Highly modified and immunoactive N-glycans of the canine heartworm

Abstract

The canine heartworm (Dirofilaria immitis) is a mosquito-borne parasitic nematode whose range is extending due to climate change. In a four-dimensional analysis involving HPLC, MALDI-TOF–MS and MS/MS in combination with chemical and enzymatic digestions, we here reveal an N-glycome of unprecedented complexity. We detect N-glycans of up to 7000 Da, which contain long fucosylated HexNAc-based repeats, as well as glucuronylated structures. While some modifications including LacdiNAc, chitobiose, α1,3-fucose and phosphorylcholine are familiar, anionic N-glycans have previously not been reported in nematodes. Glycan array data show that the neutral glycans are preferentially recognised by IgM in dog sera or by mannose binding lectin when antennal fucose and phosphorylcholine residues are removed; this pattern of reactivity is reversed for mammalian C-reactive protein, which can in turn be bound by the complement component C1q. Thereby, the N-glycans of D. immitis contain features which may either mediate immunomodulation of the host or confer the ability to avoid immune surveillance.

Comparisons of N-glycans across invertebrate phyla

“…If there is no transfer by GlcNAc-TII thereafter to the α1,6-mannose, then the glycan remains in a hybrid state (i.e., sharing aspects of oligomannosidic and complex structures); also even if GlcNAc-TIV (β1,4-specific) modifies the α1,3-mannose, then the glycan is still classified as being hybrid (Kornfeld & Kornfeld, 1985). The lower arm β1,2- and β1,4-GlcNAc residues on hybrid glycans can be modified in different ways; elongation by β1,4- N- acetylgalactosamine and β1,3- or β1,4-galactose are known in insects, molluscs, nematodes and trematodes, whether these be host or parasitic organisms (Kurz et al , 2015; Kurz et al , 2013; Martini et al , 2019; Nyame et al , 1989; Smit et al , 2015). If however, GlcNAc-TII acts and then the ‘lower’ arm β1,2-GlcNAc transferred by GlcNAc-TI is removed by a Golgi hexosaminidase such as fdl (fused lobes) in insects or HEX-2 in nematodes (Geisler & Jarvis, 2012; Gutternigg et al , 2007b), then the resulting glycans can be referred to as ‘pseudohybrid’.…”

Anionic and zwitterionic moieties as widespread glycan modifications in non-vertebrates

“…1). In the case of molluscs (specifically one gastropod, Volvarina rubella, and one bivalve, Mytilus edulis), glucuronic acid modifies antennal fucose residues [19,20]; thus far in insects, whether mosquitoes, the fruit fly, the honeybee or moth species, glucuronic acid was found attached to galactose [21][22][23][24] to form a non-sulphated form of the so-called HNK-1 epitope, while in one filarial nematode (Dirofilaria immitis) N-acetylgalactosamine residues are glucuronylated [25]. Thereby, it is interesting that D. immitis is transmitted via mosquitoes, but it is unknown whether the 'common' Nglycan modification with glucuronic acid is relevant to the parasite's lifecycle.…”

“…2 molluscs (with aminoethylphosphonate; also observed with pyruvate and phosphoethanolamine modifications), (viii) a glycosylphosphatidylinositol (GPI) anchor from the Trypanosoma cruzi NETNES protein, (ix) a mucintype O-glycan from wasp, a mucin-type and a Notch-type from Volvarina rubella, a glycosaminoglycan-like glycan from Oesophagostomum dentatum and (x) phospho-linked sugars from either Dictyostelium discoideum or Entamoeba histolytica. AEP, aminoethylphosphonate; MeAEP, methylaminoethylphosphonate; P, phosphate; PC, phosphorylcholine; PE, phosphoethanolamine; PMe, methylphosphate; Pyr, pyruvate Echinococcus granulosus [25,[110][111][112][113][114][115][116][117][118][119]; the underlying structures of the phosphorylcholine-modified Nglycans 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 [124] and on O-glycans with the composition HexNAc 2-3 HexA 1 PC 1 released from a recombinant protein expressed in lepidopteran (Sf9) cells [30].…”

Macrobrachium rosenbergii nodavirus virus‐like particles attach to fucosylated glycans in the gills of the giant freshwater prawn

“…The single‐branched HexNAc(Fuc)HexNAc extension of the core was observed in the N ‐glycomes of the lepidopteran T . ni and Lymantria dispar larvae (Stanton et al, 2017), the ruminant‐afflicting nematode Haemonchus contortus (Paschinger & Wilson, 2015), in a sulfated form of the dipteran Anopheles gambiae (Kurz et al, 2015; Kurz, King, Dinglasan, Paschinger, & Wilson, 2016), the egg/miracidia stages of helminthic parasite Schistosoma mansoni (Smit et al, 2015) and in the nematode Dirofilaria immitis (Martini et al, 2019). The HexNAc(Fuc)HexNAc extension bound with phosphoethanolamine (PE) was also found in the N ‐glycomes of the male larvae and venom of Apis mellifera , and can be further extended with βGal or α/βGalNAc (Hykollari, Malzl, Stanton, Eckmair, & Paschinger, 2019; Kubelka et al, 1993; Rendić, Wilson, & Paschinger, 2008).…”

“…mellifera (Hykollari et al, 2019; Rendić et al, 2008) and in D . immitis (Martini et al, 2019), respectively.…”