Glycosylated Form Research Articles

Expression and secretion of apoE isoforms in astrocytes and microglia during inflammation.

Neuroinflammation is a common feature in neurodegenerative diseases, modulated by the Alzheimer's disease risk factor, apolipoprotein E (APOE). In the brain, apoE protein is synthesized by astrocytes and microglia. We examined primary cultures of astrocytes and microglia from human APOE (E2, E3, and E4) targeted-replacement mice. Astrocytes secreted two species of apoE, whereas cellular apoE consisted of only one. Both forms of secreted astrocytic apoE were bound during glycoprotein isolation, and enzymatic removal of glycans produced a convergence of the two forms of apoE to a single form; thus, the two species of astrocyte-secreted apoE are differentially glycosylated. Microglia released only a single species of apoE, while cellular apoE consisted of two forms; the secreted apoE and one of the two forms of cellular apoE were glycosylated. We treated the primary glia with either endogenous (TNFα) or exogenous (LPS) pro-inflammatory stimuli. While LPS had no effect on astrocytic apoE, APOE2, and APOE3 microglia increased release of apoE; APOE4 microglia showed no effect. APOE4 microglia showed higher baseline secretion of TNFα compared to APOE2 and APOE3 microglia. TNFα treatment reduced the secretion and cellular expression of apoE only in APOE4 astrocytes. The patterns of apoE species produced by astrocytes and microglia were not affected by inflammation. No changes in APOE mRNA were observed in astrocytes after both treatments. Together, our data demonstrate that astrocytes and microglia differentially express and secrete glycosylated forms of apoE and that APOE4 astrocytes and microglia are deficient in immunomodulation compared to APOE2 and APOE3.

Aberrant RL2 O-GlcNAc antibody reactivity against serum-IgA1 of patients with colorectal cancer.

O-GlcNAcylation, a single attachment of N-acetylglucosamine (GlcNAc) on serine and threonine residues, plays important roles in normal and pathobiological states of many diseases. Aberrant expression of O-GlcNAc modification was found in many types of cancer including colorectal cancer (CRC). This modification mainly occurs in nuclear-cytoplasmic proteins; however, it can exist in some extracellular and secretory proteins. In this study, we investigated whether O-GlcNAc-modified proteins are present in serum of patients with CRC. Serum glycoproteins of CRC patients and healthy controls were enriched by wheat germ agglutinin, a glycan binding protein specifically binds to terminal GlcNAc and sialic acid. Two-dimensional gel electrophoresis, RL2 O-GlcNAc immunoblotting, affinity purification, and mass spectrometry were performed. The results showed that RL2 O-GlcNAc antibody predominantly reacted against serum immunoglobulin A1 (IgA1). The levels of RL2-reacted IgA were significantly increased while total IgA were not different in patients with CRC compared to those of healthy controls. Analyses by ion trap mass spectrometry using collision-induced dissociation and electron-transfer dissociation modes revealed one O-linked N-acetylhexosamine modification site at Ser268 located in the heavy constant region of IgA1; unfortunately, it cannot be discriminated whether it was N-acetylglucosamine or N-acetylgalactosamine because of their identical molecular mass. Although failed to demonstrate unequivocally it was O-GlcNAc, these data indicated that serum-IgA had an aberrantly increased reactivity against RL2 O-GlcNAc antibody in CRC patients. This specific glycosylated form of serum-IgA1 will expand the spectrum of aberrant glycosylation which provides valuable information to cancer glycobiology.

Magnetically immobilized edible Bacillus natto for the biotransformation of polydatin to resveratrol and its bioactivity assessment

Resveratrol is a natural polyphenolic compound widely present in plants, which has been widely used in in food additives, beverages, cosmetics and other fields. Whereas, the applicability of resveratrol is limited since it mainly exits in glycosylated form namely polydatin. In the present study, a novel biotransformation strategy was created to produce resveratrol from P. cuspidatum by magnetically immobilized Bacillus natto strains. The biotransformation conditions were statistically investigated though Box-Behnken design (BBD) with three-factor based on single-factor experiments. The optimal conditions were obtained as follows: treated temperature 37 ℃, fermented time 46 h and immobilized beads/solid ratio of 1.4 g/g. After biotransformation, the maximum biotransformation rate of polydatin reached 98.60 %, and the resveratrol yield achieved 11.27 mg/g, which was 18.18-fold to untreated one. In addition, the recovered magnetically immobilized Bacillus natto strains was still obtained 15.47-fold resveratrol yield to that of unfermented one after 3 runs. The new method established in this work could be a sustainable and effective biotransformation technology for resveratrol production from plant.

Sugar Matters: Improving In Vivo Clearance Rate of Highly Glycosylated Recombinant Plasma Proteins for Therapeutic Use.

Correct glycosylation of proteins is essential for production of therapeutic proteins as glycosylation is important for protein solubility, stability, half-life and immunogenicity. The heavily glycosylated plasma protein C1-inhibitor (C1-INH) is used in treatment of hereditary angioedema attacks. In this study, we used C1-INH as a model protein to propose an approach to develop recombinant glycoproteins with the desired glycosylation. We produced fully functional recombinant C1-INH in Chinese hamster ovary (CHO) cells. In vivo we observed a biphasic clearance, indicating different glycosylation forms. N-glycan analysis with mass spectrometry indeed demonstrated heterogeneous glycosylation for recombinant C1-INH containing terminal galactose and terminal sialic acid. Using a Ricinus Communis Agglutinin I (RCA120) column, we could reduce the relative abundance of terminal galactose and increase the relative abundance of terminal sialic acid. This resulted in a fully active protein with a similar in vivo clearance rate to plasmaderived C1-INH. In summary, we describe the development of a recombinant human glycoprotein using simple screening tools to obtain a product that is similar in function and in vivo clearance rate to its plasma-derived counterpart. The approach used here is of potential use in the development of other therapeutic recombinant human glycoproteins.

Influenza A Virus Modulates ACE2 Expression and SARS-CoV-2 Infectivity in Human Cardiomyocytes

Rationale: Influenza A virus (IAV) and SARS-CoV-2 are both acute respiratory viruses currently circulating in the human population. Prior IAV infection enhances SARS-CoV-2 infectivity and lung pathogenesis in mice; however, underlying mechanisms and the extent that co-infection leads to involvement of other organs in disease severity remains unknown. Objective: Herein, we investigated the impact of prior IAV infection on SARS-CoV-2 pathogenesis and cardiomyocyte function. Methods and Results: IAV infection in human primary lung epithelial cells, lung fibroblasts, macrophages, cardiac fibroblasts, and hiPSC-Cardiomyocytes enhances the expression of, the SARS-CoV-2 receptor ACE2. Also, we found that IAV infection enhances levels of ACE2 in the lungs of mice and humans. Interestingly, we detected poorly glycosylated ACE2 in A549 lung cells, primary lung epithelial cells, and cardiac fibroblasts. In contrast, expression of a heavily glycosylated form of ACE2 is induced by IAV infection in cardiomyocytes. In all cell types, IAV infection enhances SARS-CoV-2 spike protein binding and viral entry. However, efficient SARS-CoV-2 replication was uniquely inhibited in cardiomyocytes. Glycosylation of ACE2 correlated with enzymatic conversion of its substrate Ang II, induction of eNOS and nitric oxide production, providing a mechanistic underpinning for restricted viral replication in cardiomyocytes. Conclusions: Our results indicate that IAV-mediated induction of ACE2 is double-edged, providing increased risk for SARS-CoV-2 coinfection in human lung epithelial cells and cardiac fibroblasts , while limiting SARS-CoV-2 replication in cardiomyocytes. We conclude that differential glycosylation of ACE2 may be a molecular determinant- not of SARS-CoV-2 infection, but of replication. Funding: The work was supported by NIH grants AI 146252 and AI 146690. Declaration of Interests: The authors declare that they have no conflict of interest. Ethics Approval Statement: All procedures were approved by the OSU IACUC.We have obtained ethics approval from the Ohio State University, The Institutional Animal Care and Use Committee (#20160100-R1), Institutional Biosafety Committee (#20200135) for animal work and Institutional Review Board (#2020H0222) for human subjects.

Effects of methyl jasmonate on the monoterpenes of Muscat Hamburg grapes and wine.

The importance of monoterpenes in grape and wine aroma has compelled researchers to focus on developing methods to increase their abundance. Recent research has revealed that exogenous elicitors can increase the contents of these compounds. This study determined the effects of methyl jasmonate (MeJA) preharvest treatments on the monoterpene profiles of Muscat Hamburg grapes and wine. A total of 27 monoterpenes were identified for Muscat Hamburg grapes and wine. The contents of most of the monoterpenes (free and glycosylated forms) in the grapes and wine increased in response to MeJA. An analysis of the expression of the genes in the terpenoid biosynthesis pathway indicated that the related biosynthetic pathways were activated by MeJA. The transcript levels of some genes were consistent with monoterpene production, including VviCSLinNer, VviGwbOciF, VviPNRLin, VviGT14 and VviUGT85A1L1. The developmental expression patterns of the VviPP2B1 and VviMYB24 transcription factor genes were positively correlated with monoterpene accumulation in ripening grapes. Our results suggest that MeJA may be useful for improving the aroma quality of grapes and wines.

Improving the Recombinant Protein Expression of Human Galectin-3 in BL21 Bacterial Host System

Background: Regardless of the broad explore in the territory of glycobiology concerning structure and capacity of glycans, lectins and glycosylation forms, numerous viewpoints are still left unexplored.
 Aim: In this study, we analyzed the effect of shuttle vector on the secretion of human galectin recombinant protein.
 Methods: The galectin was expressed in E. coli BL21 by growing the bacterial culture in SOC medium and purified by nickel-based affinity chromatography due to its His-tag.
 Results: After cell lysis the protein was identified as a single 29 KDa band by 12% SDS-PAGE. Conclusion: Characterization studies clearly revealed that the purified protein was indeed galectin 3.

Conditions of enzyme-assisted extraction to increase the recovery of flavanone aglycones from pectin waste.

The citrus pectin by-product (CPB), generated from pectin industry, is a rich-source of flavanones, but not explored until now. As most of these compounds are inside vacuoles or bound to cell wall matrix, enzymatic hydrolysis was applied on their recovery, followed by hydroalcoholic and ultrasound extraction. Different parameters were studied: enzymes (β-glucosidase, tannase, and cellulase), their concentration (5, 10, and 20 U g-1 CPB), and reaction time (6, 12, and 24h). Extracts were characterized in total phenolic content (TPC), antioxidant capacity (ORAC and DPPH assays), and polyphenolic profile (HPLC-DAD). All enzymatic treatments significantly improved CPB antioxidant capacity and TPC, compared with hydroalcoholic and ultrasound extraction. β-glucosidase (5 U) for 24h was the most effective in polyphenol extraction and bioconversion, followed by β-glucosidase (5 U) for 12h and tannase (5 U) for 24h. Thus, the concentration of these enzymes was increased (10 and 20 U) to improve flavanones extraction. β-glucosidase at 20 U offered the highest amount of naringenin (77.63mg 100g-1 of CPB) and hesperetin (766.44mg 100g-1) obtained so far by biological processes. According to Person's correlation analysis, TPC and antioxidant activity were highly correlated with CPB contents of hesperetin and naringenin. The aglycone flavanones are rarely found in natural sources and have higher biological potential than their glycosylated forms. Our results indicated enzyme-assisted extraction as a good choice for recovering aglycone flavanones from CPB, and increased knowledge on the biological activity of this agroindustrial waste, amplifying their application in food and pharmaceutical field.

Differentiation of endogenous erythropoietin and exogenous ESAs by Western blotting

Doping tests for the illegal use of erythropoiesis-stimulating agents (ESAs) have been developed. We developed a new Western blotting method to detect and distinguish endogenous erythropoietin (Epo, 35-38 kDa) and exogenous ESAs (epoetin α and β, 38-42 kDa; darbepoetin α, 47-50 kDa; epoetin β pegol, 93-110 kDa). Epo and ESAs are glycoproteins and deglycosylation using peptide-N-glycosidase F shifted all Epo and ESA bands except epoetin β pegol to 22 kDa. We cut the bands of Epo and ESAs from SDS-PAGE gels and analyzed them by Liquid Chromatography/Mass Spectrometry (LC/MS). LC/MS detected all endogenous Epo and exogenous ESAs as deglycosylated 22 kDa Epo, indicating that LC/MS analysis could confirm the presence of Epo or ESA, but could not distinguish between endogenous Epo and exogenous ESAs. We propose the following Epo doping tests: 1) detect Epo or ESAs by Western blotting of the glycosylated form; 2) increase the reliability by the band shift following deglycosylation; and 3) complete confirmation of Epo or ESA by LC/MS analysis using cut gels. One of the advantages of our method is that pre-purification of samples for Epo is not required in our Western blotting.

Potential bioactive glycosylated flavonoids as SARS-CoV-2 main protease inhibitors: A molecular docking and simulation studies.

A novel coronavirus responsible of acute respiratory infection closely related to SARS-CoV has recently emerged. So far there is no consensus for drug treatment to stop the spread of the virus. Discovery of a drug that would limit the virus expansion is one of the biggest challenges faced by the humanity in the last decades. In this perspective, to test existing drugs as inhibitors of SARS-CoV-2 main protease is a good approach. Among natural phenolic compounds found in plants, fruit, and vegetables; flavonoids are the most abundant. Flavonoids, especially in their glycosylated forms, display a number of physiological activities, which makes them interesting to investigate as antiviral molecules. The flavonoids chemical structures were downloaded from PubChem and protease structure 6LU7 was from the Protein Data Bank site. Molecular docking study was performed using AutoDock Vina. Among the tested molecules Quercetin-3-O-rhamnoside showed the highest binding affinity (-9,7 kcal/mol). Docking studies showed that glycosylated flavonoids are good inhibitors for the SARS-CoV-2 protease and could be further investigated by in vitro and in vivo experiments for further validation. MD simulations were further performed to evaluate the dynamic behavior and stability of the protein in complex with the three best hits of docking experiments. Our results indicate that the rutin is a potential drug to inhibit the function of Chymotrypsin-like protease (3CL pro) of Coronavirus.

Glycosylated Siglec-6 expression in syncytiotrophoblast-derived extracellular vesicles from preeclampsia placentas

IntroductionPreeclampsia (PE) is associated with an exaggerated maternal systemic inflammatory response. Throughout gestation, the placenta releases extracellular vesicles through the syncytiotrophoblast layer (STB) into the maternal circulation and this is increased in PE. Expression of Siglec-6, a transmembrane receptor of molecular weight 50 KDa, is upregulated in PE placental tissue. MethodsHere we investigated respective abundance of Siglec-6 in PE -and normal pregnancy- (NP) derived placental lysates (PL) and syncytiotrophoblast-derived extracellular vesicles (STBEV). STBEV from PE and NP placentas were isolated through dual-lobe placental perfusion and serial ultracentrifugation. Siglec-6 was characterized by immunohistochemistry, immunoblotting, mass spectrometry (MS), and deglycosylation. ResultsImmunoblotting revealed the expected Siglec-6 (50 KDa) band present in both PE and NP PL, however an additional heavier band was observed at 70 KDa only in PE PL, but not in NP. When interrogating STBEV we saw an absence of the expected 50 KDa band but the 70 KDa was present predominantly only in the PE STBEV. Deglycosylation of PL and STBEV from PE showed that the 70 KDa and the 50 KDa bands were reduced to 48 KDa, suggesting glycosylation. Both 48 KDa and 70 KDa bands were subjected to MS, confirming Siglec-6 expression in both. DiscussionOur data shows that the inability to detect Siglec-6 in circulation might be due to the placenta secreting STBEV carrying a modified glycosylated form of Siglec-6 with a 70 KDa molecular weight, significantly and uniquely upregulated in PE STBEV.

Defending Effects of Iodide Transfer in Placental Barrier Against Maternal Iodine Deficiency.

Objective: Placental iodide transport is necessary for maintaining an adequate iodide supply to the developing fetus. We hypothesized that compounds from the placental barrier can compensate for decreases in maternal iodine intake and normalize fetal iodine levels. Methods: Pregnant rats administered different amounts of iodine (1.24, 2.5, 5, or 10 μg/day) were evaluated on gestational day (gd) 16 and 20. The iodine levels in maternal blood, amniotic fluid (AF), and placental tissue were estimated using As-Ce catalytic spectrophotometry. The protein and/or messenger RNA (mRNA) levels of sodium iodide symporter (NIS), pendrin, alpha-smooth muscle actin (α-SMA), and CD31 in the placental labyrinth, trophoblast cells isolated using laser capture microdissection (LCM), and/or fetomaternal thyroid were detected using immunoblotting, real-time polymerase chain reaction, and/or immunohistochemistry. Results: When iodine intake was reduced, iodine levels in maternal blood gradually decreased; however, placental iodine levels were not significantly different between groups on gd16 and gd20. Minimal changes were observed in AF iodine levels on gd16, and a mild decreasing trend was observed (iodine dose, 10 to 1.24 μg/day) on gd20. NIS protein, which was linearly distributed along the basolateral membrane of maternal-fetal thyroid follicles, gradually increased with decreasing iodine levels. Regarding iodine deficiency in the placental labyrinth on gd16 and gd20, pendrin and glycosylated NIS proteins were significantly upregulated in a dose-dependent manner. However, the mRNA levels were unchanged. Furthermore, the conversion of NIS protein from the nonglycosylated to the glycosylated form increased. In trophoblast cells isolated using LCM, PDS mRNA levels increased in the 1.24-μg/day group on gd16 but not NIS mRNA levels. There was a smaller α-SMA+ area in the labyrinth zone on gd16 and gd20; however, the proportional CD31+ area increased on gd16 and reduced on gd20 with decreased iodine levels. Conclusions: All mechanisms upregulating the expression of iodine transporters and changes in villous stroma and microvessel area in the placental labyrinth can promote iodide transfer from mother to fetus in iodine deficiency, especially before the onset of fetal thyroid function. Compensatory NIS protein regulation in the placenta against decreased iodine intake mainly occurs during translation and glycosylation modification after translation. Pendrin may be more important than NIS in the mediation of placental iodide transport.

Confrontation assays and mycotoxin treatment reveal antagonistic activities of Trichoderma and the fate of Fusarium mycotoxins in microbial interaction

Mycotoxins are toxic fungal metabolites, contaminating cereal grains in field or during processing and storage periods. These environmental contaminants pose great threats to humans and animals’ health due to their toxic effects. Type A trichothecenes, fumonisins and fusaric acid (FA) are commonly detected mycotoxins produced by various Fusarium species. Trichoderma spp. are promising antagonists in agriculture for their activities against plant pathogens, and also regarded as potential candidates for bioremediation of environmental contaminants. Managing toxigenic fungi by antagonistic Trichoderma is regarded as a sustainable and eco-friendly strategy for mycotoxin control. However, the metabolic activities of Trichoderma on natural occurring mycotoxins were less investigated. Our current work comprehensively explored the activities of Trichoderma against type A trichothecenes, fumonisins and FA producing Fusarium species via co-culture competition and indirect volatile assays. Furthermore, we investigated metabolism of type A trichothecenes and FA in Trichoderma isolates. Results indicated that Trichoderma were capable of bio-transforming T-2 toxin, HT-2 toxin, diacetoxyscirpenol and neosolaniol into their glycosylated forms and one Trichoderma strain could bio transform FA into low toxic fusarinol. These findings proved that Trichoderma isolates could manage toxigenic Fusarium via direct competition and volatile-mediated indirect inhibition. In addition, these antagonists possess defensive systems against mycotoxins for self-protection, which enriches our understanding on the interaction mechanism of Trichoderma spp. on toxigenic fungus.

The role of O-GlcNAcylation in immunity against infections.

SummaryMounting an effective immune response is crucial for the host to protect itself against invading pathogens. It is now well appreciated that reprogramming of core metabolic pathways in immune cells is a key requirement for their activation and function during infections. The role of several ancillary metabolic pathways in shaping immune cell function is less well understood. One such pathway, for which interest has recently been growing, is the hexosamine biosynthesis pathway (HBP) that generates uridine diphosphate N‐acetylglucosamine (UDP‐GlcNAc), the donor substrate for a specific form of glycosylation termed O‐GlcNAcylation. O‐GlcNAc is an intracellular post‐translational modification that alters the functional properties of the modified proteins, in particular transcription factors and epigenetic regulators. An increasing number of studies suggest a central role for the HBP and O‐GlcNAcylation in dictating immune cell function, including the response to different pathogens. We here discuss the most recent insights regarding O‐GlcNAcylation and immunity, and explore whether targeting of O‐GlcNAcylation could hold promise as a therapeutic approach to modulate immune responses to infections.

Uptake and Glycosylation of Smoke-Derived Volatile Phenols by Cabernet Sauvignon Grapes and Their Subsequent Fate during Winemaking.

Wine made from grapes exposed to bushfire smoke can exhibit unpleasant smoky, ashy characters, which have been attributed to the presence of smoke-derived volatile phenols, in free or glycosylated forms. Here we report the uptake and glycosylation of volatile phenols by grapes following exposure of Cabernet Sauvignon vines to smoke, and their fate during winemaking. A significant delay was observed in the conversion of volatile phenols to their corresponding glycoconjugates, which suggests sequestration, the presence of intermediates within the glycosylation pathway and/or other volatile phenol storage forms. This finding has implications for industry in terms of detecting smoke-affected grapes following vineyard smoke exposure. The potential for an in-canopy sprinkler system to mitigate the uptake of smoke-derived volatile phenols by grapes, by spraying grapevines with water during smoke exposure, was also evaluated. While “misting” appeared to partially mitigate the uptake of volatile phenols by grapes during grapevine exposure to smoke, it did not readily influence the concentration of volatile phenols or the sensory perception of smoke taint in wine. Commercial sensors were used to monitor the concentration of smoke particulate matter (PM) during grapevine exposure to low and high density smoke. Similar PM profiles were observed, irrespective of smoke density, such that PM concentrations did not reflect the extent of smoke exposure by grapes or risk of taint in wine. The sensors could nevertheless be used to monitor the presence of smoke in vineyards during bushfires, and hence, the need for compositional analysis of grapes to quantify smoke taint marker compounds.

LC-MS Based Metabolite Fingerprinting of Coccinia Plants Reveals Glycoisomerization as a Structual Diversification Strategy in Flavanoid Chemical Space

Flavonoids are ubiquitous plant metabolites with a lot of purported pharmacological properties. Emergence of advanced analytical instrumentations such as liquid chromatography hyphenated to mass spectrometry (LC–MS) has allowed for unparallel elucidation of chemical compositions of various plants. Through such analyses, various chemical modifications such as glycosylation and other forms of isomerization have been shown to be an evolutionary strategy of plants to diversify their metabolite compositions (metabolome). Herein, metabolite fingerprinting of two closely related plant species vis Coccinia grandis and Coccinia rehmannii revealed that flavonoids attached to either di- or tri-saccharides are prone to isomerization, a phenomenon termed glycoisomerization herein. Furthermore, modification known as sugar acylation by known biologically active metabolites such as derivatives of cinnamic acids (i. e., caffeic acid and coumaric acid) has been noted in C. rehmannii but not in C. grandis. The findings of the current study reveal glycosylation patterns of flavonoids as an evolutionary strategy used by plants to diversify its metabolome, a phenomenon of which the biological consequence is still to be investigated.

A Review on Luteolin: A Versatile Flavonoid

Luteolin (3′,4′,5,7-tetrahydroxy flavone) is a flavonoid belonging to the subgroup of flavone, it is a naturally occurring compound in its glycosylated form, and exists in many types of plants including fruits, vegetables, and medicinal herbs. It has been suggested in preclinical studies that flavones possesses a variety of pharmacological activities, including antioxidant, anti-inflammatory, antimicrobial and anticancer activities. Like other flavones, it has been seen that plants rich in luteolin have been used in traditional medicine for treating various diseases. The purpose of this article is to systematically explain different aspects of luteolin with respect to its biosynthesis, total chemical synthesis, structure activity relationship and the aspects of the structure making it an effective pharmacological agent, it’s pharmacokinetic aspects and its pharmacological and therapeutic activity on the human body. Here pharmacological effects of luteolin is an important aspect because of its ability to inhibit angiogenesis, to induce apoptosis, to prevent carcinogenesis in animal models thereby showing chemo-preventive and chemotherapeutic potential, show antioxidant activity, anti-inflammatory activity, anti-allergic effect, antispasmodic activity, anti-ulcer, anti-ageing , whitening activity , activity to mitigate psoriasis as well as have prominent neuroprotective effects. Keywords: Anti-allergic, anti-inflammatory, antioxidant, biosynthesis, chemoprotective, Luteolin, neuroprotective, pharmacokinetics, structure activity relationship Cite this Article Aditi Ashish Datta, Abhishek Ashish Datta. A Review on Luteolin: A Versatile Flavonoid. Research & Reviews: A Journal of Drug Formulation, Development and Production . 2020; 7(2): 33–61p.

Multi-histidine functionalized material for the specific enrichment of sialylated glycopeptides.

Sialylation, an important form of glycosylation, is involved in many biological processes and plays an important role in the development of diseases. However, due to the low abundance among various glycosylation and lack of efficient enrichment method with high specificity, the study of sialylation remains a challenge. Herein, multi-histidine modified microspheres (MHM) were synthesized to enrich sialylated glycopeptides. It was found that MHM could selectively enrich sialylated glycopeptides from over 100 times of non-sialylated glycopeptides, which indicated MHM possessed good enrichment specificity towards sialylated glycopeptides. Furthermore, MHM were utilized to the large-scale analysis of protein sialylation, and 510 intact glycopeptides were identified with over 94.5% sialylated glycopeptide specificity from 4 μL human serum. The good specificity could be attributed to the synergistic effect by the electrostatic interaction and hydrophilic interaction. Hence, MHM could provide an alternative approach for the analysis of site-specific sialylation at proteome level from complex biological samples.

C-Mannosylation Enhances the Structural Stability of Human RNase 2.

SummaryC-Mannosylation is a relatively rare form of protein glycosylation involving the attachment of an α-mannopyranosyl residue to C-2 of the indole moiety of the amino acid tryptophan. This type of linkage was initially discovered in RNase 2 from human urine but later confirmed to be present in many other important proteins. Based on NMR experiments and extensive molecular dynamics simulations on the hundred microsecond timescale we demonstrate that, for isolated glycopeptides and denatured RNase 2, the C-linked mannopyranosyl residue exists as an ensemble of conformations, among which 1C4 is the most abundant. However, for native RNase 2, molecular dynamics and NMR studies revealed that the mannopyranosyl residue favors a specific conformation, which optimally stabilizes the protein fold through a network of hydrogen bonds and which leads to a significant reduction of the protein dynamics on the microsecond timescale. Our findings contribute to the understanding of the biological role of C-mannosylation.

Influence of Different Dehydration Levels on Volatile Profiles, Phenolic Contents and Skin Hardness of Alkaline Pre-Treated Grapes cv Muscat of Alexandria (Vitis vinifera L.)

A dehydration experiment was carried out on Vitis vinifera L. cv Muscat of Alexandria (synonym Zibibbo) following the process for the production of renowned special dessert wines produced on Pantelleria island (Sicily, Italy). Harvested berries were pre-treated in a sodium hydroxide dipping solution (45 g/L, dipped for 185 s, 25 °C) to accelerate the drying process, rinsed, and dehydrated in simulated conditions (relative humidity 30%, 30 °C temperature, air speed 0.9 m/s). Three dehydration levels were achieved, corresponding to “Passolata”, “Bionda”, and “Malaga” stages (35%, 50%, and 65% of weight loss, respectively) of the Pantelleria denomination of origin (DOC). Grape skin mechanical properties, technological parameters, phenolics, and aroma profile varied considerably during dehydration. The most important aroma compounds for their olfactory impact, such as linalool, geraniol, nerol, and citronellol, especially in glycosylated forms, significantly increased in dried grapes compared to fresh ones, even if aroma profile modification occurred. A decrease in break skin force could have induced higher release of flavonoids. The findings showed relevant changes, allowing winemakers to better select the ratio of fresh and dehydrated grapes in the function of the final desired wine.