RESUMEN
Glycans constitute the most complicated post-translational modification, modulating protein activity in health and disease. However, structural annotation from tandem mass spectrometry (MS/MS) data is a bottleneck in glycomics, preventing high-throughput endeavors and relegating glycomics to a few experts. Trained on a newly curated set of 500,000 annotated MS/MS spectra, here we present CandyCrunch, a dilated residual neural network predicting glycan structure from raw liquid chromatography-MS/MS data in seconds (top-1 accuracy: 90.3%). We developed an open-access Python-based workflow of raw data conversion and prediction, followed by automated curation and fragment annotation, with predictions recapitulating and extending expert annotation. We demonstrate that this can be used for de novo annotation, diagnostic fragment identification and high-throughput glycomics. For maximum impact, this entire pipeline is tightly interlaced with our glycowork platform and can be easily tested at https://colab.research.google.com/github/BojarLab/CandyCrunch/blob/main/CandyCrunch.ipynb . We envision CandyCrunch to democratize structural glycomics and the elucidation of biological roles of glycans.
Asunto(s)
Aprendizaje Profundo , Polisacáridos , Espectrometría de Masas en Tándem , Espectrometría de Masas en Tándem/métodos , Polisacáridos/química , Polisacáridos/análisis , Glicómica/métodos , Humanos , Cromatografía Liquida/métodos , Programas Informáticos , Flujo de Trabajo , Redes Neurales de la ComputaciónRESUMEN
Humans have co-evolved with a dense community of microbial symbionts that inhabit the lower intestine. In the colon, secreted mucus creates a barrier that separates these microorganisms from the intestinal epithelium1. Some gut bacteria are able to utilize mucin glycoproteins, the main mucus component, as a nutrient source. However, it remains unclear which bacterial enzymes initiate degradation of the complex O-glycans found in mucins. In the distal colon, these glycans are heavily sulfated, but specific sulfatases that are active on colonic mucins have not been identified. Here we show that sulfatases are essential to the utilization of distal colonic mucin O-glycans by the human gut symbiont Bacteroides thetaiotaomicron. We characterized the activity of 12 different sulfatases produced by this species, showing that they are collectively active on all known sulfate linkages in O-glycans. Crystal structures of three enzymes provide mechanistic insight into the molecular basis of substrate specificity. Unexpectedly, we found that a single sulfatase is essential for utilization of sulfated O-glycans in vitro and also has a major role in vivo. Our results provide insight into the mechanisms of mucin degradation by a prominent group of gut bacteria, an important process for both normal microbial gut colonization2 and diseases such as inflammatory bowel disease3.
Asunto(s)
Bacteroides/enzimología , Colon/metabolismo , Colon/microbiología , Microbioma Gastrointestinal , Mucinas/metabolismo , Sulfatasas/metabolismo , Acetilgalactosamina/química , Acetilgalactosamina/metabolismo , Animales , Colon/química , Cristalografía por Rayos X , Femenino , Galactosa/metabolismo , Humanos , Masculino , Ratones , Modelos Moleculares , Especificidad por Sustrato , Sulfatasas/químicaRESUMEN
The modern pattern of the Asian monsoon is thought to have formed around the Oligocene/Miocene transition and is generally attributed to Himalaya-Tibetan Plateau (H-TP) uplift. However, the timing of the ancient Asian monsoon over the TP and its response to astronomical forcing and TP uplift remains poorly known because of the paucity of well-dated high-resolution geological records from the TP interior. Here, we present a precession-scale cyclostratigraphic sedimentary section of 27.32 to 23.24 million years ago (Ma) during the late Oligocene epoch from the Nima Basin to show that the South Asian monsoon (SAM) had already advanced to the central TP (32°N) at least by 27.3 Ma, which is indicated by cyclic arid-humid fluctuations based on environmental magnetism proxies. A shift of lithology and astronomically orbital periods and amplified amplitude of proxy measurements as well as a hydroclimate transition around 25.8 Ma suggest that the SAM intensified at ~25.8 Ma and that the TP reached a paleoelevation threshold for enhancing the coupling between the uplifted plateau and the SAM. Orbital short eccentricity-paced precipitation variability is argued to be mainly driven by orbital eccentricity-modulated low-latitude summer insolation rather than glacial-interglacial Antarctic ice sheet fluctuations. The monsoon data from the TP interior provide key evidence to link the greatly enhanced tropical SAM at 25.8 Ma with TP uplift rather than global climate change and suggest that SAM's northward expansion to the boreal subtropics was dominated by a combination of tectonic and astronomical forcing at multiple timescales in the late Oligocene epoch.
RESUMEN
Breast milk is abundant with functionalized milk oligosaccharides (MOs) to nourish and protect the neonate. Yet we lack a comprehensive understanding of the repertoire and evolution of MOs across Mammalia. We report â¼400 MO-species associations (>100 novel structures) from milk glycomics of nine mostly understudied species: alpaca, beluga whale, black rhinoceros, bottlenose dolphin, impala, L'Hoest's monkey, pygmy hippopotamus, domestic sheep, and striped dolphin. This revealed the hitherto unknown existence of the LacdiNAc motif (GalNAcß1-4GlcNAc) in MOs of all species except alpaca, sheep, and striped dolphin, indicating the widespread occurrence of this potentially antimicrobial motif in MOs. We also characterize glucuronic acid-containing MOs in the milk of impala, dolphins, sheep, and rhinoceros, previously only reported in cows. We demonstrate that these GlcA-MOs exhibit potent immunomodulatory effects. Our study extends the number of known MOs by >15%. Combined with >1900 curated MO-species associations, we characterize MO motif distributions, presenting an exhaustive overview of MO biodiversity.
Asunto(s)
Antílopes , Camélidos del Nuevo Mundo , Delfines , Stenella , Humanos , Femenino , Recién Nacido , Animales , Bovinos , Ovinos , Leche Humana , OligosacáridosRESUMEN
Caenorhabditis elegans is a frequently employed genetic model organism and has been the object of a wide range of developmental, genetic, proteomic, and glycomic studies. Here, using an off-line MALDI-TOF-MS approach, we have analyzed the N-glycans of mixed embryos and liquid- or plate-grown L4 larvae. Of the over 200 different annotatable N-glycan structures, variations between the stages as well as the mode of cultivation were observed. While the embryonal N-glycome appears less complicated overall, the liquid- and plate-grown larvae differ especially in terms of methylation of bisecting fucose, α-galactosylation of mannose, and di-ß-galactosylation of core α1,6-fucose. Furthermore, we analyzed the O-glycans by LC-electrospray ionization-MS following ß-elimination; especially the embryonal O-glycomes included a set of phosphorylcholine-modified structures, previously not shown to exist in nematodes. However, the set of glycan structures cannot be clearly correlated with levels of glycosyltransferase transcripts in developmental RNA-Seq datasets, but there is an indication for coordinated expression of clusters of potential glycosylation-relevant genes. Thus, there are still questions to be answered in terms of how and why a simple nematode synthesizes such a diverse glycome.
Asunto(s)
Caenorhabditis , Animales , Caenorhabditis/metabolismo , Fucosa/metabolismo , Proteómica , Cromatografía Líquida de Alta Presión , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Caenorhabditis elegans/metabolismo , Polisacáridos/metabolismo , GlicómicaRESUMEN
Fasciola hepatica is a global helminth parasite of humans and their livestock. The invasive stage of the parasite, the newly excysted juvenile (NEJs), relies on glycosylated excreted-secreted (ES) products and surface/somatic molecules to interact with host cells and tissues and to evade the host's immune responses, such as disarming complement and shedding bound antibody. While -omics technologies have generated extensive databases of NEJs' proteins and their expression, detailed knowledge of the glycosylation of proteins is still lacking. Here, we employed glycan, glycopeptide, and proteomic analyses to determine the glycan profile of proteins within the NEJs' somatic (Som) and ES extracts. These analyses characterized 123 NEJ glycoproteins, 71 of which are secreted proteins, and allowed us to map 356 glycopeptides and their associated 1690 N-glycan and 37 O-glycan forms to their respective proteins. We discovered abundant micro-heterogeneity in the glycosylation of individual glycosites and between different sites of multi-glycosylated proteins. The global heterogeneity across NEJs' glycoproteome was refined to 53 N-glycan and 16 O-glycan structures, ranging from highly truncated paucimannosidic structures to complex glycans carrying multiple phosphorylcholine (PC) residues, and included various unassigned structures due to unique linkages, particularly in pentosylated O-glycans. Such exclusive glycans decorate some well-known secreted molecules involved in host invasion, including cathepsin B and L peptidases, and a variety of membrane-bound glycoproteins, suggesting that they participate in host interactions. Our findings show that F. hepatica NEJs generate exceptional protein variability via glycosylation, suggesting that their molecular portfolio that communicates with the host is far more complex than previously anticipated by transcriptomic and proteomic analyses. This study opens many avenues to understand the glycan biology of F. hepatica throughout its life-stages, as well as other helminth parasites, and allows us to probe the glycosylation of individual NEJs proteins in the search for innovative diagnostics and vaccines against fascioliasis.
Asunto(s)
Fasciola hepatica , Animales , Humanos , Fasciola hepatica/fisiología , Proteómica , Secretoma , Glicoproteínas/metabolismo , Polisacáridos/metabolismo , Glicoproteínas de Membrana/metabolismoRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is one of the most common causes of cancer-related deaths worldwide, accounting for 90% of primary pancreatic tumors with an average 5-year survival rate of less than 10%. PDAC exhibits aggressive biology, which, together with late detection, results in most PDAC patients presenting with unresectable, locally advanced, or metastatic disease. In-depth lipid profiling and screening of potential biomarkers currently appear to be a promising approach for early detection of PDAC or other cancers. Here, we isolated and characterized complex glycosphingolipids (GSL) from normal and tumor pancreatic tissues of patients with PDAC using a combination of TLC, chemical staining, carbohydrate-recognized ligand-binding assay, and LC/ESI-MS2. The major neutral GSL identified were GSL with the terminal blood groups A, B, H, Lea, Leb, Lex, Ley, P1, and PX2 determinants together with globo- (Gb3 and Gb4) and neolacto-series GSL (nLc4 and nLc6). We also revealed that the neutral GSL profiles and their relative amounts differ between normal and tumor tissues. Additionally, the normal and tumor pancreatic tissues differ in type 1/2 core chains. Sulfatides and GM3 gangliosides were the predominant acidic GSL along with the minor sialyl-nLc4/nLc6 and sialyl-Lea/Lex. The comprehensive analysis of GSL in human PDAC tissues extends the GSL coverage and provides an important platform for further studies of GSL alterations; therefore, it could contribute to the development of new biomarkers and therapeutic approaches.
Asunto(s)
Glicoesfingolípidos , Neoplasias Pancreáticas , Humanos , Cromatografía Liquida , Cromatografía en Capa Delgada , Gangliósidos/química , Glicoesfingolípidos/análisis , Glicoesfingolípidos/química , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/fisiopatología , Sulfoglicoesfingolípidos/química , Carcinoma Ductal Pancreático/diagnóstico , Carcinoma Ductal Pancreático/fisiopatología , Espectrometría de Masas en Tándem , Biomarcadores de Tumor/metabolismoRESUMEN
Simple organisms are often considered to have simple glycomes, but plentiful paucimannosidic and oligomannosidic glycans overshadow the less abundant N-glycans with highly variable core and antennal modifications; Caenorhabditis elegans is no exception. By use of optimized fractionation and assessing wildtype in comparison to mutant strains lacking either the HEX-4 or HEX-5 ß-N-acetylgalactosaminidases, we conclude that the model nematode has a total N-glycomic potential of 300 verified isomers. Three pools of glycans were analyzed for each strain: either PNGase F released and eluted from a reversed-phase C18 resin with either water or 15% methanol or PNGase Ar released. While the water-eluted fractions were dominated by typical paucimannosidic and oligomannosidic glycans and the PNGase Ar-released pools by glycans with various core modifications, the methanol-eluted fractions contained a huge range of phosphorylcholine-modified structures with up to three antennae, sometimes with four N-acetylhexosamine residues in series. There were no major differences between the C. elegans wildtype and hex-5 mutant strains, but the hex-4 mutant strains displayed altered sets of methanol-eluted and PNGase Ar-released pools. In keeping with the specificity of HEX-4, there were more glycans capped with N-acetylgalactosamine in the hex-4 mutants, as compared with isomeric chito-oligomer motifs in the wildtype. Considering that fluorescence microscopy showed that a HEX-4::enhanced GFP fusion protein colocalizes with a Golgi tracker, we conclude that HEX-4 plays a significant role in late-stage Golgi processing of N-glycans in C. elegans. Furthermore, finding more "parasite-like" structures in the model worm may facilitate discovery of glycan-processing enzymes occurring in other nematodes.
Asunto(s)
Caenorhabditis elegans , beta-N-Acetilhexosaminidasas , Animales , Acetilgalactosamina/metabolismo , beta-N-Acetilhexosaminidasas/metabolismo , Caenorhabditis elegans/metabolismo , Glicosilación , Hexosaminidasas/metabolismo , Metanol , Polisacáridos/metabolismoRESUMEN
Sulfated glycans are ubiquitous nutrient sources for microbial communities that have coevolved with eukaryotic hosts. Bacteria metabolize sulfated glycans by deploying carbohydrate sulfatases that remove sulfate esters. Despite the biological importance of sulfatases, the mechanisms underlying their ability to recognize their glycan substrate remain poorly understood. Here, we use structural biology to determine how sulfatases from the human gut microbiota recognize sulfated glycans. We reveal seven new carbohydrate sulfatase structures spanning four S1 sulfatase subfamilies. Structures of S1_16 and S1_46 represent novel structures of these subfamilies. Structures of S1_11 and S1_15 demonstrate how non-conserved regions of the protein drive specificity toward related but distinct glycan targets. Collectively, these data reveal that carbohydrate sulfatases are highly selective for the glycan component of their substrate. These data provide new approaches for probing sulfated glycan metabolism while revealing the roles carbohydrate sulfatases play in host glycan catabolism.
Asunto(s)
Microbioma Gastrointestinal , Sulfatasas , Bacterias/metabolismo , Humanos , Polisacáridos/química , Sulfatasas/química , Sulfatos/químicaRESUMEN
Helicobacter pylori colonizes the stomach of half of the human population. Most H. pylori are located in the mucus layer, which is mainly comprised by glycosylated mucins. Using mass spectrometry, we identified 631 glycans (whereof 145 were fully characterized and the remainder assigned as compositions) on mucins isolated from 14 Helicobacter spp.-infected and 14 Helicobacter spp.-noninfected stomachs. Only six identified glycans were common to all individuals, from a total of 60 to 189 glycans in each individual. An increased number of unique glycan structures together with an increased intraindividual diversity and larger interindividual variation were identified among O-glycans from Helicobacter spp.-infected stomachs compared with noninfected stomachs. H. pylori strain J99, which carries the blood group antigen-binding adhesin (BabA), the sialic acid-binding adhesin (SabA), and the LacdiNAc-binding adhesin, bound both to Lewis b (Leb)-positive and Leb-negative mucins. Among Leb-positive mucins, H. pylori J99 binding was higher to mucins from Helicobacter spp.-infected individuals than noninfected individuals. Statistical correlation analysis, binding experiments with J99 wt, and J99ΔbabAΔsabA and inhibition experiments using synthetic glycoconjugates demonstrated that the differences in H. pylori-binding ability among these four groups were governed by BabA-dependent binding to fucosylated structures. LacdiNAc levels were lower in mucins that bound to J99 lacking BabA and SabA than in mucins that did not, suggesting that LacdiNAc did not significantly contribute to the binding. We identified 24 O-glycans from Leb-negative mucins that correlated well with H. pylori binding whereof 23 contained α1,2-linked fucosylation. The large and diverse gastric glycan library identified, including structures that correlated with H. pylori binding, could be used to select glycodeterminants to experimentally investigate further for their importance in host-pathogen interactions and as candidates to develop glycan-based therapies.
Asunto(s)
Infecciones por Helicobacter , Helicobacter pylori , Humanos , Adhesinas Bacterianas/metabolismo , Adhesión Bacteriana , Mucinas Gástricas/metabolismo , Mucosa Gástrica/metabolismo , Helicobacter pylori/metabolismo , Polisacáridos/metabolismoRESUMEN
Changes in glycosphingolipid structures have been shown to occur during the development of several types of human cancers, generating cancer-specific carbohydrate structures that could be used as biomarkers for diagnosis and therapeutic targeting. In this study, we characterized nonacid glycosphingolipids isolated from a human gastric adenocarcinoma by mass spectrometry, enzymatic hydrolysis, and by binding with a battery of carbohydrate-recognizing ligands. We show that the majority of the complex nonacid glycosphingolipids had type 2 (Galß4GlcNAc) core chains (neolactotetraosylceramide, the Lex, H type 2, x2, and the P1 pentaosylceramides, and the Ley, A type 2, and neolacto hexaosylceramides). We also found glycosphingolipids with type 1 (Galß3GlcNAc) core (lactotetraosylceramide and the H type 1 pentaosylceramide) and globo (GalαGal) core chains (globotriaosylceramide and globotetraosylceramide). Interestingly, we characterized two complex glycosphingolipids as a P1 heptaosylceramide (Galα4Galß4GlcNAcß3Galß4GlcNAcß3Gal ß4Glcß1Cer) and a branched P1 decaosylceramide (Galα4Gal ß4GlcNAcß3(Galα4Galß4GlcNAcß6)Galß4GlcNAcß3Galß4Glc ß1Cer). These are novel glycosphingolipid structures and the first reported cases of complex glycosphingolipids larger than pentaosylceramide carrying the P1 trisaccharide. We propose that these P1 glycosphingolipids may represent potential biomarkers for the early diagnosis of gastric cancer.
Asunto(s)
Adenocarcinoma , Neoplasias Gástricas , Adenocarcinoma/diagnóstico , Biomarcadores , Carbohidratos , Glicoesfingolípidos/metabolismo , Humanos , Neoplasias Gástricas/diagnósticoRESUMEN
Despite sulfated O-linked glycans being abundant on ovarian cancer (OC) glycoproteins, their regulation during cancer development and involvement in cancer pathogenesis remain unexplored. We characterized O-glycans carrying sulfation on galactose residues and compared their expression with defined sulfotransferases regulated during OC development. Desialylated sulfated oligosaccharides were released from acidic glycoproteins in the cyst fluid from one patient with a benign serous cyst and one patient with serous OC. Oligosaccharides characterized by LC-MSn were identified as core 1 and core 2 O-glycans up to the size of decamers and with 1 to 4 sulfates linked to GlcNAc residues and to C-3 and/or C-6 of Gal. To study the specificity of the potential ovarian sulfotransferases involved, Gal3ST2 (Gal-3S)-, Gal3ST4 (Gal-3S)-, and CHST1 (Gal-6S)-encoding expression plasmids were transfected individually into CHO cells also expressing the P-selectin glycoprotein ligand-1/mouse immunoglobulin G2b (PSGL-1/mIg G2b) fusion protein and the human core 2 transferase (GCNT1). Characterization of the PSGL-1/mIg G2b O-glycans showed that Gal3ST2 preferentially sulfated Gal on the C-6 branch of core 2 structures and Gal3ST4 preferred Gal on the C-3 branch independently if core-1 or -2. CHST1 sulfated Gal residues on both the C-3 (core 1/2) and C-6 branches of core 2 structures. Using serous ovarian tissue micro array, Gal3ST2 was found to be decreased in tissue classified as malignant compared with tissues classified as benign or borderline, with the lowest expression in poorly differentiated malignant tissue. Neither Gal3ST4 nor CHST1 was differentially expressed in benign, borderline, or malignant tissue, and there was no correlation between expression level and differentiation stage. The data displays a complex sulfation pattern of O-glycans on OC glycoproteins and that aggressiveness of the cancer is associated with a decreased expression of the Gal3ST2 transferase.
Asunto(s)
Adenoma/metabolismo , Cistadenocarcinoma Seroso/metabolismo , Neoplasias Ováricas/metabolismo , Polisacáridos/metabolismo , Sulfotransferasas/metabolismo , Animales , Células CHO , Cricetulus , Femenino , Humanos , Mucinas/metabolismo , Sulfatos/metabolismo , Sulfotransferasas/genéticaRESUMEN
Alterations in protein glycosylation in colorectal cancer (CRC) have been extensively studied using cell lines as models. However, little is known about their O-glycome and the differences in glycan biosynthesis in different cell types. To provide a better understanding of the variation in O-glycosylation phenotypes and their association with other molecular features, an in-depth O-glycosylation analysis of 26 different CRC cell lines was performed. The released O-glycans were analysed on porous graphitized carbon nano-liquid chromatography system coupled to a mass spectrometer via electrospray ionization (PGC-nano-LC-ESI-MS/MS) allowing isomeric separation as well as in-depth structural characterization. Associations between the observed glycan phenotypes with previously reported cell line transcriptome signatures were examined by canonical correlation analysis. Striking differences are observed between the O-glycomes of 26 CRC cell lines. Unsupervized principal component analysis reveals a separation between well-differentiated colon-like and undifferentiated cell lines. Colon-like cell lines are characterized by a prevalence of I-branched and sialyl Lewis x/a epitope carrying glycans, while most undifferentiated cell lines show absence of Lewis epitope expression resulting in dominance of truncated α2,6-core sialylated glycans. Moreover, the expression of glycan signatures associates with the expression of glycosyltransferases that are involved in their biosynthesis, providing a deeper insight into the regulation of glycan biosynthesis in different cell types. This untargeted in-depth screening of cell line O-glycomes paves the way for future studies exploring the role of glycosylation in CRC development and drug response leading to discovery of novel targets for the development of anti-cancer antibodies.
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Diferenciación Celular , Glicómica/métodos , Polisacáridos/análisis , Secuencia de Carbohidratos , Línea Celular Tumoral , Cromatografía Líquida de Alta Presión , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Glicosilación , Glicosiltransferasas/genética , Glicosiltransferasas/metabolismo , Humanos , Fenotipo , Polisacáridos/metabolismo , Análisis de Componente Principal , Espectrometría de Masas en TándemRESUMEN
Glycan biosynthesis simulation research has progressed remarkably since 1997, when the first mathematical model for N-glycan biosynthesis was proposed. An O-glycan model has also been developed to predict O-glycan biosynthesis pathways in both forward and reverse directions. In this work, we started with a set of O-glycan profiles of CHO cells transiently transfected with various combinations of glycosyltransferases. The aim was to develop a model that encapsulated all the enzymes in the CHO transfected cell lines. Due to computational power restrictions, we were forced to focus on a smaller set of glycan profiles, where we were able to propose an optimized set of kinetics parameters for each enzyme in the model. Using this optimized model we showed that the abundance of more processed glycans could be simulated compared to observed abundance, while predicting the abundance of glycans earlier in the pathway was less accurate. The data generated show that for the accurate prediction of O-linked glycosylation, additional factors need to be incorporated into the model to better reflect the experimental conditions.
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Polisacáridos , Animales , Células CHO , Simulación por Computador , Cricetinae , Cricetulus , Glicosilación , Polisacáridos/metabolismoRESUMEN
Among the earliest deuterostomes, the echinoderms are an evolutionary important group of ancient marine animals. Within this phylum, the holothuroids (sea cucumbers) are known to produce a wide range of glycoconjugate biopolymers with apparent benefits to health; therefore, they are of economic and culinary interest throughout the world. Other than their highly modified glycosaminoglycans (e.g. fucosylated chondroitin sulfate and fucoidan), nothing is known about their protein-linked glycosylation. Here we used multistep N-glycan fractionation to efficiently separate anionic and neutral N-glycans before analyzing the N-glycans of the black sea cucumber (Holothuria atra) by MS in combination with enzymatic and chemical treatments. These analyses showed the presence of various fucosylated, phosphorylated, sialylated, and multiply sulfated moieties as modifications of oligomannosidic, hybrid, and complex-type N-glycans. The high degree of sulfation and fucosylation parallels the modifications observed previously on holothuroid glycosaminoglycans. Compatible with its phylogenetic position, H. atra not only expresses vertebrate motifs such as sulfo- and sialyl-Lewis A epitopes but displays a high degree of anionic substitution of its glycans, as observed in other marine invertebrates. Thus, as for other echinoderms, the phylum- and order-specific aspects of this species' N-glycosylation reveal both invertebrate- and vertebrate-like features.
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Holothuria/metabolismo , Polisacáridos/química , Sulfatos/química , Animales , Conformación de Carbohidratos , Secuencia de Carbohidratos , Cromatografía Líquida de Alta Presión , Ecosistema , Glicósido Hidrolasas/metabolismo , Glicosilación , Holothuria/clasificación , Filogenia , Polisacáridos/clasificación , Polisacáridos/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Echinoderms are among the most primitive deuterostomes and have been used as model organisms to understand chordate biology because of their close evolutionary relationship to this phylogenetic group. However, there are almost no data available regarding the N-glycomic capacity of echinoderms, which are otherwise known to produce a diverse set of species-specific glycoconjugates, including ones heavily modified by fucose, sulfate, and sialic acid residues. To increase the knowledge of diversity of carbohydrate structures within this phylum, here we conducted an in-depth analysis of N-glycans from a brittle star (Ophiactis savignyi) as an example member of the class Ophiuroidea. To this end, we performed a multi-step N-glycan analysis by HPLC and various exoglyosidase and chemical treatments in combination with MALDI-TOF MS and MS/MS. Using this approach, we found a wealth of hybrid and complex oligosaccharide structures reminiscent of those in higher vertebrates as well as some classical invertebrate glycan structures. 70% of these N-glycans were anionic, carrying either sialic acid, sulfate, or phosphate residues. In terms of glycophylogeny, our data position the brittle star between invertebrates and vertebrates and confirm the high diversity of N-glycosylation in lower organisms.
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Glicómica/métodos , Polisacáridos/química , Estrellas de Mar/metabolismo , Animales , Secuencia de Carbohidratos , Cromatografía Líquida de Alta Presión , Glicósido Hidrolasas/metabolismo , Glicosilación , Oligosacáridos/química , Filogenia , Polisacáridos/clasificación , Polisacáridos/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Estrellas de Mar/clasificaciónRESUMEN
As part of a systematic investigation of the glycosphingolipids in human tissues, acid and non-acid glycosphingolipids from human thyroid and parathyroid glands were isolated and characterized with mass spectrometry and binding of carbohydrate-recognizing ligands, with a focus on complex compounds. The glycosphingolipid patterns of the human parathyroid and thyroid glands were very similar. The major acid glycosphingolipids were sulfatide and the gangliosides GM3, GD3, GD1a, GD1b, GT1b and Neu5Ac-neolactotetraosylceramide, and the major non-acid glycosphingolipids were globotriaosylceramide and globoside. We also found neolactotetra- and neolactohexaosylceramide, the x2 glycosphingolipid, and complex glycosphingolipids with terminal blood group O and A determinants in both tissues. A glycosphingolipid with blood group Leb determinant was identified in the thyroid gland, and the parathyroid sample had a glycosphingolipid with terminal blood group B determinant. Immunohistochemistry demonstrated the expression of blood group A antigens in both the thyroid and parathyroid glands. A weak cytoplasmatic expression of the GD1a ganglioside was present in the thyroid, while the parathyroid gland had a strong GD1a expression on the cell surface. Thus, the glycosylation of human thyroid and parathyroid glands is more complex than previously appreciated. Our findings provide a platform for further studies of alterations of cell surface glycosphingolipids in thyroid and parathyroid cancers.
Asunto(s)
Glicoesfingolípidos/análisis , Glándulas Paratiroides/química , Glándula Tiroides/química , Antígenos de Grupos Sanguíneos/metabolismo , Cromatografía en Capa Delgada , Gangliósidos/química , Humanos , Ligandos , Espectrometría de Masas , Especificidad de Órganos , Glándulas Paratiroides/inmunología , Glándula Tiroides/inmunologíaRESUMEN
Medullary thyroid carcinoma (MTC) accounts for only 1-2% of thyroid cancers; however, metastatic MTC is a mortal disease with no cure. In this study, glycosphingolipids were isolated from human MTCs and characterized by mass spectrometry and binding of carbohydrate recognizing ligands. The tissue distribution of selected compounds was investigated by immunohistochemistry. The amount of acid glycosphingolipids in the MTCs was higher than in the normal thyroid glands. The major acid glycosphingolipid was the GD3 ganglioside. Sulfatide and the gangliosides GM3 and GD1a were also present. The majority of the complex non-acid glycosphingolipids had type 2 (Galß4GlcNAc) core chains, i.e., the neolactotetraosylceramide, the Lex, H type 2 and x2 pentaosylceramides, the Ley and A type 2 hexaosylceramides, and the A type 2 heptaosylceramide. There were also compounds with globo (GalαGalß4Glc) core, i.e., globotriaosylceramide, globotetraosylceramide, the Forssman pentaosylceramide, and the Globo H hexaosylceramide. Immunohistochemistry demonstrated an extensive expression av Ley in the MTC cells and also a variable intensity and prevalence of Globo H and Lex. One individual with multiple endocrine neoplasia type 2B expressed the Forssman determinant, which is rarely found in humans. This study of human MTC glycosphingolipids identifies glycans that could serve as potential tumor-specific markers.
Asunto(s)
Carcinoma Neuroendocrino/metabolismo , Glicoesfingolípidos/aislamiento & purificación , Neoplasias de la Tiroides/metabolismo , Biomarcadores de Tumor/análisis , Carcinoma Neuroendocrino/diagnóstico , Glicoesfingolípidos/análisis , Humanos , Inmunohistoquímica , Espectrometría de Masas , Glándula Tiroides/metabolismo , Neoplasias de la Tiroides/diagnósticoRESUMEN
The FORS histo-blood group system is the most recently discovered carbohydrate-based human blood group system. FORS is a rare blood group system, and most individuals have naturally occurring anti-FORS1 antibodies in plasma. Screening for anti-FORS1 antibodies is often done by hemagglutination assays using FORS1-expressing sheep erythrocytes, since FORS1-positive human erythrocytes are most often not available. Here, we have characterized the non-acid glycosphingolipids from sheep erythrocytes and isolated subfractions, with mass spectrometry, binding of antibodies and lectins, and by enzymatic hydrolysis. This demonstrated the presence of Forssman and Galili pentaosylceramides, and a Galili heptaosylceramide. Two complex glycosphingolipids recognized by human anti-FORS1 antibodies were characterized as a Forssman neolacto hybrid hexaosylceramide (GalNAcα3GalNAcß3Galß4GlcNAcß3Galß4Glcß1Cer) and a Forssman Galili hybrid heptaosylceramide (GalNAcα3GalNAcß3Galα3Galß4GlcNAcß3Galß4Glcß1Cer). These are novel glycosphingolipid structures, and to our knowledge, the first case of an elongated Galili antigen. Thus, the anti-Forssman antibodies in human serum bind not only to the classical Forssman pentaosylceramide (GalNAcα3GalNAcß3Galα4Galß4Glcß1Cer), but also when the GalNAcα3GalNAcß3 sequence is presented on a neolacto core chain and even on a Galili carbohydrate sequence.
Asunto(s)
Anticuerpos/química , Eritrocitos/química , Glicoesfingolípidos/análisis , Animales , Anticuerpos/inmunología , Eritrocitos/inmunología , Glicoesfingolípidos/inmunología , Humanos , OvinosRESUMEN
Animal bioprosthetic heart valves (BHV) are used to replace defective valves in patients with valvular heart disease. Especially young BHV recipients may experience a structural valve deterioration caused by an immune reaction in which α-Gal and Neu5Gc are potential target antigens. The expression of these and other carbohydrate antigens in animal tissues used for production of BHV was explored. Protein lysates of porcine aortic and pulmonary valves, and porcine, bovine and equine pericardia were analyzed by Western blotting using anti-carbohydrate antibodies and lectins. N-glycans were released by PNGase F digestion and O-glycans by ß-elimination. Released oligosaccharides were analyzed by liquid chromatography - tandem mass spectrometry. In total, 102 N-glycans and 40 O-glycans were identified in animal heart tissue lysates. The N- and O-glycan patterns were different between species. α-Gal and Neu5Gc were identified on both N- and O-linked glycans, N,N´-diacetyllactosamine (LacdiNAc) on N-glycans only and sulfated O-glycans. The relative amounts of α-Gal-containing N-glycans were higher in bovine compared to equine and porcine pericardia. In contrast to the restricted number of proteins carrying α-Gal and LacdiNAc, the distribution of proteins carrying Neu5Gc-determinants varied between species and between different tissues of the same species. Porcine pericardium carried the highest level of Neu5Gc-sialylated O-glycans, and bovine pericardium the highest level of Neu5Gc-sialylated N-glycans. The identified N- and O-linked glycans, some of which may be immunogenic and remain in BHVs manufactured for clinical use, could direct future genetic engineering to prevent glycan expression rendering the donor tissues less immunogenic in humans.