RESUMO
A multitude of animal species undergo prolonged fasting events at regularly occurring life history stages. During such periods of food deprivation, individuals need to suppress their appetite. The satiety signalling gut hormone ghrelin has received much attention in this context in studies looking at mammalian systems. In wild birds, however, knowledge on the ghrelin system and its role during extended fasts is still scarce. In this study, we collected plasma samples for measurements of circulating ghrelin concentrations from adult southern rockhopper penguins (Eudyptes chrysocome chrysocome) during the three to four week-long moult-fast that they repeat annually to replace their feathers. We further sampled chicks before and after feeding bouts and non-moulting adults. Circulating ghrelin levels did not differ significantly between fed and unfed chicks but chicks had significantly lower plasma ghrelin levels compared to adults. Furthermore, penguins in late moult (i.e. individuals at the end of the prolonged fasting bout) had higher ghrelin levels compared to non-moulting adults. Our results show elevated levels of circulating ghrelin during moult and generally lower levels of ghrelin in chicks than in adults regardless of feeding state. Given the scarcity or absence of knowledge on the function of ghrelin in seabirds and in fasting birds in general, our results add greatly to our understanding of the avian ghrelin system.
Assuntos
Grelina , Muda , Spheniscidae , Animais , Grelina/sangue , Spheniscidae/sangue , Spheniscidae/fisiologia , Muda/fisiologia , Masculino , Jejum , Feminino , Privação de Alimentos/fisiologia , Apetite/fisiologia , Comportamento Alimentar/fisiologiaRESUMO
We found major seasonal changes of polyunsaturated fatty acids (PUFAs) in muscular phospholipids (PL) in a large non-hibernating mammal, the red deer (Cervus elaphus). Dietary supply of essential linoleic acid (LA) and α-linolenic acid (ALA) had no, or only weak influence, respectively. We further found correlations of PL PUFA concentrations with the activity of key metabolic enzymes, independent of higher winter expression. Activity of the sarcoplasmic reticulum (SR) Ca++-ATPase increased with SR PL concentrations of n-6 PUFA, and of cytochrome c oxidase and citrate synthase, indicators of ATP-production, with concentrations of eicosapentaenoic acid in mitochondrial PL. All detected cyclic molecular changes were controlled by photoperiod and are likely of general relevance for mammals living in seasonal environments, including humans. During winter, these changes at the molecular level presumably compensate for Arrhenius effects in the colder peripheral body parts and thus enable a thrifty life at lower body temperature.
RESUMO
Urbanisation is one of the biggest environmental challenges of our time, yet we still lack an integrative understanding of how cities affect behaviour, physiology and parasite susceptibility of free-living organisms. In this study, we focus on carotenoids, strictly dietary micronutrients that can either be used as yellow-red pigments, for integument colouration (signalling function), or as antioxidants, to strengthen the immune system (physiological function) in an urban predator, the Eurasian kestrel (Falco tinnunculus). Kestrels are specialised vole hunters but shift to avian prey in cities where diurnal rodents are not sufficiently available. This different foraging strategy might determine the quantity of carotenoids available. We measured integument colouration, circulating carotenoids in the blood and ectoparasite burden in kestrels along an urban gradient. Our results showed that nestlings that were raised in more urbanised areas displayed, unrelated to their ectoparasite burden, a paler integument colouration. Paler colours were furthermore associated with a lower concentration of circulating carotenoids. These findings support the hypothesis that the entire urban food web is carotenoid deprived and only prey of low quality with low carotenoid content is available (e.g. fewer carotenoids in urban trees, insects, small birds and finally kestrels). The alternative hypothesis that nestlings allocate carotenoids to reduce physiological stress and/or to cope with parasites rather than invest into colouration could not be supported. Our study adds to existing evidence that urban stressors negatively affect carotenoid production in urban areas, a deficiency that dissipate into higher trophic levels.
Assuntos
Aves Predatórias , Urbanização , Animais , Carotenoides , Pele , Cadeia AlimentarRESUMO
The approaches for analysis of N-glycans have radically altered in the last 20 years or so. Due to increased sensitivity, mass spectrometry has become the predominant method in modern glycomics. Here, we summarize recent studies showing that the improved resolution and detection by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has contributed greatly to the discovery of a large range of anionic and zwitterionic N-glycan structures across the different kingdoms of life, whereby MALDI-TOF MS in negative mode is less widely performed than in positive mode. However, its use enables the detection of key fragments indicative of certain sugar modifications such as sulfate, (methyl) phosphate, phosphoethanolamine, (methyl)aminoethylphosphonate, glucuronic, and sialic acid, thereby enabling certain isobaric glycan variations to be distinguished. As we also discuss in this review, complementary approaches such as negative-mode electrospray ionization-MS/MS, Fourier-transform ion cyclotron resonance MS, and ion mobility MS yield, respectively, cross-linkage fragments, high accuracy masses, and isomeric information, thus adding other components to complete the jigsaw puzzle when defining unusual glycan modifications from lower organisms.
Assuntos
Ácido N-Acetilneuramínico , Espectrometria de Massas em Tandem , Animais , Invertebrados/química , Fosfatos , Polissacarídeos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Açúcares , SulfatosRESUMO
Glycosylation is a highly diverse set of co- and posttranslational modifications of proteins. For mammalian glycoproteins, glycosylation is often site-, tissue-, and species-specific and diversified by microheterogeneity. Multitudinous biochemical, cellular, physiological, and organismic effects of their glycans have been revealed, either intrinsic to the carrier proteins or mediated by endogenous reader proteins with carbohydrate recognition domains. Furthermore, glycans frequently form the first line of access by or defense from foreign invaders, and new roles for nucleocytoplasmic glycosylation are blossoming. We now know enough to conclude that the same general principles apply in invertebrate animals and unicellular eukaryotes-different branches of which spawned the plants or fungi and animals. The two major driving forces for exploring the glycomes of invertebrates and protists are (i) to understand the biochemical basis of glycan-driven biology in these organisms, especially of pathogens, and (ii) to uncover the evolutionary relationships between glycans, their biosynthetic enzyme genes, and biological functions for new glycobiological insights. With an emphasis on emerging areas of protist glycobiology, here we offer an overview of glycan diversity and evolution, to promote future access to this treasure trove of glycobiological processes.
Assuntos
Glicoproteínas/metabolismo , Polissacarídeos/metabolismo , Animais , Evolução Biológica , Glicômica , Glicosilação , Humanos , Processamento de Proteína Pós-Traducional , ProteômicaRESUMO
Zwitterionic modifications of glycans, such as phosphorylcholine and phosphoethanolamine, are known from a range of prokaryotic and eukaryotic species and are recognized by mammalian antibodies and pentraxins; however, defined saccharide ligands modified with these zwitterionic moieties for high-throughput studies are lacking. In this study, we prepared and tested example mono- and disaccharides 6-substituted with either phosphorylcholine or phosphoethanolamine as bovine serum albumin neoglycoconjugates or printed in a microarray format for subsequent assessment of their binding to lectins, pentraxins, and antibodies. C-Reactive protein and anti-phosphorylcholine antibodies bound specifically to ligands with phosphorylcholine, but recognition by concanavalin A was abolished or decreased as compared with that to the corresponding nonzwitterionic compounds. Furthermore, in array format, the phosphorylcholine-modified ligands were recognized by IgG and IgM in sera of either non-infected or nematode-infected dogs and pigs. Thereby, these new compounds are defined ligands which allow the assessment of glycan-bound phosphorylcholine as a target of both the innate and adaptive immune systems in mammals.
Assuntos
Proteína C-Reativa/metabolismo , Glicoconjugados/metabolismo , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Componente Amiloide P Sérico/metabolismo , Animais , Ascaríase/diagnóstico , Ascaríase/veterinária , Ascaris , Sequência de Carboidratos , Bovinos , Dirofilaria immitis , Dirofilariose/diagnóstico , Cães , Etanolaminas/síntese química , Etanolaminas/imunologia , Etanolaminas/metabolismo , Glicoconjugados/síntese química , Glicoconjugados/imunologia , Humanos , Ligantes , Fosforilcolina/análogos & derivados , Fosforilcolina/imunologia , Fosforilcolina/metabolismo , Ligação Proteica , Soroalbumina Bovina/química , SuínosRESUMO
BACKGROUND: Previous glycophylogenetic comparisons of dipteran and lepidopteran species revealed variations in the anionic and zwitterionic modifications of their N-glycans; therefore, we wished to explore whether species- and order-specific glycomic variations would extend to the hymenoptera, which include the honeybee Apis mellifera, an agriculturally- and allergologically-significant social species. METHODS: In this study, we employed an off-line liquid chromatography/mass spectrometry approach, in combination with enzymatic and chemical treatments, to analyse the N-glycans of male honeybee larvae and honeybee venom in order to facilitate definition of isomeric structures. RESULTS: The neutral larval N-glycome was dominated by oligomannosidic and paucimannosidic structures, while the neutral venom N-glycome displayed more processed hybrid and complex forms with antennal N-acetylgalactosamine, galactose and fucose residues including Lewis-like epitopes; the anionic pools from both larvae and venom contained a wide variety of glucuronylated, sulphated and phosphoethanolamine-modified N-glycans with up to three antennae. In comparison to honeybee royal jelly, there were more fucosylated and fewer Man4/5-based hybrid glycans in the larvae and venom samples as well as contrasting antennal lengths. CONCLUSIONS: Combining the current data on venom and larvae with that we previously published on royal jelly, a total honeybee N-glycomic repertoire of some 150 compositions can be proposed in addition to the 20 previously identified on specific venom glycoproteins. SIGNIFICANCE: Our data are indicative of tissue-specific modification of the core and antennal regions of N-glycans in Apis mellifera and reinforce the concept that insects are capable of extensive processing to result in rather complex anionic oligosaccharide structures.
Assuntos
Venenos de Abelha/metabolismo , Abelhas/metabolismo , Proteínas de Insetos/metabolismo , Animais , Glicosilação , Masculino , Especificidade de ÓrgãosRESUMO
We regret to inform of an incomplete affiliation in this article.
RESUMO
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.
Assuntos
Dirofilaria immitis/imunologia , Dirofilariose/imunologia , Glicômica/métodos , Interações Hospedeiro-Parasita/imunologia , Polissacarídeos/imunologia , Animais , Proteína C-Reativa/imunologia , Proteína C-Reativa/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Complemento C1q/imunologia , Complemento C1q/metabolismo , Dirofilaria immitis/química , Dirofilariose/parasitologia , Cães , Feminino , Glicosilação , Vigilância Imunológica/imunologia , Masculino , Polissacarídeos/química , Polissacarídeos/metabolismo , Ligação Proteica , Espectrometria de Massas em Tandem/métodosRESUMO
N-Glycans are posttranslational modifications of proteins attached to the amide side chains of asparagine residues, with possible heterogeneity due to different structures being possible at the same glycosylation site. In contrast to the mammalian systems, invertebrate N-glycosylation presents a challenge in analysis as there exist unfamiliar epitopes and a high degree of structural and isomeric variation between different species. A simple analytical approach to analyze N-glycans on specific glycoproteins is presented, which involves a combination of tryptic peptide mass spectrometry and "off-line" RP-HPLC MALDI-TOF MS/MS complemented by blotting to recognize specific epitopes. An additional N-glycan enrichment and labeling step can facilitate the analysis of single structures and even provide isomeric separation of N-glycans from specific proteins.
Assuntos
Glicoproteínas , Proteoma , Proteômica , Animais , Western Blotting , Eletroforese em Gel de Poliacrilamida , Mapeamento de Epitopos , Epitopos/química , Epitopos/imunologia , Epitopos/metabolismo , Glicoproteínas/metabolismo , Invertebrados , Peptídeos/metabolismo , Polissacarídeos/química , Proteólise , Proteômica/métodos , Especificidade da Espécie , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Royal jelly has received attention because of its necessity for the development of queen honeybees as well as claims of benefits on human health; this product of the hypopharyngeal glands of worker bees contains a large number of proteins, some of which have been claimed to have various biological effects only in their glycosylated state. However, although there have been glycomic and glycoproteomic analyses in the past, none of the glycan structures previously defined would appear to have potential to trigger specific biological functions. In the current study, whole royal jelly as well as single protein bands were subject to off-line LC-MALDI-TOF MS glycomic analyses, complemented by permethylation, Western blotting and arraying data. Similarly to recent in-depth studies on other insect species, previously overlooked glucuronic acid termini, sulfation of mannose residues and core ß-mannosylation of the N-glycans were found; additionally, a relatively rare zwitterionic modification with phosphoethanolamine is present, in contrast to the phosphorylcholine occurring in lepidopteran species. Indicative of tissue-specific remodelling of glycans in the Golgi apparatus of hypopharyngeal gland cells, only a low amount of fucosylated or paucimannosidic glycans were detected as compared with other insect samples or even bee venom. The unusual modifications of hybrid and multiantennary structures defined here may not only have a physiological role in honeybee development, but represent epitopes recognized by pentraxins with roles in animal innate immunity.
Assuntos
Ácidos Graxos/química , Glicoproteínas/metabolismo , Polissacarídeos/química , Polissacarídeos/isolamento & purificação , Animais , Ânions , Bovinos , Fracionamento Químico , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Etanolaminas/metabolismo , Fucose/metabolismo , Ácido Glucurônico/metabolismo , Glicosídeo Hidrolases/metabolismo , Glicosilação , Isomerismo , Manose/metabolismo , Proteoma/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Sulfatos/metabolismo , Espectrometria de Massas em TandemRESUMO
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; IIß 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.
Assuntos
Dioclea/química , Lectinas de Plantas/imunologia , Especificidade de Anticorpos , Epitopos/química , Epitopos/imunologia , Hemaglutinação , Humanos , Lectinas de Plantas/química , Polissacarídeos/química , Polissacarídeos/imunologiaRESUMO
The unusual nature of the N-glycans of the cellular slime mould Dictyostelium discoideum has been revealed by a number of studies, primarily based on examination of radiolabeled glycopeptides but more recently also by MS. The complexity of the N-glycomes of even glycosylation mutants is compounded by the occurrence of anionic modifications, which also present an analytical challenge. In this study, we have employed hydrophilic interaction anion exchange (HIAX) HPLC in combination with MALDI-TOF MS/MS to explore the anionic N-glycome of the M31 (modA) strain, which lacks endoplasmic reticulum α-glucosidase II, an enzyme conserved in most eukaryotes including Homo sapiens. Prefractionation with HIAX chromatography enabled the identification of N-glycans with unusual oligo-α1,2-mannose extensions as well as others with up to four anionic modifications. Due to the use of hydrofluoric acid treatment, we were able to discriminate isobaric glycans differing in the presence of sulphate or phosphate on intersected structures as opposed to those carrying GlcNAc-phosphodiesters. The latter represent biosynthetic intermediates during the pathway leading to formation of the methylphosphorylated mannose epitope, which may have a similar function in intracellular targeting of hydrolases as the mannose-6-phosphate modification of lysosomal enzymes in mammals. In conclusion, HIAX in combination with MS is a highly sensitive approach for both fine separation and definition of neutral and anionic N-glycan structures.
Assuntos
Cromatografia por Troca Iônica/métodos , Dictyostelium/química , Glicômica/métodos , Polissacarídeos/análise , Polissacarídeos/química , Cromatografia Líquida de Alta Pressão/métodos , Dictyostelium/metabolismo , Hexoses/análise , Hexoses/química , Interações Hidrofóbicas e Hidrofílicas , Manose/análise , Manose/química , Fosfatos/análise , Fosfatos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Sulfatos/análise , Sulfatos/químicaRESUMO
BACKGROUND: Insects are significant to the environment, agriculture, health and biotechnology. Many of these aspects display some relationship to glycosylation, e.g., in case of pathogen binding or production of humanised antibodies; for a long time, it has been considered that insect N-glycosylation potentials are rather similar and simple, but as more species are glycomically analysed in depth, it is becoming obvious that there is indeed a large structural diversity and interspecies variability. METHODS: Using an off-line LC-MALDI-TOF MS approach, we have analysed the N-glycomes of two lepidopteran species (the cabbage looper Trichoplusia ni and the gypsy moth Lymantria dispar) as well as of the commonly-used T. ni High Five cell line. RESULTS: We detected not only sulphated, glucuronylated, core difucosylated and Lewis-like antennal fucosylated structures, but also the zwitterion phosphorylcholine on antennal GlcNAc residues, a modification otherwise familiar from nematodes; in L. dispar, N-glycans with glycolipid-like antennae containing α-linked N-acetylgalactosamine were also revealed. CONCLUSION: The lepidopteran glycomes analysed not only display core α1,3-fucosylation, which is foreign to mammals, but also up to 5% anionic and/or zwitterionic glycans previously not found in these species. SIGNIFICANCE: The occurrence of anionic and zwitterionic glycans in the Lepidoptera data is not only of glycoanalytical and evolutionary interest, but is of biotechnological relevance as lepidopteran cell lines are potential factories for recombinant glycoprotein production.
Assuntos
Lepidópteros/metabolismo , Lepidópteros/fisiologia , Polissacarídeos/metabolismo , Animais , Linhagem Celular , Glicolipídeos , Glicoproteínas/metabolismo , Glicosilação , Mariposas/metabolismo , Mariposas/fisiologia , Fosforilcolina/metabolismo , Sulfatos/metabolismoRESUMO
N-glycans from invertebrates and protists have often unusual structures which present analytical challenges. Both core and antennal modifications can be quite different from the more familiar vertebrate glycan motifs; thereby, contrary to the concept that "simple" organisms have "simple" N-glycans, rather complex oligosaccharides structures, including zwitterionic and anionic ones, have been found in a range of species. Thus, to facilitate the optimized elucidation of the maximal possible range of structures, the analytical workflow for glycomics of these organisms should include sequential release and fractionation steps. Peptide:N-glycosidase F is sufficient to isolate N-glycans from fungi and some protists, but in most invertebrates core α1,3-fucose is present, so release of the glycans from glycopeptides with peptide:N-glycosidases A is required. Subsequent solid-phase extraction with graphitized carbon and reversed phase resins enables different classes of N-glycans to be separated prior to high-pressure liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Depending on the types and numbers of glycans present, either reversed- or normal-phase HPLC (or both in series) enable even single isomeric or isobaric structures to be separated prior to MALDI-TOF MS and MS/MS. The use of enzymatic or chemical treatments allows further insights to be gained, although some glycan modifications (especially methylation) are resistant. Using a battery of methods, sometimes up to 100 structures from a single organism can be assigned, a complexity which raises evolutionary questions regarding the function of these glycans.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Glicômica/métodos , Glicopeptídeos/química , Polissacarídeos/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Western Blotting/métodos , Glicopeptídeos/isolamento & purificação , Invertebrados , Polissacarídeos/isolamento & purificação , Extração em Fase Sólida/métodosRESUMO
N-glycosylation is an essential set of post-translational modifications of proteins; in the case of filamentous fungi, N-glycans are present on a range of secreted and cell wall proteins. In this study, we have compared the glycans released by peptide/N-glycosidase F from proteolysed cell pellets of three Penicillium species (P. dierckxii, P. nordicum and P. verrucosum that all belong to the Eurotiomycetes). Although the major structures are all within the range Hex(5-11)HexNAc(2) as shown by mass spectrometry, variations in reversed-phase chromatograms and MS/MS fragmentation patterns are indicative of differences in the actual structure. Hydrofluoric acid and mannosidase treatments revealed that the oligomannosidic glycans were not only in part modified with phosphoethanolamine residues and outer chain och1-dependent mannosylation, but that bisecting galactofuranose was present in a species-dependent manner. These data are the first to specifically show the modification of N-glycans in fungi with zwitterionic moieties. Furthermore, our results indicate that mere mass spectrometric screening is insufficient to reveal the subtly complex nature of N-glycosylation even within a single fungal genus.
Assuntos
Glicômica/métodos , Manose/metabolismo , Oligossacarídeos/metabolismo , Penicillium/metabolismo , Polissacarídeos/metabolismo , Cromatografia de Fase Reversa , Etanolaminas/metabolismo , Glicosilação , Ácido Fluorídrico/metabolismo , Manosidases/metabolismo , Penicillium/classificação , Processamento de Proteína Pós-Traducional , Especificidade da Espécie , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em TandemRESUMO
In this study, we have performed the first mass spectrometric analysis of N-glycans of the M31 mutant strain of the cellular slime mould Dictyostelium discoideum, previously shown to have a defect in glucosidase II. Together with glucosidase I, this enzyme mediates part of the initial processing of N-glycans; defects in either glucosidase are associated with human diseases and result in an accumulation of incorrectly processed oligosaccharides which are not, or only poor, substrates for a range of downstream enzymes. To examine the effect of the glucosidase II mutation in Dictyostelium, we employed off-line LC-MALDI-TOF MS in combination with chemical and enzymatic treatments and MS/MS to analyze the neutral and anionic N-glycans of the mutant as compared to the wild type. The major neutral species were, as expected, of the composition Hex10-11 HexNAc2-3 with one or two terminal glucose residues. Consistent with the block in processing of neutral N-glycans caused by the absence of glucosidase II, fucose was apparently absent from the N-glycans and bisecting N-acetylglucosamine was rare. The major anionic oligosaccharides were sulfated and/or methylphosphorylated forms of Hex8-11 HexNAc2-3 , many of which surprisingly lacked glucose residues entirely. As anionic N-glycans are considered to be mostly associated with lysosomal enzymes in Dictyostelium, we hypothesise that glycosidases present in the acidic compartments may act on the oligosaccharides attached to such slime mould proteins. Furthermore, our chosen analytical approach enabled us, via observation of diagnostic negative-mode MS/MS fragments, to determine the fine structure of the methylphosphorylated and sulfated N-glycans of the M31 glucosidase mutant in their native state.
Assuntos
Dictyostelium/genética , Glicômica/métodos , Polissacarídeos/análise , Polissacarídeos/química , Proteínas de Protozoários/genética , alfa-Glucosidases/genética , Sequência de Aminoácidos , Cromatografia Líquida , Dictyostelium/química , Dictyostelium/metabolismo , Espectrometria de Massas , Dados de Sequência Molecular , Mutação , Polissacarídeos/metabolismo , Alinhamento de SequênciaRESUMO
The eastern oyster (Crassostrea virginica) has become a useful model system for glycan-dependent host-parasite interactions due to the hijacking of the oyster galectin CvGal1 for host entry by the protozoan parasite Perkinsus marinus, the causative agent of Dermo disease. In this study, we examined the N-glycans of both the hemocytes, which via CvGal1 are the target of the parasite, and the plasma of the oyster. In combination with HPLC fractionation, exoglycosidase digestion, and fragmentation of the glycans, mass spectrometry revealed that the major N-glycans of plasma are simple hybrid structures, sometimes methylated and core α1,6-fucosylated, with terminal ß1,3-linked galactose; a remarkable high degree of sulfation of such glycans was observed. Hemocytes express a larger range of glycans, including core-difucosylated paucimannosidic forms, whereas bi- and triantennary glycans were found in both sources, including structures carrying sulfated and methylated variants of the histo-blood group A epitope. The primary features of the oyster whole hemocyte N-glycome were also found in dominin, the major plasma glycoprotein, which had also been identified as a CvGal1 glycoprotein ligand associated with hemocytes. The occurrence of terminal blood group moieties on oyster dominin and on hemocyte surfaces can account in part for their affinity for the endogenous CvGal1.
Assuntos
Sistema ABO de Grupos Sanguíneos/metabolismo , Proteínas Sanguíneas/metabolismo , Crassostrea/metabolismo , Galectinas/metabolismo , Hemócitos/metabolismo , Polissacarídeos/metabolismo , Sistema ABO de Grupos Sanguíneos/química , Alveolados/fisiologia , Animais , Proteínas Sanguíneas/química , Crassostrea/química , Crassostrea/parasitologia , Epitopos/química , Epitopos/metabolismo , Galectinas/química , Hemócitos/química , Hemócitos/parasitologia , Interações Hospedeiro-Parasita/fisiologia , Polissacarídeos/químicaRESUMO
N-glycans modify the great majority of all secreted and plasma membrane proteins, which themselves constitute one-third to one-half of the proteome. The ultimate definition of the glycoproteome would be the identification of all the N-glycans attached to all the modified asparaginyl sites of all the proteins, but glycosylation heterogeneity makes this an unachievable goal. However, mass spectrometry in combination with other methods does have the power to deeply mine the N-glycome of Dictyostelium, and characterize glycan profiles at individual sites of glycoproteins. Recent studies from our laboratories using mass spectrometry-based methods have confirmed basic precepts of the N-glycome based on prior classical methods using radiotracer methods, and have extended the scope of glycan diversity and the distribution of glycan types across specific glycoprotein attachment sites. The protocols described here simplify studies of the N-glycome and -glycoproteome, which should prove useful for interpreting mutant phenotypes, conducting interstrain and interspecies comparisons, and investigating glycan functions in glycoproteins of interest.
Assuntos
Dictyostelium/metabolismo , Glicoproteínas/isolamento & purificação , Polissacarídeos/isolamento & purificação , Proteômica/métodos , Proteínas de Protozoários/isolamento & purificação , Sequência de Carboidratos , Cromatografia Líquida de Alta Pressão , Glicoproteínas/química , Glicoproteínas/metabolismo , Glicosilação , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/química , Polissacarídeos/química , Polissacarídeos/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em TandemRESUMO
The HL241 mutant strain of the cellular slime mold Dictyostelium discoideum is a potential model for human congenital disorder of glycosylation type IL (ALG9-CDG) and has been previously predicted to possess a lower degree of modification of its N-glycans with anionic moieties than the parental wild-type. In this study, we first showed that this strain has a premature stop codon in its alg9 mannosyltransferase gene compatible with the occurrence of truncated N-glycans. These were subject to an optimized analytical workflow, considering that the mass spectrometry of acidic glycans often presents challenges due to neutral loss and suppression effects. Therefore, the protein-bound N-glycans were first fractionated, after serial enzymatic release, by solid phase extraction. Then primarily single glycan species were isolated by mixed hydrophilic-interaction/anion-exchange or reversed-phase HPLC and analyzed using chemical and enzymatic treatments and MS/MS. We show that protein-linked N-glycans of the mutant are of reduced size as compared to those of wild-type AX3, but still contain core α1,3-fucose, intersecting N-acetylglucosamine, bisecting N-acetylglucosamine, methylphosphate, phosphate, and sulfate residues. We observe that a single N-glycan can carry up to four of these six possible modifications. Due to the improved analytical procedures, we reveal fuller details regarding the N-glycomic potential of this fascinating model organism.