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Carbohydrates and glycosylation*

Patricia M. Berninsone§
Biology Department, University of Nevada, Reno, Reno, NV 89557 USA

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Table of Contents

1. Introduction
2. Glycosylation pathways in C. elegans
3. Glycosaminoglycans
3.1. Chondroitin
3.2. Heparan sulfate
3.3. Heparan sulfate and chondroitin modified proteins
4. N-linked glycans
4.1. Diversity of N-glycan structures in C. elegans
4.2. N-glycosylated proteins in C. elegans
5. O-linked glycans
5.1. O-GalNAc linked (mucin-type) glycans
5.2. O-GlcNAc linked
6. Glycolipids
7. Chitin
8. Conclusions
9. References
A. Appendix 1


The C. elegans genome contains sequences similar to a large number of mammalian genes implicated in the assembly, processing, and modification of glycans. In recent years, spectacular progress has been made in developing and refining tools to obtain structural information with small amounts of material, increasing our understanding of glycan structural complexity in this organism. These approaches have revealed novel N- and O-glycan structures in C. elegans, as well as a high degree of conservation in glycosaminoglycan structure. In parallel, studies in which glycan structure is perturbed by genetic manipulation have begun to reveal the roles of specific carbohydrate moieties in developmental and physiological processes. This review summarizes recent work elucidating the fine structure of complex carbohydrates in C. elegans as well as genetic studies that have uncovered novel roles for complex carbohydrates in developmental processes.

1. Introduction

Complex carbohydrates are involved in multiple biological processes, from protein folding, oligomerization and stability, to the immune response and host-pathogen interactions (Varki, 1993). Glycoconjugates also play important roles in developmental processes, as revealed by the pathology of human diseases caused by abnormal glycosylation (Freeze and Aebi, 2005) and genetic studies in model organisms (Haltiwanger and Lowe, 2004).

The structural diversity of naturally occurring glycans is determined by complex biosynthetic pathways, which typically vary among species and appear to be modulated by factors that can differ greatly among cell types. Unlike nucleic acids and proteins, oligosaccharide chains are often assembled in non-linear, branched structures that are synthesized without the use of a template. In principle, each monosaccharide can generate an α or a β linkage to one of several positions on another monosaccharide or to another type of molecule, having the potential to produce an astounding molecular complexity. Moreover, individual sugar moieties within a chain can be modified by phosphorylation, sulfation, methylation, O-acetylation, or fatty acylation, further increasing the combinatorial possibilities for oligosaccharide diversity (Varki et al., 1999). In addition to the complexity of glycan moieties per se, it is not possible to predict with certainty whether a protein will be modified by oligosaccharides, even when glycosylation motifs have been observed for a number of glycopeptide bonds (Spiro, 2002). Another source of variation is microheterogeneity, in which the structure of the oligosaccharide attached to each glycosylation site may vary (Jones et al., 2005).The focus of this review will be on the contributions that genetic and biochemical analysis in C. elegans have made to our understanding of the role of glycans in development.

2. Glycosylation pathways in C. elegans

The C. elegans genome contains sequences similar to a large number of mammalian genes implicated in the assembly, processing and modification of a variety of glycans (Schachter, 2004; see Appendix 1). Although only a limited number of these genes have been functionally characterized, the degree of homology suggests that C. elegans possesses a repertoire of enzymes for the synthesis of a variety of complex carbohydrates. The occurrence of N-glycans [reviewed in Schachter (2004)], GalNAc-O-Ser/Thr glycans (Guerardel et al., 2001), glycosaminoglycans (Toyoda et al., 2000; Yamada et al., 1999), glycolipids (Gerdt et al., 1999; Gerdt et al., 1997) and chitin (Veronico et al., 2001) has been verified in C. elegans in structural or biochemical studies. Although C. elegans N- and O-glycans have common features with vertebrate glycans in terms of their core glycan biosynthesis, their terminal structures show significant differences. Among them, C. elegans glycans lack sialic acid (Bacic et al., 1990) and contain unusual fucose additions, O-methylated fucose and mannose and phosphorylcholine substitutions (Cipollo et al., 2002; Haslam and Dell, 2003). In recent years, spectacular progress has been made in developing and refining tools to define the structures of glycans. Our understanding of the role of glycoconjugates in the development of C. elegans to date is largely due to the identification of mutations in genes involved in their biosynthesis.

3. Glycosaminoglycans

Proteoglycans consist of a core protein and one or more covalently attached glycosaminoglycan (GAG) chains. GAG chains are attached to serine residues in core proteins by a common “tetrasaccharide linkage” region. Structural studies have confirmed that C. elegans synthesizes this ubiquitous linker region (Guerardel et al., 2001). GAGs are linear polysaccharides, composed of repeating disaccharide units consisting of an amino sugar (GlcNAc or GalNAc) and an uronic acid (GlcA and IdoA; see Figure 1). Different classes of GAGs are defined by the composition of the disaccharides: heparan sulfate (HS) has a repeating disaccharide of GlcNAc-α1,4-GlcA-β1,4, whereas chondroitin and chondroitin sulfate (CS) have GalNAc-α1,4-GlcAβ1,3. Monosaccharides within the GAG polymers can be modified in several ways: GlcNAc N-deacetylation and N-sulfation, uronic acid epimerization and O-sulfation at different positions. These modifications result in an enormous molecular complexity that increases the capacity of GAGs to interact with proteins through varied arrangements of sulfated sugar residues (Turnbull et al., 2001). C. elegans produces heparan sulfate containing all modifications previously described in other organisms (Toyoda et al., 2000; Yamada et al., 1999). In contrast, chondroitin appears not to be secondarily modified in C. elegans (Toyoda et al., 2000).

figure 1

Figure 1. Structure of C. elegans chondroitin and heparan sulfate and their linkage tetrasaccharide to proteins. Considerable variations occur in the positions of heparan sulfate sulfations and epimerization (IdoA). [Based on the compositions described in Guerardel et al. (2001); Toyoda et al. (2000); Yamada et al. (1999).]

3.1. Chondroitin

A screen for C. elegans mutants defective in vulva morphogenesis identified 8 sqv (squashed vulva) genes (Herman et al., 1999) required for various steps of chondroitin biosynthesis (see Figure 2). With the exception of sqv-5, the sqv genes control the biosynthesis of both heparan sulfate and chondroitin (Berninsone et al., 2001; Bulik et al., 2000; Hwang and Horvitz, 2002; Hwang and Horvitz, 2002; Hwang et al., 2003). SQV-5 is a bifunctional glycosyltransferase that functions in the initiation and elongation of chondroitin, but not heparan sulfate chains (Hwang et al., 2003; Mizuguchi et al., 2003), suggesting that defects in the biosynthesis of chondroitin account for the defects caused by mutations in all sqv genes.

In all the sqv mutants, the vulval extracellular space fails to expand during vulval morphogenesis (Herman et al., 1999). Strong sqv mutations also cause maternal-effect lethality: most progeny of homozygous mutants fail to initiate cytokinesis. These mutant eggs fail to form the fluid-filled extracellular space between the egg and the eggshell (Herman and Horvitz, 1999; Hwang and Horvitz, 2002). The ability of extracellular chondroitin to interact with water and generate osmotic pressure might be implicated in the expansion of the extracellular spaces in the C. elegans embryo and vulva; among other possibilities (Hwang et al., 2003).

3.2. Heparan sulfate

The EXT gene family members are glycosyltransferases required for the biosynthesis of heparan sulfate chains. Mutations in EXT1 and EXT2 lead to hereditary multiple exostoses. Only two homologs of the EXT family exist in C. elegans: rib-1 and rib-2. rib-2 is essential for embryonic development (Morio et al., 2003) and encodes a unique α1,4-N-acetylglucosaminyltransferase involved in both the biosynthetic initiation and elongation of heparan sulfate (Kitagawa et al., 2001). The biochemical activity of RIB-1 remains elusive.

figure 2

Figure 2. Model of the function of the SQV proteins in the biosynthesis of heparan and chondroitin chains. SQV-4 (UDP-glucose dehydrogenase) synthesizes UDP-glucuronic acid (Hwang and Horvitz, 2002) and SQV-1 catalyzes the synthesis of UDP-xylose by decarboxylation of UDP-glucuronic acid (Hwang and Horvitz, 2002). SQV-7 transports UDP-glucuronic acid, UDP-galactose and UDP-N-acetylgalactosamine into the Golgi lumen (Berninsone et al., 2001). sqv-6, sqv-3, sqv-2 and sqv-8 encode the xylosyltransferase, galactosyltransferase I, galactosyltransferase II and glucuronyltransferase I, respectively, involved in the synthesis of the common “core tetrasaccharide” linkage region (Bulik et al., 2000; Herman and Horvitz, 1999; Hwang et al., 2003). SQV-5 is required for the polymerization of chondroitin chains (Hwang et al., 2003; Mizuguchi et al., 2003). Reprinted by permission from Macmillan Publishers Ltd: Nature 423, 439–443, copyright (2003).

Contrasting with the essential nature of heparan sulfate chain synthesis (Morio et al., 2003), C. elegans mutants lacking three heparan sulfate modifying enzymes are viable and fertile. hse-5, hst-6 and hst-2 mutants (HSE-5: glucuronyl C5-epimerase, HST-6: heparan sulfate 6O-sulfotransferase and HST-2: 2O-sulfotransferase) exhibit distinct as well as overlapping axonal and cellular guidance defects in specific neurons (Bulow and Hobert, 2004). hst-2 mutants also display cell migration defects (Kinnunen et al., 2005). These HS modifications are required for the function of the slt-1/sax-3 signaling system in some, but not all cellular contexts (Bulow and Hobert, 2004).

Defects in heparan sulfate synthesis or specific modifications disrupt neuroblast migrations. C-5 epimerized and 6-O-sulfated heparan sulfate chains attached to the cell surface proteoglycans syndecan (SDN-1) and glypican (GPN-1) are required for the activity of KAL-1, the C. elegans anosmin ortholog, in embryonic neuroblast migration (Hudson et al., 2006). These in vivo analyses underscore the importance of molecular diversity encoded by HS chains and argue that each individual modification provides information required for axon patterning and cell migration.

3.3. Heparan sulfate and chondroitin modified proteins

The C. elegans genome encodes several putative heparan sulfate proteoglycan (HSPG) core proteins: the cell surface HSPGs SDN-1/syndecan (Minniti et al., 2004; Rhiner et al., 2005) and GPN-1/glypican (Hudson et al., 2006) and the basement membrane HSPGs UNC-52/perlecan (Rogalski et al., 1993) and CLE-1/collagen XVIII (Ackley et al., 2001). SDN-1 (Minniti et al., 2004) and GPN-1 (Hudson et al., 2006) have been shown to be modified by heparan sulfate chains.

Nine chondroitin proteoglycan core proteins (CPG-1 to CPG-9) were identified in C. elegans using a combination of biochemical purification and mass spectrometry (Olson et al., 2006). None of these proteins have homologs in vertebrates or other invertebrates, but all of them have sequence motifs required for initiation of glycosaminoglycan biosynthesis similar to those found in vertebrates. Simultaneous RNAi depletion of two of these core proteins (CPG-1/C07G2.1a and CPG-2/B0280.5) resulted in multinucleate single cell embryos (Olson et al., 2006), resembling the embryonic phenotype resulting from inhibiting the SQV-5 chondroitin synthase (Hwang et al., 2003; Mizuguchi et al., 2003). This study shows that the chondroitin proteoglycans encoded by cpg-1 and cpg-2 play a crucial role in embryonic cytokinesis.

4. N-linked glycans

N-linked glycans are complex and diverse sugar chains attached to an asparagine residue of a polypeptide chain within the consensus peptide sequence Asn-X-Ser/Thr. The yeast and mammalian biosynthetic pathways have been elucidated in great detail (Herscovics, 1999; Kornfeld and Kornfeld, 1985). The initial steps of this pathway are conserved in most eukaryotes and involve the synthesis of a lipid-linked oligosaccharide precursor structure that is transferred en bloc to nascent proteins in the ER. Transfer of Glc3Man9GlcNAc2 to Asn is followed by glucose trimming in the ER. Subsequent cycles of glucose re-addition and removal participate in quality control of protein-folding. The processed high-mannose Man5GlcNAc2-Asn N-glycan serves as a substrate for the diversification of N-glycans in the Golgi. In vertebrates, these reactions generate a large repertoire of hybrid and complex N-glycan subtypes (see Figure 3, top panel).

figure 3

Figure 3. Comparative overview of the major types of vertebrate N-glycan subtypes and some representative C. elegans N-glycans. Top panel: Vertebrate diversification in the Golgi apparatus generates high-mannose, hybrid and complex N-glycan subtypes. Most cell surface and secreted N-glycans are of the complex subtype. Vertical arrows indicate locations of branch formation in diversification, not all of which occur on a single N-glycan. (Adapted from (Varki et al., 1999) Bottom panel: The main classes of C. elegans N-glycans include high-mannose (up to Man9GlcNAc2), pauci-mannosidic and hybrid type. Note that this scheme only includes some of the documented structures.

4.1. Diversity of N-glycan structures in C. elegans

Whereas the N-glycan biosynthetic pathway appears to be fairly well conserved in C. elegans (see Appendix 1), the resulting N-glycan structures are qualitatively and quantitatively different than those found in mammals, as shown by structural analysis of N-glycans enzymatically released from C. elegans glycoproteins (see Figure 3, reviewed in Haslam and Dell, 2003; Schachter, 2004). C. elegans predominantly contains oligomannose Man5-9GlcNAc2-Asn glycans (which are present in vertebrates) and large amounts of paucimannose Man3-4GlcNAc2-Asn glycans (which are not usually present in vertebrates; Altmann et al., 2001; Cipollo et al., 2002; Haslam and Dell, 2003; Haslam et al., 2002; Natsuka et al., 2002; Schachter et al., 2002; Zhang et al., 2003; see Figure 3, bottom panel). Complex and hybrid N-glycans, which are very abundant in vertebrates, are either absent or present in low levels in C. elegans (Cipollo et al., 2002; Haslam et al., 2002; Natsuka et al., 2002), although this organism has at least some of the biosynthetic components required for the synthesis of high mannose and abbreviated mammalian-type complex glycans. GnTI (N-acetylglucosaminyltransferase I) initiates and is essential for the conversion of Man5GlcNAc2-Asn to hybrid and complex N-glycans. Three genes encoding enzymatically active GnTI (gly-12, gly-13 and gly-14) exist in C. elegans (Chen et al., 1999). GnTII (gly-20; Chen et al., 2002) and GnTV (gly-2; Warren et al., 2002; Warren et al., 2002), which initiate the formation of the N-glycan “antennae”, are also expressed in C. elegans. A model to explain this apparent discrepancy has been proposed, based on the expression in C. elegans of a N-acetylglucosaminidase, which removes GnTI-added GlcNAc, thus preventing the formation of complex glycan and resulting in the formation of large amounts of “paucimannose” N-glycans (Zhang et al., 2003).

Unusually modified N-glycans have been detected in C. elegans. Fuc-rich glycans, which carry terminal Fuc on the antennae as well as the core, sometimes contain O-methylated Fuc and O-methylated Man. Phosphorylcholine (PCho)-substituted N-glycans have been identified in a high mannose structure carrying three PCho groups (Cipollo et al., 2002) and on complex type glycans where PCho is attached at GlcNAc (Haslam and Dell, 2003). This last structure has been previously found in several parasitic nematodes (Haslam et al., 1999; Haslam et al., 1997; Morelle et al., 2000).

The structural studies summarized above identified N-glycans extracted from mixed stage C. elegans cultures. These preparations are expected to be enriched in glycans from the largest and most represented developmental stages. A recent study (Cipollo et al., 2005) has shown that the N-glycan profile of each developmental stage appears to be unique. The general trend of the number and abundance of N-glycans was Dauer~L1>Adult~L4>L3~L2. Dauer larvae contain complex glycans with higher molecular masses than those of other stages, and a higher abundance of high molecular weight PCho-containing glycans was observed in L1 and Dauer larvae. This study also documents the structures of novel N-glycans, e.g., difucosyl PCho glycans in which PCho substitutes both core and terminally linked GlcNAc.

4.2. N-glycosylated proteins in C. elegans

The rather strict consensus peptide sequence Asn-X-Ser/Thr for N-glycosylation has been supported by numerous structural, mutagenic and in vitro approaches (Spiro, 2002). Although this sequence occurs frequently in proteins, N-linked glycosylation does not occur at every potential glycosylation site, most probably due to conformational factors (Apweiler et al., 1999). Thus, it is not possible to predict with certainty if a protein is in fact N-glycosylated. Microheterogeneity in N-glycan chains can also occur among different molecules of the same protein (Jones et al., 2005).

Recently developed methods have allowed the identification of 304 C. elegans proteins containing N-glycans and the simultaneous determination of the glycosylation sites (Fan et al., 2004; Fan et al., 2005; Kaji et al., 2003). These studies employed lectin affinity chromatography to isolate glycopeptides generated by tryptic digestion of C. elegans protein fractions, followed by a variety of mass spectrometry approaches. All but four of the N-glycosylation sites identified correspond to the conventional Asn-X-Ser/Thr. Three peptides contain Asn-X-Cys (Fan et al., 2005; Kaji et al., 2003). The identified N-glycoproteins comprise soluble and hydrophobic proteins, and many of them are extracellular matrix components which have been implicated in cell adhesion or are components of basement membranes. Among them, PAT-2, INA-1 (the two C. elegans integrin α subunits), PAT-3 (the integrin β subunit), T22A3.8, EPI-1, LAM-1, C54D1.5 (the four laminin subunit gene products), NID-1 (nidogen) and DGN-1 (dystroglycan) contain at least one N-linked glycan [see Fan et al. (2005); Kaji et al., (2003) for complete lists].

In individual studies, some C. elegans proteins have been demonstrated to contain N-linked oligosaccharides. GLP-1, a member of the LIN-12/Notch family of receptor proteins essential for distal tip cell (DTC) control of germline proliferation (Austin and Kimble, 1987) is a glycoprotein (Crittenden et al., 1994).

MIG-17, a member of the ADAM (a disintegrin and metalloprotease) family, regulates directional migration of the distal tip cell (DTC) (Nishiwaki et al., 2000). Sensitivity to N-glycanase indicated that MIG-17 is N-glycosylated (Nishiwaki et al., 2004). A mutation in mig-23 causes defective DTC migration and genetically interacts with mig-17. MIG-23 is a membrane-bound nucleoside diphosphatase (NDPase) required for glycosylation and proper localization of MIG-17 (Nishiwaki et al., 2004). NDPase activities in the lumen of the Golgi apparatus generate nucleoside monophosphates required for translocation of nucleotide sugars from the cytosol into the Golgi lumen (Berninsone et al., 1994). Nucleotide sugars are the activated donors for glycosylation reactions in the lumen of the endoplasmic reticulum and Golgi apparatus (Hirschberg et al., 1998). Genetic studies in S.cerevisiae showed that guanosine diphosphatase, an NDPase, is required for protein and sphingolipid glycosylation in the Golgi lumen (Abeijon et al., 1993). Hence, MIG-23 likely affects DTC migration through its role in the transport of nucleotide sugars into the lumen of the Golgi apparatus. Although the mig-23 mutation may affect the glycosylation of other proteins, MIG-23 appears to regulate DTC migration through glycosylation and correct localization of MIG-17.

5. O-linked glycans

5.1. O-GalNAc linked (mucin-type) glycans

The GalNAc α-Ser/Thr linkage has been considered a hallmark of mucins where it occurs in clusters. However, this linkage has also been found in a wide variety of other proteins (Hang and Bertozzi, 2005). No primary amino acid consensus sequence has emerged for mucin-type O-linked glycosylation. In general, glycosylation of Thr is preferred over Ser (Elhammer et al., 1993) and the linkage is found in clusters of Ser/Thr residues with a βturn near Pro and at a distance from charged amino acids (Hang and Bertozzi, 2005).

C. elegans expresses a family of functional polypeptide N-acetylgalactosaminyltransferases (ppGalNacT) involved in the initiation of mucin-type O-glycosylation (Hagen and Nehrke, 1998; see Appendix 1). A C. elegans core 1 β3-GalT or T-synthase has been recently characterized (Ju et al., 2006). This enzyme adds Gal to GalNAcα1-Ser/Thr to generate the core 1 structure Galβ1-3GalNAcα1-Ser/Thr. This is the most common of O-glycan structures in vertebrates and is a precursor to more complex O-glycans. Two families of unusual mucin-type O-glycans were identified in C. elegans (Guerardel et al., 2001). Both families were characterized by the presence of multiple β-Glc residues in non-reducing terminal positions, a type of substitution without precedent in O-glycans. One of these families presented either type-1 core Galβ1,3GalNAc or one of three novel core types: Galβ1,6[Galβ1,3]GalNAc, Glcβ1-6[Galβ1,3]GalNAc and [Glcβ1-6(Glcβ1,4)Galβ1-3)]GalNAc. Some of the O-glycans contain the GlcAβ1,3 Galβ1,3GalNAc trisaccharide sequence. The second mucin type O-glycan characterized is unusual in that it presented a GlcNAc-ol in the terminal position. This suggests that it would be linked to the protein through a GlcNAc and not a GalNAc. The unusual features of these structures suggest the expression in C. elegans of novel glycosyltransferase activities. For example, a novel β1,6GlcT acting on the GalNAc residue of the core has been cloned and expressed (Warren et al., 2002). The enzyme is encoded by the gly-1 gene and is homologous to the vertebrate β1,6GlNAcT that acts on the GalNAc residue of O-glycan core 1 (Galβ1,3GlcNAc) to make O-glycan core 2.

The large amounts of O-glycans purified in this study suggest that C. elegans expresses an abundance of highly O-glycosylated proteins. Studies on the parasitic nematode Toxocara canis have shown that it secretes a family of highly O-glycosylated mucin type glycoproteins (Loukas et al., 2000; Dell et al., 1999; Khoo et al., 1991). In C. elegans, let-653 encodes a mucin-type glycoprotein (Jones and Baillie, 1995). Mutations in let-653 result in larval death and vacuole formation anterior to the pharyngeal bulb, suggesting malfunction or deformation of the excretory/secretory apparatus. Biochemical characterization of a C. elegans L1-specific surface antigen suggested that it contains O-linked carbohydrates (Hemmer et al., 1991). This antigen is not detected on the surface of L1 animals carrying mutations in the srf-3 gene, which encodes a Golgi UDP-Gal/UDP-GlcNAc transporter implicated in glycosylation of surface components (Hoflich et al., 2004). Loss of SRF-3 activity affects both N-linked and O-linked glycosylation (Cipollo et al., 2004).

5.2. O-GlcNAc linked

O-linked GlcNAc at Ser and Thr residues is an evolutionary conserved modification emerging as a key regulator of nuclear and cytoplasmic protein activity (Love and Hanover, 2005). The GlcNAc transferase (OGT) responsible for this modification was the first glycopeptide-forming enzyme to be localized outside the secretory apparatus (Lubas et al., 1997). O-GlcNAc addition may compete with O-phosphorylation for certain Ser/Thr target sites, suggesting a potential regulatory cycle in which cytosolic β-N-acetylglucosaminidase plays a key role (Comer and Hart, 2001). O-GlcNAc addition is driven in part by the levels of UDP-GlcNAc derived from the hexosamine biosynthetic pathway, a nutrient-sensing pathway implicated in cellular signaling (Hanover, 2001). In mammals, the OGT gene is essential for embryonic and stem cell development and produces multiple transcripts (Hanover et al., 2003; O'Donnell et al., 2004; Shafi et al., 2000). O-GlcNAc addition has been implicated in mammalian insulin resistance (Hanover, 2001; McClain et al., 2002; Vosseller et al., 2002; Wells et al., 2001) histone remodeling, transcription, proliferation, apoptosis and proteosomal degradation.

In C. elegans, ogt-1 and oga-1 encode the single orthologs of OGT (Lubas et al., 1997) and O-GlcNAcase (Forsythe et al., 2006), respectively. Putative null alleles of ogt-1 and oga-1 are viable and fertile, allowing the examination of the role of O-GlcNAc in this organism. Nuclear pore complexes in the ogt-1 mutant strain lack O-GlcNAc but are capable of transporting transcription factors into the nucleus at normal rates. Conversely, oga-1 null mutants accumulate O-GlcNAc on nuclear pores and other proteins. A dramatic elevation in the levels of trehalose and glycogen was detected in ogt-1 and oga-1 mutants with a concomitant decrease in triglyceride storage (Hanover et al., 2005). The ogt-1 deletion allele suppresses dauer formation induced by the partial loss of daf-2, encoding an insulin-like receptor (Riddle, 1977). In comparable conditions, the oga-1 null mutant enhances dauer formation of the daf-2 mutant. Thus, the nematode hexosamine-signaling pathway interacts with the insulin-like-signaling pathway, as suggested in mammals. The insulin-like-signaling pathway modulates lipid and carbohydrate storage in C. elegans (Kimura et al., 1997). These studies implicate O-GlcNAc cycling by OGT-1 and OGA-1 in modulating insulin-like signaling and/or parallel pathways regulating dauer formation in C. elegans.

6. Glycolipids

A glycosphingolipid consists of an oligosaccharide usually attached via glucose or galactose to the terminal primary hydroxyl group of the lipid moiety ceramide. Most mammalian glycosphingolipids contain a Gal-β1,4-Glc-β1-ceramide core, which is extended by the stepwise addition of further monosaccharides giving rise to several defined core structures (Varki et al., 1999).

C. elegans glycosphingolipids belong to the invertebrate-specific arthro-series, which contain the invertebrate-specific Man-β1,4-Glc-β1-ceramide core, extended by addition of neutral sugars. They share structural homology with the equivalent structures from the porcine parasitic nematode, Ascaris suum (Gerdt et al., 1997; Gerdt et al., 1999; Lochnit et al., 1997). Some of these glycosphingolipids are modified by phosphorylcholine (PCho), an antigenic determinant frequently detected in parasitic nematodes (Lochnit et al., 1998; Maizels et al., 1987).

A role for glycosphingolipids as receptors for the Bacillus thuringensis (Bt) crystal toxin was revealed by genetic studies in C. elegans. The crystal (Cry) proteins produced by Bt are pore forming toxins that are lethal to insects and nematodes but generally innocuous to vertebrates. The potential problem associated with long-term use of transgenic crops expressing Cry proteins to control insect pests is the development of resistance among target populations. Mutations in the bre (Bt resistant) genes cause Bt resistance in C. elegans (Griffitts et al., 2003; Griffitts et al., 2001; Marroquin et al., 2000). The bre genes encode four glycosyltransferases that act in a single pathway and are required for the uptake of toxin into intestinal cells. (Griffitts et al., 2003; Griffitts et al., 2001). BRE-2 and BRE-4 synthesize the Galβ1,3GlcNAc and GalNAcβ1,4GlcNAc moieties, respectively, which are commonly found on glycolipids and glycoproteins. bre-3 and bre-5 are the C. elegans homologs of the Drosophila genes egghead and brainiac, respectively, which encode consecutive glycosyltransferases involved in the synthesis of the carbohydrate chains on glycosphingolipids (Griffitts et al., 2003). Lipid and glycoprotein analysis of bre mutant animals demonstrated that the BRE enzymes are required to synthesize the carbohydrate chains of glycolipids and that Bt toxin specifically binds glycolipids (Griffitts et al., 2005).

Mutations in bre-5 and other genes involved in glycosphingolipid biosynthesis also suppress the egg-laying defect associated with elevated lin-12 activity (Katic et al., 2005). Brainiac modulates Notch signaling in Drosophila (Goode et al., 1996; Goode and Perrimon, 1997) suggesting that the importance of glycosphingolipids for proper LIN-12/Notch signaling might be functionally conserved. Genetic evidence suggests that bre-5 functions prior to LIN-12 activation by ligand-induced ectodomain shedding (Katic et al., 2005).

7. Chitin

Chitin, a linear polymer of β1,4 linked GlcNAc, is one of the main components of the fungal cell wall and the exoskeletons of arthropods. The eggshells of many free-living and parasitic nematodes contain chitin (Brydon et al., 1987; Wharton, 1980). In addition, this polysaccharide has been detected in the feeding apparatus of the strongyloid nematode Oesophagostomum dentatun (Neuhaus et al., 1997). In C. elegans, chitin is detected in the eggshell and in elaborate patterns in the pharyngeal lumen walls of adult and larvae (Zhang et al., 2005). Two genes encoding chitin synthases, chs-1 and chs-2, exhibit different temporal expression patterns: chs-1 is expressed in later larval stages and adults, while chs-2 expression is restricted to a short period before each molt, but not in adults (Veronico et al., 2001). Maternal chs-1 gene activity is likely involved in the production of eggshell chitin, as shown by RNAi experiments and analysis of chs-1 null mutants. chs-2 is expressed in the pharynx and inactivation of chs-2 by RNAi revealed visible defects in the grinder (Zhang et al., 2005). Hence, chs-1 and chs-2 play different and non-overlapping roles in development.

8. Conclusions

Assignment of biological functions to glycans and glycan modified acceptors in any multicellular organism is a difficult task. The extreme complexity of glycan structures is compounded by the fact that many yet unidentified acceptors might be modified by the same type of glycan. In addition, very little is known about how the expression of these molecules is regulated during development and differentiation. C. elegans and small model organisms are likely to provide platforms where perturbations of glycan structure can be analyzed in the context of development and differentiation. Sensitive techniques for glycan analysis can be applied to mutants to obtain qualitative and quantitative information about their glycosylation phenotypes. Identification of the specific acceptor(s) carrying the abnormal glycan structures represents an additional challenge, yet these studies may help understand the molecular mechanisms linking glycosylation defects with specific phenotypes. Despite the enormous difference in complexity with mammals, the recent genetic and structural studies suggest that C. elegans is a promising model system for investigating basic roles of glycans in development.

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Zhang, Y., Foster, J.M., Nelson, L.S., Ma, D., and Carlow, C.K. (2005). The chitin synthase genes chs-1 and chs-2 are essential for C. elegans development and responsible for chitin deposition in the eggshell and pharynx, respectively. Dev. Biol. 285, 330–339. Abstract Article

Zheng, Q., Van Die, I., and Cummings, R.D. (2002). Molecular cloning and characterization of a novel alpha 1,2-fucosyltransferase (CE2FT-1) from Caenorhabditis elegans. J. Biol. Chem. 277, 39823–39832. Abstract Article

A. Appendix 1

This Appendix includes putative glycosylation genes based on homology to genes from other species. Activity refers to presumed biochemical function, except those entries indicated with *, for which the C. elegans gene has been expressed and its biochemical activity has been demonstrated. Adapted from Schachter (2004).

Activity C. elegans gene Mutant phenotype RNAi phenotype Reference(s)
Genes involved in synthesis and modification of GAG (Xyl-Ser O-glycans)
UDP-GlcA decarboxylase * D2096.4 (sqv-1) Squashed vulva, hermaphrodite sterile WT Hwang and Horvitz, 2002
βgal β1,3-GalT II * Y110A2AL.14 (sqv-2) Squashed vulva, hermaphrodite sterile Clr, Cyk, Egl, Sma Hwang et al., 2003; Wang et al., 2005
Xyl-prot β1,4-GalT * R10E11.4 (sqv-3) Squashed vulva, hermaphrodite sterile   Bulik et al., 2000
UDP-Glc dehydrogenase * F29F11.1 (sqv-4) Squashed vulva, hermaphrodite sterile Emb Hwang and Horvitz, 2002
β1,4GalNAcT I/II (chondroitin synthase) * T24D11.1a,b (sqv-5) Squashed vulva, hermaphrodite sterile   Hwang et al., 2003; Mizuguchi et al., 2003
Polypeptide β-XylT * Y50D4C.4 (sqv-6) Squashed vulva, hermaphrodite sterile Cyk Hwang et al., 2003
Gal-Gal-Xyl-S β1,3 GlcAT * ZK1307.5 (sqv-8) Squashed vulva, hermaphrodite sterile   Bulik et al., 2000
Heparan sulfate synthesis F12F6.3 (rib-1) Abnormal WT Kitagawa et al., 2001
α1,4-GnT I/II (heparan sulfate synthesis) * K01G5.6 (rib-2) Abnormal WT Kitagawa et al., 2001
GlcAT/GlcT/GalT? B0310.5   WT  
  F08G5.5   WT  
D-GlcA C5-epimerase B0285.5 (hse-5) Axon, cell guidance defects WT Bulow and Hobert, 2004
Heparan sulfate N-deacetylase/N-sulfotransferase F08B4.6 (hst-1) Abnormal WT/Emb, Ste  
Heparan sulfate 2O-sulfotransferase C34F6.4 (hst-2) Axon, cell guidance defects Mig, Egl Bulow and Hobert, 2004; Kinnunen et al., 2005
Heparan sulfate 6O-sulfotransferase Y34B4A.3 (hst-6) Axon, cell guidance defects WT Bulow and Hobert, 2004
Genes involved in N-glycan synthesis
Synthesis of the lipid linked precursor
ALG7 T08D2.2   WT  
  Y60A3A.13   WT  
ALG1 T26A5.4   WT/Emb,Ste  
ALG2 T23F2.1   WT/Abn  
ALG3 K09E4.2   WT  
ALG6 C08B11.8   WT  
ALG8 C08H9.3   WT/Emb, Lva  
ALG9 C14A4.3   WT  
ALG10 T24D1.4   WT  
Lec35/Monosaccharide-P-dolichol utilization F38E1.9   Let, Gro, Bmd, Lva  
Oligosaccharyltransferase complex
OST1/ribophorin T22D1.4   WT/ Abn Wang et al., 2005
Ribophorin II M01A10.3   WT/Abn  
OST3 ZK686.3   WT/Abn  
WBP1/DDOST/Ost48 T09A5.11   Emb, Gro, Lon, Lvl, Unc  
OST2/DAD-1 F57B10.10 (dad-1)   Emb, Gro, Lva, Unc DAD-1 suppresses programmed cell death (Sugimoto et al., 1995)
STT3 T12A2.2   Emb, Lva, Lon, Ste  
Processing glycosydases
α1,2 glycosidase I F13H10.4   WT/Abn  
α1,3 glycosidase II F40F9.6a,b   Emb, Lva  
α 1,2 mannosidase I C47E12.3   WT  
  T03G11.4   WT  
  ZC410.3   WT/Emb  
  ZC506.1   WT  
α 3,6 mannosidase II F48C1.1a,b   WT  
Quality control glycosyltransferases
Glycoprotein GlcT F26H9.8   WT  
  F48E3.3   WT  
N-acetylglycosaminyltransferases specific for the synthesis of complex N-glycans
β 1,2 GnT I * B0416.6 (gly-13) WT WT Chen et al., 1999
  F48E3.1a,b (gly-12) WT Abn  
  M01F1.1 (gly-14) WT WT  
β 1,2 GnT II * C03E10.4 (gly-20) WT WT Chen et al., 2002
β 1,6 GnT V * C55B7.2 (gly-2) WT WT/Emb Warren et al., 2002
N-Glycan core fucosyltransferase
Gn-Asn-X α 1,6 FucT C10F3.6 (fut-8)   WT Paschinger et al., 2005
Gal(NAc)β1,4Gn (Fuc to Gn) α1,3FucT * K08F8.3 (fut-1) WT WT Paschinger et al., 2004
Genes specific for GalNAc Ser/Thr O-glycan synthesis
Polypeptide GalNAcT * ZK688.8 (gly-3)   WT Hagen and Nehrke, 1998
  Y116F11B.12.a,b (gly-4)   WT Wang et al., 2005
  Y39E4B.12a,b,c (gly-5)   WT  
  H38K22.5a (gly-6)   WT  
  Y46H3A.6 (gly-7)   WT  
  Y66A7A.6 (gly-8)   WT  
  Y47D3A.23(gly-9)   WT  
  Y45F10D.3 (gly-10)   WT  
  Y75B8A.9 (gly-11)   WT  
GalNAc α 1-O-S/T β 1,3GalT (O-glycan core 1)
Core 1 β3-Gal-T or T-synthase* C38H2.2   WT Ju et al., 2006
  C02H6.1   WT  
  C16D9.6   WT  
  T22B11.2   WT  
  Y38C1AB.1   WT  
  Y38C1AB.5   WT  
  ZC250.2   WT  
  C17A2.3   WT  
Homology to O-glycan core 1 β1,3 GalT/core 2 β1,6 GnT
  T09E11.6   WT  
Genes for GalNAc Ser/Thr O-glycan synthesis and possibly other pathways
Gal β1,3GalNAca1-O-S/T (Gn to GalNAc) β1,6-GnT (O-glycan core 2); homologous to β1,6 IGnT Warren et al., 2001
  F44F4.6 (gly-1) WT WT Warren et al., 2002; gly-1 does not encode core 2 β1,6 GnT, but rather a β1,6 GlcT
  C54C8.11 (gly-15)   WT  
  T15D6.2 (gly-16) WT WT  
  T15D6.3 (gly-17)   WT  
  F22D6.11 (gly-18) WT WT  
  F22D6.12 (gly-19)   WT  
  T09E11.9   WT  
  R07B7.6   WT  
  F30A10.4   WT  
Other galactosyltransferases
βGn β1,3-GalT F14B6.6   WT/Gro  
  K08D12.5   WT  
  C47F8.3   WT  
  C47F8.6   WT  
  T09E11.11   WT  
  T09F5.1   WT  
  E03H4.11   WT  
  T15D6.5   WT  
  F14B6.4   WT  
  T09E11.10 (β1,3-GnT?)   WT  
  C47F8.5 (β1,3-GnT?)   WT  
  C54C8.3 (β1,3-GnT?)   WT  
βGn β1,4-GalT W02B12.11   WT  
Other N-acetylhexosaminyltransferases
Chitin Gn-T T25G3.2 (chi-1) Emb Emb. Gro,Ste Veronico et al., 2001; Zhang et al., 2005
  F48A11.1 (chs-2)   Bmd, Emb, Gro, Unc Veronico et al., 2001; Zhang et al., 2005
Galβ1,4Gnb β1,3-GnT C18G1.3   WT  
O-Gn transferase * K04G7.3 (ogt-1) Viable; suppressed constitutive dauer formation   Hanover et al., 2005
Galβ1,4GlcCer β1,3-GnT F48F7.3   WT  
Protein O-Man β1,2-GnT1 M70.4a,b   WT  
Genes involved in the biosynthesis of glycosphingolipids
βGn β1,3-GalT Y39E4B.9 (bre-2) Bt resistant WT Griffitts et al., 2005
Glcb1-Cer β1,4-Man-T B0464.3 (bre-3) Bt resistant   Griffitts et al., 2005
GnbR β1,4-GalNAcT Y73E7A.7 (bre-4) Bt resistant   Griffitts et al., 2005; Kawar et al., 2002
Manβ1,4GlcR β1,3-GnT T12G3.8 (bre-5) Bt resistant Suppresses Egl defect associated with elevated lin-12 activity.   Griffitts et al., 2005; Katic et al., 2005
Cer GlcT F59G1.1a,b,c,d   WT  
  F20B4.6   WT  
Other fucosyltransferases
  EGAP9.2 (fut-2) WT WT Zheng et al., 2002
  C06E1.7   WT  
  C14C6.3   WT  
  C17A2.4   WT  
  C18G1.8   WT  
  EGAP9.3   WT  
  F08A8.5   WT  
  F11A5.5   WT  
  F17B5.4   WT  
  F31F4.11   WT  
  F31F4.17   WT  
  K06H6.6   WT  
  T26E4.3   WT  
  T26E4.4   WT  
  T26E4.5   WT  
  T26H5.8   WT  
  T28A8.2   WT  
  T28F2.1   WT  
  W07G4.2   WT  
  Y5H2B.1   WT  
  F41D3.6 (galectin?)   WT  
Galβ1,4/3Gn (Fuc to Gn) α1,3/4-FucT (Lewis x or a)
  F59E12.13 (fut-3) WT WT  
  K12H6.3 (fut-4) WT WT  
  T05A7.10 (fut-5)      
  T05A7.5 (fut-6) WT WT  
Protein O-FucT C15C7.7   WT Wang et al., 2005
  K10G9.3 (pad-2)   Egl, Bmd, Let,Evl Menzel et al., 2004
Nucleotide sugar transport / antiporter cycle
UDP-GlcA/UDP-Gal/UDP-GalNAc transporter * C52E12.3 (sqv-7) Squashed vulva WT/Emb Berninsone et al., 2001
UDP-Gal/UDP-GlcNAc transporter * M02B1.1 (srf-3) Abnormal surface reactivity   Hoflich et al., 2004
GDP-Fuc transporter * C50F4.14   WT Luhn et al., 2001
Putative nucleotide sugar transporter F15B10.1   WT  
Putative nucleotide sugar transporter ZC250.3   WT/Abn  
Putative nucleotide sugar transporter K02E10.5   WT  
UDP-GlcNAc/UDP-GalNAc transporter* C03H5.2   WT/Ste Caffaro et al., 2006
Putative nucleotide sugar transporter ZK896.9   WT  
E-NTPDase * R07E4.4 (mig-23) Defective DTC migration WT Nishiwaki et al., 2004
E-NTPDase * K08H10.4 (uda-1)   WT Uccelletti et al., 2004
E-NTPDase * C33H5.14   WT Uccelletti et al., 2004
E-NTPDase F08C6.6   WT  
Nucleotide sugar synthesis
UDP-Gn 2 epimerase D2007.5   WT  
GDP-Man pyrophosphorylase C15F1.4 (ppp-1)   Abn  
  Y47D9A.1a,b   WT  
P-Man mutase 2 F52B11.2   WT/Gro  
P-Glc mutase/P-Man mutase F21D5.1   Emb, Osm  
  R05F9.6   WT/Emb  
  Y43F4B.5a,b   WT/Emb/Gro  
P-Man isomerase C05C8.7   WT/Emb, Lva, Sck, Ste  
  ZK632.4   WT  
UDP-Glu/UDP-Gal 4-epimerase C47B2.6   WT  
Gal-1-phosphate uridyltransferase Y55D5A.2      
  ZK1058.3   WT  
GDP-Man 4,6-dehydratase F56H6.5   WT/ Abn  
UDP-galactopyranose mutase H04M03.4      
Other glycosidases
α-mannosidase F55D10.1   WT  
Endo-βN-acetylglucosaminidase * F01F1.10a,b   WT  
O-Gn selective β-Gnase T20B5.3 (oga-1)   WT Forsythe et al., 2006

*Edited by Patricia Kuwabara. Last revised October 17, 2006. Published December 18, 2006. This chapter should be cited as: Berninsone, P.M. Carbohydrates and glycosylation (December 18, 2006), WormBook, ed. The C. elegans Research Community, WormBook, doi/10.1895/wormbook.1.125.1, http://www.wormbook.org.

Copyright: © 2006 Patricia M. Berninsone. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

§To whom correspondence should be addressed. E-mail: berninsone@unr.edu

[] Abbreviations:
Cer: ceramide
Fuc: fucose
FucT: fucosyltransferase
GAG: glycosaminoglycan
Gal: galactose
GalNAc: N-acetylgalactosamine
GalNAcT: N-acetylgalactosiminyltransferase
GalT: galactosyltransferase
Glc: glucose
GlcA: glucuronic acid
GlcAT: glucuronyltransferase
GlcNAc: N-acetylglucosamine
GlcT: glucosyltransferase
GnT: N-acetylglucosamilyltransferase
IdoA: iduronic acid
HS: heparan sulfate
HSPG: heparan sulfate proteoglycan
Man: mannose
ManT: mannosyltransferase
PCho: phosphorylcholine
P-glu: phosphoglucose
P-man: phosphomannose
Xyl: xylose
XylT: xylosyltransferase

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