During the human-infectious stage of the helminthic life cycle, parasites engage in a series of actions designed to evade the host immune response (1). Initially, defensive manoeuvres such as tegument moulting and membrane turnover were described (2, 3), but it is now understood that parasites can further promote their longevity by directly manipulating host systems, via the release of biologically active excretory/secretory molecules (E/S). The evolution of molecular parasitology in recent years has enabled an increasingly precise characterisation of these molecules.

Herein we examine nematode phosphorylcholine (PC)-containing glycoconjugates, in the context of the structure and function of these unique molecules. The first suggestion of the relevance of PC in host-parasite interactions came with discovery of PC on nematode carbohydrate-containing molecules, and also anti-PC antibodies in rats exposed to Nippostrongylus brasiliensis and Haemonchus contortus, following anecdotal reports of similar results from Ascaris suum-infected rats (4). In vivo studies revealed anti-PC antibody complexes were in fact abundant in the sera of mice exposed to Brugia malayi and B. pahanghi, as well as jirds, chimpanzees and human subjects suffering filarial parasitosis (5–10). Analysis of the E/S profile of the adult rodent filarial nematode Acanthocheilonema viteae revealed that it is dominated by one PC-containing glycoprotein, ES-62 (4, 11). Homologues of this protein have since been detected in other filarial and also non-filarial nematodes species, although it is unknown whether all of these ES-62 homologues contain PC groups (12–17).

ES-62 is the most characterised of the PC-bearing helminthic molecules, and accordingly the one which forms the basis for most of our understanding of the structure and function of nematode PC-glycans. ES-62 originates from the anterior oesophageal cells of A. viteae as a homotetrameric protein (18, 19). By subjecting ES-62 to N-glycosidase F, it was demonstrated that PC is attached to the protein backbone via N-linked glycans, a finding confirmed by examination of ES-62 following exposure of A. viteae to tunicamycin, an inhibitor of N-type glycosylation (20, 21). Monomeric ES-62 contains four potential N-linked glycosylation sites at residues 213, 254, 344 and 400 respectively (22). Fast atom bombardment mass spectroscopy enabled the resolution of three associated N-type glycan structures: a high mannose N-glycan (Man_5-9GlcNAc₂), a truncated oligosaccharide, trimmed to the trimannosyl core and fucosylated (Fuc₁Man₃GlcNAc_3-6), and a novel glycan, which is similarly truncated and may be fucosylated or not, and which acquires between 1-4 antenna GlcNAc residues (Fuc_0-1Man₃GlcNAc_3-6) to which PC is attached ( Figure 1 ). More recently, employment of nano-flow liquid chromatography followed by electrospray ionization mass spectrometry by North et al. (23) revealed that each of the N-linked glycosylation sites of ES-62 can accommodate PC-bearing glycans. Furthermore, each glycan’s structure was determined to contain up to five PC groups: four from antennary GlcNAc residues, and a fifth attaching to core GlcNAc. It has been estimated that the secreted tetrameric ES-62 can bear up to 72 PC groups, with the molecule’s structure ensuring the bulk of these are positioned for receptor engagement.

Molecules closely mimicking the original ES-62 PC-glycan structures ( Figure 1 ) have been detected in anthropophilic filarial species B. malayi, Onchocerca volvulus, Wucheria bancrofti and Loa loa, and additionally in B. pahangi (feline), O. gibsoni (bovine) and Dirofilaria immitis (canine) species (12, 13, 17, 19). Outside of filaria, these PC-glycan structures also remain relatively consistent in parasitic nematodes; thus Trichinella spiralis produces glycans which similarly appear to bear PC moieties likely attached to GlcNAc residues on a trimmed trimannosyl core, although this is followed by further GalNAc transferase activity to extend the antenna (24). A. suum (HexNAc_3-5Hex_3-4Fuc_0-1PC_1-2) and Trichuris suis (Hex₃HexNAc_4-5Fuc₂PC) additionally produce a number of comparable glycan structures to the ES-62 PC-glycans (15, 25). Fascinatingly, similar PC-modified N-glycans have also been described in free-living species including Caenorhabditis elegans (Hex₃HexNAc₃Fuc_0-1PC) and Pristionchus pacificus (Hex₃HexNAc₅Fuc_0-1PC_1-3), with attachment occurring on non-reducing GlcNAc or LacDiNac motifs. This suggests that such PC-N-glycans may be conserved throughout the phylum (26, 27), although the PC-containing structures in free-living nematodes are tri-antennary rather than tetra-antennary (tetra-antennary N-glycans don’t appear to be formed in free living species) as observed for ES-62. Moreover, PC-substituted glycosphingolipids have been reported in embryonic-stage C. elegans, as well as in A. viteae, O. volvulus, O. ochengi, Litomosoides sigmodontis and A. suum species (28–30).

Employment of a combination of intracellular trafficking inhibitors, oligosaccharide processing inhibitors, pulse-chase radiolabelling experiments and FAB-MS analysis has permitted characterisation of the intracellular processing events which attach PC to newly produced glycans in A. viteae and C. elegans (23, 30–33). It is within the medial Golgi that the process of PC attachment to maturing N-glycans occurs ( Figure 2 ). Initially, an antennary GlcNAc residue is attached to the glycan structure by GlcNAc Transferase I. PC is subsequently transferred from a donor, likely phosphatidycholine, via a C-6 phosphodiester linkage of the antenna GlcNAc residue. Two mannose sugars are then trimmed from the core by Mannosidase II. Next, GlcNAc Transferase II adds a second terminal GlcNAc residue and then additional PC may be added. As yet, the PC-transferase enzyme involved in this process remains uncharacterised.

PC on nematode glycoproteins has immunomodulatory properties as first shown by its inhibition of lymphocyte proliferation (34, 35). The mechanisms behind such activity have been extensively studied using ES-62 and to a lesser degree, PC alone or conjugated to proteins such as bovine serum albumin (BSA), or small molecule analogues (SMAs) of ES-62 based on its PC moiety ( Figure 3A ) (36). For example, the PC-containing molecules inhibit B cell receptor (BCR)-stimulated phosphoinositide-3-kinase (PI3K) and protein kinase C activities (35–37), as well as reducing the phosphorylation of Igβ and the adaptor Shc1 resulting in reduced Erk1/2 MAPK activation (37, 38). This appears to reflect that ES-62 promotes recruitment of the protein tyrosine phosphatase, SHP-1 resulting in rapid dephosphorylation of these BCR-stimulated substrates (38). Furthermore, ES-62 terminates ongoing Erk1/2 activity upon BCR stimulation by recruitment of dual specificity phosphatase Dusp2, which dephosphorylates their threonine and tyrosine activation motifs.

Such immunomodulatory actions have also been observed with innate immune system cells. Thus, like ES-62, PC-based SMAs 11a and 12b inhibit FcϵRI-mediated signalling and degranulation, as well as proinflammatory cytokine secretion ( Figure 3B ) (39–42). This can in part be explained by reduced calcium mobilisation and protein kinase C alpha (PKCα) protein expression, actions also reproduced by pre-treatment with PC-BSA (39–41). Moreover, antigen-presenting cells (APCs) like dendritic cells and macrophages, that educate adaptive immunity are also desensitised in response to ES-62 and PC ( Figure 3C ) (43, 44). Thus here, PC-ovalbumin and SMAs (11a, 12b) inhibit NF-κB mediated pro-inflammatory cytokine secretion stimulated via MyD88-dependent toll-like receptors (TLRs; TLR4, LPS; TLR9, CpG; TLR1/2 heterodimer, PAM₃CSK₄) (43–50). ES-62 also supresses the synergistic NF-κB-mediated pro-inflammatory cytokine secretion observed in response to these stimuli when tested in combination with interferon gamma (IFNγ) (44, 51). In contrast, TLR3 signalling, which is independent of MyD88, is unaffected by PC-ovalbumin or ES-62 (43, 44, 52–54). This selectivity reflects that MyD88 protein expression is reduced in the presence of ES-62 or SMAs, 11a & 12b (50, 55–58) and as this downregulation is also recapitulated in certain mast cell subsets (41) and lymphocytes (50, 55–59), it provides a unifying primary target for PC/ES-62 action in limiting chronic inflammatory responses.

PC, either as part of ES-62 or attached to ovalbumin, appears to “signal” through TLR4 (43, 44). Unlike LPS, the most defined TLR4 ligand, PC maintains its activity in the context of a proline to histidine substitution at position 712 on the receptor (43, 44, 60, 61). This residue sits within a cytoplasmic domain required for interaction with the signalling adaptor Mal (itself required for TLR4 interaction with MyD88), which suggests PC signals through a different mechanism to LPS (62, 63). Indeed, SMAs 11a and 12b interact directly with the TIR domain of MyD88, inhibiting homodimer formation (56). As ES-62 is internalised in both mast cells and macrophages, it is possible that the parent molecule also interacts with MyD88 through its PC moieties (40, 64).

Complement is an innate mechanism that recognises microbial PAMPs and facilitates microbicidal activity; it is also intensely activated by PC, which has bound to C-reactive protein (65–68). PC directly attached to proteins also activates complement in vitro; but complement activation drops considerably when PC is attached at the end of either synthetic flexible linkers, or importantly, to glycans such as in ES-62 (69, 70). This functionality may benefit parasitic worms by sequestering components of the complement cascade, resulting in a low complement activation state.

PC on the glycans of ES-62 is also active in vivo, for example, it alters the subclass of antibody produced in response to ES-62 in mice, in that removal of PC results in the generation of IgG2a antibodies that are absent when the intact molecule is employed (71). Moreover, exploring the potential of a potent anti-inflammatory molecule like ES-62 as a biologic intervention in human disease has been dependent upon robust pre-clinical evaluation using in vivo experimentation. In fact, ES-62, and the drug-like SMAs represent the only stand-alone nematode PC-products that have been tested in vivo, although the suppression of rodent arthritis by A. suum extract is suspected to be attributed to a homologous PC-bearing molecule (72). These treatments have been demonstrated to be safely tolerated throughout the summarised experiments below, with potency illustrated in the resolution of a range of inflammatory conditions, irrespective of phenotype.

Anti-PC antibodies have been found to reduce inflammation in ischemic mouse models and are plentiful in human subjects at low risk of atherosclerosis (92–94). In addition, in the context of rheumatic disease, an inverse correlation between anti-PC antibodies and, organ damage and disease activity in SLE patients has been demonstrated (95, 96). Although the exact mechanism(s) underlying the protective effects of anti-PC antibodies is still under investigation, anti-inflammatory and cardioprotective benefits of anti-PC antibodies in SLE and also Sjögren’s syndrome and mixed connective tissue disease are associated with regulatory T cell polarisation, oxidised low density lipoprotein uptake inhibition and enhanced apoptotic cell clearance (96–99). Interestingly, IgM anti-PC antibodies are greatly increased in response to ES-62 in murine models of lupus and in high calorie diet (HCD)-fed mice (82, 89). These antibodies were not cross-reactive to oxidised low density lipoprotein nor conformed to the T15 idiotype (characteristic of most of the protective anti-PC antibodies reported) (82, 93, 100, 101). Nevertheless, machine learning identified anti-PC IgM levels as the best predictor of effective ES-62 treatment in HCD-fed mice (89). Thus, overall, it would perhaps be premature to rule out a protective role for ES-62-induced anti-PC antibodies in terms of protecting the host against obesity-accelerated ageing at this stage. Also of interest, natural IgM antibodies appear to promote a regulatory phenotype of B cells, which then reduce inflammation in vivo (102). Natural antibodies are secreted by B1 B cells independently of T cell stimulation, have limited diversity and recognise multiple antigens (103–107). Indeed, ES-62 stimulates B1 B cell activation in vivo and increases regulatory B cell numbers in several in vivo models (74, 76, 81, 87, 108).

Increasingly sophisticated analytical procedures are being applied to the elucidation of the structure of nematode PC-containing glycoconjugates. This has revealed a general uniformity across the nematode phylum and also that these novel carbohydrate structures may have not one, but multiple, PC groups attached. PC-containing nematode glycoconjugates possess immunomodulatory properties such that they are potentially therapeutic but full exploitation of this with respect to PC-containing glycoproteins such as ES-62 is handicapped by an inability to produce fully active recombinant forms, as no convenient protein expression system exists that encodes the (unidentified) requisite PC transferase enzyme (109–112). PC-based SMAs can alternatively circumvent this problem and may represent the form in which active nematode molecules make it to the clinic. At the same time, three issues which remain to be resolved are: (i) increased understanding of the molecular complexity underlying the range of immunomodulatory activities of secreted PC-containing proteins like ES-62. For example, we have learned of how ES-62 is reliant on expression of TLR4 for PC-dependent activity against macrophages and dendritic cells (46), but it appears that additional receptors may exist in cells such as lymphocytes with which ES-62 can interact in a PC-dependent manner (66). Moreover, ES-62’s modulation of immune cell function can be associated with effects on multiple cell signalling pathways, e.g., in mast cells, ES-62 impacts on cross talk amongst TLR4-, ST2- and FcϵRI-dependent pathways (113), highlighting the level of complexity that may need to be dissected. (ii) a greater understanding of the function of PC-glycoconjugates found as internal rather than secreted products of nematodes – this is unlikely to relate to immunomodulation because as described earlier the structures are found within free-living species in addition to parasitic species. One possibility relates to growth and development. Consistent with this, chemical blockage of production of PC-containing glycosphingolipids by targeting enzymes upstream of PC addition impairs embryonic development in C. elegans, although a direct role for PC has not been shown (114). (iii) the identity of the aforementioned nematode enzyme which transfers PC to carbohydrate – as such structures are absent from mammals, this enzyme if identified could offer a potential novel drug target. Interestingly, we have shown previously that enzymes acting upstream of the PC transferase in C. elegans can be knocked down by RNAi and that this results in reduced transfer of PC to proteins (115). We believe this approach could be employed in investigating the identity of the PC transferase when applied to genes of likely related function based on sequence homology in nematode genomes. Similarly, CRISPR (112) knockdown of potential related genes offers a more recent but similar approach, as do proteomics approaches focusing on the site of PC transfer, the Golgi. Of benefit to these analyses should be an in vitro assay of PC transferase activity developed by Cipollo and colleagues (33).

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