593 Regulation of signal transduction pathways in development by glycosylation Robert S Haltiwanger Recent studies from several laboratories have provided evidence that cell surface complex carbohydrates play key roles in the regulation of developmentally relevant signal transduction events The demonstration that Fringe a known modifier of Notch function is a fucose specific N acetylglucosaminyltransferase provided strong evidence that the Notch signaling pathway could be regulated by alterations of O fucose structures More recently the demonstration that O fucose modification of Cripto is essential for Nodaldependent signaling provides further evidence of a role for glycosylation in signal transduction These and other examples provide a new paradigm for the regulation of signal transduction events by glycosylation Address Department of Biochemistry and Cell Biology Institute for Cell and Developmental Biology State University of New York at Stony Brook Stony Brook New York 11794 5215 USA Current Opinion in Structural Biology 2002 12 593 598 0959 440X 02 see front matter 2002 Elsevier Science Ltd All rights reserved Abbreviations CFC Cripto FRL 1 Cryptic CSL CBF1 Suppressor of hairless Lag 1 EGF epidermal growth factor GlcNAc N acetylglucosamine GPI glycosyl phosphatidyl inositol O FucT 1 GDP fucose protein O fucosyltransferase 1 TGF transforming growth factor uPA urinary type plasminogen activator Introduction The discovery of the complexity and diversity of complex carbohydrates on the cell surface led researchers over 30 years ago to hypothesize that glycoconjugates play roles in communication between cells and in the transfer of information from the outside of the cell to the inside 1 As this type of communication is essential for numerous stages of development specific carbohydrate modifications were proposed to play roles in particular biological events at the cell surface Over the years numerous observations have supported this concept Early on many of the stage specific embryonic antigens e g SSEA 1 SSEA 3 SSEA 4 HNK 1 were demonstrated to be specific carbohydrate structures 2 For instance SSEA 1 is the Lewis x oligosaccharide SSEA 3 and SSEA 4 are glycolipids of the globo series and HNK 1 is a sulfated glycan The expression of unique glycan structures at specific stages implied a particular function for that structure The demonstration that the presence or absence of polysialic acid alters homotypic interactions of the neural cell adhesion molecule NCAM added further support 2 More recently the demonstration of embryonic lethality resulting from the genetic ablation of several glycosyltransferases has revealed that particular carbohydrate structures are essential for development to proceed past certain stages 3 Nonetheless with the exception of NCAM identifying examples of particular carbohydrate structures on specific proteins mediating such effects was for many years elusive Recent work has begun to identify some of these molecules The first examples came with the demonstration that cell surface heparan sulfate proteoglycans play an essential role in Wnt hedgehog FGF fibroblast growth factor and TGF transforming growth factor superfamily pathways More recently O fucose modifications of epidermal growth factor EGF like repeats have been shown to modulate Notch TGF family Nodal and urinarytype plasminogen activator uPA signal transduction Several excellent recent reviews have been written concerning the role of heparan sulfate proteoglycans in development 4 6 and thus will not be considered further here This review will focus on the recent studies of the O fucose modifications of EGF repeats of Notch Cripto and uPA Involvement of O fucose modifications of EGF repeats in signal transduction O linked carbohydrate modifications of EGF repeats Fucose O linked to serine or threonine was first observed over 25 years ago as amino acid fucosides isolated from human urine 7 The first protein reported to bear O fucose was uPA 8 quickly followed by tissue type plasminogen activator and several clotting factors factors VI IX and XII 9 Comparison of the sequences surrounding the sites of O fucose modification on these proteins showed the fucose to be localized to a putative consensus sequence within EGF repeats EGF repeats are small approximately 40 amino acid protein motifs originally observed in epidermal growth factor They are defined by the presence of six conserved cysteine residues that form three disulfide bonds Figure 1 10 EGF repeats occur in dozens of cell surface and secreted proteins and are known to play roles in protein protein interactions The O fucose modification occurs between the second and third conserved cysteines of the putative consensus sequence C2XXGGS TC3 9 Proteins predicted to be modified with O fucose based on the presence of this consensus sequence have been demonstrated to bear the modification 11 14 indicating that it can be used to make accurate predictions about whether a protein will bear O fucose Table 1 Recent work has suggested that the originally proposed consensus site is too narrow and that O fucose modifications occur more broadly than predicted 14 As a result a broader consensus site C2X3 5S TC3 has recently been proposed 594 Carbohydrates and glycoconjugates Figure 1 Table 1 O fucose modified proteins C Proteins known to be modified with O fucose 5 2 3SiaT Blood clotting dissolution Urokinase uPA 8 Tissue type plasminogen activator tPA 46 Desmodus bat salivary plasminogen activator 47 Factor VII 48 Factor IX 49 Factor XII 50 6 Fringe X X2 4 X X Sialic acid 2 3 Galactose 1 4 GlcNAc 1 3 Fucose O S 3 1 4GalT1 O FucT 1 N Current Opinion in Structural Biology EGF repeat modified with the O fucose tetrasaccharide A representation of an EGF repeat based on EGF1 from factor VII 18 modified with the O fucose tetrasaccharide is shown The conserved cysteines of the EGF repeat are in yellow and are numbered and the disulfide bonds between them C1 C3 C2 C4 and C5 C6 are shown The serine shown or threonine modified with O fucose is in blue and the other amino acids between C2 and C3 are shown as X The number of Xs can vary between 3 and 5 14 The enzymes responsible for the addition of each saccharide are indicated O FucT 1 15 17 Fringe 30 37 4GalT1 39 and either 2 3SiaT shown or 2 6SiaT 11 30 Adapted from 30 The enzyme responsible for the addition of O fucose to EGF repeats GDP fucose protein O fucosyltransferase 1 O FucT 1 has been identified and