniofacial skeleton

Abstract

UDP-xylose synthase (Uxs1) is strongly conserved from bacteria to humans, but because no mutation has been studied in any animal, we do not understand its roles in development. Furthermore, no crystal structure has been published. Uxs1 synthesizes UDP-xylose, which initiates glycosaminoglycan attachment to a protein core during proteoglycan formation. Crystal structure and biochemical analyses revealed that an R233H substitution mutation in zebrafish uxs1 alters an arginine buried in the dimer interface, thereby destabilizing and, as enzyme assays show, inactivating the enzyme. Homozygous uxs1 mutants lack Alcian blue-positive, proteoglycan-rich extracellular matrix in cartilages of the neurocranium, pharyngeal arches, and pectoral girdle. Transcripts for uxs1 localize to skeletal domains at hatching. GFP-labeled neural crest cells revealed defective organization and morphogenesis of chondrocytes, perichondrium, and bone in uxs1 mutants. Proteoglycans were dramatically reduced and defectively localized in uxs1 mutants. Although col2a1a transcripts over-accumulated in uxs1 mutants, diminished quantities of Col2a1 protein suggested a role for proteoglycans in collagen secretion or localization. Expression of col10a1, indian hedgehog, and patched was disrupted in mutants, reflecting improper chondrocyte/perichondrium signaling. Up-regulation of sox9a, sox9b, and runx2b in mutants suggested a molecular mechanism consistent with a role for proteoglycans in regulating skeletal cell fate. Together, our data reveal time-dependent changes to gene expression in uxs1 mutants that support a signaling role for proteoglycans during at least two distinct phases of skeletal development. These investigations are the first to examine the effect of mutation on the structure and function of Uxs1 protein in any vertebrate embryos, and reveal that Uxs1 activity is essential for the production and organization of skeletal extracellular matrix, with consequent effects on cartilage, perichondral, and bone morphogenesis.

Dual Roles of O-Glucose Glycans Redundant with Monosaccharide O-Fucose on Notch in Notch Trafficking

“…Mutants of other genes required for GAG synthesis and maturation, such as toutvelu and sulfateless, are also recessive lethal at the same developmental stage as Uxs 1 , which supports our idea that GAG synthesis is disrupted in the Uxs 1 mutant (29,41). Oxt is another enzyme required for GAG synthesis (38). Although an oxt mutant has not been reported, the knockdown of oxt in Drosophila by RNA interference was reported (22).…”

“…In mammals, UDP-xylose synthesis fully depends on UDP-xylose synthase 1, which encodes their only UDP-xylose synthase (37). The Drosophila ortholog of UDP-xylose synthase 1 is UDPxylose synthase (Uxs; CG7979), the only gene in the Drosophila genome that encodes UDP-xylose synthase (38). To generate potential Uxs-null mutants, we used imprecise P-element excision.…”

“…1C). The Uxs 1 mutant has a 1201-base pair deletion of the Uxs genomic DNA locus, which removes its putative initiation codon and more than half of its coding region, corresponding to the N-terminal half of the Uxs protein (including a nucleotide-binding site) (38) (Fig. 1C).…”

On the Roles and Regulation of Chondroitin Sulfate and Heparan Sulfate in Zebrafish Pharyngeal Cartilage Morphogenesis

“…A number of genes involved in PG biosynthesis have been shown to be crucial for the development of the jaw and the pharyngeal arches in zebrafish, including glypican 4 (gpc4/knypek) (9,10), UDP-glucuronic acid decarboxylase 1 (uxs1) (11,12), exostosin 2 (ext2/dackel) (12)(13)(14)(15), exostoses (multiple)-like 3 (extl3/boxer) (15), udp-glucose dehydrogenase (ugdh/jekyll) (16,17), and solute carrier family 35, member B2 (slc35b2/ pincher, reported as papst1) (13). From previous studies of these genes, it stands relatively clear that PGs are required for the intercalation of chondrocytes during the formation of rodlike cartilage structures.…”

Antisense expression of Gossypium hirsutum UDP-glucuronate decarboxylase in Arabidopsis leads to changes in cell wall components

“…From various species of bacteria, 826 predicted UXS enzymes have been identified, suggesting that xylose is more prevalent in bacterial glycans (Coyne et al, 2011). In zebrafish, UXS1 activity is essential for functional deposition of proteoglycans in the extracellular matrix (Eames et al, 2010). The crystal structure, molecular dynamics, and response pathways of UXS have also been analyzed in humans (Eixelsbergeret al, 2012).…”