The human immunodeficiency virus (HIV) envelope protein (Env) mediates viral entry into host cells and is the primary target for the humoral immune response. Env is extensively glycosylated, and ...these glycans shield underlying epitopes from neutralizing antibodies. The glycosylation of Env is influenced by the type of host cell in which the virus is produced. Thus, HIV is distinctly glycosylated by CD4+ T cells, the major target cells, and macrophages. However, the specific differences in glycosylation between viruses produced in these cell types have not been explored at the molecular level. Moreover, it remains unclear whether the production of HIV in CD4+ T cells or macrophages affects the efficiency of viral spread and resistance to neutralization. To address these questions, we employed the simian immunodeficiency virus (SIV) model. Glycan analysis implied higher relative levels of oligomannose-type N-glycans in SIV from CD4+ T cells (T-SIV) compared to SIV from macrophages (M-SIV), and the complex-type N-glycans profiles seem to differ between the two viruses. Notably, M-SIV demonstrated greater infectivity than T-SIV, even when accounting for Env incorporation, suggesting that host cell-dependent factors influence infectivity. Further, M-SIV was more efficiently disseminated by HIV binding cellular lectins. We also evaluated the influence of cell type-dependent differences on SIV's vulnerability to carbohydrate binding agents (CBAs) and neutralizing antibodies. T-SIV demonstrated greater susceptibility to mannose-specific CBAs, possibly due to its elevated expression of oligomannose-type N-glycans. In contrast, M-SIV exhibited higher susceptibility to neutralizing sera in comparison to T-SIV. These findings underscore the importance of host cell-dependent attributes of SIV, such as glycosylation, in shaping both infectivity and the potential effectiveness of intervention strategies.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Glycosylation, especially
-glycosylation, is one of the most common protein modifications, with immense importance at the molecular, cellular, and organismal level. Thus, accurate and reliable
...-glycan analysis is essential in many areas of pharmaceutical and food industry, medicine, and science. However, due to the complexity of the cellular glycosylation process, in-depth glycoanalysis is still a highly challenging endeavor. Contamination of samples with oligosaccharide impurities (OSIs), typically linear glucose homo-oligomers, can cause further complications. Due to their physicochemical similarity to
-glycans, OSIs produce potentially overlapping signals, which can remain unnoticed. If recognized, suspected OSI signals are usually excluded in data evaluation. However, in both cases, interpretation of results can be impaired. Alternatively, sample preparation can be repeated to include an OSI removal step from samples. However, this significantly increases sample amount, time, and effort necessary. To overcome these issues, we investigated the option to enzymatically degrade and thereby remove interfering OSIs as a final sample preparation step. Therefore, we screened ten commercially available enzymes concerning their potential to efficiently degrade maltodextrins and dextrans as most frequently found OSIs. Of these enzymes, only dextranase from
and glucoamylase P from
enabled a degradation of OSIs within only 30 min that is free of side reactions with
-glycans. Finally, we applied the straightforward enzymatic degradation of OSIs to
-glycan samples derived from different standard glycoproteins and various stem cell lysates.
Background
Pig‐derived tissues could overcome the shortage of human donor organs in transplantation. However, the glycans with terminal α‐Gal and Neu5Gc, which are synthesized by enzymes, encoded by ...the genes GGTA1 and CMAH, are known to play a major role in immunogenicity of porcine tissue, ultimately leading to xenograft rejection.
Methods
The N‐glycome and glycosphingolipidome of native and decellularized porcine pericardia from wildtype (WT), GGTA1‐KO and GGTA1/CMAH‐KO pigs were analyzed by multiplexed capillary gel electrophoresis coupled to laser‐induced fluorescence detection.
Results
We identified biantennary and core‐fucosylated N‐glycans terminating with immunogenic α‐Gal‐ and α‐Gal‐/Neu5Gc‐epitopes on pericardium of WT pigs that were absent in GGTA1 and GGTA1/CMAH‐KO pigs, respectively. Levels of N‐glycans terminating with galactose bound in β(1‐4)‐linkage to N‐acetylglucosamine and their derivatives elongated by Neu5Ac were increased in both KO groups. N‐glycans capped with Neu5Gc were increased in GGTA1‐KO pigs compared to WT, but were not detected in GGTA1/CMAH‐KO pigs. Similarly, the ganglioside Neu5Gc‐GM3 was found in WT and GGTA1‐KO but not in GGTA1/CMAH‐KO pigs. The applied detergent based decellularization efficiently removed GSL glycans.
Conclusion
Genetic deletion of GGTA1 or GGTA1/CMAH removes specific epitopes providing a more human‐like glycosylation pattern, but at the same time changes distribution and levels of other porcine glycans that are potentially immunogenic.
Sulfate modification of N-glycans is important for several biological functions such as clearance of pituitary hormones or immunoregulation. Yet, the prevalence of this N-glycan modification and its ...functions remain largely unexplored. Characterization of N-glycans bearing sulfate modifications is hampered in part by a lack of enzymes that enable site-specific detection of N-glycan sulfation. In this study, we used functional metagenomic screening to identify enzymes that act upon sulfated N-acetylglucosamine (GlcNAc). Using multiplexed capillary gel electrophoresis with laser-induced fluorescence detection (xCGE-LIF) -based glycoanalysis we proved their ability to act upon GlcNAc-6-SO
on N-glycans.
Our screen identified a sugar-specific sulfatase that specifically removes sulfate from GlcNAc-6-SO
when it is in a terminal position on an N-glycan. Additionally, in the absence of calcium, this sulfatase binds to the sulfated glycan but does not remove the sulfate group, suggesting it could be used for selective isolation of sulfated N-glycans. Further, we describe isolation of a sulfate-dependent hexosaminidase that removes intact GlcNAc-6-SO
(but not asulfated GlcNAc) from a terminal position on N-glycans. Finally, the use of these enzymes to detect the presence of sulfated N-glycans by xCGE-LIF is demonstrated.
The present study demonstrates the feasibility of using functional metagenomic screening combined with glycoanalytics to discover enzymes that act upon chemical modifications of glycans. The discovered enzymes represent new specificities that can help resolve the presence of GlcNAc-6-SO
in N-glycan structural analyses.
•Introduction of a smart new glycoanalytical approach.•Combination of cost-effective, widespread, and easy-to-handle analytical tools.•Tagging of N-glycans via reversible (removable) labeling.•Power ...of approach exemplified by hybrid and multiply-sialylated complex‐N-glycans.•Detection and annotation of often overlooked N-glycan motifs—O-acetate, sulfate.
As the roles of glycans in health and disease continue to be unraveled, it is becoming apparent that glycans’ immense complexity cannot be ignored. To fully delineate glycan structures, we developed an integrative approach combining a set of cost-effective, widespread, and easy-to-handle analytical methods. The key feature of our workflow is the exploitation of a removable fluorescent label—exemplified by 9-fluorenylmethyl chloroformate (Fmoc)—to bridge the gap between diverse glycoanalytical methods, especially multiplexed capillary gel electrophoresis with laser-induced fluorescence detection (xCGE-LIF) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Through the detailed structural analysis of selected, dauntingly complex N-glycans from chicken ovalbumin, horse serum, and bovine transferrin, we illustrate the capabilities of the presented strategy. Moreover, this approach “visualizes” N-glycans that have been difficult to identify thus far—such as the sulfated glycans on human immunoglobulin A—including minute changes in glycan structures, potentially providing useful new targets for biomarker discovery.
The negatively charged nonulose sialic acid (Sia) is essential for murine development in vivo. In order to elucidate the impact of sialylation on differentiation processes in the absence of maternal ...influences, we generated mouse embryonic stem cell (mESC) lines that lack CMP‐Sia synthetase (CMAS) and thereby the ability to activate Sia to CMP‐Sia. Loss of CMAS activity resulted in an asialo cell surface accompanied by an increase in glycoconjugates with terminal galactosyl and oligo‐LacNAc residues, as well as intracellular accumulation of free Sia. Remarkably, these changes did not impact intracellular metabolites or the morphology and transcriptome of pluripotent mESC lines. Moreover, the capacity of Cmas−/− mESCs for undirected differentiation into embryoid bodies, germ layer formation and even the generation of beating cardiomyocytes provides first and conclusive evidence that pluripotency and differentiation of mESC in vitro can proceed in the absence of (poly)sialoglycans.
Sialic acid and development. Ablation of the CMAS gene in murine embryonic stem cells (mESC) resulted in the complete elimination of sialylation, demonstrating that CMAS is the sole sialic acid activating enzyme. Strikingly, asialo mESC similar to wild type undergo germ layer formation during early embryonic development in vitro.
Human induced pluripotent stem‐cell‐derived cardiomyocytes (hiPSC CMs) may be used in regenerative medicine for individualized tissue transplants in the future. For application in patients, the ...generated CMs have to be highly pure and well characterized. In order to overcome the prevalent scarcity of CM‐specific markers, we quantitatively assessed cell‐surface‐exposed sialo‐glycoproteins and N‐glycans of hiPSCs, CM progenitors, and CMs. Applying a combination of metabolic labeling and specific sialo‐glycoprotein capture, we could highly enrich and quantify membrane proteins during cardiomyogenic differentiation. Among them we identified a number of novel, putative biomarkers for hiPSC CMs. Analysis of the N‐glycome by capillary gel electrophoresis revealed three novel structures comprising β1,3‐linked galactose, α2,6‐linked sialic acid and complex fucosylation; these were highly specific for hiPSCs. Bisecting GlcNAc structures strongly increased during differentiation, and we propose that they are characteristic of early, immature CMs.
Differentiating hearts: Human induced pluripotent stem cells, cardiac progenitors, and stem‐cell‐derived cardiomyocytes were analyzed and compared at the sialo‐glycoproteomic, proteomic, and N‐glycomic levels. This approach led to the identification of novel potential biomarkers for these cell types.
Several health benefits, associated with human milk oligosaccharides (HMOS), have been revealed in the last decades. Further progress, however, requires not only the establishment of a simple ...“routine” method for absolute quantification of complex HMOS mixtures but also the development of novel synthesis strategies to improve access to tailored HMOS. Here, we introduce a combination of salvage-like nucleotide sugar-producing enzyme cascades with Leloir-glycosyltransferases in a sequential pattern for the convenient tailoring of stable isotope-labeled HMOS. We demonstrate the assembly of 13C6galactose into lacto-N- and lacto-N-neo-type HMOS structures up to octaoses. Further, we present the enzymatic production of UDP-15NGlcNAc and its application for the enzymatic synthesis of 13C6/15Nlacto-N-neo-tetraose for the first time. An exemplary application was selected—analysis of tetraose in complex biological mixtures—to show the potential of tailored stable isotope reference standards for the mass spectrometry-based quantification, using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) as a fast and straightforward method for absolute quantification of HMOS. Together with the newly available well-defined tailored isotopic HMOS, this can make a crucial contribution to prospective research aiming for a more profound understanding of HMOS structure-function relations.
Abstract
Sialyl-Lewis x (sLe
x
, CD15s) is a tetra-saccharide on the surface of leukocytes required for E-selectin-mediated rolling, a prerequisite for leukocytes to migrate out of the blood vessels. ...Here we show using flow cytometry that sLe
x
expression on basophils and mast cell progenitors depends on fucosyltransferase 6 (
FUT6
). Using genetic association data analysis and qPCR, the cell type-specific defect was associated with single nucleotide polymorphisms (SNPs) in the
FUT6
gene region (tagged by rs17855739 and rs778798), affecting coding sequence and/or expression level of the mRNA. Heterozygous individuals with one functional
FUT6
gene harbor a mixed population of sLe
x+
and sLe
x-
basophils, a phenomenon caused by random monoallelic expression (RME). Microfluidic assay demonstrated
FUT6
-deficient basophils rolling on E-selectin is severely impaired.
FUT6
null alleles carriers exhibit elevated blood basophil counts and a reduced itch sensitivity against insect bites.
FUT6
-deficiency thus dampens the basophil-mediated allergic response in the periphery, evident also in lower IgE titers and reduced eosinophil counts.
Facilitated by substantial advances in analytical methods, plasma N-glycans have emerged as potential candidates for biomarkers. In the recent years, several investigations could link aberrant plasma ...N-glycosylation to numerous diseases. However, due to often limited specificity and sensitivity, only a very limited number of glycan biomarkers were approved by the authorities up to now. The inter-individual heterogeneity of the plasma N-glycomes might mask disease related changes in conventional large cross-sectional cohort studies, with a one-time sampling approach. But, a possible benefit of longitudinal sampling in biomarker discovery could be, that already small changes during disease progression are revealed, by monitoring the plasma N-glycome of individuals over time.
To evaluate this, we collected blood plasma samples of five healthy donors over a time period of up to six years (min. 1.5years). The plasma N-glycome was analyzed by xCGE-LIF, to investigate the intra-individual N-glycome variability over time. It is shown, that the plasma N-glycome of an individual is remarkably stable over a period of several years, and that observed small longitudinal changes are independent from seasons, but significantly correlated with lifestyle and environmental factors. Thus, the potential of future longitudinal biomarker discovery studies could be demonstrated, which is a further step towards personalized diagnostics. This article is part of a Special Issue entitled “Glycans in personalised medicine” Guest Editor: Professor Gordan Lauc.
•Longitudinal sampling of individuals plasma N-glycomes for personalized diagnostics.•Plasma N-glycomics with minimal invasive sampling (single blood droplets) is shown.•Longitudinal monitoring of individuals allows detection of small event related changes.•Time-series from individuals can be beneficial compared to cross-sectional large-scale studies.
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