Fig. 2. (A) Sequence comparison of Nti and human GDP-fucose O-fucosyltransferase (O-FucT-1). The identical amino acids are reversed, conserved changes are shaded. The proteins are 42% identical and 75% similar. The position of the 4R6 mutation is shown in red (replace Lysine in Nti by stop in 4R6). The putative type II transmembrane domain is underlined. The positions of the potential N-glycosylation (*) site, and the EXD motif that is important for glycosyltransferase activity (#), are also shown. (B,C) In situ hybridisation to whole-mount embryos stained with a nti cDNA probe revealed that nti mRNA is uniformly expressed in stage 3 embryos (B), and gradually disappears by stage 11 (C). nti mRNA is not detectable after stage 14 (not shown). (D) Wg expression in wing imaginal disc was recovered in nti mutant cells with hs-CG12366 (hs-nti; lack of GFP shown by green, arrowhead), indicating that nti is CG12366. (E) Expression of a nti inverted repeat RNA driven by ptc-GAL4 generates wing phenotypes (vein thickening and wing nicking indicated by arrow and arrowhead, respectively) that resemble wings containing nti homozygous mutant clones (Fig. 1F), indicating that nti-IR is indeed blocking nti function. (B-D) Anterior is towards the left and dorsal is upwards. (F) A schematic diagram of the tetrasaccharide, Sia-2, 3-Gal-ß1, 4-GlcNAc-ß1, 3-Fuc, attached to a EGF repeat of Notch. Nti adds O-linked fucose, and O-fucose residues is further elongated by Fng, ß1, 3-N-acetylglucosaminyltransferase. The N-acetylglucosamine (GlcNAc) is further elongated by an endogenous ß1, 4-galactosyltransferase and a ß2, 3-sialyltransferase. (G) Model for Fng-dependent and Fng-independent glycosylation of a Notch EGF repeat. (Upper) O-fucose residue attached by Nti is elongated to yield the tetrasaccharide in Fng-dependent manner. (Lower) Without Fng function, EGF repeats of Notch is O-fucosylated by Nti.