Glycosylated Form Research Articles

Characterization of Italian Grape Ale beers obtained with different additions of Malvasia di Candia Aromatica must and marcs

In 2015, a new style of fruit beer, called Italian Grape Ale (IGA), was introduced by the Beer Judge Certification Program. The style of this beer is defined as a union between beer (wort or final beer) and grape or grape must (from red or white cultivar). This study aimed to characterize the chemical composition and sensorial properties of IGA beers obtained by adding grape juice or marc. To this aim, different amounts of grape juice or marcs (10 % and 20 %) from cv. Malvasia di Candia Aromatica (MCA) were blended with Golden Ale wort before primary fermentation. The addition of MCA grape marc and juice resulted in a significant increase in total fermentable sugar concentration, organic acids, and higher titratable acidity, both in the wort and in the final beer. Furthermore, the CIELab colour space parameters were strongly influenced by MCA, with the additions of marc leading to the most pronounced colour changes in beer, producing a dark yellow hue. Regarding the volatile profile of IGA beers, the MCA additions modulated the profile of IGA beers with key markers that consisted mainly of terpenes (both free and glycosylated forms), higher alcohols and esters. These compounds, as confirmed by sensory analysis, enhanced the aroma of beer samples with floral and fruit notes, making them easily distinguishable from IGA traditional beers. These findings are among the first data available on the effect of white aromatic grape juice and marc in brewing, both from a chemical and sensory point of view.

Ultradeep O-GlcNAc proteomics reveals widespread O-GlcNAcylation on tyrosine residues of proteins

As a unique type of glycosylation, O-linked β-N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation) on Ser/Thr residues of proteins was discovered 40 y ago. O-GlcNAcylation is catalyzed by two enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which add and remove O-GlcNAc, respectively. O-GlcNAcylation is an essential glycosylation that regulates the functions of many proteins in virtually all cellular processes. However, deep and site-specific characterization of O-GlcNAcylated proteins remains a challenge. We developed an ultradeep O-GlcNAc proteomics workflow by integrating digestion with multiple proteases, two mass spectrometric approaches (i.e., electron-transfer/higher-energy collision dissociation [EThcD] and HCD product-dependent electron-transfer/higher-energy collision dissociation [HCD-pd-EThcD]), and two data analysis tools (i.e., MaxQuant and Proteome Discoverer). The performance of this strategy was benchmarked by the analysis of whole lysates from PANC-1 (a pancreatic cancer cell line). In total, 2,831 O-GlcNAc sites were unambiguously identified, representing the largest O-GlcNAc dataset of an individual study reported so far. Unexpectedly, in addition to confirming known sites and identifying many other sites of Ser/Thr modification, O-GlcNAcylation was found on 121 tyrosine (Tyr) residues of 93 proteins. In vitro enzymatic assays showed that OGT catalyzes the transfer of O-GlcNAc onto Tyr residues of peptides and OGA catalyzes its removal. Taken together, our work reveals widespread O-GlcNAcylation on Tyr residues of proteins and that Tyr O-GlcNAcylation is mediated by OGT and OGA. As another form of glycosylation, Tyr O-GlcNAcylation is likely to have important regulatory roles.

Improving the bioactivity of water-soluble alfalfa saponins using biotransformation.

Medicago sativa L. is gaining attention as a sustainable plant-based food protein. Alfalfa saponins (ASs) typically exist in a glycosylated form in nature, which has poor cell membrane permeability, while the deglycosylated saponins may show better bioactivity. The AS was deglycosylated by β-glucosidase from Aspergillus niger, and the chemical structures and biological activities, including in vivo assays, of AS and deglycosylated AS (DAS) were determined. The results showed that the half maximal inhibitory concentration for 2,2-diphenyl-1-picrylhydrazyl inhibition of DAS was 29.5µg/mL, demonstrating a significantly higher reducing capacity compared to AS (p<0.05). The DAS induced 33.8% antibacterial activity against Escherichia coli and enhanced the proliferation of human airway epithelial cells (BEAS-2B) at a concentration of 125µg/mL. In vivo experiments on C57BL/6 mice fed a high-fat diet demonstrated that high-level DAS treatment produced significantly greater hypolipidemic effects compared to AS (p<0.05). Thus, the AS can be deglycosylated, which leads to an improvement in biological activity, particularly since the DAS exhibits significantly enhanced hypolipidemic activity. PRACTICAL APPLICATION: Alfalfa saponins were deglycosylated by β-glucosidase from Aspergillus niger, which contributed to increased bioactivity, particularly its hypolipidemic activity.

Metabolic Control of Glycosylation Forms for Establishing Glycan-Dependent Protein Interaction Networks.

Protein-protein interactions (PPIs) provide essential insights into the complex molecular mechanisms and signaling pathways within cells that regulate development and disease-related phenotypes. However, for membrane proteins, the impact of various forms of glycosylation has often been overlooked in PPI studies. In this study, we introduce a novel approach, glycan-dependent affinity purification followed by mass spectrometry (GAP-MS), to assess variations in PPIs for any glycoprotein of interest under different glycosylation conditions. As a proof of principle, we selected four glycoproteins-BSG, CD44, EGFR, and SLC3A2-as baits to compare their co-purified partners across five metabolically controlled glycan conditions. The findings demonstrate the capability of GAP-MS to identify PPIs influenced by altered glycosylation states, establishing a foundation for systematically exploring the Glycan-Dependent Protein Interactome (GDPI) for other glycoproteins of interest.

Unveiling the Power of Flax Lignans: From Plant Biosynthesis to Human Health Benefits.

Flax (Linum usitatissimum L.) is the richest plant source of lignin secondary metabolites. Lignans from flax have been applied in the fields of food, medicine, and health due to their significant physiological activities. The most abundant lignan is secoisolariciresinol, which exists in a glycosylated form in plants. After ingestion, it is converted by human intestinal flora into enterodiol and enterolactone, which both have physiological roles. Here, the basic structures, contents, synthesis, regulatory, and metabolic pathways, as well as extraction and isolation methods, of flax lignans were reviewed. Additionally, the physiological activity-related mechanisms and their impacts on human health, from the biosynthesis of lignans in plants to the physiological activity effects observed in animal metabolites, were examined. The review elucidates that lignans, as phenolic compounds, not only function as active substances in plants but also offer significant nutritional values and health benefits when flax is consumed.

Inhibitory effects of extracts from Eucalyptus gunnii on α-synuclein amyloid fibrils.

Amyloid fibril formation is associated with various amyloidoses, including neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Despite the numerous studies on the inhibition of amyloid formation, the prevention and treatment of a majority of amyloid-related disorders are still challenging. In this study, we investigated the effects of various plant extracts on amyloid formation of α-synuclein. We found that the extracts from Eucalyptus gunnii are able to inhibit amyloid formation, and to disaggregate preformed fibrils, in vitro. The extract itself did not lead to cell damage. In the extract, miquelianin, which is a glycosylated form of quercetin and has been detected in the plasma and the brain, was identified and assessed to have a moderate inhibitory activity, compared to the effects of ellagic acid and quercetin, which are strong inhibitors for amyloid formation. The properties of miquelianin provide insights into the mechanisms controlling the assembly of α-synuclein in the brain.

Influence of O-GlcNAcylation on KGN cell function

O-GlcNAcylation is a unique form of post-translational glycosylation that affects a variety of cytoplasmic and nuclear proteins of cells. Aberrant O-GlcNAcylation is characteristic of many cancers, and impacts cell proliferation, tumorigenicity and metabolism. O-GlcNAcylation occurs in granulosa cells of ovarian follicles, its expression differs between small (3-5 mm) and large (>8.5 mm) antral follicles, and its manipulation in vitro alters granulosa cell proliferation and metabolism. Here, the aim was to assess whether O-GlcNAcylation similarly occurs in cells from a type of granulosa cell tumor, specifically KGN cells, knowing these cells share functional features of granulosa cells of mature, preovulatory follicles (e.g., FSH-responsiveness and estradiol production). The immortal KGN cell line was utilized to conduct cell culture experiments for the detection and manipulation of O-GlcNAcylation. The cells were grown to confluency in serum containing medium and then sub-cultured in serum-free conditions for immunodetection of O-GlcNAcylation (n = 8 expts.), for cell proliferation (n = 3 expts) and for metabolism assays (n = 12 expts.). The KGN cells were also treated without or with small molecule inhibitors to directly enhance or impair O-GlcNAcylation. Immunoblotting confirmed O-GlcNAc expression in KGN cells, as well as the efficacy of Thiamet-G and OSMI-1 to augment (P < 0.05) and inhibit O-GlcNAcylation (P < 0.05), respectively. Only the inhibition of O-GlcNAcylation compromised KGN cell proliferation (P < 0.05), resulting in a 25 % reduction in proliferation compared to control conditions over a 72 h culture period. Seahorse XFe96 analysis measured effects of O-GlcNAcylation on cellular respiration in the KGN cells. Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) provided indices of glycolysis and oxidative phosphorylation, respectively. During a glycolysis stress test, high glucose increased ECAR and decreased OCR (P < 0.05); oligomycin did not further affect ECAR (P > 0.05), but impaired OCR (P < 0.05); and 2-deoxy-d-glucose decreased ECAR (P < 0.05) without affecting OCR (P > 0.05). Comparatively, a mitochondrial stress test revealed oligomycin increased ECAR (P < 0.05) with a compensatory decrease in OCR (P < 0.05); FCCP increased both ECAR and OCR (P < 0.05); and rotenone + antimycin A decreased both ECAR and OCR (P < 0.05). Manipulation of O-GlcNAcylation in the KGN cells had no effect on ECAR (P > 0.05), but inhibited OCR (P < 0.05). Collectively, the results indicate O-GlcNAcylation occurs in KGN cells, its inhibition impairs cell proliferation, and while KGN cells rely upon both glycolysis and oxidative phosphorylation for cellular respiration, manipulation of O-GlcNAcylation acutely perturbs only oxidative phosphorylation, an effect observed previously with granulosa cells of large antral follicles.

Advances in the structural basis for angiotensin-1 converting enzyme (ACE) inhibitors.

Human somatic angiotensin-converting enzyme (ACE) is a key zinc metallopeptidase that plays a pivotal role in the renin-angiotensin-aldosterone system (RAAS) by regulating blood pressure and electrolyte balance. Inhibition of ACE is a cornerstone in the management of hypertension, cardiovascular diseases, and renal disorders. Recent advances in structural biology techniques have provided invaluable insights into the molecular mechanisms underlying ACE inhibition, facilitating the design and development of more effective therapeutic agents. This review focuses on the latest advancements in elucidating the structural basis for ACE inhibition. High-resolution crystallographic studies of minimally glycosylated individual domains of ACE have revealed intricate molecular details of the ACE catalytic N- and C-domains, and their detailed interactions with clinically relevant and newly designed domain-specific inhibitors. In addition, the recently elucidated structure of the glycosylated form of full-length ACE by cryo-electron microscopy (cryo-EM) has shed light on the mechanism of ACE dimerization and revealed continuous conformational changes which occur prior to ligand binding. In addition to these experimental techniques, computational approaches have also played a pivotal role in elucidating the structural basis for ACE inhibition. Molecular dynamics simulations and computational docking studies have provided atomic details of inhibitor binding kinetics and energetics, facilitating the rational design of novel ACE inhibitors with improved potency and selectivity. Furthermore, computational analysis of the motions observed by cryo-EM allowed the identification of allosteric binding sites on ACE. This affords new opportunities for the development of next-generation allosteric inhibitors with enhanced pharmacological properties. Overall, the insights highlighted in this review could enable the rational design of novel ACE inhibitors with improved efficacy and safety profiles, ultimately leading to better therapeutic outcomes for patients with hypertension and cardiovascular diseases.

LacdiNAc to LacNAc: remodelling of bovine α-lactalbumin N-glycosylation during the transition from colostrum to mature milk.

α -Lactalbumin, an abundant protein present in the milk of most mammals, is associated with biological, nutritional and technological functionality. Its sequence presents N-glycosylation motifs, the occupancy of which is species-specific, ranging from no to full occupancy. Here, we investigated the N-glycosylation of bovine α-lactalbumin in colostrum and milk sampled from four individual cows, each at 9 time points starting from the day of calving up to 28.0 d post-partum. Using a glycopeptide-centric mass spectrometry-based glycoproteomics approach, we identified N-glycosylation at both Asn residues found in the canonical Asn-Xxx-Ser/Thr motif, i.e. Asn45 and Asn74 of the secreted protein. We found similar glycan profiles in all four cows, with partial site occupancies, averaging at 35% and 4% for Asn45 and Asn74, respectively. No substantial changes in occupancy occurred over lactation at either site. Fucosylation, sialylation, primarily with N-acetylneuraminic acid (Neu5Ac), and a high ratio of N,N'-diacetyllactosamine (LacdiNAc)/N-acetyllactosamine (LacNAc) motifs were characteristic features of the identified N-glycans. While no substantial changes occurred in site occupancy at either site during lactation, the glycoproteoform (i.e. glycosylated form of the protein) profile revealed dynamic changes; the maturation of the α-lactalbumin glycoproteoform repertoire from colostrum to mature milk was marked by substantial increases in neutral glycans and the number of LacNAc motifs per glycan, at the expense of LacdiNAc motifs. While the implications of α-lactalbumin N-glycosylation on functionality are still unclear, we speculate that N-glycosylation at Asn74 results in a structurally and functionally different protein, due to competition with the formation of its two intra-molecular disulphide bridges.

The major surface protein of malaria sporozoites is GPI-anchored to the plasma membrane

Glycosylphosphatidylinositol (GPI) anchor protein modification in Plasmodium species is well known and represents the principal form of glycosylation in these organisms. The structure and biosynthesis of GPI anchors of Plasmodium spp. has been primarily studied in the asexual blood stage of P. falciparum and is known to contain the typical conserved GPI structure of EtN-P-Man3GlcN-PI. Here, we have investigated the circumsporozoite protein (CSP) for the presence of a GPI-anchor. CSP is the major surface protein of Plasmodium sporozoites, the infective stage of the malaria parasite. While it is widely assumed that CSP is a GPI-anchored cell surface protein, compelling biochemical evidence for this supposition is absent. Here, we employed metabolic labeling and mass-spectrometry based approaches to confirm the presence of a GPI anchor in CSP. Biosynthetic radiolabeling of CSP with [3H]-palmitic acid and [3H]-ethanolamine, with the former being base-labile and therefore ester-linked, provided strong evidence for the presence of a GPI anchor on CSP, but these data alone were not definitive. To provide further evidence, immunoprecipitated CSP was analyzed for presence of myo-inositol (a characteristic component of GPI anchor) using strong acid hydrolysis and GC-MS for a highly sensitive and quantitative detection. The single ion monitoring (SIM) method for GC-MS analysis confirmed the presence of the myo-inositol component in CSP. Taken together, these data provide confidence that the long-assumed presence of a GPI anchor on this important parasite protein is correct.

Development of Cyclodextrin-Based Mono and Dual Encapsulated Powders by Spray Drying for Successful Preservation of Everlasting Flower Extract.

The study aimed to develop encapsulation systems to maintain the preservation of everlasting (Helichrysum plicatum) flower extract polyphenols. Spray-dried encapsulates were formulated using β-cyclodextrin (BCD) and 2-hydroxypropyl-β-cyclodextrin (HPBCD) as supramolecular hosts, and their macromolecule mixtures with the conventional carriers, maltodextrin (MD) and whey protein (WP). The obtained microparticles were comparatively assessed regarding technological, physicochemical, and phytochemical properties. The highest yields were achieved by combining cyclodextrins with whey protein (73.96% for WP+BCD and 75.50% for WP+HPBCD compared to 62.48% of pure extract). The extract-carrier interactions and thermal stability were evaluated by FTIR and DSC analysis, suggesting successful entrapment within the carriers. Carriers reduced the particle diameter (3.99 to 4.86 μm compared to 6.49 μm of pure extract), classifying all encapsulates as microsystems. Carrier blends made the particle size distribution uniform, while SEM analysis revealed the production of more spherical and less aggregated particles. The HPBCD provided the highest encapsulation efficiency, with the highest content of detected aglycones and slightly lower values of their glycosylated forms. An analysis of the dual macromolecule encapsulation systems revealed the highest bioactive preservation potential for SHE+MD+BCD and SHE+WP+HPBCD. Overall, macromolecule combinations of cyclodextrins and conventional biopolymers in the spray-drying process can enhance the functional properties of H. plicatum extract.

The Impact of Glycosylation on the Functional Activity of CD133 and the Accuracy of Its Immunodetection.

The membrane glycoprotein CD133 (prominin-1) is widely regarded as the main molecular marker of cancer stem cells, which are the most malignant cell subpopulation within the tumor, responsible for tumor growth and metastasis. For this reason, CD133 is considered a promising prognostic biomarker and molecular target for antitumor therapy. Under normal conditions, CD133 is present on the cell membrane in glycosylated form. However, in malignancies, altered glycosylation apparently leads to changes in the functional activity of CD133 and the availability of some of its epitopes for antibodies. This review focuses on CD133's glycosylation in human cells and its impact on the function of this glycoprotein. The association of CD133 with proliferation, differentiation, apoptosis, autophagy, epithelial-mesenchymal transition, the organization of plasma membrane protrusions and extracellular trafficking is discussed. In this review, particular attention is paid to the influence of CD133's glycosylation on its immunodetection. A list of commercially available and custom antibodies with their characteristics is provided. The available data indicate that the development of CD133-based biomedical technologies should include an assessment of CD133's glycosylation in each tumor type.

Application of hydrophobic eutectic solvent in efficient biotransformation of total flavonoids of Herba Epimedii

Icaritin, a hydrolysate from total flavonoids of Epimedii (TFE), which has better anti-hepatoma activity than its glycosylated form. In this work, immobilized enzymes 4LP-Tpebgl3@Na-Y and DtRha@ES-107 were used to hydrolyze TFE to prepare icaritin. Five different hydrophobic deep eutectic solvents (HDES) were prepared and the most ideal HDES was successfully selected, which was composed of dodecyl alcohol and thymol with the molar ratio of 2:1. The relative enzyme activity of 4LP-Tpebgl3@Na-Y and DtRha@ES-107 was about 102.4 % and 112.5 %, respectively. In addition, the thermal and binding stability of 4LP-Tpebgl3@Na-Y and DtRha@ES-107 in HDES was not affected negatively. In the biphasic system composed of 50 % (v/v) HDES and Na2HPO4-citric acid buffer (50 mM, pH 5.5), 4LP-Tpebgl3@Na-Y (1.0 U/mL) and TFE (1 g/L) were reacted at 80 °C for 1 h, and then reacted with DtRha@ES-107 (20 U/mL) at 80 °C for 2 h. Finally, TFE was completely converted to 301.8 mg/L icaritin (0.82 mM). After 10 cycles, 4LP-Tpebgl3@Na-Y/DtRha@ES-107 still maintained 84.1 % original activity. In this study, we developed an efficient methodology for icaritin preparation through the integration of enzymatic catalysis and adsorption separation, presenting a viable approach for large-scale, cost-effective production of icaritin.

Production of N-glycosylated alcohol dehydrogenase in Escherichia coli

N-glycosylation of recombinant proteins using bacterial glycosylation system has proven to be a valuable although developing tool ultimately applicable to various industries. When used for enzyme engineering, it offers the possibility of increased stability or immobilization route and thus increasing effectiveness of e.g. biotransformation or other biocatalysis procedures. One such promising enzyme is alcohol dehydrogenase (ADH) for use in redox biotransformation reactions. Given the current possibilities of recombinant enzyme production, including major advances in glycoengineering and glycoprotein production in bacterial organisms, the aim of this work was the production of thermotolerant ADH from Rhodococcus ruber (RrADH) in glycosylated form in Escherichia coli. We have successfully developed a dual plasmid expression system enabling glycosylation of target proteins utilizing a glyco-tag approach. We were able to produce RrADH in soluble form and at the same time we detected a bacterial glycan conjugated to RrADH as well as the activity of the enzyme. The glycan bound to recombinant enzyme can be used for oriented covalent immobilization of the enzyme, which would increase the potential for its practical application in biotransformation of various compounds.

Sialyltransferase ST3GAL6 silencing reduces α2,3-sialylated glycans to regulate autophagy by decreasing HSPB8-BAG3 in the brain with hepatic encephalopathy.

End-stage liver diseases, such as cirrhosis and liver cancer caused by hepatitis B, are often combined with hepatic encephalopathy (HE); ammonia poisoning is posited as one of its main pathogenesis mechanisms. Ammonia is closely related to autophagy, but the molecular mechanism of ammonia's regulatory effect on autophagy in HE remains unclear. Sialylation is an essential form of glycosylation. In the nervous system, abnormal sialylation affects various physiological processes, such as neural development and synapse formation. ST3 β‍-galactoside α2,‍3-sialyltransferase 6 (ST3GAL6) is one of the significant glycosyltransferases responsible for adding α2,3-linked sialic acid to substrates and generating glycan structures. We found that the expression of ST3GAL6 was upregulated in the brains of mice with HE and in astrocytes after ammonia induction, and the expression levels of α2,3-sialylated glycans and autophagy-related proteins microtubule-associated protein light chain 3 (LC3) and Beclin-1 were upregulated in ammonia-induced astrocytes. These findings suggest that ST3GAL6 is related to autophagy in HE. Therefore, we aimed to determine the regulatory relationship between ST3GAL6 and autophagy. We found that silencing ST3GAL6 and blocking or degrading α2,3-sialylated glycans by way of Maackia amurensis lectin-II (MAL-II) and neuraminidase can inhibit autophagy. In addition, silencing the expression of ST3GAL6 can downregulate the expression of heat shock protein β8 (HSPB8) and Bcl2-associated athanogene 3 (BAG3). Notably, the overexpression of HSPB8 partially restored the reduced autophagy levels caused by silencing ST3GAL6 expression. Our results indicate that ST3GAL6 regulates autophagy through the HSPB8-BAG3 complex.

Glycosylation Modulation Dictates Trafficking and Interaction of SARS-CoV-2 S1 Subunit and ACE2 in Intestinal Epithelial Caco-2 Cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mainly targets the upper respiratory tract. It gains entry by interacting with the host cell receptor angiotensin-converting enzyme 2 (ACE2) via its heavily glycosylated spike glycoprotein. SARS-CoV-2 can also affect the gastrointestinal tract. Given the significant role of glycosylation in the life cycle of proteins and the multisystem target of SARS-CoV-2, the role of glycosylation in the interaction of S1 with ACE2 in Caco-2 cells was investigated after modulation of their glycosylation patterns using N-butyldeoxynojirimycin (NB-DNJ) and 1-deoxymannojirimycin (dMM), in addition to mutant CHO cells harboring mutations at different stages of glycosylation. The data show a substantial reduction in the interactions between the altered glycosylation forms of S1 and ACE2 in the presence of NB-DNJ, while varied outcomes resulted from dMM treatment. These results highlight the promising effects of NB-DNJ and its potential use as an off-label drug to treat SARS-CoV-2 infections.

Chrysin bonded to β-d-glucose tetraacetate enhances its protective effects against the neurotoxicity induced by aluminum in Swiss mice.

To evaluate whether the glycosylation of chrysin (CHR) enhances its protective effects against aluminum-induced neurotoxicity. To compare the antioxidant, anticholinesterase, and behavioral effects of CHR with its glycosylated form (CHR bonded to β-d-glucose tetraacetate, denoted as LQFM280), we employed an integrated approach using both in vitro (SH-SY5Y cells) and in vivo (aluminum-induced neurotoxicity in Swiss mice) models. LQFM280 demonstrated higher antioxidant activity than CHR in both models. Specifically, LQFM280 exhibited the ability to exert antioxidant effects in the cytoplasm of SH-SY5Y cells, indicating its competence in traversing neuronal membranes. Remarkably, LQFM280 proved more effective than CHR in recovering memory loss and counteracting neuronal death in the aluminum chloride mice model, suggesting its increased bioavailability at the brain level. The glycosylation of CHR with β-d-glucose tetraacetate amplifies its neuroprotective effects, positioning LQFM280 as a promising lead compound for safeguarding against neurodegenerative processes involving oxidative stress.

Protein glycosylation in cardiovascular health and disease.

Protein glycosylation, which involves the attachment of carbohydrates to proteins, is one of the most abundant protein co-translational and post-translational modifications. Advances in technology have substantially increased our knowledge of the biosynthetic pathways involved in protein glycosylation, as well as how changes in glycosylation can affect cell function. In addition, our understanding of the role of protein glycosylation in disease processes is growing, particularly in the context of immune system function, infectious diseases, neurodegeneration and cancer. Several decades ago, cell surface glycoproteins were found to have an important role in regulating ion transport across the cardiac sarcolemma. However, with very few exceptions, our understanding of how changes in protein glycosylation influence cardiovascular (patho)physiology remains remarkably limited. Therefore, in this Review, we aim to provide an overview of N-linked and O-linked protein glycosylation, including intracellular O-linked N-acetylglucosamine protein modification. We discuss our current understanding of how all forms of protein glycosylation contribute to normal cardiovascular function and their roles in cardiovascular disease. Finally, we highlight potential gaps in our knowledge about the effects of protein glycosylation on the heart and vascular system, highlighting areas for future research.

Efficient Expression in Leishmania tarentolae (LEXSY) of the Receptor-Binding Domain of the SARS-CoV-2 S-Protein and the Acetylcholine-Binding Protein from Lymnaea stagnalis.

Leishmania tarentolae (LEXSY) system is an inexpensive and effective expression approach for various research and medical purposes. The stated advantages of this system are the possibility of obtaining the soluble product in the cytoplasm, a high probability of correct protein folding with a full range of post-translational modifications (including uniform glycosylation), and the possibility of expressing multi-subunit proteins. In this paper, a LEXSY expression system has been employed for obtaining the receptor binding domain (RBD) of the spike-protein of the SARS-CoV-2 virus and the homopentameric acetylcholine-binding protein (AChBP) from Lymnaea stagnalis. RBD is actively used to obtain antibodies against the virus and in various scientific studies on the molecular mechanisms of the interaction of the virus with host cell targets. AChBP represents an excellent structural model of the ligand-binding extracellular domain of all subtypes of nicotinic acetylcholine receptors (nAChRs). Both products were obtained in a soluble glycosylated form, and their structural and functional characteristics were compared with those previously described.

The expression system influences stability, maturation efficiency, and oligomeric properties of the potassium-chloride co-transporter KCC2

The neuron-specific K+/Cl− co-transporter 2, KCC2, which is critical for brain development, regulates γ-aminobutyric acid-dependent inhibitory neurotransmission. Consistent with its function, mutations in KCC2 are linked to neurodevelopmental disorders, including epilepsy, schizophrenia, and autism. KCC2 possesses 12 transmembrane spans and forms an intertwined dimer. Based on its complex architecture and function, reduced cell surface expression and/or activity have been reported when select disease-associated mutations are present in the gene encoding the protein, SLC12A5. These data suggest that KCC2 might be inherently unstable, as seen for other complex polytopic ion channels, thus making it susceptible to cellular quality control pathways that degrade misfolded proteins. To test these hypotheses, we examined KCC2 stability and/or maturation in five model systems: yeast, HEK293 cells, primary rat neurons, and rat and human brain synaptosomes. Although studies in yeast revealed that KCC2 is selected for endoplasmic reticulum-associated degradation (ERAD), experiments in HEK293 cells supported a more subtle role for ERAD in maintaining steady-state levels of KCC2. Nevertheless, this system allowed for an analysis of KCC2 glycosylation in the ER and Golgi, which serves as a read-out for transport through the secretory pathway. In turn, KCC2 was remarkably stable in primary rat neurons, suggesting that KCC2 folds efficiently in more native systems. Consistent with these data, the mature glycosylated form of KCC2 was abundant in primary rat neurons as well as in rat and human brain. Together, this work details the first insights into the influence that the cellular and membrane environments have on several fundamental KCC2 properties, acknowledges the advantages and disadvantages of each system, and helps set the stage for future experiments to assess KCC2 in a normal or disease setting.