Special Issue: the Magic of the Sugar Code

Supplementary material

Special issue: The magic of the sugar code

Glycan variation and evolution in the eukaryotes

Anthony P Corfield and Monica Berry

Mucin Research Group, University of Bristol, School of Clinical Sciences, Bristol Royal Infirmary, Bristol BS2 8HW,

United Kingdom. Email [email protected]; Tel: +44(0) 117 9868479

*University of Bristol, School of Physics, NSQI, Tyndall Avenue, Bristol BS8 1FD, UK. E-mail: [email protected]; Tel: +44 (0)117 3940014

Eukaryotic phylogeny and representative organisms

The main groups of organisms forming the Eukaryotes are considered here, with regard to glycan structure and glycoconjugate function. A phylogenetic tree for the Eukarya is shown in Figure 1. It shows species that have had their genomes sequenced and the pertinent groups they belong to. Information on selected organisms belonging to the main phyla is given below.

Deuterostomia

The Deuterostomes comprise several major phyla, the sea urchins and starfish, (Echinodermata); the acorn worms

(Hemichordata); the ascidians (Urochrodata); amphioxus (Cephalochordata) and the large group including fish, amphibians, reptiles, birds and mammals (Vertebrata). A further superphylum (Protostomia) incorporates the corals and sponges (Porifera); anemones and hydra (Cnidara); segmented worms (Annelida), clams, oysters, snails and slugs

(Mollusca) and insects, spiders and crustacae (Arthropoda). Examples from a few of these phyla have been selected for presentation here, in order to illustrate glycobiology with well studied model organisms. This is essentially a brief snapshot, as a comprehensive appraisal is not within the scope of this review.

In sea urchins (Echinodermata) the eggs are surrounded by a gel layer of high molecular weight fucose sulfate [1], 1-3 or 1-4 linked fucose polymers in different species [2, 3]. These polymers bind to the sperm acrosome plasma membrane C-type lectin receptors and induce fertilization. In addition, polysialic acid glycans present on the egg and potentiate the acrosome reaction. Several features of the Echinodermata are shared with Vertebrata, where the extracellular matrix gel layer of the ovum, the zona pellucida, has three mucin type glycoproteins ZP1-3. These O- glycosylated glycoproteins are targets for sperm plasma membrane receptors and induce the acrosome reaction.

Xenopus laevis, the African clawed toad, also expresses lectins, which bind to mucin-like egg gel O-glycosylated glycoproteins. These, however, may play a role in pathogen binding as part of innate immune defence. Further support for the role of glycans in echinoderms has come from the identification of a number of lectins with specificity for galactose, sialic acids, and fucoidins. They function in embryogenesis and innate defence, binding to a variety of cells and other molecules in the extracellular matrix [2].

Xenopus laevis is a well-established model animal for embryonic and fertilization research. As with the echinoderms, the eggs have been used as a valuable expression system [2]. Both hyaluronan and chitin have been employed as target glycan polymers for glycan signaling. Hyaluronan is synthesized from the UDP-sugar substrates, UDP-N-acetyl-D- glucosamine and UDP-D-glucuronic acid and primers by hyaluronan synthase (HAS), which has homology with the

Xenopus gene DG42.

A number of other examples of glycomic evolution have been identified with the Metazoa. The N-glycan glycosyltransferase UDP-GlcNAc 3-D-mannoside 1-2-N-acetylglucosaminyltransferase I (GnTI, encoded by Mgat1) has been shown to occur at a similar time to the emergence of the metazoa [4], while phylogenetic studies of the sialidase family demonstrated orthologs in teleost fishes, amphibians, reptiles, birds and mammals and allowed the evolution from the ancestral sialidase, NEU1, to be plotted [5]. Similarly, the evolution of a galactose α2-3- sialyltransferase, detected in Ciona intestinalis and Takifugu rubripes, which sialylates Galβ1-3GalNAc structures on glycoproteins, could also be mapped [6].

Clues to glycan evolution can be found in Xenopus tropicalis, which expresses over 20 different gel-forming mucins, in contrast with the smaller number, five or fewer, found in mammals [7]. Xenopus tropicalis MUC2 homologues have unique cysteine rich, PTS (proline-threonine-serine) domains, which alternate with SEA (Sea urchin sperm protein

Enterokinase and Agrin) domains. In addition, the MUC4-like glycoprotein found in Xenopus contains PTS domains, together with adhesion-associated (AMOP), nidogen (NIDO) and Von Willebrand-D (VWD) domains [7]. Its secreted oviduct glycoproteins are rich in sialyl-Lea, which implies an 1-4-fucosyltransferase activity, previously thought to be restricted to primates [8].

A variety of studies have probed the glycobiology of the teleost zebrafish (Danio rerio). O-glycans and sialyl- oligosaccharides were detected in cells during gastrulation, and changes in cell-surface glycans occurred during mitosis

[9]. Glycomic analysis has disclosed discrete patterns [10]: a polysialytransferase, St8Sia3, showed phenotypes linked with segmentation and somite formation [11]. Polysialic acids (PSA) also play important roles in Zebrafish central nervous system development [12]: regions of neuronal rearrangement contain polysialyltransferases STX (St8sia2) and

PST (St8sia4) homologues, whose target is the polysialylated neural cell adhesion molecule (NCAM). Additionally, neuronal plasticity in the cerebellum may be regulated by the array of these transferases [13]. The cell adhesion molecule, siglec-4 was identified in the zebrafish genome: siglecs bind to sialic acids and are restricted to the nervous system in both teleosts and mammals [14]. A number of other Zebrafish glycan related genes have shed light on similarities between mammals and other vertebrates. These include orthologs of the mosaic sperm protein zonadhesin, which shows a tissue distribution reflecting continuing evolution with major function in the gut in the fish and a reproductive role in later mammals [15].

The sialomucin endolyn (CD 164), a highly glycosylated membrane protein that shuttles between the cell surface and lysosomes in mammalian kidneys, is necessary for normal zebrafish kidney function through apical targeting and endocytosis [16]. The extracellular adaptor protein matrilin-3 contains Von Willebrand-A (VWA) and epidermal growth factor-like (EGF) repeats. Its exon- intron organization is highly similar to the mammalian genes, including a

PTS domain, which suggests mucin-like properties. Developmental patterns and tissue distribution of matrilin 3 in zebrafish are similar to those reported in mammals [17].

Among the Deuterostomes, vertebrates have attracted continuous interest because of their relevance to human biology; the most common model organisms being mammals and rodents, especially the mouse [2]. Other vertebrates have been studied due to commercial interest: improvements in animal health and welfare, in conjunction with animal product management e.g. milk and egg production. Continuing efforts are being made to increase breeding efficiency through better understanding of fertilization processes in cattle and horses.

The fungi include yeasts, molds, puffballs and mushrooms. They have attracted wide interest due to their ease of manipulation for experimental analysis of glycosylation processes and their value in the production of antibiotics and foodstuffs. Moreover, pathogenic species, especially Candida and Cryptococcus, have focused attention on their glycans metabolic pathways [18]. The use of yeasts for glycoengineering is a valuable tool in the production of glycosylated recombinant proteins [19, 20].

Glycosylation of proteins is a major feature in yeasts and shows distinct variation in the range of monosaccharides and associated glycan sequences [21]. Although there are many advantages in the use of yeasts as models for glycosylation, important differences from mammalian glycosylation have limited their use. These include the inability to synthesize sialic acids, complex N-glycans, glycosaminoglycans, mucin type O-glycans or single -O-linked N-acetyl-D- glucosamine on glycoproteins, as well as the lack of complex glycosphingolipid biosynthesis.

The storage polysaccharide glycogen, found as granules in fungal cells, is very similar to the polymer found in vertebrates. In both clades glycogen is covalently attached to the glycogenin protein at its reducing terminus. Glycogen is a glucose homopolymer consisting of long chains of Glc1-4Glc disaccharide repeats and branches formed by 1-

6Glc linkages along the main Glc1-4Glc backbone. The polymer is formed by stepwise addition of glucose from

UDP-Glc to primer oligosaccharides of maltotetraose (Glc1-4Glc4) or larger in the glycogen synthase complex.

Glycogenin acts as an autocatalytic primer molecule with UDP-Glc as the donor. The degradation of glycogen, glycogenolysis, takes place through the action of glycogen phosphorylase, which releases Glc-1-phosphate units that are subsequently recycled through salvage pathways.

A rich variety of O-glycosylation pathways are active in the Fungi, generating a plethora of O-glycan structures. These glycosylation programmes are linked with secretory protein stability and solubility, with resistance to proteolytic degradation and with activity as sorting determinants. In filamentous strains O-glycosylation is important in the regulation of fungal morphology, hyphal development and differentiation [22].

Systems biological assessment of N- and O-linked glycans in filamentous fungi showed that the cytoplasmic, endoplasmic reticulum (ER), and Golgi pathways for N-glycosylation are highly conserved in this group and lead to the formation of high mannose, but not complex N-glycans. Unusual structures such as galactofuranoses and phosphodiesters are also formed [23]. O-glycosylation of serine and threonine acceptor sites in fungal proteins, mainly through mannosyltransferases, is highly conserved in the filamentous fungi, with similarity to that of the baker’s yeast,

Saccharomyces cerevisiae.

Study of the fungal cell wall has demonstrated a complex, cross-linked organization comprised of a variety of polymers including mannans and phosphomannans, glucans, chitins, gluco- galacto-and rhamnomannans. N-linked mannans contain an 1-6 linked backbone, while the glucans have a mixture of 1-3 > 1-6 linked glucose. Peripheral additions to the backbones are typical for each organism and include 1-2 and 1-3 branches, phospho-1-2 mannan chains and short galactan glycans [21]. The synthesis of the chitins found in the fungi is tightly regulated through multiple chitin synthases and is closely linked to fungal morphological locations, cell growth and developmental programmes [21]. A number of the fungi possess glycosylphosphatidylinositol- (GPI) anchored proteins with the typical mannan and glucan glycans reported above. Saccharomyces spp. have GPI-anchors containing phosphoethanolamine substitutions

(compared to the mammal equivalent), while Aspergillus strains lack this modification [21].

Viridiplantae

The green plants, perhaps up to 50% of all global living matter, have a substantial impact on our understanding and use of glycobiology and glycomics. A wide range of engineered glycomic products and specific proteins with glycobiological value are sourced from green plants. Plant lectins have contributed greatly to glycobiological and biomedical research. They impact day-to-day life through the production of large-scale nutritional sources; through the secure and reproducible isolation of medically valuable products, and use in biofuel synthesis [24, 25].

Many of the glycan structures found in plants are shared with animals, but unique glycans play a crucial role in plant structure and function. The plant cell wall has been the focus of wide chemical, biochemical, biophysical and biological studies. It must have the strength to tolerate the internal turgor pressure of the cell, enable dynamic changes, which occur during growth and also allow interaction with the cellular location and environment. The primary wall comprises up to 90% polysaccharide as cellulose, hemicellulose and pectin, with protein forming the remaining 10%. The cellulose microfibrils are organized in a network of hemicelluloses, such as xyloglucans, and pectins. Hydrogen binding between the cross-linking xyloglucans and cellulose microfibrils is integrated with the pectins, which exist as hydrated and adhesive components. Secondary cell walls are found associated with specialized structural cells, similar to the primary walls, but with a higher cellulose content, and a matrix composed of 1-4 xylans with lignin cross-linking.

Cellulose, the most plentiful biopolymer in the natural world, is a linear polymer of 1-4 linked glucose, typically of 36 glucose units. The chains form hydrogen bonds with each other as they are synthesized by the cellulose synthase complex. Six synthase subunits are spatially arranged at the plasma membrane to enable the synthesis of parallel cellulose fibres in register with the cellular cytoskeleton. The glucose units are added stepwise by a glucosyltransferase, employing cytoplasmic UDP-glucose, produced from sucrose by reversal of sucrose synthase, as the donor. The formation of cellulose fibres is facilitated by hemicelluloses, which coat and cross-link them. The hemicelluloses are, typically, xyloglucans, polymers with a repeating 1-4 glucan tetramer, with 1-6 xylose linked to the first three glucose units. These xylose unit may be extended by 1-2 galactose and subsequently an 1-2 fucose. The cellulose- xyloglucan grid is assembled in a network of pectins. The pectins are structurally complex plant wall polysaccharides made up of a linear backbone of 1-4 linked galacturonic acid, or rhamnogalacturonic acid. These exist as homogalacturonan, which may be 6-methylated and/or acetylated; rhamnoglacturonan-I, which may be further substituted by D-galactan, D-galacto-L-arabinan, and L-arabinan side chains or 6-methylated and/or acetylated. Some pectins contain xylogalacturonan sequences, with single xylose substitution, and a rhamnogalacturonan II structure where the rhamnose is found in complex side-chains [24, 25].

Starch, the plant-specific analogue of glycogen, is a storage polymer consisting of a long 1-4Glc backbone with 1-

6Glc side arm branches.

The biosynthesis of N-glycans is plants is well documented, and the basic properties of the biosynthetic pathway; dolichol oligosaccharide formation and transfer, and correct folding of the products, have been described in all plants. A striking difference from vertebrate N-glycans is the absence of sialylation. Hallmarks of the Viridiplantae, such as lack of phospho-mannose residues on high-mannose glycans, addition of 2-N-acetyl-D-glucosamine to peripheral mannoses, followed by Golgi mediated transfer of xylose in 2 linkage to the branch point mannose in the trimannosyl core with a -linkage to the chitobiose unit attached to asparagine on the plant peptide do not have counterparts in other studied clades. 1-3 fucosylation of the N-acetyl-D-glucosamine residue attached to the plant peptide asparagine is also a common feature in plant N-glycans. The Golgi mediated 2-xylosyl and 1-3 fucosyl transfers yield plant specific N- glycan structures [24]. O-glycosylation in plants varies considerably from vertebrate systems: mucins are not detected in plants. The main O- glycosylated products are hydroxyproline rich glycoproteins (HRGP) associated with the plant cell wall. The proline residues in these glycoproteins are converted to hydroxyproline, as in collagen. Chains of up to 4 arabinose units are linked to the hydroxyproline residues in these glycoproteins. Arabinogalactans are also found in the HRGP and containing galactose-O-hydroxyproline and galactose-O-serine links with glycan chains mainly composed of a 1-

3galactan backbone and extensive 1-6 galactosyl branch points. Arabinose is substituted on the periphery of the galactan skeleton. These glycoproteins have a structural and stabilizing role in plant cell walls, but have also been shown to function in signaling, cell proliferation and expansion, development and somite embryogenesis [24, 25].

Lectins, proteins with binding specificity for glycan structures, have been isolated, characterised and employed in a wide range of glycobiological analytical techniques, and have also been assessed for their roles in plant biology. The broad range of lectins, their properties and uses, have been described in detail elsewhere and will not be covered here

[26-31].

Plant models for glycosylation have been developed in recent years; Arabidopsis thaliana has been widely used to examine defects in glycobiological pathways [32-34] and is a complementary system to the mammalian CHO cell lines

[35]. Plants have also been adopted as a choice for de novo glycoengineering, designed to by-pass the difficulties encountered using vertebrate or mammalian cells. Strategies have included gene transfer into plant cells in order to synthesize selected glycoconjugate targets. For the synthesis of mucin-type glycoproteins, genes to synthesize nucleotide-sugar donors, such as UDP-GalNAc, and a target mucin acceptor, e.g. MUC1 and MUC16 have been engineered in Arabidopsis and tobacco cells. Transfer of a bacterial GlcNAc C4 epimerase to generate GalNAc, and the human GalNAc transferases GalNAc-T2 and GalNAc-T4 led to the incorporation of multiple GalNAc residues at serine sites in the MUC1 acceptor [36]. It was found necessary to use inhibitors to dampen endogenous activities, which otherwise led to proline hydroxylation and the incorporation of arabinose in the glycoprotein targets [37].

Strategies for the expression of the sialic acid pathway in plants and the synthesis of sialylated, mammalian type glycoproteins have also been reported [38]. These examples demonstrate the application of glycoengineering for the preparation of selected recombinant glycoproteins.

Nematoda

The nematodes comprise around 10,000 species, and are commonly known in relation to parasitic diseases. Many studies have been carried on Caenorhabditis elegans as a model organism due to its small and completely sequenced genome and ease of handling [39]. Over 300 carbohydrate-related genes are expressed including over 240 glycosyltransferases, glycosidases, polysaccharide lyases, and carbohydrate esterases. The C elegans glycoconjugates show many similarities to the well-known vertebrate components, but with C elegans specific characteristics. The main differences lie in the absence of sialic acids and enzymes for its metabolism, variable glycolipid core structures and truncated N-glycans..

The N-glycans are biosynthesized in mammalian-like pathways as far as GlcNAc1Man5GlcNAc2-Asn, but are then processed to generate paucimannose N-glycans, with three or fewer mannose residues. These N-glycans also display

1-3 fucosylation of the N-acetyl-D-glucosamine residue attached to the asparagine, as found in plants, in addition to

1-6 fucosylation of the same GlcNAc, common to vertebrates. Further nematode variations comprise the linkage of phosphorylcholine to the peripheral and core GlcNAc residues [40] and 1-4 linked galactose on either or both of the

1-3 or 1-6 linked fucose units.

C elegans O-glycans are core 1 (Gal1-3GalNAc-Ser/Thr) based, in common with vertebrates. Multiple ppGalNAc transferases create this core disaccharide. Nematode-specific features include the GlcA1-3GalNAc disaccharide and extended chains containing -glucose and -glucuronic acid, 1-2 linked fucose and 2-O-methylated fucose [41].

Mucins, commonly found in vertebrates, are not synthesized by C elegans. Defence against nematode infection appears to be mediated through these mucin glycoproteins in vertebrate systems (see below). C elegans possesses a significant variety of proteins that bind glycans: 28 putative galectins, C-, R- and L-type lectins and a mannose-6-phosphate receptor, but no I-type lectins, in keeping with the lack of sialic acid in nematodes.

Other nematodes also rely on glycan structures for important functional activities. Plant-parasitic nematodes synthesize and secrete effector proteins which have similar structure to the plant proteins and which are regulated by arabinosylated forms [42]. The cattle parasite Dictyocaulus viviparous forms high mannose, truncated and bi-, tri-and tetra-antennary complex N-glycans, which carry the Lewisx epitopes. These epitopes may play a role in parasite-host interactions, possibly as immunomodulators [43]. Parelaphostrongylus tenuis, the deer brain worm, contains complex

N-glycans with a terminal Gal1-3Gal1-4GlcNAc sequence, which acts as a mimic for a common vertebrate glycan

Gal1-3Gal, and may block immune detection by the host [44].

Binding properties and the molecular structure of vertebrate gastrointestinal mucins together with the detection of a nematode serine protease that can effectively degrade mucus gels suggests a role for mucins in the protection against the murine parasite Trichuris muris (closely related to the human parasite whipworm Trichuris trichiura) [45, 46]. This role has been further refined showing that the induction of a single mucin, MUC5AC, protects against infection by this nematode [47] and that the mucins form part of a coordinated immune response to nematode infection [45]. Similarly, intestinal nematode infection with Nippostrongylus brasiliensis, triggers an early upregulation of MUC2 and sialyltransferases, followed by a late upregulation of MUC3 and MUC4 together with increased 3-O-sulphotransferase expression. Further sulphotransferase increases were seen after the worms were expelled, indicating a continuing mucosal response during recovery [48]. The parasitic nematode Haemonchus contortus infects lambs. The animals can be protected by vaccination against excretory/secretory (ES) glycoproteins, yielding a response to glycan epitopes, in particular the GalNAc1-4 (Fuc1-

3)GlcNAc, fucosylated LacdiNAc antigen [49]. The same glycan functions in man against the parasite Schistosoma mansoni, in response to glycans on the eggs of these helminth parasitic worms.

Arthropoda

The arthropods include spiders, centipedes and millipedes, insects and crustaceans. This clade contains one of the most studied organisms, the fruit fly Drosophila melanogaster [50].

Chitin, a linear polymer composed of 1-4 linked GlcNAc units, is the characteristic molecule for arthropods, the major constituent of the cuticular exoskeleton in all members of this phylum. In addition, the insect midgut peritrophic membrane is also coated with a chitin-protein complex [51, 52].

The main N-glycans found in the arthropods are high- or paucimannose type. The enzymatic machinery leading to complex N-glycans also exists, although these represent minor components As noted for other phyla, there is an overlap in N-glycan structures with vertebrates, but typical arthropod structures are also found [50]. The chitobiose core may be substituted by two fucose residues in 1-3 and 1-6 on the N-acetyl-D-glucosamine residue attached to the asparagine.

The Fuc1-3GlcNAc is restricted to neural tissue in most arthropods. Embryonic expression of bi- and tri-antennary, sialylated and fucosylated complex N-glycans are closer to mammals, but their low abundance contrasts with mammalian tissues. The action of an arthropod -hexosaminidase removing GlcNAc from the 1-3Man branch of the trimannosyl core effectively prevents formation of extended complex or hybrid N-glycans.

Insects O-glycosylate a range of secreted, intracellular and cell surface proteins. These include the core 1 disaccharide

Gal1-3GalNAc-Ser/Thr. Mucin-like proteins carry this glycan in both insects and vertebrates. Branched structures, such as cores 2 or 4 with extended chains, have not been reported. In D melanogaster core-1 disaccharide containing glycoproteins have developmental significance. D melanogaster possesses many ppGalNAc transferases, which initiate the O-glycan chains. These are critical for life, as deletion of transferases is lethal [53].

The Notch and Cripto/FRL/Criptic families are important examples of glycosylated molecules involved in cell-fate pathways. In vertebrates, the complete tetrasaccharide chains, Neu5Ac2-3/6Gal1-4GlcNAc1-3Fuc--O-Ser/Thr mediate the interaction of Notch with its ligands. Differential binding properties of Notch with its extracellular ligands

Delta and Serrate/Jagged are governed by Fuc--O-Ser/Thr and GlcNAc1-3Fuc--O-Ser/Thr glycosylation and act as a regulator of the Notch receptor, or change its ligand preference. Extension of the GlcNAc1-3Fuc--O-Ser/Thr disaccharide does not occur in D melanogaster .

Insects synthesize a family of neutral and acidic arthroglycosphingolipids. These are based on a mactosylceramide core,

Man1-4Glc-ceramide and are extended by a GlcNAc and subsequent GalNAc and Gal units with GlcA providing a negative charge; typical structures such as GlcA1-3Gal1-3GalNAc1-4GlcNAc1-3Gal1-3GalNAc1-

4GalNAc1-4GlcNAc1-3Man1-4Glc-cer are found. Some of the GlcNAc residues in the neutral glycolipids are enhanced by addition of ethanolamine.

Heparin and chondroitin sulphates have been identified in Drosophila and other insects and show close identity to their vertebrate counterparts. Heparan sulphates contained mono, doi and tri sulphated disaccharides and N- 2-O and 6-O sulphated units. In contrast, chondroitin sulphates are mostly non-sulphated, with some 4- and 6-O sulphated forms [50,

54]. Hyaluronan is not synthesized by the athropods.

Intracellular lectins act in ER quality control, trafficking through the initial compartments of the secretory pathway and protein folding [50]. Screening of the Drosophila genome for carbohydrate binding proteins revealed the presence of all groups of lectins known in vertebrates. Galectins, C-type and I-type lectins have been reported [50]. The galectins and

C-type lectins have been linked with innate immune defence to pathogen infection in Drosophila and other insects.

There is currently limited information regarding the mechanisms of action in these processes.

Acknowledgements

We wish to thank Prof Hans-Joachim Gabius for his guidance and encouragement during the preparation of this review.

We are also grateful to Dr Pascal Gagneux and Prof. P.T. Pollard for permission to reproduce the Phylogenetic Tree in

Figure 1. Due to editorial constraints it is not possible to cite all of the relevant literature linked with the broad topic of

Eukaryote Glycosylation. We therefore acknowledge the contributions made by authors whose work is not referenced here.

Literature

1. Mourao, P.A. (2007) A carbohydrate-based mechanism of species recognition in sea urchin fertilization. Braz

J Med Biol Res. 40 5-17.

2. Freeze, H.H., V.D. Vacquier, and J.D. Esko (2009) Deuterostomes, in Essentials of Glycobiology A. Varki, et al., Editors. Cold Spring Harbor (NY) 363-373.

3. Vilela-Silva, A.C., et al. (2002) Sulfated fucans from the egg jellies of the closely related sea urchins

Strongylocentrotus droebachiensis and Strongylocentrotus pallidus ensure species-specific fertilization. J Biol Chem.

277, 379-387.

4. Schachter, H. (2010) Mgat1-dependent N-glycans are essential for the normal development of both vertebrate and invertebrate metazoans. Semin Cell Dev Biol. 21, 609-615.

5. Giacopuzzi, E., et al. (2012) New insights on the sialidase protein family revealed by a phylogenetic analysis in metazoa. PLoS One. 7, e44193. 6. Lehmann, F. et al. (2008) The evolution of galactose alpha2,3-sialyltransferase: Ciona intestinalis ST3GAL

I/II and Takifugu rubripes ST3GAL II sialylate Galbeta1,3GalNAc structures on glycoproteins but not glycolipids.

Glycoconj J. 25, 323-334.

7. Lang, T., G.C. Hansson, and T. Samuelsson (2007) Gel-forming mucins appeared early in metazoan evolution.

Proc Natl Acad Sci U S A. 104, 16209-16214.

8. Guerardel, Y. et al. (2003) Identification of the blood group Lewis(a) determinant in the oviducal mucins of

Xenopus tropicalis. FEBS Lett. 554, 330-336.

9. Baskin, J.M., et al. (2010) Visualizing enveloping layer glycans during zebrafish early embryogenesis. Proc

Natl Acad Sci U S A 107, 10360-10365.

10. Guerardel, Y., et al. (2006) Glycomic survey mapping of zebrafish identifies unique sialylation pattern.

Glycobiology 16, 244-257.

11. Bentrop, J., et al. (2008) Molecular evolution and expression of zebrafish St8SiaIII, an alpha-2,8- sialyltransferase involved in myotome development. Dev Dyn. 237, 808-818.

12. Marx, M., U. Rutishauser, and M. Bastmeyer (2001) Dual function of polysialic acid during zebrafish central nervous system development. Development 128, 4949-4958.

13. Rieger, S., K. Volkmann, and R.W. Koster (2008) Polysialyltransferase expression is linked to neuronal migration in the developing and adult zebrafish. Dev Dyn. 237, 276-285.

14. Lehmann, F., et al. (2004) Evolution of sialic acid-binding proteins: molecular cloning and expression of fish siglec-4. Glycobiology 14, 959-968.

15. Hunt, P.N., et al. (2005) Expression and genomic organization of zonadhesin-like genes in three species of fish give insight into the evolutionary history of a mosaic protein. BMC Genomics 6, 165.

16. Mo, D., et al. (2012) Apical targeting and endocytosis of the sialomucin endolyn are essential for establishment of zebrafish pronephric kidney function. J Cell Sci.125, 5546-5554.

17. Ko, Y.P., et al. (2005) Zebrafish (Danio rerio) matrilins: shared and divergent characteristics with their mammalian counterparts. Biochem J. 386, 367-379.

18. Varki, A. and R. Schauer (2009) Sialic Acids, in Essentials of Glycobiology A. Varki, et al., Editors. Cold

Spring Harbor (NY) 199-217.

19. De Pourcq, K., K. De Schutter, and N. Callewaert (2010) Engineering of glycosylation in yeast and other fungi: current state and perspectives. Appl Microbiol Biotechnol. 87, 1617-1631.

20. Kruszewska, J.S., et al. (2008) Alterations in protein secretion caused by metabolic engineering of glycosylation pathways in fungi. Acta Biochim Pol. 55, 447-456. 21. Cummings, R.D. and T.L. Doering (2009) Fungi, in Essentials of Glycobiology, A. Varki, et al., Editors. Cold

22. Goto, M. (2007) Protein O-glycosylation in fungi: diverse structures and multiple functions. Biosci Biotechnol

Biochem. 71, 1415-1427.

23. Deshpande, N., et al. (2008) Protein glycosylation pathways in filamentous fungi. Glycobiology 18, 626-637.

24. Etzler, M.E. and D. Mohnen (2009) Viridiplantae, in Essentials of Glycobiology, A. Varki, et al., Editors. Cold

25. Wilson, I.B. (2002) Glycosylation of proteins in plants and invertebrates. Curr Opin Struct Biol. 12, 569-577.

26. Abbott, K.L. and J.M. Pierce (2010) Lectin-based glycoproteomic techniques for the enrichment and identification of potential biomarkers. Methods Enzymol. 2010. 480: p. 461-76.

27. Gabius, H.J., et al. (2009) The sugar code: functional lectinomics. Biochim Biophys Acta 1572, 165-17.

28. Lis, H. and N. Sharon (1998) Lectins: Carbohydrate-Specific Proteins That Mediate Cellular Recognition.

Chemical Reviews 98, 637-674.

29. Rudiger, H. (1998) Plant lectins - More than just tools for glycoscientists:Occurrence, structure and possible functions of plant lectins. Acta Anat. 161, 130-152.

30. Sharon, N. (1993) Lectin-carbohydrate complexes of plants and animals: An atomic view. Trends in

Biochemical Sciences, 18, 221-226.

31. Sharon, N. (1994) When lectin meets oligosaccharide. Nature Struct Biol. 1, 843-845.

32. Hartweck, L.M., C.L. Scott, and N.E. Olszewski (2002) Two O-linked N-acetylglucosamine transferase genes of Arabidopsis thaliana L. Heynh. have overlapping functions necessary for gamete and seed development. Genetics,

161, 12719-12791.

33. Kim, Y.C., et al. (2013) Identification and origin of N-linked beta-D-N-acetylglucosamine monosaccharide modifications on Arabidopsis proteins. Plant Physiol. 161, 455-464.

34. Rips, S., et al. (2014) Multiple N-Glycans Cooperate in the Subcellular Targeting and Functioning of

Arabidopsis KORRIGAN1. Plant Cell, 26, 3792-3808.

35. Patnaik, S.K. and P. Stanley (2006) Lectin-resistant CHO glycosylation mutants. Methods Enzymol. 416, 159-

36. Yang, Z., et al. (2012) Engineering mammalian mucin-type O-glycosylation in plants. J Biol Chem. 287,

11911-11923.

37. Yang, Z., et al. (2012) Toward stable genetic engineering of human O-glycosylation in plants. Plant Physiol.

160, 450-463. 38. Castilho, A., et al. (2010) In planta protein sialylation through overexpression of the respective mammalian pathway. J Biol Chem. 285, 15923-15930.

39. Cummings, R.D. and J.D. Esko (2009) Nematoda, in Essentials of Glycobiology, A. Varki, et al., Editors. Cold

40. Houston, K.M., et al. (2008) Gene inactivation confirms the identity of enzymes involved in nematode phosphorylcholine-N-glycan synthesis. Mol Biochem Parasitol. 157, 88-91.

41. Guerardel, Y., et al. (2001) The nematode Caenorhabditis elegans synthesizes unusual O-linked glycans: identification of glucose-substituted mucin-type O-glycans and short chondroitin-like oligosaccharides. Biochem J. 357,

167-182.

42. Chen, S., et al. (2015) In Planta Processing and Glycosylation of a Nematode CLAVATA3/Endosperm surrounding region-Like Effector and Its Interaction with a Host CLAVATA2-Like Receptor to Promote Parasitism.

Plant Physiol. 167, 262-272.

43. Haslam, S.M., et al. (2000) Structural characterization of the N-glycans of Dictyocaulus viviparus: discovery of the Lewis(x) structure in a nematode. Glycobiology 10, 223-229.

44. Duffy, M.S., et al. (2006) Protein glycosylation in Parelaphostrongylus tenuis--first description of the

Galalpha1-3Gal sequence in a nematode. Glycobiology 16, 854-862.

45. Hasnain, S.Z., et al. (2013) A new role for mucins in immunity: insights from gastrointestinal nematode infection. Int J Biochem Cell Biol, 45, 364-374.

46. Hasnain, S.Z., et al. (2012) Serine protease(s) secreted by the nematode Trichuris muris degrade the mucus barrier. PLoS Negl Trop Dis. 6, e1856.

47. Hasnain, S.Z., et al. (2011) Muc5ac: a critical component mediating the rejection of enteric nematodes. J Exp

Med. 208, 893-900.

48. Yamauchi, J., et al. (2006) Altered expression of goblet cell- and mucin glycosylation-related genes in the intestinal epithelium during infection with the nematode Nippostrongylus brasiliensis in rat. Apmis 114, 270-278.

49. Vervelde, L., et al. (2003) Vaccination-induced protection of lambs against the parasitic nematode

Haemonchus contortus correlates with high IgG antibody responses to the LDNF glycan antigen. Glycobiology 13, 795-

50. Tiemeyer, M., S.B. Selleck, and J.D. Esko (2009) Arthropoda, in Essentials of Glycobiology, A. Varki, et al.,

Editors. Cold Spring Harbor (NY) 347-362.

51. Sarauer, B.L., C. Gillott, and D. Hegedus (2003) Characterization of an intestinal mucin from the peritrophic matrix of the diamondback moth, Plutella xylostella. Insect Mol Biol. 12, 333-343. 52. Wang, P. and R.R. Granados (2000) Calcofluor disrupts the midgut defense system in insects. Insect Biochem

Mol Biol. 30, 135-143.

53. Tran, D.T., et al. (2012) Multiple members of the UDP-GalNAc: polypeptide N- acetylgalactosaminyltransferase family are essential for viability in Drosophila. J Biol Chem. 287, 5243-5252.

54. Lin, X. (2004) Functions of Heparan Sulfate Proteoglycans in Cell Signalling During Development.

Development. 131, 6009 - 6021.