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During Leishmania transmission sand flies inoculate parasites and saliva into the skin of vertebrates. Saliva has anti-haemostatic and anti-inflammatory activities that evolved to facilitate bloodfeeding, but also modulate the host's immune responses. Sand fly salivary proteins have been extensively studied, but the nature and biological roles of protein-linked glycans remain overlooked. Here, we characterised the profile of N-glycans from the salivary glycoproteins of Lutzomyia longipalpis, vector of visceral leishmaniasis in the Americas. In silico predictions suggest half of Lu. longipalpis salivary proteins may be N-glycosylated. SDS-PAGE coupled to LC–MS analysis of sand fly saliva, before and after enzymatic deglycosylation, revealed several candidate glycoproteins. To determine the diversity of N-glycan structures in sand fly saliva, enzymatically released sugars were fluorescently tagged and analysed by HPLC, combined with highly sensitive LC–MS/MS, MALDI-TOF–MS, and exoglycosidase treatments. We found that the N-glycan composition of Lu. longipalpis saliva mostly consists of oligomannose sugars, with Man5GlcNAc2 being the most abundant, and a few hybrid-type species. Interestingly, some glycans appear modified with a group of 144 Da, whose identity has yet to be confirmed. Our work presents the first detailed structural analysis of sand fly salivary glycans. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The carbohydrate specificities of Dioclea grandiflora lectins DGL-I1 and DGL-II, and Galactia lindenii lectin II (GLL-II) were explored by use of remodeled glycoproteins as well as by the lectin hemagglutinating activity against erythrocytes from various species with different glycomic profiles. The three lectins exhibited differences in glycan binding specificity but also showed overlapping recognition of some glycotopes (i.e. Tα glycotope for the three lectins; IIβ glycotope for DGL-II and GLL-II lectins); in many cases the interaction with distinct glycotopes was influenced by the structural context, i.e., by the neighbouring sugar residues. Our data complement and expand the existing knowledge about the binding specificity of these three Diocleae lectins, and taken together with results of previous studies, allow us to suggest a functional map of the carbohydrate recognition which illustrate the impact of modification of basic glycotopes enhancing, permiting, or inhibiting their recognition by each lectin. [ABSTRACT FROM AUTHOR]

Modern glycan analysis is primarily based on mass spectrometry, where instruments based on electrospray or matrix-assisted laser desorption ionization are currently the most frequently used. In the present study, electrospray ionization (ESI) coupled with a high-resolution Fourier transform mass spectrometer (LTQ Orbitrap) and matrix-assisted laser desorption/ionization (MALDI) coupled with a time-of-flight (TOF/TOF) detector were used to analyze two N-glycan standards with intact free reducing ends (disialo biantennary and asialo triantennary) and representative PA-labeled human serum N-glycan structures isolated by hydrophilic interaction anion-exchange chromatography (HIAX), confirmed by 1H NMR analysis and consequently compared with the ProteinScape Glycome database. Different combinations of ion sources with fragmentation devices results in various fragmentation patterns and adducts. Also, the effect of sample derivatization on the acquired signals is discussed. Compared to the MALDI technique, free glycans did not lose labile sialic acids easily in the ESI source. On the other hand, fluorescent PA-labeling leads to improved core fragmentation and signal intensities; linkage-specific ethyl esterification leads to reduced adduct and fragment formation and enhanced stability of sialic acids in the MALDI ion source. Thereby, both methods have their advantages and disadvantages in terms of detection, fragmentation and robustness. [ABSTRACT FROM AUTHOR]

We regret to inform of an incomplete affiliation in this article. [ABSTRACT FROM AUTHOR]

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. The glycome of parasites can have immunomodulatory properties or help to avoid immune surveillance, but details are unknown. Here, Martini et al. characterize the N-glycome of the canine heartworm, reveal an unprecedented complexity, particularly in anionic N-glycans, and determine recognition by components of the immune system. [ABSTRACT FROM AUTHOR]