Molecular genetic analysis of the glycosyltransferase Fringe in Drosophila

Abstract

Fringe proteins are ␤1,3-N-acetylglucosaminyltransferases that modulate signaling through Notch receptors by modifying Olinked fucose on epidermal growth factor domains. Fringe is highly conserved, and comparison among 18 different Fringe proteins from 11 different species identifies a core set of 84 amino acids that are identical among all Fringes. Fringe is only distantly related to other glycosyltransferases, but analysis of the predicted Drosophila proteome identifies a set of four sequence motifs shared among Fringe and other putative ␤1,3-glycosyltransferases. To gain functional insight into these conserved sequences, we genetically and molecularly characterized 14 point mutations in Drosophila fringe. Most nonsense mutations act as recessive antimorphs, raising the possibility that Fringe may function as a dimer. Missense mutations identify two distinct motifs that are conserved among ␤1,3-glycosyltransferases, and that can be modeled onto key motifs in the crystallographic structures of bovine ␤1,4-galactosyltransferase 1 and human glucuronyltransferase I. Other missense mutations map to amino acids that are conserved among Fringe proteins, but not among other glycosyltransferases, and thus may identify structural motifs that are required for unique aspects of Fringe activity. Although the Notch pathway mediates a wide range of cell fate decisions, the influence of fringe ( fng) genes on Notch signaling has been best studied during the development of the Drosophila wing. In the developing wing imaginal disk, FNG inhibits the ability of Serrate to activate Notch, and potentiates the ability of Delta to activate Notch. These effects of FNG position a stripe of Notch activation along the border between dorsal and ventral cells, which is essential for the subsequent patterning, growth, and compartmentalization of the wing (reviewed in refs. 5 and 6).Drosophila and vertebrate FNG proteins are highly conserved and possess an unusual ␤1,3-N-acetylglucosaminyltransferase activity that elongates O-linked fucose within EGF domains (7,8). Although FNG is not closely related to other glycosyltransferases, it does share a few short sequence motifs with certain other glycosyltransferases (9), and we report here that it is most closely related to members of a ␤1,3-glycosyltransferase (␤3GT) superfamily. These enzymes use a variety of sugar donors and acceptors, but all transfer the sugar from a UDP-sugar donor, creating a ␤ linkage between the 1 carbon of the donor and the 3 carbon of the acceptor. With the exception of site-directed mutations in a DDD motif of FNG (7,8,10,38), the functional requirements for conserved sequence motifs in FNG or other ␤3GTs have not been assessed. Moreover, the only crystallographic structure for a ␤3GT determined to date is that of human glucuronyltransferase I (GlcAT-I), which does not share significant primary sequence similarity with FNG.Here, we employ two complementary approaches to identify functionally important amino acids within FNG: sequence conservation, and genetic and mole...

DrosophilaGlial Glutamate Transporter Eaat1 Is Regulated by Fringe-Mediated Notch Signaling and Is Essential for Larval Locomotion

“…Fly stocks were obtained from the Bloomington Stock Center and the Vienna Drosophila RNAi Center (VDRC), or alternative published sources as noted: P{EPgy2}Eaat1 EY20741 ; Df(2L)30A-C (here denoted Eaat1 Df ); UAS-mCD8::RFP; UAS-Eaat1; UAS-Pros RNAi (VDRC ID 101477); Eaat1-Gal4 (Rival et al, 2004); htl-Gal4 (Shishido et al, 1997); Repo-Gal4 (Sepp and Auld, 2003); tub-Gal80 ts (McGuire et al, 2003); UAS-N ICD (Go et al, 1998); UAS-Hairless (Maier et al, 1999); UAS-Tom (Lai et al, 2000); UAS-Eaat1::GFP (Rival et al, 2004); UAS-mCD8::GFP (Lee and Luo, 1999); UAS-nuclearGFP (van Meyel et al, 2003); Su(H)-lacZ (Griffiths et al, 2007); and fng L73 and fng 13 (Irvine and Wieschaus, 1994;Correia et al, 2003). To create CG31235-nuclear green fluorescent protein (nGFP) and CG31235-Gal4, we PCR amplified 3004 base pairs (bp) of regulatory sequences located immediately upstream of CG31235 using the primers 5Ј-CATAGTGCATTGGTGAGGTGT-3Ј and 5Ј-TACTGGGTGCGCGTTAGGTC-3Ј.…”

Abnormal vertebral segmentation and the notch signaling pathway in man

“…The proband's parents, with normal spinal anatomy, were both heterozygous for the mutant allele. The phenylalanine residue substituted is highly conserved (Correia et al, 2003) and close to the active site of the enzyme. The mutation created a novel MseI restriction enzyme site, which was used to confirm the sequencing results in the pedigree.…”

Requirement for a core 1 galactosyltransferase in the Drosophila nervous system

“…We also note that four of the putative core 1 GalTs (CG3119, CG2975, CG18558, and CG2983) are located in close proximity to each other on the second chromosome (Table 1), suggesting that they arose from relatively recent duplication events. By BLASTP (Correia et al, 2003) or ClustalW analysis, CG9520 is both the closest Drosophila homologue of human core 1 GalT1, and the closest homologue of most of the putative Drosophila core 1 GalTs, and thus appears to represent the ancestral core 1 GalT in Drosophila (Fig. 1).…”

“…However, based on bioinformatic analysis, we suggested that Drosophila are likely to encode at least eight distinct core 1 GalT domains, which at that time were annotated as seven distinct genes (CG9520, CG8708, CG2975, CG3119, CG7440, CG13904, CG18558), one of which (CG13904) encoded two tandemly repeated core 1 GalT domains (Correia et al, 2003). Another gene, CG2983, shares a high degree of overall sequence similarity with the other putative core 1 GalTs, but appears to lack critical amino acids within the most highly conserved sequence motifs (Correia et al, 2003). In a subsequent refinement of the Drosophila genome annotation, CG13904 has been split into two adjacent genes, CG34056 and CG34057 (Drysdale and Crosby, 2005).…”

“…There exists a large family of enzymes that can catalyze the attachment of GalNAc to protein in mammals (ppGalNAcTs; Ten Hagen et al, 2003a), but only a single core 1 Galactosyltransferase (core 1 GalT) has been identified (Ju et al, 2002a). Conversely, bioinformatics analysis suggests that Drosophila possess large families both of ppGalNAcTs (Ten Hagen et al, 2003b), and of core 1 GalTs (Correia et al, 2003). Biochemical studies have confirmed that several of these ppGalNAcTs and at least four of the core 1 GalTs possess the expected enzymatic activity (Ten Hagen et al, 2003b;Muller et al, 2005).…”