The embryonic development is a complex process in which many signaling pathways and growth factors are involved. Most receptors are glycosylated proteins and, therefore changes in the glycosylation machinery can affect the function of these glycoproteins. Moreover, changes in glycosylation occur during the ontogenesis and cell differentiation. Nucleotide sugar transporters deliver activated sugars for glycosyltransferases and therefore, mutations of NSTs usually affect the function of many transferases and have pleiotropic effects. The first malformation caused by a mutation in an NST gene has been described in C. elegans. Squashed vulva (sqv) mutants are defective in epithelial invaginations and vulva formation during ontogeny (Herman et al., 1999).
On molecular level it has been shown to affect glycosaminoglycan biosynthesis. Eight sqv mutants have been isolated, all involved in glycosaminoglycan biosynthesis or the biosyntheses of nucleotide sugars. The sqv-7 gene encodes a multi membrane spanning protein and has been proposed to be an NST (Herman et al., 1999). The protein has been expressed in Saccharomyces cerevisiae and in-vitro transport assay indicated activity for GlcA, Gal and UDP-GalNAc (Berninsone et al., 2001). Glycosaminoglycan sidechains of proteoglycans in the extracellular matrix at the cell surfaces are implicated in the signaling functions of various heparin-binding growth factors and chemokines, and play critical roles in the development. The
Introduction
sqv-7 transporter is thought to deliver activated sugars for glycosyltransferases involved in glycosaminoglycan biosynthesis.
In Drosophila melanogaster, Notch is a receptor involved in determination of the dorsal-ventral boundary in the wing and eye imaginal discs during development. Fringe, a fucose-specific GlcNAc transferase, modifies Notch by elongating oligosaccharide chains attached to the O-linked fucose residues on the receptor. Changes in glycosylation modulate the interaction of Notch with its ligands (Blair, 2000). A defect in the fringe (fng) gene leads to mutant phenotypes including nicked wing margin, thickened wing vein, rough eye, duplicated notal macrochaetae and fused leg segments, due to aberrant boundary formations. Mutation in another gene, "fringe connection" (frc), also produces fng like phenotypes including nicked wing margin, thickened wing vein, rough eye and fused leg segments. It has been shown that frc encodes an NST with substrate specificity for UDP-GlcA and UDP-GlcNAc (Goto et al., 2001;Selva et al., 2001).
The slalom phenotype in Drosophila has been shown to associate with mutation in the sll gene. Sll is essential for Drosophila, however viable transheterozygote mutants with reduced sll activity have been investigated. On the molecular level, alterations in Wg, Hh TGF-β signaling pathways have been observed. Wg and Hh exhibit gradient expressions, which are engaged in a patterning of the ventral embryonic epidermis, development of wing imaginal disc and wing veins. Expressed phenotype and comparison with other Drosophila mutants, have suggested defects in glycosaminoglycan biosynthesis. Hydrophobicity analyses and BLAST search have indicated homology of sll with NSTs. In-vitro transport assays, have demonstrated specific uptake of PAPS (Luders et al., 2003). Based on homology with sll, the human PAPS transporter has been identified (Kamiyama et al., 2003). Recently, a second human PAPS transporter with tissue specific expression has been described (Kamiyama et al., 2006). Mutations in the PAPS transporter affect biosynthesis of sulfated glycosaminoglycans, which play important roles in many signaling pathways.
In human, diseases caused by mutations in genes involved in glycosylation processes are known as Congenital Disorders of Glycosylation (CDG). The first CDG caused by a mutation in an NST has been described in patients with a new type of Leukocyte Adhesion Deficiency syndrome (LAD) (Etzioni et al., 1992). The LAD-II patients exhibit severe psychomotor, mental and growth retardation and suffer from recurrent infections. Biochemically this syndrome has
been characterized by a generalized lack of fucosylated glycoconjugates, including immunologically important selectin ligands, which play role in the initial interaction of leukocytes with the endothelium before extravasation (Marquardt 1999). Studies of cultured cells from a patient revealed that the transport of GDP-fucose into isolated Golgi-enriched microsomal vesicles is reduced (Lubke et al., 1999). The defect in fucose metabolism could be partially corrected in patient by oral supplementation of fucose (Marquardt et al., 1999). Two independent groups succeeded in cloning of the first GDP-Fuc transporter by using a complementation cloning strategy in which restoration of fucosylation was detected by fucose specific lectin staining. LAD patient fibroblasts have been transfected with a human retroviral cDNA library and a single clone able to restore fucosylation has been identified. In Golgi enriched vesicles, the protein was able to restore the GDP-Fuc import and two missense mutation have been identified in a patient’s genome(Lubke et al., 2001). In the same time, twelve out of sixteen cDNAs from C. elegans that encode hydrophobic proteins with significant homology to known nucleotide sugar transporters, were isolated and transfected into fibroblasts from a LAD II patient. One of these clones recovered the expression of fucosylated glycoconjugates. Further, the human homolog of this transporter has been identified and verified to complement the defect in LAD II fibroblasts. In this study, C. elegans and human GDP-Fuc transporters have been characterized (Luhn et al., 2001). Recently, fibroblast from LAD II patients were applied for cloning of Drosophila GDP-Fuc transporter (Luhn et al., 2004).
The bovine Complex Vertebral Malformation (CVM) is a recessively identified inherited disorder with onset during fetal development, leading to frequent abortion of fetuses or perinatal death, and vertebral anomalies (Agerholm et al., 2001;Agerholm et al., 2004a;Agerholm et al., 2004b;Etzioni et al., 1992;Nielsen et al., 2003). CVM has been characterized by misshapen and fused vertebrae around the cervico–thoracic junction. The extensive use of a limited number of elite bulls in cattle breeding leaded to rapid spread of this syndrome. By genetic mapping analyses, the defect has been restricted to an area of the genome where the gene of the bovine homolog of the human UDP-GlcNAc transporter (SLC35A3 according to the new systematic of human NSTs (Ishida and Kawakita, 2004)) is located. The gene function has been confirmed by complementation of the mnn2-2 mutant of K. lactis lacking UDP-GlcNAc transport (Abeijon et al., 1996b;Abeijon et al., 1996a). The wild type sequence restores the UDP-GlcNAc transport in yeast while the mutation V180D was not (Thomsen et al., 2006).
Introduction
Malformations caused by mutations in NST genes clearly demonstrate the importance of these transporters in ontogenesis of multicellular organisms.