Deglycosylated Form Research Articles

Different Effects of Deglycosylation on the Lactose Sensing Ability of Mesophilic and Thermophilic Cellobiose Dehydrogenases.

The development of an efficient lactose biosensor employing cellobiose dehydrogenases (CDHs) for monitoring and precise control of the lactose levels in dairy-based products is extremely important for the health of lactose-intolerant population. In this study, the mesophilic (Nc_CDH) and thermophilic (Ct_CDH-A, Ct_CDH-B) CDHs were successfully obtained by heterologous expression and treated with α-1,2-mannosidase and endoglycosidase H to prepare the deglycosylated forms (Nc_dCDH, Ct_dCDH-A, and Ct_dCDH-B); then, the effects of deglycosylation on the catalytic activity in solution and electrochemical performance on electrodes for lactose detection were systematically investigated. In solution, Nc_dCDH was more stable and had a higher Vmax value and lower KM value than Nc_CDH at different temperatures and pH values. In contrast, deglycosylation had adverse effects on the stability of Ct_CDH-A and Ct_CDH-B. When the CDHs mixed with multi-walled carbon nanotubes were dropped and immobilized on electrodes, with regard to Nc_CDH, in the presence of the same concentration of lactose, the detection current of the electrode modified with Nc_dCDH was higher than that of the electrode modified with Nc_CDH, and it had a lower detection limit (2.006mM) and higher sensitivity (39.37μA.mmol.L-1.cm-2). However, with respect to the thermophilic CDHs, the sensitivity was lowered and the detection limit was increased after deglycosylation. The discrepancy may result from two reasons: N-glycosylation may play a more crucial role in thermostability and structural stability of thermophilic CDHs, and the distribution sites of glycosylated residues may affect the electron transfer kinetics. This study is a step toward using CDH as an electron transfer-based lactose biosensor.

Enhancing the anti-fatigue effect of ginsenoside extract by Bifidobacterium animalis subsp. lactis CCFM1274 fermentation through biotransformation of Rb1, Rb2 and Rc to Rd

The conversion of major ginsenosides into minor ginsenosides has been demonstrated to promote their absorption in vivo and enhance their bioavailability and bioactivity. In this study, we aimed to screen the strain that transforms the less efficient glycosylated ginsenosides (Rb1, Rc, and Rb2) into the highly efficient de-glycosylated form (Rd). The anti-fatigue ability and corresponding mechanism of fermented ginsenoside extracts (F-GE) from Rd-producing bacteria were evaluated using an exercise-induced model. Our results showed that Bifidobacterium animalis subsp. lactis CCFM1274 was capable of converting Rb1, Rc, and Rb2 into Rd in vitro. The anti-fatigue activity of ginsenoside extracts (GE) was significantly increased by CCFM1274 fermentation, characterized by an improvement in exhaustive swimming time (7.2 ± 0.8 to 10.7 ± 1.8 min) and histopathological changes. Moreover, F-GE treatment showed superior effects than GE in reducing the content of blood lactic acid (16.3 ± 3.6 to 13.7 ± 1.0 mmol/L), blood urea nitrogen (9.4 ± 0.6 to 5.2 ± 1.1 mmol/L), and malondialdehyde (9.3 ± 1.3 to 7.7 ± 2.1 nmol/mg), increasing the level of glycogen (1.1 ± 0.3 to 1.4 ± 0.3 mg/g), and superoxide dismutase activity (429.8 ± 107.8 to 671.3 ± 95.1 U/mg), which were associated with the upregulation of MCT1, LDHB, PDK4, GLUT4, Nrf2, and HO-1. Therefore, CCFM1274, which improved the bioactivity of ginsenoside, exhibited unique applicative potential for ginseng-based products to alleviate exercise fatigue.

Impact of Glycosylation on Protein-Protein Self-Interactions of Monoclonal Antibodies.

Protein self-interactions measured via second osmotic virial coefficients (B22) and dynamic light scattering interaction parameter values (kD) are often used as metrics for assessing the favorability of protein candidates and different formulations during monoclonal antibody (MAb) product development. Model predictions of B22 or kD typically do not account for glycans, though glycosylation can potentially impact experimental MAb self-interactions. To the best of our knowledge, the impact of MAb glycosylation on the experimentally measured B22 and kD values has not yet been reported. B22 and kD values of two fully deglycosylated MAbs and their native (i.e., fully glycosylated) counterparts were measured by light scattering over a range of pH and ionic strength conditions. Significant differences between B22 and kD of the native and deglycosylated forms were observed at a range of low to high ionic strengths used to modulate the effect of electrostatic contributions. Differences were most pronounced at low ionic strength, indicating that electrostatic interactions are a contributing factor. Though B22 and kD values were statistically equivalent at high ionic strengths where electrostatics were fully screened, we observed protein-dependent qualitative differences, which indicate that steric interactions may also play a role in the observed B22 and kD differences. A domain-level coarse-grained molecular model accounting for charge differences was considered to potentially provide additional insight but was not fully predictive of the behavior across all of the solution conditions investigated. This highlights that both the level of modeling and lack of inclusion of glycans may limit existing models in making quantitatively accurate predictions of self-interactions.

The Protective Role of Oleuropein Aglycone against Pesticide-Induced Toxicity in a Human Keratinocytes Cell Model.

The extensive use of agricultural pesticides to improve crop quality and yield significantly increased the risk to the public of exposure to small but repeated doses of pesticides over time through various routes, including skin, by increasing the risk of disease outbreaks. Although much work was conducted to reduce the use of pesticides in agriculture, little attention was paid to prevention, which could reduce the toxicity of pesticide exposure by reducing its impact on human health. Extra virgin olive oil (EVOO), a major component of the Mediterranean diet, exerts numerous health-promoting properties, many of which are attributed to oleuropein aglycone (OleA), the deglycosylated form of oleuropein, which is the main polyphenolic component of EVOO. In this work, three pesticides with different physicochemical and biological properties, namely oxadiazon (OXA), imidacloprid (IMID), and glyphosate (GLYPHO), were compared in terms of metabolic activity, mitochondrial function and epigenetic modulation in an in vitro cellular model of human HaCaT keratinocytes to mimic the pathway of dermal exposure. The potential protective effect of OleA against pesticide-induced cellular toxicity was then evaluated in a cell pre-treatment condition. This study showed that sub-lethal doses of OXA and IMID reduced the metabolic activity and mitochondrial functionality of HaCaT cells by inducing oxidative stress and altering intracellular calcium flux and caused epigenetic modification by reducing histone acetylation H3 and H4. GLYPHO, on the other hand, showed no evidence of cellular toxicity at the doses tested. Pretreatment of cells with OleA was able to protect cells from the damaging effects of the pesticides OXA and IMID by maintaining metabolic activity and mitochondrial function at a controlled level and preventing acetylation reduction, particularly of histone H3. In conclusion, the bioactive properties of OleA reported here could be of great pharmaceutical and health interest, as they could be further studied to design new formulations for the prevention of toxicity from exposure to pesticide use.

Distribution, biosynthesis and therapeutic potential of lignans.

Lignans have long been known for their abundant therapeutic properties due to their polyphenolic structure. Linseed is the richest plant source of lignans and has been studied widely for their properties. The most prevalent lignan, secoisolariciresinol diglucoside (SDG), is consumed with linseed and converted into mammalian lignans, enterodiol (END) and enterolactone (ENL), by the gut microbiota. SDG can easily be assessed using HPLC and its deglycosylated form viz secoisolariciresinol can be asses using GC-MS techniques. Variety of extraction and analysis methods has been reported for plant lignans. SDG is known to have therapeutic properties including anti-oxidant, anti-cancerous, anti-inflammatory, modulation of gene expression, anti-diabetic, estrogenic and anti-estrogenic. Despite a large number of bioactivities, strong evidences for the underlying mechanisms for most of the properties are still unknown. SDG is most studied for its anti-cancerous properties. But the use of lignans as anti-carcinogenic agent is limited and commercially not reported due to challenges of purification at commercial level, rapid metabolism, untargeted delivery and toxic compounds associated with lignans. Exploration of more prominent and active derivatives of SDG and their targeted drug delivery should be an important research toward the use of bioactive lignans of linseed.

Effects of N-linked glycans of bilirubin oxidase on direct electron transfer-type bioelectrocatalysis

Bilirubin oxidase from Myrothecium verrucaria (mBOD) is a promising enzyme for catalyzing the four-electron reduction of dioxygen into water and realizes direct electron transfer (DET)-type bioelectrocatalysis. It has two N-linked glycans (N-glycans), and N472 and N482 are known as binding sites. Both binding sites located on opposite side of the type I (T1) Cu, which is the electrode-active site of BOD. We investigated the effect of N-glycans on DET-type bioelectrocatalysis by performing electrochemical measurements using electrodes with controlled surface charges. Two types of BODs with different N-glycans, mBOD and recombinant BOD overexpressed in Pichia pastoris (pBOD), and their deglycosylated forms (dg-mBOD and dg-pBOD) were used in this study. Kinetic analysis of the steady-state catalytic waves revealed that both size and composition of N-glycans affected the orientation of adsorbed BODs on the electrodes. Interestingly, the most favorable orientation was achieved with pBOD, which has the largest N-glycans. Furthermore, the effect of the orientation control by the N-glycans is cooperative with electrostatic interaction.

The deglycosylated form of 1E12 inhibits platelet activation and prothrombotic effects induced by VITT antibodies.

In order to improve the safety of COVID-19 vaccines, there is an urgent need to unravel the pathogenesis of vaccine-induced immune thrombotic thrombocytopenia (VITT), a severe complication of recombinant adenoviral vector vaccines used to prevent COVID-19, and likely due to anti-platelet factor 4 (PF4) IgG antibodies. In this study, we demonstrated that 1E12, a chimeric anti-PF4 antibody with a human Fc fragment, fully mimics the effects of human VITT antibodies, as it activates platelets to a similar level in the presence of platelet factor 4 (PF4). Incubated with neutrophils, platelets and PF4, 1E12 also strongly induces NETosis, and in a microfluidic model of whole blood thrombosis, it triggers the formation of large platelet/leukocyte thrombi containing fibrin(ogen). In addition, a deglycosylated form of 1E12 (DG-1E12), which still binds PF4 but no longer interacts with Fcγ receptors, inhibits platelet, granulocyte and clotting activation induced by human anti-PF4 VITT antibodies. This strongly supports that 1E12 and VITT antibodies recognize overlapping epitopes on PF4. In conclusion, 1E12 is a potentially important tool to study the pathophysiology of VITT, and for establishing mouse models. On the other hand, DG-1E12 may help the development of a new drug that specifically neutralizes the pathogenic effect of autoimmune anti-PF4 antibodies, such as those associated with VITT.

The Deglycosylated Form of 1E12, a Monoclonal Anti-PF4 IgG, Strongly Inhibits Antibody-Triggered Cellular Activation in Vaccine-Induced Thrombotic Thrombocytopenia, and Is a Potential New Treatment for Vιττ

▪IntroductionVaccine-induced thrombotic thrombocytopenia (VITT) is a severe complication of recombinant adenoviral vector vaccines used to prevent COVID-19, likely due to anti-platelet factor 4 (PF4) IgG antibodies. The specificity and platelet-activating activity of VITT antibodies strikingly resemble that of antibodies detected in “autoimmune” heparin-induced thrombocytopenia (HIT), but their features remain poorly characterized. In particular, a better knowledge of these antibodies should help to understand the mechanisms leading to hypercoagulability and the particular thrombotic events observed in VITT, but rarely in typical HIT. We have recently developed a chimeric IgG1 anti-PF4 antibody, 1E12, which strongly mimics “autoimmune” HIT antibodies in terms of specificity and cellular effects. Therefore, we assessed whether 1E12 could mimic VITT antibodies. We then evaluated the capability of DG-1E12, a deglycosylated form of 1E12 unable to bind FcγR, to inhibit cellular activation induced by VITT antibodies.Methods and ResultsUsing a PF4-sensitized serotonin release assay (PF4-SRA) (Vayne C, New Engl J Med, 2021), we demonstrated that 1E12 (5 and 10 μg/mL) strongly activated platelets, with a pattern similar to that obtained with human VITT samples (n=7), i.e. in a PF4-dependent manner and without heparin. This platelet activation was inhibited by low heparin concentration (0.5 IU/mL), an effect also observed with VITT samples. Serotonin release induced by 1E12 was also fully inhibited by IV-3, a monoclonal antibody blocking FcγRIIa, or by IdeS, a bacterial protease that cleaves IgG and strongly inhibits the binding of IgG antibodies to FcγRIIa. This inhibitory effect of IV-3 and IdeS strongly supports that interactions between pathogenic anti-PF4 IgG and FcγRIIa play a central role in VITT.Incubation of 1E12 or VITT samples with isolated neutrophils (PMN) and platelets with PF4 (10 µg/mL) strongly induced DNA release and NETosis, supporting that PMN are involved in the processes leading to thrombosis in VITT. Furthermore, when whole blood from healthy donors incubated with 1E12 or VITT plasma was perfused in capillaries coated with von Willebrand Factor, numerous large platelet/leukocyte aggregates containing fibrin(ogen) were formed.To investigate whether 1E12 and VITT antibodies recognize overlapping epitopes on PF4, we then performed competitive assays with a deglycosylated form of 1E12 (DG-1E12), still able to bind PF4 but not to interact with Fcγ receptors. In PF4-SRA, pre-incubation of DG-1E12 (50 µg/mL) dramatically reduced platelet activation induced by VITT antibodies, which was fully abrogated for 9 of the 14 VITT samples tested. Additional experiments using a whole blood PF4-enhanced flow cytometry assay recently designed for VITT diagnosis (Handtke et al, Blood 2021), confirmed that DG-1E12 fully prevented platelet activation induced by VITT antibodies. Moreover, when platelets and neutrophils were pre-incubated with DG-1E12 (100 µg/mL), NETosis and thus DNA release, nuclear rounding, and DNA decondensation induced by VITT antibodies were completely inhibited. Finally, DG-1E12 (100 µg/mL) also fully abolished VITT antibody-mediated thrombus formation in whole blood in vitro under vein flow conditions. Comparatively, DG-1E12 did not inhibit ALB6, a murine monoclonal anti-CD9 antibody, which also strongly activates platelets in a FcγRIIa-dependent manner.ConclusionsOur results show that 1E12 exhibits features similar to those of human VITT antibodies in terms of specificity, affinity and cellular effects, and could therefore be used as a model antibody to study the pathophysiology of VITT. Our data also demonstrate that DG-1E12 prevents blood cell activation and thrombus formation induced by VITT antibodies, likely due to the competitive effect of its Fab fragment on antibody binding to PF4. DG-1E12 may allow the development of a new drug neutralizing the pathogenic effect of autoimmune anti-PF4 antibodies, such as those associated with VITT. DisclosuresThiele: Bristol Myers Squibb: Honoraria, Other; Pfizer: Honoraria, Other; Bayer: Honoraria; Chugai Pharma: Honoraria, Other; Novo Nordisk: Other; Novartis: Honoraria; Daichii Sankyo: Other. Pouplard: Stago: Research Funding. Greinacher: Macopharma: Honoraria; Biomarin/Prosensa: Other, Research Funding; Sagent: Other, Research Funding; Rovi: Other, Research Funding; Gore inc.: Other, Research Funding; Bayer Healthcare: Other, Research Funding; Paringenix: Other, Research Funding; BMS: Honoraria, Other, Research Funding; MSD: Honoraria, Other, Research Funding; Boehringer Ingelheim: Honoraria, Other, Research Funding; Aspen: Honoraria, Other, Research Funding; Portola: Other; Ergomed: Other; Instrument Laboratory: Honoraria; Chromatec: Honoraria. Gruel: Stago: Other: symposium fees, Research Funding. Rollin: Stago: Research Funding.

Structural model and functional properties of an exo-polygalacturonase from Neosartorya glabra

A purified exo-polygalacturonase of Neosartorya glabra (EplNg) was successfully characterized. EplNg native presented 68.2 kDa, with 32% carbohydrate content. The deglycosylated form showed 46.3 kDa and isoelectric point of 5.4. The identity of EplNg was confirmed as an exo-polygalacturonase class I (EC 3.2.1.67) using mass spectrometry and Western-Blotting. Capillary electrophoresis indicated that only galacturonic acid was released by the action of EplNg on sodium polypectate, confirming an exoenzyme character. The structural model confers that EplNg has a core formed by twisted parallel β-sheets structure. Among twelve putative cysteines, ten were predicted to form disulfide bridges. The catalytic triad predicted is composed of Asp223, Asp245, and Asp246 aligned along with a distance in 4–5 Å, suggesting that EplNg probably does not perform the standard inverting catalytic mechanism described for the GH28 family. EplNg was active from 30 to 90 °C, with maximum activity at 65 °C, pH 5.0. The Km and Vmax determined using sodium polypectate were 6.9 mg·mL−1 and Vmax 690 μmol·min−1.mg−1, respectively. EplNg was active and stable over a wide range of pH values and temperatures, confirming the interesting properties EplNg and provide a basis for the development of the enzyme in different biotechnological processes.

Retinoprotective Effects Of Crocin And Crocetin via Anti-Angiogenic Mechanism in High Glucose-Induced Human Retinal Pigment Epithelium Cells.

Diabetic retinopathy (DR) is one of the most common side effects of diabetes. We aimed to investigate the effects of crocin and crocetin (as a deglycosylated form of crocin in blood stream) in gene expression or protein levels of vascular endothelial growth factor (VEGF), vascular endothelial growth factor-receptor1 (VEGFR-1), matrix metalloproteinases2 (MMP-2), matrix metalloproteinases9 (MMP-9) and thrombospondin-2 (TSP-2) in high glucose cell culture media. The retinal pigment epithelium (RPE) cells were exposed to high glucose (HG, 30 mM glucose concentration) and normal glucose (NG, 24.5 mM mannitol + 5.5 mM glucose) for six days. RPE cells were treated in four treatment groups (crocin, crocetin, Bevacizumab, and crocin + Bevacizumab). Gene expressions were measured using quantitative real-time PCR, and protein levels were evaluated by western blot. Findings showed that VEGF gene expression and protein level significantly decreased in all treatment groups. In addition, reduction in VEGFR1 gene expression was significantly higher in Bevacizumab and crocin + Bevacizumab groups than other groups. Only crocin and crocetin could reduce the gene levels of MMP-2 and MMP-9. In addition, TSP-2 protein levels increased when HG cells were exposed to crocin or crocin + Bevacizumab groups. Our data showed that crocin and crocetin have anti-VEGF function similar to Bevacizumab, act as an anti-angiogenic agent. Also, crocin and crocetin could decrease MMP-2 and MMP-9 gene levels being inflammatory and angiogenesis factors. As a result, crocin and crocetin have protective effects against angiogenesis and inflammation in DR.

Genetic modification to design a stable yeast-expressed recombinant SARS-CoV-2 receptor binding domain as a COVID-19 vaccine candidate

BackgroundCoronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has now spread worldwide to infect over 110 million people, with approximately 2.5 million reported deaths. A safe and effective vaccine remains urgently needed. MethodWe constructed three variants of the recombinant receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein (residues 331–549) in yeast as follows: (1) a “wild type” RBD (RBD219-WT), (2) a deglycosylated form (RBD219-N1) by deleting the first N-glycosylation site, and (3) a combined deglycosylated and cysteine-mutagenized form (C538A-mutated variant (RBD219-N1C1)). We compared the expression yields, biophysical characteristics, and functionality of the proteins produced from these constructs. Results and conclusionsThese three recombinant RBDs showed similar secondary and tertiary structure thermal stability and had the same affinity to their receptor, angiotensin-converting enzyme 2 (ACE-2), suggesting that the selected deletion or mutations did not cause any significant structural changes or alteration of function. However, RBD219-N1C1 had a higher fermentation yield, was easier to purify, was not hyperglycosylated, and had a lower tendency to form oligomers, and thus was selected for further vaccine development and evaluation. General significanceBy genetic modification, we were able to design a better-controlled and more stable vaccine candidate, which is an essential and important criterion for any process and manufacturing of biologics or drugs for human use.

Glycosylation of β1 subunit plays a pivotal role in the toxin sensitivity and activation of BK channels

Background:The accessory β1 subunits, regulating the pharmacological and biophysical properties of BK channels, always undergo post-translational modifications, especially glycosylation. To date, it remains elusive whether the glycosylation contributes to the regulation of BK channels by β1 subunits. Methods:Herein, we combined the electrophysiological approach with molecular mutations and biochemical manipulation to investigate the function roles of N-glycosylation in β1 subunits.Results: The results show that deglycosylation of β1 subunits through double-site mutations (β1 N80A/N142A or β1 N80Q/N142Q) could significantly increase the inhibitory potency of iberiotoxin, a specific BK channel blocker. The deglycosylated channels also have a different sensitivity to martentoxin, another BK channel modulator with some remarkable effects as reported before. On the contrary to enhancing effects of martentoxin on glycosylated BK channels under the presence of cytoplasmic Ca2+, deglycosylated channels were not affected by the toxin. However, the deglycosylated channels were surprisingly inhibited by martentoxin under the absence of cytoplasmic Ca2+, while the glycosylated channels were not inhibited under this same condition. In addition, wild type BK (α+β1) channels treated with PNGase F also showed the same trend of pharmacological results to the mutants. Similar to this modulation of glycosylation on BK channel pharmacology, the deglycosylated forms of the channels were activated at a faster speed than the glycosylated ones. However, the V1/2 and slope were not changed by the glycosylation.Conclusion: The present study reveals that glycosylation is an indispensable determinant of the modulation of β1-subunit on BK channel pharmacology and its activation. The loss of glycosylation of β1 subunits could lead to the dysfunction of BK channel, resulting in a pathological state.

Germinated black soybean fermented with Lactobacillus pentosus SC65 alleviates DNFB-induced delayed-type hypersensitivity in C57BL/6N mice

Ethnopharmacological relevanceRhynchosia nulubilis (black soybean) has many applications in oriental medicine. It is traditionally used to treat disease related with high blood pressure, diabetes, inflammation, and osteoporosis. Furthermore, fermented soybean foods have traditionally been used for immunity enhancement in East Asia. However, the anti-inflammatory effects of germinated R. nulubilis (GR) against delayed-type hypersensitivity (DTH) are not fully understood. Aim of studyThis study aimed to investigate the anti-inflammatory effects of germinated Rhynchosia nulubilis (GR) fermented with the lactic acid bacterium Lactobacillus pentosus SC65 (GR-SC65) isolated from pickled burdock. Materials and methodsWe investigated the effects of GR-SC65 (300 mg/kg/day) on ear thickness and immune cell infiltration in DNFB-induced DTH in mice. We used dexamethasone (3 mg/kg) as a reference drug. Changes in infiltration of CD4+ and CD8+ T cells and NK cells were examined by immunohistochemistry. In addition, we investigated cytokine and chemokine production related to DTH using reverse transcription-polymerase chain reaction. We also investigated DTH-related cytokine production using lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. ResultsTwo lactic acid bacterial strains (Lactobacillus pentosus SC65 and Pediococcus pentosaceus ON81A) were selected for fermenting GR due to their high 2,2–diphenyl–1–picrylhydrazyl (DPPH) radical-scavenging activity. The total polyphenol contents (TPCs) in GR-SC65 and GR-ON81A were higher than that in unfermented GR (∗∗∗P < 0.001 vs. GR). Content of daidzein, glycitein, and genistein, the deglycosylated form of isoflavonoids, was higher in GR-SC65 than in unfermented GR. The ethanol extracts of GR-SC65 exerted a stronger anti-inflammatory activity than GR by inhibiting pro-inflammatory cytokines, such as tumor necrosis factor (TNF), interleukin-6 (IL-6), and interleukin-1β (IL-1β) in LPS-induced RAW264.7 macrophages. GR-SC65 reduced 1-fluoro-2,4-dinitrofluorobenzene (DNFB)-induced ear swelling and hyperplasia as well as vascular permeability. Fewer infiltrated CD4+ and CD8+ T cells were observed in the ear tissue of the GR-SC65-treated mice than those of the unfermented GR-treated mice. Furthermore, fewer infiltrated NK cells were observed in the GR-SC65 treated mice, than in the GR-treated mice. GR-SC65 significantly diminished the levels of CCL5 and COX-2 mRNAs and increased the level of IL-10 mRNA. ConclusionsThese data suggest that GR-SC65 can be used as a health supplement or a prophylactic against delayed-type hypersensitive inflammatory disease.

Fibrinogen-clotting enzyme, pictobin, from Bothrops pictus snake venom. Structural and functional characterization

A thrombin-like enzyme, pictobin, was purified from Bothrops pictus snake venom. It is a 41-kDa monomeric glycoprotein as showed by mass spectrometry and contains approx. 45% carbohydrate by mass which could be removed with N-glycosidase. Pictobin coagulates plasma and fibrinogen, releasing fibrinopeptide A and induces the formation of a friable/porous fibrin network as visualized by SEM. The enzyme promoted platelet aggregation in human PRP and defibrination in mouse model and showed catalytic activity on chromogenic substrates S-2266, S-2366, S-2160 and S-2238. Pictobin interacts with the plasma inhibitor α2-macroglobulin, which blocks its interaction with fibrinogen but not with the small substrate BApNA. Heparin does not affect its enzymatic activity. Pictobin cross reacted with polyvalent bothropic antivenom, and its deglycosylated form reduced its catalytic action and antivenom reaction. In breast and lung cancer cells, pictobin inhibits the fibronectin-stimulated migration. Moreover, it produces strong NADH oxidation, mitochondrial depolarization, ATP decrease and fragmentation of mitochondrial network. These results suggest by first time that a snake venom serinprotease produces mitochondrial dysfunction by affecting mitochondrial dynamics and bioenergetics. Structural model of pictobin reveals a conserved chymotrypsin fold β/β hydrolase. These data indicate that pictobin has therapeutic potential in the treatment of cardiovascular disorders and metastatic disease.

The occurrence of nonglycosylated forms of N-glycoprotein upon proteasome inhibition does not confirm cytosolic deglycosylation.

N-Glycosylated substrates that undergo ER-associated degradation (ERAD) are often deglycosylated by the cytosolic peptide:N-glycanase (Ngly1) during their proteasomal degradation in the cytosol. Consequently, the presence of non- or deglycosylated forms of such proteins after treatment with proteasome inhibitors is widely used as evidence for cytosolic deglycosylation by Ngly1. However, in this study, the accumulation of nonglycosylated RTA∆m, a model ERAD substrate, was still observed in mouse cells lacking cytosolic de-N-glycosylating enzymes, when treated with proteasome inhibitors. It was found that RTA∆m is normally partially N-glycosylated, while the nonglycosylated form is rapidly degraded by proteasomes in the cytosol. Our results suggest that the occurrence of 'nonglycosylated' ERAD substrates upon treatment with proteasome inhibitors is not necessarily a clue for cytosolic deglycosylation.

N-GlycositeAtlas: a database resource for mass spectrometry-based human N-linked glycoprotein and glycosylation site mapping

BackgroundN-linked glycoprotein is a highly interesting class of proteins for clinical and biological research. The large-scale characterization of N-linked glycoproteins accomplished by mass spectrometry-based glycoproteomics has provided valuable insights into the interdependence of glycoprotein structure and protein function. However, these studies focused mainly on the analysis of specific sample type, and lack the integration of glycoproteomic data from different tissues, body fluids or cell types.MethodsIn this study, we collected the human glycosite-containing peptides identified through their de-glycosylated forms by mass spectrometry from over 100 publications and unpublished datasets generated from our laboratory. A database resource termed N-GlycositeAtlas was created and further used for the distribution analyses of glycoproteins among different human cells, tissues and body fluids. Finally, a web interface of N-GlycositeAtlas was created to maximize the utility and value of the database.ResultsThe N-GlycositeAtlas database contains more than 30,000 glycosite-containing peptides (representing > 14,000 N-glycosylation sites) from more than 7200 N-glycoproteins from different biological sources including human-derived tissues, body fluids and cell lines from over 100 studies.ConclusionsThe entire human N-glycoproteome database as well as 22 sub-databases associated with individual tissues or body fluids can be downloaded from the N-GlycositeAtlas website at http://nglycositeatlas.biomarkercenter.org.

The structure of Acinetobacter-secreted protease CpaA complexed with its chaperone CpaB reveals a novel mode of a T2SS chaperone–substrate interaction

Members of the Acinetobacter baumannii-calcoaceticus complex are nosocomial pathogens frequently causing multidrug-resistant infections that are increasing at alarming rates. A. baumannii has become the Gram-negative bacterium with the highest rate of multidrug resistance. As such, it is categorized by the World Health Organization as a critical priority for the research and development of new antimicrobial therapies. The zinc-dependent metalloendopeptidase CpaA is a predominant substrate of the type II secretion system (T2SS). CpaA is also a virulence factor of medically relevant Acinetobacter strains that specifically degrade the human glycoprotein coagulation factor XII and not its deglycosylated form, but the mechanism for this specificity is unknown. CpaB is a membrane-anchored T2SS chaperone that interacts with CpaA and is required for its stability and secretion. Here, we report the crystal structure of the CpaAB complex at 2.6-Å resolution, revealing four glycan-binding domains in CpaA that were not predicted from its primary sequence and may explain CpaA's glycoprotein-targeting activity. The structure of the complex identified a novel mode for chaperone-protease interactions in which the protease surrounds the chaperone. The CpaAB organization was akin to zymogen inactivation, with CpaB serving as a prodomain that inhibits catalytically active CpaA. CpaB contains a C-terminal tail that appears to block access to the CpaA catalytic site, and functional experiments with truncated variants indicated that this tail is dispensable for CpaA expression and secretion. Our results provide new insight into the mechanism of CpaA secretion and may inform the future development of therapeutic strategies for managing Acinetobacter infections.

Cytokine production and signalling in human THP-1 macrophages is dependent on Toxocara canis glycans

The effect of Toxocara canis antigens on cytokine production by human THP-1 macrophages was studied in vitro. Toxocara Excretory–Secretory products (TES) and recombinant mucins (Tc-MUC-2, Tc-MUC-3, Tc-MUC-4, and Tc-MUC-5) as well as deglycosylated forms of these antigens were used in the study. TES products stimulated macrophages to produce the innate proinflammatory IL-1β, IL-6, and TNF-α cytokines regardless of the presence of glycans. Recombinant mucins induced glycan-dependent cytokine production. Sugar moieties led to at least 3-fold higher production of regulatory IL-10 as well as proinflammatory cytokines. The presence of glycans on mucins also affected the downstream signalling pathways in stimulated cells. The most prominent difference was noted in AKT and AMPK kinase activation. AKT phosphorylation was observed in cells stimulated with glycosylated mucins, whereas treatment with deglycosylated antigens led to AMPK phosphorylation. MAP kinase family members such as JNK and p38 and c-Jun transcription factor were phosphorylated in both cases what suggests that toll-like receptor signalling may be involved in mucin-treated macrophages. This pathway is however modified by other signalling molecules as only mucins containing intact sugars significantly induced the production of cytokines.

Application of high-throughput, capillary-based Western analysis to modulated cleavage of the cellular prion protein

The cellular prion protein (PrPC) is a glycoprotein that is processed through several proteolytic pathways. Modulators of PrPC proteolysis are of interest because full-length PrPC and its cleavage fragments differ in their propensity to misfold, a process that plays a key role in the pathogenesis of prion diseases. PrPC may also act as a receptor for neurotoxic, oligomeric species of other proteins that are linked to neurodegeneration. Importantly, the PrPC C-terminal fragment C1 does not contain the reported binding sites for these oligomers. Western blotting would be a simple end point detection method for cell-based screening of compound libraries for effects on PrPC proteolysis or overall expression level. However, traditional Western blotting methods provide unreliable quantification and have only low throughput. Consequently, we explored capillary-based Western technology as a potential alternative; we believe that this study is the first to report analysis of PrPC using such an approach. We successfully optimized the detection and quantification of the deglycosylated forms of full-length PrPC and its C-terminal cleavage fragments C1 and C2, including simultaneous quantification of β-tubulin levels to control for loading error. We also developed and tested a method for performing all cell culture, lysis, and deglycosylation steps in 96-well microplates prior to capillary Western analysis. These advances represent steps along the way to the development of an automated, high-throughput screening pipeline to identify modulators of PrPC expression levels or proteolysis.

Various N-glycoforms differentially upregulate E-NTPDase activity of the NTPDase3/CD39L3 ecto-enzymatic domain.

The GDA1/CD39 ecto-nucleoside triphosphate diphosphosphohydrolase (E-NTPDase) superfamily is a group of eight heavily glycosylated ecto-enzymes that hydrolyze extracellular nucleosides di- and tri-phosphates in the presence of divalent cations, to generate the monophosphate derivatives. This catalytic process differentially regulates a complex array of purinergic signaling responses. NTPDase3/CD39L3is dominantly expressed in pancreatic islet cells, where it may regulate insulin secretion, and has seven N-linked glycosylation sites with four close to five highly conserved domains called "apyrase conserved regions" (ACRs). In a manner similar to CD39, NTPDase3/CD39L3 uses ATP as its preferential substrate and also possesses significant activities toward other triphosphate and diphosphate nucleosides. To understand the mechanism of the ecto-NTPDase activity and substrate specificity, potentially impacted by N-glycans, we have generated soluble enzymatic domains of NTPDase3/CD39L3 in human embryotic kidney cells with four different glycan modifications. These include mannose5-9 glycans with kifunesine treatment, single GlcNAc-Asn by treatment with EndoH, de-glycosylated form by treatment with PNGaseF, and wild-type glycans. Our functional data indicate that the non-glycosylated NTPDase3/CD39L3 ecto-enzymatic domain retains activity, but that N-glycan attachments, such as the GlcNAc-Asn, substantially upregulate specific NTPDase activity by 2-20 fold. Both the Vmax and the Km on di- or tri-phosphate nucleosides are substantially and differentially altered by the glycan attachments. Structural modeling analysis based on putative structures derived from bacterial-originated CD39 domain proteins suggests that N-glycan modifications at Asn149 next to ACR2 and/or Asn454, N-terminal to ACR5 have critical roles in regulating the catalytic pocket of NTPDase3/CD39L3. Our data provide both new insights into the enzymatic mechanisms of NTPDase family members and further evidence that N-glycans directly modulate functional ectonucleotidase activities.