Abstract
Notch signaling is a component of a wide variety of developmental processes in many organisms. Notch activity can be modulated by O-fucosylation (mediated by protein O-fucosyltransferase-1) and Fringe, a beta1,3-N-acetylglucosaminyltransferase that modifies O-fucose in the context of epidermal growth factor-like (EGF) repeats. Fringe was initially described in Drosophila, and three mammalian homologues have been identified, Manic fringe, Lunatic fringe, and Radical fringe. Here for the first time we have demonstrated that, similar to Manic and Lunatic, Radical fringe is also a fucose-specific beta1,3-N-acetylglucosaminyltransferase. The fact that three Fringe homologues exist in mammals raises the question of whether and how these enzymes differ. Although Notch contains numerous EGF repeats that are predicted to be modified by O-fucose, previous studies in our laboratory have demonstrated that not all O-fucosylated EGF repeats of Notch are further modified by Fringe, suggesting that the Fringe enzymes can differentiate between them. In this work, we have sought to identify specificity determinants for the recognition of an individual O-fucosylated EGF repeat by the Fringe enzymes. We have also sought to determine differences in the biochemical behavior of the Fringes with regard to their in vitro enzymatic activities. Using both in vivo and in vitro experiments, we have found two amino acids that appear to be important for the recognition of an O-fucosylated EGF repeat by all three mammalian Fringes. These amino acids provide an initial step toward defining sequences that will allow us to predict which O-fucosylated EGF repeats are modified by the Fringes.
Highlights
Many of these epidermal growth factor-like (EGF) repeats contain consensus sites for modification by O-fucose (a process mediated by the protein O-fucosyltransferase-1 (O-FucT-1) [2, 3]
3 The abbreviations used are: EGF, epidermal growth factor-like; Bnz-fuc, benzyl-␣-Ofucose; Mfng, Manic fringe; Lfng, Lunatic fringe; Rfng, Radical fringe; Drosophila Fringe (Dfng), Drosophila fringe; O-FucT-1, protein O-fucosyltransferase-1; CHO, Chinese hamster ovary; HPAEC, high pH anion exchange chromatography; Ni-NTA, nickel-nitrilotriacetic acid; many of these EGF repeats contain consensus sites for modification by O-fucose (a process mediated by the protein O-fucosyltransferase-1 (O-FucT-1) [2, 3]
We have demonstrated that Rfng is a fucose-specific 1,3-N-GlcNAc transferase (similar to Lfng and Mfng [4]), and we have sought to determine the differences in MS/MS, mass spectrometry/mass spectrometry; HPLC, high pressure liquid chromatography
Summary
Preparation of Constructs—Amino acids 93–129 of human factor IX and amino acids 106 –147 of human factor VII were cloned into pSecTag (Invitrogen) using the HindIII and XhoI restriction sites. pSecTag encodes an Ig signal sequence for secretion and C-terminal Myc and hexahistidine tags. Metabolic Labeling—Lec1-CHO cells were transiently transfected with pSecTag constructs encoding the first EGF repeat of human clotting factors VII or IX or versions of these plasmids containing point mutants as described previously [6]. EGF repeats were further purified by reversed phase HPLC on a 250 ϫ 4.6-mm Dynamax C18 column (Rainin) as previously described [23]. The O-fucosylated EGF repeat was purified by reversed phase HPLC as described above using a 90-min 0 – 80% acetonitrile gradient in 0.1% trifluoroacetic acid. To confirm fucosylation of the EGF repeats, samples of HPLC fractions containing the EGF repeats were dried in the SpeedVac (Savant) and resuspended in 20% acetonitrile and 0.1% formic acid. Other Methods—Analysis of the Rfng product was performed essentially as described previously [2, 3], except that the HPAEC analysis was performed using an isocratic gradient at 26 mM NaOH
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