Anionic and zwitterionic moieties as widespread glycan modifications in non-vertebrates
Glycan structures in non-vertebrates are highly variable; it can be assumed that this is a product of evolution and speciation, not that it is just a random event. However, in animals and protists, there is a relatively limited repertoire of around ten monosaccharide building blocks, most of which are neutral in terms of charge. While two monosaccharide types in eukaryotes (hexuronic and sialic acids) are anionic, there are a number of organic or inorganic modifications of glycans such as sulphate, pyruvate, phosphate, phosphorylcholine, phosphoethanolamine and aminoethylphosphonate that also confer a ‘charged’ nature (either anionic or zwitterionic) to glycoconjugate structures. These alter the physicochemical properties of the glycans to which they are attached, change their ionisation when analysing them by mass spectrometry and result in different interactions with protein receptors. Here, we focus on N-glycans carrying anionic and zwitterionic modifications in protists and invertebrates, but make some reference to O-glycans, glycolipids and glycosaminoglycans which also contain such moieties. The conclusion is that ‘charged’ glycoconjugates are a widespread, but easily overlooked, feature of ‘lower’ organisms.
KeywordsGlycomics Glycans Glucuronic acid Phosphorylcholine Phosphoethanolamine Sulphate Nematode Insect Mollusc
Glycans modify a range of proteins and lipids, thereby altering their properties and their interactions. All cells have a glycan coat and so the exact nature of glycan structures can determine a wide range of cell-cell, gamete-gamete, host-pathogen, vector-pathogen and host-symbiont interactions; it can be assumed that the glycomic repertoire of a species is formed by evolutionary processes and can define speciation . The basic building blocks of glycans are monosaccharides with hexoses, deoxyhexoses, pentoses and N-acetylhexosamines being very common regardless of whether the glycoconjugate is of animal, plant, protist, fungal or bacterial origin, although the monosaccharide repertoires of bacteria and plants are the most diverse. In addition to these ‘uncharged’ sugars, which can be modified also by moieties such as methyl groups, there are ‘charged’ saccharide and non-saccharide elements. Thereby, hexuronic and sialic acids on glycoconjugates are derived from corresponding uridine diphosphate or cytidine monophosphate donors for the relevant glycosyltransferases; in the case of sulphates, phosphate esters and phosphonates, other activated known or unknown donors are required by the enzymes that perform ‘post-glycosylational modifications’ of glycans.
While sialic and hexuronic acids are well-known in vertebrates as components of glycosaminoglycans, glycoproteins and glycolipids [2, 3, 4], sulphates are widespread on glycans of many species [5, 6]. Phosphates are known in the context of the mannose-6-phosphate marker for trafficking of lysosomal enzymes , but phosphodiesters are less common: exceptions being that phosphoethanolamine is a component of glycosylphosphatidylinositol anchors  and ribitol phosphodiesters are part of the modification of dystroglycan . This contrasts with the invertebrates: sialic acids are generally rare, but phosphodiesters and phosphonates have been found on glycoconjugates in a number of species. Hexuronic acids are certainly found in invertebrates, but are less familiar or absent from fungi and protists. In plants, charged modifications (especially hexuronic acids) are on polysaccharides [10, 11], but not on N-glycans.
As N-glycans are the most commonly analysed glycoconjugates and most specific structural information in non-vertebrates is on this class of structure, we will concentrate on these. We also mention the occurrence of anionic and zwitterionic modifications in O-glycan, glycolipid and glycosaminoglycan structures, but exclude glucuronylated and sulphated small metabolites from our discussion. It is probably impossible to be exhaustive in citing all relevant literature and there are other recent reviews which also address neutral glycans in various invertebrates [12, 13, 14]. We hope readers will appreciate the overall conclusion that hexuronic and sialic acid, sulphate and phospho-based moieties in non-vertebrates can no longer be overlooked, but indeed vastly increase the range of possible glycan structures in a large number of species.
Glucuronic acid is probably the most common hexuronic acid; its activated form (UDP-GlcA) derives from the action of a dehydrogenase on UDP-Glc . In turn, UDP-GlcA can be decarboxylated into UDP-Xyl; thus, any organism capable of making and transferring xylose can also theoretically have glucuronylated glycoconjugates. Indeed, glucuronic acid is known from a variety of glycoconjugates from different species. In terms of mass spectrometric analyses, glucuronic acid has the same mass increment (Δm/z 176 Da) as a methylated hexose. However, hexuronic acids, but not methylhexoses, enable ionisation in both positive and negative MALDI-TOF MS modes; also, aiding definition of these modifications are glucuronidases, which can be used in the analysis of glucuronylated glycans, as well as the enrichment of such structures in the ‘acidic’ fraction upon graphitised carbon-based solid phase extraction . Other known hexuronic acids, include galacturonic acid and iduronic acid, which are respectively components of plant cell wall polysaccharides and animal glycosaminoglycans [17, 18].
O-glycans from insects can also be modified with glucuronic acid [22, 29, 30], whether these be of the ‘mucin-type’ (e.g., GlcAβ1,3Galβ1,3GalNAc, Galβ1,3[GlcAβ1,4]GalNAc or ones carrying Hex1HexNAc1HexA1 repeats; the latter found in the lepidopteran HighFive cell line and in mosquitoes) or the ‘Notch-type’ (GlcNAcβ1,3[GlcAβ1,4]Fuc; see Fig. 1); recently, it was shown that O-glycan glucuronylation is important for neuromuscular junction formation in Drosophila . Glucuronic acid is also present on O-glycans from Caenorhabditis elegans , while a circulating antigen from the trematode Schistosoma mansoni is a βGalNAc-based threonine-linked polysaccharide with β1,3GlcA side chains . Monoglucuronylated O-glycans with reducing terminal mannose have been described from the fungus Trichoderma reesii .
Chondroitin and heparan chains of proteoglycans with varying degrees of sulphation can also be considered as O-glycans, as these primarily consist of HexA1HexNAc1 glycosaminoglycan repeats attached via a GlcAβ1,3Galβ1,3Galβ1,4Xyl tetrasaccharide linker (see Fig. 1) in probably all animals, including cnidarian, annelid, nematode and insect species [35, 36, 37, 38]. The structurally-related dermatan sulphate containing iduronic acid is also found in echinoderms , while hyaluronic acid occurring as (GlcAβ3GlcNAcβ4)n repeats is apparently absent from invertebrates . In terms of genetic model organisms, glycosaminoglycans from Caenorhabditis and Drosophila have a number of roles in development as shown by studies on various mutants lacking key enzymes in chondroitin and heparan biosynthesis [41, 42]. Probably the best characterised invertebrate β1,3-glucuronyltransferases are three enzymes from Drosophila with potential to act in N-, O-, and lipid glycan or glycosaminoglycan biosynthesis .
In most invertebrates, it can be safely concluded that sialic acids are absent, as neither the relevant monosaccharides have been detected by modern methods nor the genetic capacity to make or transfer them is present. Insects constitute an exception as these do have sialyltransferase genes; however, only in Drosophila is there proper evidence as to it being attached to glycans . On the other hand, as discussed below, primitive deuterostomes such as echinoderms have multiple sialyltransferase genes and a number of studies have proven the occurrence of sialylated glycans. However, the presence of plant genes with homology to sialyltransferases  also throws up questions as to the real enzymatic and biological role of such proteins in organisms for which the occurrence of sialic acid on glycans is not proven; indeed, more recently it has been proposed that these plant sialyltransferase-like enzymes may transfer either 2-keto-3-deoxy-D-lyxo-heptulosaric acid (DHA) or 2-keto-3-deoxy-D-manno-octulosonic acid (KDO) to rhamnogalacturonan-II . In the case of trypanosomatids, trans-sialylation results in parasites coating their mucins with host-derived sialic acid . Various reports claiming that sialic acid is present in other invertebrate organisms, however, must be taken with caution, as mere HPLC peaks or lectin blotting data are not conclusive proofs and can be due to, e.g., lectin cross-reactivity or the presence of contaminants; only mass spectrometric proof of the structure, in the absence of deuterostome components in media or food, is an acceptable demonstration of sialylation.
Sialylation in insects has long been controversial. One early report suggested the presence of polysialic acid in Drosophila , but the only sialylated glycans found by mass spectrometry contain a single residue of N-acetylneuraminic acid attached to galactose on N-glycans [44, 49] (see example in Fig. 1). The single Drosophila sialyltransferase has been functionally and biochemically characterised [50, 51]. In other insect species, sialyltransferase genes have been identified and parts of the CMP-sialic acid biosynthesis pathway revealed [52, 53]; however, only upon glycoengineering can it be safely concluded that Lepidoptera will express sialic acid on recombinant glycoproteins .
In the deuterostome lineage, even in organisms evolutionarily more primitive than the vertebrates, there is quite definitely an expansion of the sialyltransferase gene family. For instance, multiple sialyltransferases are encoded by the genome of a cephalochordate (Branchiostoma belcheri); both N-acetylneuraminic acid (Neu5Ac) or N-glycolylneuraminic acid (Neu5Gc) were found to be expressed in a tissue-specific manner in this organism, while methylated 3-deoxy-D-glycero-D-galacto-2-nonulosonic acid (KDN; a non-acylated sialic acid) was found on ovary O-glycans . In echinoderms, a number of α2,3-, α2,6- and α2,8-sialyltransferase homologues, yet to be enzymatically characterised, occur in the sea urchin Strongylocentrotus purpuratus [56, 57, 58]; NeuAc and NeuGc are found in a number of echinoderm species, also in O-acetylated and O-sulphated forms. In starfish and sea cucumbers, a large percentage of these sialic acids are additionally methylated at O-8, whereas in other echinoderms this position can be sulphated [59, 60]. Relevant metabolic enzymes for the generation of methylated NeuGc are the CMP-NeuAc hydroxylase, as also found in non-human mammals, and an 8-O-methyltransferase [61, 62].
Specific examples of defined sialylated structures in echinoderms include a mucin-type O-glycan from the sperm flagella of the sea urchin (Hemicentrotus pulcherrimus) which contains terminal sulphated O-8 α2,9-linked polyNeu5Ac linked to the protein backbone via GalNAc [63, 64], a motif also present in two other sea urchins (Strongylocentrotus purpuratus and Str. franciscanus). On other sea urchin sperm and egg O-linked glycoproteins, α2,8-sialic acid and α2,5-sialic acid were found [65, 66]. Gangliosides (i.e., lactosyl- or glucosylceramides carrying one or more NeuAc or NeuGc residues, reportedly also in fucosylated, methylated or sulphated forms; see example in Fig. 1) represent another class of sialylated glycoconjugates studied in species of various echinoderm classes [67, 68, 69, 70, 71], such as sea cucumbers (Stichopus chloronotus, Sti. japonicus and Holothuria leucospilota), starfish (Linckia laevigata, Asterias amurensis, Luidia maculata, Asterina pectinifera and Acanthaster planci), sea urchins (Str. intermedius and H. pulcherrimus), a feather star (Comanthus japonica) and a brittle star (Ophiocoma scolopendrina).
The presence of pyruvate on N-glycans is known from yeast species, specifically 4,6-ketal-linked to outer chain galactose residues , whereby it should also be noted that some bacterial and seaweed polysaccharides are also pyruvylated. The pvg1 gene, a pyruvyltransferase, is one of five necessary for pyruvylation in the fission yeast, whereby the pvg3 gene encodes the galactosyltransferase required for transfer of the underlying β1,3-linked galactose residue [73, 74]. Pyruvate was also found on glycolipids of the sea hare, which is a mollusc  and on the glyconectin polysaccharides isolated from some Porifera sponges .
Probably sulphation is most familiar from glycosaminoglycans (e.g., heparan, chondroitin and keratan sulphates), but some of these are either absent or undersulphated in many invertebrates ; on the other hand, some marine organisms have highly sulphated glycosaminoglycans, sometimes also with fucosyl modifications . Indeed, an increased degree of sulphation on various glycoconjugates seems to occur in the context of marine or saline environments [79, 80]. Especially glycosaminoglycans from sharks, hagfish, king crabs, squid, molluscs, sea cucumbers and sea squirts are characterized by oversulphated complex structures which carry more sulphated residues per disaccharide unit than the terrestrial vertebrates on their chondroitin and dermatan sulphates , which contain only one sulphate group per disaccharide unit (one, two or three sulphates being possible, of course, on the disaccharide units of vertebrate heparan sulphate). Sulphated fucans and galactans from echinoderms, distinct from glycosaminoglycans, may have roles in fertilisation [81, 82]. Also, although sulphated polysaccharides have not been described in vascular plants (angiosperms), sulphated galactans and fuc(oid)ans have been isolated from marine sea grass species and algae [79, 80], while sulphoquinovosyldiacylglycerol is an important anionic glycolipid component of plant chloroplasts . Sulphated O-glycans with mannose at the reducing terminus have been described from a coral .
In terms of N-glycans, sulphation of α-linked mannose and/or core α1,6-fucose is known from arthropods (specifically a lobster as well as insects; see examples in Fig. 1) and from Dictyostelium [21, 85, 86, 87, 88], while sulphation of galactose is a feature of the Eastern oyster . Based on studies with radioactive SO4 labelling and PNGase F treatment, the occurrence of sulphated N-linked oligosaccharide chains on glycoproteins involved in sea urchin embryonic skeleton formation was postulated ; these glycan chains, recognised by the 1223 antibody, apparently bind calcium , but their exact structures were not defined. Sulphated ‘mucin-type’ O-glycans are known from a marine snail as well as from dipteran species, whereby the major structures are based on the core 1 Galβ1,3GalNAc motif [20, 21]. More unusual are the sulphated O-glycans with Hex-HexNAc-HexA repeats found in mosquito larvae and a recombinant protein expressed in HighFive cells [21, 30]. As mentioned above, 8-O-sulphated sialic acid has been found on sea urchin O-glycans and glycolipids [63, 70]. Generally, the sulphate on glycans originates from 3′-phosphoadenosine-5′-phosphosulfate as the activated donor; however, other than sulphotransferases involved in glycosaminoglycan synthesis [92, 93, 94, 95, 96], probably no other invertebrate glycan-modifying sulphotransferases have been either biochemically or biologically characterised.
Phosphate and methylphosphate
Phosphorylcholine and phosphoethanolamine
Other than phosphate, zwitterionic phosphodiesters modify glycoconjugates from a range of prokaryotic and eukaryotic organisms. Phosphoethanolamine (PE; also known as aminoethylphosphate or ethanolamine phosphate; Δm/z 123 Da) and its tri-N-methylated form, phosphorylcholine (PC; Δm/z 165 Da), have been described on N-glycans, O-glycans, glycolipids and glycosaminoglycans from protists, fungi, insects, annelids and nematodes as well as on lipopolysaccharides from various bacteria. Phosphoethanolamine is also a component of eukaryotic glycosylphosphatidylinositol (GPI) anchors .
In nematodes and in one cestode it appears that GlcNAc or GalNAc residues of N-glycans and glycolipids can be modified with phosphorylcholine (see examples in Fig. 2), as shown by studies on C. elegans, Ascaris suum, Trichuris suis, Haemonchus contortus, Oesophagostomum dentatum, Pristionchus pacificus, Trichinella spiralis, various filarial worms (including Dirofilaria immitis) and Echinococcus granulosus [25, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119]; the underlying structures of the phosphorylcholine-modified N-glycans from nematodes vary, but multiple residues (in the context of a single GlcNAc, chito-oligomer or LacdiNAc-type motifs) on two, three or four antennae are possible. Phosphorylcholine is also found on annelid and nematode glycolipids [120, 121, 122, 123], on glycosaminoglycan-like structures from at least one nematode  and on O-glycans with the composition HexNAc2-3HexA1PC1 released from a recombinant protein expressed in lepidopteran (Sf9) cells . In fungi, phosphorylcholine is found to substitute either galactofuranose on glycosylinositol-phosphoceramide glycolipids  or mannose in a peptidophosphogalactomannan .
Phosphorylcholine was found on core ‘GalFuc’ moieties of N-glycans of the gastropod Volvarina rubella  as well as on the antennae of a not insignificant modification of N-glycans from Lepidoptera, including cell lines used in biotechnology such as Trichoplusia ni HighFive . In other insects, it is rather phosphoethanolamine that is found on N-glycans of the honeybee  and the O-glycans of wasps ; on the other hand, N-glycans from Diptera were not yet found to contain zwitterionic modifications, even though phosphoethanolamine linked to GlcNAc is a component of glycolipids from dipteran insects such as Drosophila and Calliphora [128, 129] (see example in Fig. 2) or on O-glycans from mosquito larvae with the composition HexNAc1-2Hex1HexA2PE1 . Phosphoethanolamine is also present on mannose residues of N-glycans from Penicillium species and Trichomonas vaginalis [103, 130] and has been reported as a modification of glycolipids from the bivalve, Corbicula sandai .
At the analytical level, phosphoethanolamine yields signals in both positive and negative modes of MALDI-TOF MS, while the phosphorylcholine is only (and excellently) detectable in positive mode ; however, both are sensitive to hydrofluoric acid hydrolysis. The enzymatic basis for addition of these moieties is far from resolved, but conceivably CDP-choline/ CDP-ethanolamine or phosphatidylcholine/ phosphatidylethanolamine could be the donor substrates for PC/PE-transferases [132, 133, 134]. Both phosphorylcholine and phosphoethanolamine are ligands for pentraxins (e.g., human C-reactive protein and serum amyloid P), which are components of the innate immune system ; at least phosphorylcholine modifications on glycolipids and glycoproteins are associated with immunomodulatory activity, as especially shown for the Acanthocheilonema viteaeES-62 protein, which is an excretory-secretory protein of a rodent nematode parasite, whereby the Toll-like receptor 4 (TLR4) may be involved in the relevant signalling pathway .
Phosphonates differ from phosphates in that there is a carbon-phosphorus bond without any intermediate oxygen atom. In eukaryotes, the first demonstrations of the 2-aminoethylphosphonate and N-methyl-2-aminoethylphosphonate modifications of glycans (Δm/z 107 and 121 Da) came from studies on glycolipids of gastropods, such as the sea hare Aplysia kurodai [137, 138, 139, 140, 141, 142]; as shown in Fig. 2, one such glycolipid is reported to also contain pyruvate and phosphoethanolamine residues . In other studies, a GPI anchor from Trypanosoma cruzi (the causative agent of Chagas’ disease), N-glycans from locust and O-glycans from jellyfish were found to be modified with aminoethylphosphonate [143, 144, 145]. Most recently, methylaminoethylphosphonate and aminoethylphosphonate were detected on N-glycans as well as O-fucose-based structures from a marine snail , while there is also evidence that oligomannosidic glycans from Euglena gracilis, a free-living protist, carry aminoethylphosphonate .
Predominantly, it seems that either mannose or GlcNAc residues can be modified by these phosphonates; like phosphorylcholine and phosphoethanolamine, these can be cleaved by hydrofluoric acid. Aminoethylphosphonates were detected in both positive and negative ion modes of FAB- and MALDI-TOF-MS. Neither the recognition of aminoethylphosphonate by pentraxins nor the routes for its transfer to glycan substrates is known.
Mass alone is not enough
In this brief overview of anionic and zwitterionic glycans from various lower eukaryotes, it is obvious that the glycosylation in protists and invertebrates is highly variable. Of course, our knowledge is only based on those organisms or phyla which have been investigated; very many phyla (and naturally huge numbers of species) have never been studied (or adequately so) as to their glycomes. Mere mass spectrometric profiling, without doing deep analyses based on fractionated glycomes, will also not show the true extent of the glycostructural possibilities. As many anionic or zwitterionic glycans with potential biological activity occur in low amounts, they are often overseen if, e.g., the whole N-glycome is ‘shot’ from a single spot on a MALDI plate; certainly the isomeric structures will not be easily distinguished. However, as technologies and methodologies evolve, there is potential for even more variants and/or commonalities between species.
Open access funding provided by Austrian Science Fund (FWF). Work in our laboratory relevant to this review has been funded by the Austrian Science Fund (FWF; grants P21946 and P25058 to K.P. and P23922 and P29466 to I.B.H.W.) and the European Union (Glycopar EU FP7 Marie Curie Initial Training Network; PITN-GA-2013-608295). We thank Daniel Malzl for help in preparing figures.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflicts of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
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