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Neuromyelitis optica spectrum disorders (NMOSDs) are rare autoimmune-mediated chronic inflammatory disorders involving the central nervous system. The concept of NMOSDs has widened up recently. Its diagnostic criteria have broadened the knowledge of this particular disease including the atypical cases where antiaquaporin-4 antibody is negative and anti-myelin oligodendrocyte glycoprotein (MOG) antibody is positive. Myelin oligodendrocyte glycoprotein is a protein expressed on the outer surface of myelin sheath and oligodendrocytes of the central nervous system. The detection of anti-MOG-Ab is emerging evidence, and thus, research on the role of this antibody in such seronegative cases is still underway. Early diagnosis and adequate therapy is essential in such cases. Here, we report an eight-year-old girl who was diagnosed with NMOSDs and found to have anti-MOG antibodies in place of AQP-4 antibodies.

In 2020 and 2021, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel coronavirus, caused a global pandemic. Vaccines are expected to reduce the pressure of prevention and control, and have become the most effective strategy to solve the pandemic crisis. SARS-CoV-2 infects the host by binding to the cellular receptor angiotensin converting enzyme 2 (ACE2) via the receptor-binding domain (RBD) of the surface spike (S) glycoprotein. In this study, a candidate vaccine based on a RBD recombinant subunit was prepared by means of a novel glycoengineered yeast Pichia pastoris expression system with characteristics of glycosylation modification similar to those of mammalian cells. The candidate vaccine effectively stimulated mice to produce high-titer anti-RBD specific antibody. Furthermore, the specific antibody titer and virus-neutralizing antibody (NAb) titer induced by the vaccine were increased significantly by the combination of the double adjuvants Al(OH)3 and CpG. Our results showed that the virus-NAb lasted for more than 6 months in mice. To summarize, we have obtained a SARS-CoV-2 vaccine based on the RBD of the S glycoprotein expressed in glycoengineered Pichia pastoris, which stimulates neutralizing and protective antibody responses. A technical route for fucose-free complex-type N-glycosylation modified recombinant subunit vaccine preparation has been established.

Active lesions in the small bowel (SB) have been independently associated with poorer prognoses in patients with Crohn's disease (CD)1; however, there has been a lack of accurate and convenient screening methods. Past studies have found that serum levels of the glycoprotein leucine-rich α2 glycoprotein (LRG) correlates with endoscopic activity in ulcerative colitis,2,3 and this is now available for routine clinical use as a biomarker in patients with inflammatory bowel disease in Japan. LRG has not yet been thoroughly verified in CD, and we investigated whether it can be used as a serum biomarker for detecting SB mucosal activity in patients with CD.

AimsP-selectin is a key surface adhesion molecule for the interaction of platelets with leukocytes. We have shown previously that the N-terminal domain of S. aureus extracellular fibrinogen-binding protein (Efb) binds to P-selectin and interferes with platelet-leukocyte aggregate formation. Here, we aimed to identify the minimal Efb motif required for binding platelets and to characterise its ability to interfering with the formation of platelet-leukocyte aggregates.Methods and ResultsUsing a library of synthetic peptides, we mapped the platelet-binding site to a continuous 20 amino acid stretch. The peptide Efb68-87 was able to bind to resting and, to a greater extent, thrombin-stimulated platelets in the absence of fibrinogen. Dot blots, pull-down assays and P-selectin glycoprotein ligand-1 (PSGL-1) competitive binding experiments identified P-selectin as the cellular docking site mediating Efb68-87 platelet binding. Accordingly, Efb68-87 did not bind to other blood cells and captured platelets from human whole blood under low shear stress conditions. Efb68-87 did not affect platelet activation as tested by aggregometry, flow cytometry and immunoblotting, but inhibited the formation of platelet-leukocyte aggregates (PLAs). Efb68-87 also interfered with the platelet-dependent stimulation of neutrophil extracellular traps (NETs) formation in vitro.ConclusionsWe have identified Efb68-87 as a novel selective platelet-binding peptide. Efb68-87 binds directly to P-selectin and inhibits interactions of platelets with leukocytes that lead to PLA and NET formation. As PLAs and NETs play a key role in thromboinflammation, Efb68-87 is an exciting candidate for the development of novel selective inhibitors of the proinflammatory activity of platelets.

BACKGROUND: A portion of individuals with hemophilia A develop neutralizing antibodies called inhibitors to glycoprotein factor VIII (FVIII). There are multiple risk factors that contribute to the risk of inhibitor formation. However, knowledge of the role of FVIII asparagine (N)-linked glycosylation in FVIII immunity is limited. OBJECTIVE: To evaluate the effect of site-specific N-linked glycan removal on FVIII biochemical properties, endocytosis by murine bone marrow-derived dendritic cells (BMDCs), and antibody responses. METHODS: Four recombinant B domain deleted (BDD) FVIII variants with single-site amino acid substitutions to remove N-linked glycans were produced for experimental assays. RESULTS: BDD FVIII-N41G, FVIII-N239A, FVIII-N1810A, and FVIII-N2118A with confirmed removal of N-linked glycans and similar glycosylation profiles to BDD FVIII were produced. There were no differences in thrombin activation or von Willebrand factor binding of FVIII variants compared to BDD FVIII, however, reduced FVIII expression, activity, and specific activity was observed with all variants. BDD FVIII-N41G and FVIII-N1810A had reduced uptake by BMDCs, but there were no differences in antibody development in immunized hemophilia A mice compared to BDD FVIII. Half of a repertoire of 12 domain-specific FVIII MAbs had significantly reduced binding to ≥1 FVIII variant with a 50% decrease in A1 domain MAb 2-116 binding to FVIII-N239A. CONCLUSIONS: Modifications of FVIII N-linked glycans reduced FVIII endocytosis by BMDCs and binding of domain-specific FVIII MAbs, but did not alter de novo antibody production in hemophilia A mice suggesting that N-glycans do not significantly contribute to inhibitor formation.

Background and Aims Development of a prophylactic hepatitis C virus (HCV) vaccine will require accurate and reproducible measurement of neutralizing breadth of vaccine-induced antibodies. Currently available HCV panels may not adequately represent the genetic and antigenic diversity of circulating HCV strains, and the lack of standardization of these panels makes it difficult to compare neutralization results obtained in different studies. Here, we describe the selection and validation of a genetically and antigenically diverse reference panel of 15 HCV pseudoparticles (HCVpp) for neutralization assays. Methods We chose 75 envelope (E1E2) clones to maximize representation of natural polymorphisms observed in circulating HCV isolates, and 65 of these clones generated functional HCVpp. Neutralization sensitivity of these HCVpp varied widely. HCVpp clustered into 15 distinct groups based on patterns of relative sensitivity to seven broadly neutralizing monoclonal antibodies (bNAbs). We used these data to select a final panel of 15 antigenically representative HCVpp. Results Both the 65 and 15 HCVpp panels span four tiers of neutralization sensitivity, and neutralizing breadth measurements for seven bNAbs were nearly equivalent using either panel. Differences in neutralization sensitivity between HCVpp were independent of genetic distances between E1E2 clones. Conclusions Neutralizing breadth of HCV antibodies should be defined using viruses spanning multiple tiers of neutralization sensitivity, rather than panels selected solely for genetic diversity. We propose that this multi-tier reference panel could be adopted as a standard for the measurement of neutralizing antibody potency and breadth, facilitating meaningful comparisons of neutralization results from vaccine studies in different laboratories.

Glycoproteins play vital roles in living organisms and often serve as biomarkers for some disease. However, due to the low content of glycoprotein in biological fluids, selective detection of glycoproteins is still a challenging issue that needs to be addressed. In this study, molecularly imprinted colloidal array with multi-boronic acid sites for glycoprotein detection under physiological pH was proposed. Monodispersed glycoprotein imprinted particles (SiO2@PEI/MIPs) was first prepared based on surface imprinting strategy using horseradish peroxidase (HRP) as template, and polyethyleneimine (PEI) was used to increase the number of boronic acid groups. The binding experiment indicated that the SiO2@PEI/MIPs hold satisfactory adsorption capacity (1.41 μmol/g), rapid adsorption rate (40 min) and preferable selectivity toward HRP. Then the SiO2@PEI/MIPs was assembled into close-packed colloidal array to construct a label free optical sensor (denoted as GICA). Benefiting from the high ordered photonic crystal structure, binding of HRP onto the GICA could be directly readout from the changes in structure color and diffracted wavelength. The structure color of the GICA changed from bright blue to yellow with the diffraction wavelength red shifted 59 nm when the HRP concentration increased from 2.5 to 15 μmol/L. Importantly, the GICA was capable of detecting HRP from human serum samples. All those results indicated the potential of the GICA for naked-eye detection of glycoprotein.

BACKGROUND: Mast cells are involved in many distinct pathological conditions, suggesting that they recognize and respond to various stimuli and thus require a rich repertoire of cell surface proteins. However, mast cell surface proteomes have not been comprehensively characterized. OBJECTIVE: We aimed to further characterize the mast cell surface proteome to obtain a better understanding of how mast cells function in health and disease. METHODS: We enriched for glycosylated surface proteins expressed in mouse bone marrow-derived cultured mast cells (BMCMCs) and identified them using mass spectrometry analysis. The presence of novel surface proteins in mast cells was validated by qPCR and flow cytometry analysis in BMCMCs and peritoneal mast cells (PMCs). We developed a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) gene editing approach to disrupt genes of interest in BMCMCs. RESULTS: The glycoprotein enrichment approach resulted in the identification of 1270 proteins in BMCMCs, 378 of which were localized to the plasma membrane. The most common protein classes among plasma membrane proteins are small GTPases, receptors and transporters. One such cell surface protein is CD98 heavy chain (CD98hc) encoded by the Slc3a2 gene. Slc3a2 gene disruption resulted in a significant reduction in CD98hc expression, adhesion, and proliferation. CONCLUSIONS: Our study indicates that glycoprotein enrichment coupled with mass spectrometry can be used to identify novel surface molecules in mast cells. Moreover, we found that CD98hc plays an important role in mast cell function.

Human herpes simplex viruses (HSVs) belong to the Herpesviridae family. At present, no vaccine or curative treatment is available for the prevention of HSV infections. Here, we review the cell surface receptors that are recognized by HSV's glycoprotein B, glycoprotein C, glycoprotein D, and the glycoprotein H – glycoprotein L complex to facilitate entry into host cells. These receptors include heparan sulfate (HS), herpesvirus entry mediator (HVEM), and nectin-1/-2, 3-O-sulfated heparan sulfate (3-OS HS).

There is an urgent need to understand the functional effects of mutations in emerging variants of SARS-CoV-2. Variants of concern (alpha, beta, gamma and delta) acquired four patterns of spike glycoprotein mutations that enhance transmissibility and immune evasion: 1) mutations in the N-terminal domain (NTD), 2) mutations in the Receptor Binding Domain (RBD), 3) mutations at interchain contacts of the spike trimer, and 4) furin cleavage site mutations. Most distinguishing mutations among variants of concern are exhibited in the NTD, localized to sites of high structural flexibility. Emerging variants of interest such as mu, lambda and C.1.2 exhibit the same patterns of mutations as variants of concern. There is a strong likelihood that SARS-CoV-2 variants will continue to emerge with mutations in these defined patterns, thus providing a basis for the development of next line antiviral drugs and vaccine candidates.

All available SARS-CoV-2 spike protein crystal and cryo-EM structures have shown missing electron densities for cytosolic C-terminal regions (CTR). Generally, the missing electron densities point towards the intrinsically disordered nature of the protein region (IDPR). This curiosity has led us to investigate the cytosolic CTR of the spike glycoprotein of SARS-CoV-2 in isolation. The spike CTR is supposed to be from 1235 to 1273 residues or 1242–1273 residues based on our used prediction. Therefore, we have demonstrated the structural conformation of cytosolic region and its dynamics through computer simulations up to microsecond timescale using OPLS and CHARMM forcefields. The simulations have revealed the unstructured conformation of cytosolic region. Further, we have validated our computational observations with circular dichroism (CD) spectroscopy-based experiments and found its signature spectra at 198 nm. We believe that our findings will surely help in understanding the structure-function relationship of the spike protein's cytosolic region., Graphical abstract Image 1

Background The binding of the A1 domain of von Willebrand factor (VWF) to platelet receptor glycoprotein (GP)Ibα defines the VWF activity in hemostasis. Recent studies suggest that sequences flanking A1 form cooperatively an autoinhibitory module (AIM) that reduces the accessibility of the GPIbα binding site on A1. Application of a tensile force induces unfolding of the AIM. Desialylation induces spontaneous binding of plasma VWF to platelets. Most O-glycans in VWF are located around the A1 domain. Removing certain O-glycans in the flanking sequences by site-directed mutagenesis enhances A1 binding to GPIbα and produces an effect similar to type 2B von Willebrand disease in animals. Objectives To understand if and how desialylation of O-glycans in the flanking sequences increases A1 activity. Methods A recombinant AIM-A1 fragment encompassing VWF residues 1238-1493 and only O-glycans was treated with neuraminidase to produce desialylated protein. The glycan structure, dynamics, stability, and function of the desialylated protein was characterized by biochemical and biophysical methods and compared to the sialylated fragment. Results Asialo-AIM-A1 exhibited increased binding activity and induced more apparent platelet aggregation than its sialylated counterpart. It exhibited a lower melting temperature, and increased hydrogen-deuterium exchange rates at residues near the secondary GPIbα binding site and the N-terminal flanking sequence. Asialo-AIM-A1 is less mechanically stable than sialo-AIM-A1, with its unstressed unfolding rate approximately 3-fold greater than the latter. Conclusions Desialylation of O-glycans around A1 increases its activity by destabilizing the AIM.

Protein glycosylation is one of the ubiquitous post-translational modifications in eukaryotic cells, which play important roles in plant growth and adverse response. In this study, we performed the first comprehensive wheat plasma membrane N-glycoproteome analysis under drought stress via glycopeptide HILIC enrichment and LC-MS/MS identification. In total, 414 glycosylated sites corresponding to 407 glycopeptides and 312 unique glycoproteins were identified, of which 173 plasma membrane glycoproteins with 215 N-glycosylation sites were significantly regulated by drought stress. Functional enrichment analysis reveals that the significantly regulated N-glycosylation proteins were particularly related to protein kinase activity involved in the reception and transduction of extracellular signal and plant cell wall remolding. The motifs and sequence structures analysis showed that the significantly regulated N-glycosylation sites were concentrated within [NxT] motif, and 79.5% of them were located on the random coil that is always on the protein surface and flexible regions, which could facilitate protein glycosylated modification and enhance protein structural stability via reducing protein flexibility. PNGase F enzyme digestion and glycosylation site mutation further indicated that N-glycosylated modification could increase protein stability. Therefore, N-glycosylated modification is involved in plant adaptation to drought stress by improving the stability of cell wall remodeling related plasma membrane proteins.

During HIV-1 infection, the envelope glycoprotein subunit gp41 folds into a six-helix bundle structure (6HB) formed by the interaction between its N-terminal (NHR) and C-terminal (CHR) heptad-repeats, promoting viral and cell membranes fusion. A highly preserved, hydrophobic pocket (HP) on the NHR surface is crucial in 6HB formation and, therefore, HP-binding compounds constitute promising therapeutics against HIV-1. Here, we investigated the conformational and dynamic properties of the HP using a rationally designed single-chain protein (named covNHR) that mimics the gp41 NHR structure. We found that the fluorescent dye 8-anilino-naphtalene- 1-sulfonic acid (ANS) binds specifically to the HP, suggesting that ANS derivatives may constitute lead compounds to inhibit 6HB formation. ANS shows different binding modes to the HP, depending on the occupancy of other NHR pockets. Moreover, in presence of a CHR peptide bound to the N-terminal pockets in gp41, two ANS molecules can occupy the HP showing cooperative behavior. This binding mode was assessed using molecular docking and molecular dynamics simulations. The results show that the HP is conformationally flexible and connected allosterically to other NHR regions, which strongly influence the binding of potential ligands. These findings could guide the development of small-molecule HIV-1 inhibitors targeting the HP., Spanish State Research Agency, SRA/ 10.13039/501100011033 (grants BIO2016-76640-R and PID2019.107515RB.C21), ERDF/ESF

Influenza A viruses (IAVs) exploit host glycans in airway epithelial mucosa to gain entry and initiate infection. Rapid detection of changes in IAV specificity towards host glycan classes can provide early indication of virus transmissibility and infection potential. IAVs use hemagglutinins (HA) to bind sialic acids linked to larger glycan structures and a switch in HA specificity from α2,3-to α2,6-linked sialoglycans is considered a prerequisite for viral transmission from birds to humans. While the changes in HA structure associated with the evolution of binding phenotype have been mapped, the influence of glycan receptor presentation on IAV specificity remains obscured. Here, we describe a glycan array platform which uses synthetic mimetics of mucin glycoproteins to model how receptor presentation in the mucinous glycocalyx, including glycan type and valency of the glycoconjugates and their surface density, impact IAV binding. We found that H1N1 virus produced in embryonated chicken eggs, which recognizes both receptor types, exclusively engaged mucin-mimetics carrying α2,3-linked sialic acids in their soluble form. The virus was able utilize both receptor structures when the probes were immobilized on the array; however, increasing density in the mucin-mimetic brush diminished viral adhesion. Propagation in mammalian cells produced a change in receptor pattern recognition by the virus, without altering its HA affinity, toward improved binding of α2,6-sialylated mucin mimetics and reduced sensitivity to surface crowding of the probes. Application of a support vector machine (SVM) learning algorithm as part of the glycan array binding analysis efficiently characterized this shift in binding preference and may prove useful to study the evolution of viral responses to a new host.

The Paramyxoviridae family includes enveloped single-stranded negative-sense RNA viruses such as measles, mumps, human parainfluenza, canine distemper, Hendra, and Nipah viruses, which cause a tremendous global health burden. The ability of paramyxoviral glycoproteins to merge viral and host membranes allows entry of the viral genome into host cells, as well as cell–cell fusion, an important contributor to disease progression. Recent molecular and structural advances in our understanding of the paramyxovirus membrane fusion machinery gave rise to various therapeutic approaches aiming at inhibiting viral infection, spread, and cytopathic effects. These therapeutic approaches include peptide mimics, antibodies, and small molecule inhibitors with various levels of success at inhibiting viral entry, increasing the potential of effective antiviral therapeutic development.

Recent emergence of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transpired into pandemic coronavirus disease 2019 (COVID-19). SARS-CoV-2 has been rapidly transmitted across the globe within a short period of time, with more than 106 million cases and 2.3 million deaths. The continuous rise in worldwide cases of COVID-19, transmission dynamics of SARS-CoV-2 including re-infections and enormous case-fatality rates emphasizes the urgent need of potential preventive and therapeutic measures. The development of effective therapeutic and preventive measures relies on understanding the molecular and cellular mechanism of replication exhibited by SARS-CoV-2. The structure of SARS-CoV-2 is ranging from 90/120 nm that comprises surface viral proteins including spike, envelope, membrane which are attached in host lipid bilayer containing the helical nucleocapsid comprising viral RNA. Spike (S) glycoprotein initiates the attachment of SARS-CoV-2 with a widely expressed cellular receptor angiotensin-converting enzyme 2 (ACE2), and subsequent S glycoprotein priming via serine protease TMPRSS2. Prominently, comprehensive analysis of structural insights into the crucial SARS-CoV-2 proteins may lead us to design effective therapeutics molecules. In the present article, we will emphasize the mechanistic insights of SARS-CoV-2 replication including insights into the structural perspective of crucial SARS-CoV-2 proteins.

Summary Disease outbreaks caused by Ebola virus and other filoviruses highlight the urgent need for an in-depth understanding of the role of antibody responses in recovery. In this Personal View we aim to discuss the controversial biological role of antibodies during natural filovirus infections in humans. Survival during natural human filovirus infections correlates with the magnitude of the process of antibodies binding to the filovirus glycoprotein and neutralising the virus. Despite the severity of the disease, highly potent monoclonal antibodies have been isolated from survivors of natural filovirus infections, suggesting that the magnitude of the antibody response is insufficient for prevention of severe disease. Unlike natural infections, filovirus vaccines, which express the viral glycoprotein, do induce protective concentrations of antibodies, albeit only when administered at very high doses. Multiple mechanisms by which filoviruses can delay and reduce the antibody response have been identified in the past decade. Furthermore, subneutralising antibody concentrations have been shown to enhance filovirus infections of immune cells bearing Fc receptors. Understanding the role of antibody responses during natural filovirus infections is important for the development of safe and potent vaccines and antibody-based treatments.

Background and aims Information regarding inflammation and cardiovascular disease (CVD) risk in type 1 diabetes (T1D) or preeclampsia (PE) is scarce. We assessed differences in inflammation markers according to the presence of both conditions and their association with atherosclerosis. Methods and results We recruited 112 women without CVD and last pregnancy ≥5 years previously (n = 28 per group): a)T1D and PE; b)T1D without PE; c)PE without T1D; and d)Controls (without T1D or PE). Groups were matched by several CVD risk factors, and diabetes duration and retinopathy in T1D. Carotid intima-media thickness (IMT) and plaque presence (IMT ≥1.5 mm) were assessed by ultrasonography. Inflammatory markers included classical variables (leucocytes and high-sensitivity C-reactive protein [hsCRP]) and glycoproteins by nuclear magnetic resonance (1H-NMR) spectroscopy (GlycA, GlycB, GlycF and the height/width [H/W] ratios of GlycA and GlycB). The age of the participants was 44.9 ± 7.8 years, and 20.5% harbored plaque. There were no differences in inflammatory markers among the four study groups. Overall, in multivariate-adjusted models, all 1H-NMR-glycoproteins (except GlycB) were positively associated with IMT measures (IMT of bulb and maximum-IMT of any carotid segment; p Conclusions High 1H-NMR-glycoprotein concentrations have a negative impact on carotid atherosclerosis among women with preeclampsia, regardless of T1D status.

Discrimination of glycoproteins and cell types is a significant but difficult issue. Herein, we presented a novel fluorescence sensor array for the detection and identification of glycoproteins and cancer cells based on the specific affinity between boronic acid-containing carbon dots (BA-CDs) and cis-diol residues of polysaccharides. The differential binding affinity of three BA-CDs to various glycoproteins resulted in a different fluorescence turn-on signal pattern caused by aggregation-enhanced emission (AEE), along with negligible response from other proteins. Therefore, BA-CDs encompassing sensing elements and signal indicator into one can enable a fast and accurate discrimination of glycoproteins with simple and easy operation. Seven glycoproteins could be well discriminated at a very low concentration of 10 nmol/L. The discriminating capability of glycoproteins is not sacrificed in both human urine and serum. Notably, different glycoprotein compositions of cancer cells provide more recognizable features for identification of cancer cells, comparing to the total protein. Five cell types could be identified in 15 min at a low concentration of 1000 cells/mL. This method is fast, accurate, and easy operation, and has a potential application in cancer diagnosis.

Human Immunodeficiency Virus type-1 (HIV-1) is the causative agent of AIDS. Its entry step is mediated by the envelope glycoprotein (Env). During the entry process, Env vastly changes its conformation. While non-liganded Env tends to have a closed structure, receptor-binding of Env opens its conformation, which leads to virus-cell membrane fusion. Single-molecule fluorescence resonance energy transfer (smFRET) imaging allows observation of these conformational changes on the virion surface. Nascent HIV-1 particles incorporate multiple host transmembrane proteins, some of which inhibit the entry process. The Env structure or its dynamics may determine the effectiveness of these antiviral mechanisms. Here, we review recent findings about the Env conformation changes on virus particles and inhibition of Env activities by virion-incorporated host transmembrane proteins.

Protein glycosylation is the most diverse and omnipresent protein modification. Glycosylation provides glycoproteins with important structural and functional properties to facilitate critical biological processes. Despite the significance of protein glycosylation, the investigation of glycoproteome, especially O-linked glycoproteome, remains elusive due to the lack of a comprehensive methodology to conform with the diversity of O-linked glycoforms of O-linked glycoproteins. In recent years, mass spectrometry has become an indispensable tool for the characterization of O-linked glycosylated proteins across biological systems. We herein highlight the recent developments in MS-based O-linked glycoproteomic technologies, quantitative data acquisition strategy and bioinformatic tools, with a special focus on mucin-type O-linked glycosylation.

During oncogenesis, several alterations occur within cells, one of them being the abnormal glycosylation of proteins, resulting in the formation of glycoproteins with aberrant glycan structures, which can be secreted into the blood stream. Their specific association to tumour cells makes them useful indicators (biomarkers) of the oncogenic process and their detection in blood can be employed in different stages of tumour development for early detection, prognosis and therapeutic drug monitoring. Due to the importance of detecting cancer-associated glycoproteins with aberrant glycosylation in blood or serum, analytical methodologies with improved performance are required to ameliorate the laboratorial tests currently used for the detection of these analytes. Microfluidics was created to facilitate the implementation of simple and point-of-care analysis, away from a centralized laboratory. The massive use of microfluidic systems in clinical settings can be seen in pregnancy tests and diabetes control, for example. But what about other clinical domains, such as the detection of glycoproteins with aberrant glycans secreted by tumour cells? Are microfluidic systems helpful in this case? This review analyses the requirements of a microfluidic assay for the detection of low-abundant blood/serum cancer-associated glycoproteins with abnormal glycans and the progresses that have been made in the last years to develop integrated microfluidic devices for this particular application. The diverse microfluidic systems found in literature present, in general, the same analytical performance as the conventional assays but have additional advantages, namely a reduction in assay times, a decrease of sample and reagent consumption and lower costs. The review will also focus on the improvements that are still needed for better biosensing of this type of cancer biomarkers using microfluidic devices.

Proteoglycosylation is the addition of monosaccharides or glycans to the protein peptide chain. This is a common post-translational modification of proteins with a variety of biological functions. At present, more than half of all biopharmaceuticals in clinic are modified by glycosylation. Most glycoproteins are potential drug targets and biomarkers for disease diagnosis. Therefore, in-depth study of glycan structure of glycoproteins will ultimately improve the sensitivity and specificity of glycoproteins for clinical disease detection. With the deepening of research, the function and application value of glycans and glycosylation has gradually emerged. This review systematically introduces the latest research progress of glycans and glycosylation. It encompasses six cancers, four viruses, and their latest discoveries in Alzheimer's disease, allergic diseases, congenital diseases, gastrointestinal diseases, inflammation, and aging.

Electrophoresis is one of the major techniques to analyze macromolecular structure and interaction. Its capability depends on the sensitivity and specificity of the staining methods. We have here examined silver staining of proteins and nucleic acids separated by agarose native gel electrophoresis. By comparing five commercial kits, we identified Silver Stain Plus from Bio-Rad most adequate, as it provided little background staining and reasonable band staining. One of the disadvantages of the Silver Stain Plus kit is its variable staining of glycoproteins as tested with several model samples, including hen egg white proteins, α1-acid glycoprotein and SARS-CoV-2 Spike protein. One of the advantages of silver staining is its ability to stain nucleic acids as demonstrated here for a model nucleic acid with two kits. It was then used to monitor the removal of nucleic acids from the affinity-purified maltose binding protein and monoclonal antibody. It also worked well on staining proteins on agarose gels prepared in the vertical mode, although preparation of the vertical agarose gels required technological modifications described in this report. With the silver staining method optimized here, it should be possible in the future to analyze biological samples that may be available in limited quantity.

The HIV-1 derived gp145 protein is being investigated by research groups as preclinical studies have shown high promise for this protein as a vaccine against HIV. However, one of the main challenges with manufacturing this promising protein has been ascribed to the low yield obtained in mammalian cell cultures. Significant improvements in gp145 production are needed to address this issue to test the gp145 protein as a potentially effective, safe, and affordable HIV vaccine. Here we describe the application of a novel expression technology to create GMP-grade CHO cell lines expressing approximately 50 μg/ml in non-optimized fed-batch culture, which is an order of magnitude higher than that obtained in existing processes. Top producing clones show a high degree of similarity in the glycosylation patterns of the purified protein to the reference standard. Conformational integrity and functionality was demonstrated via high-affinity binding to soluble CD4, using a panel of antibodies including VRC01, F105, Hk20, PG9 and 17b. In summary, we were able to generate CHO cell lines expressing HIV gp145 with significantly higher overall expression yields than currently accessible, and high product quality that could potentially be suitable for future studies assessing the efficacy and safety of gp145-based HIV vaccines.

Self-amplifying mRNA (saRNA) represents a promising platform for nucleic acid delivery of vaccine immunogens. Unlike plasmid DNA, saRNA does not require entry into the nucleus of target cells for expression, having the capacity to drive higher protein expression compared to mRNA as it replicates within the cytoplasm. In this study, we examined the potential of stabilized native-like HIV-1 Envelope glycoprotein (Env) trimers to elicit immune responses when delivered by saRNA polyplexes (PLXs), assembled with linear polyethylenimine. We showed that Venezuelan equine encephalitis virus (VEEV) saRNA induces a stronger humoral immune response to the encoded transgene compared to Semliki Forest virus saRNA. Moreover, we characterized the immunogenicity of the soluble and membrane-bound ConSOSL.UFO Env design in mice and showed a faster humoral kinetic and an immunoglobulin G (IgG)2a skew using a membrane-bound design. The immune response generated by PLX VEEV saRNA encoding the membrane-bound Env was then evaluated in larger animal models including macaques, in which low doses induced high IgG responses. Our data demonstrated that the VEEV saRNA PLX nanoparticle formulation represents a suitable platform for the delivery of stabilized HIV-1 Env and has the potential to be used in a variety of vaccine regimens., Graphical abstract, Aldon, McKay, and colleagues evaluated the potential of formulated VEEV and SFV self-amplifying mRNA (saRNA) for the delivery of HIV-1 Env stabilized trimers. The authors demonstrate great potential for PEI-polyplex formulated VEEV-saRNA platform, which elicits strong humoral responses at low doses in non-human primates.

CD59 is a small glycoprotein modified with a glycophosphatidylinositol (GPI) anchor that prevents the formation of the membrane attack complex, thereby protecting host cells from lysis. A previous study identified that cell surface CD59 staining required the intramembrane protease signal peptide peptidase-like 3 (SPPL3). However, the effect of SPPL3 on the staining of CD59 remains unknown. This study shows that SPPL3 is essential for the surface labeling of CD59 but not of major GPI-anchored proteins. Surface CD59 staining requires the intramembrane protease activity of SPPL3 and SPPL3-mediated suppression of the (neo)lacto-series glycosphingolipids (nsGSLs)-but not N-glycan-synthesis pathway. The abundance of nsGSLs may affect complement-dependent cytotoxicity by altering the abundance or accessibility of cell surface CD59.

The analysis of endogenous glycoproteins and glycopeptides in human body fluids is of great importance for screening and discovering disease biomarkers with clinical significance. However, the presence of interfering substances makes the direct quantitative detection of low-abundance glycoproteins and glycopeptides in human body fluids one of the great challenges in analytical chemistry. Magnetic solid phase extraction (MSPE) has the advantages of easy preparation, low cost and good magnetic responsiveness. Magnetic adsorbents are the core of MSPE technology, and magnetic adsorbents based on different functional materials are widely used in the quantitative analysis of glycoproteins and glycopeptides in human body fluids, making it possible to analyze glycoproteins and glycopeptides with low abundance as well as multiple types, which provides a technical platform for screening and evaluating glycoproteins and glycopeptides in body fluids as disease biomarkers. In this paper, we focus on the recent advances in the application of MSPE technology and magnetic adsorbents for the separation and enrichment of glycoproteins and glycopeptides in human body fluids, and the future trends and application prospects in this field are also presented.

N-glycosylation is a major post-translational modification of proteins and involved in many diseases, however, the state and role of N-glycosylation in cartilage degeneration of osteonecrosis of femoral head (ONFH) remain unclear. The aim of this study is to identify the glycoproteins of ONFH hip cartilage. Cartilage tissues were collected from nine patients with ONFH and nine individuals with traumatic femoral neck fracture. Cartilage glycoproteins were identified by glycoproteomics based on LC-MS/MS. The differentially N-glycoproteins including glycosites were identified in ONFH and controls. A total of 408 N-glycoproteins with 444 N-glycosites were identified in ONFH and control cartilage. Among them, 104 N-glycoproteins with 130 N-glycosites were significantly differential in ONFH and control cartilage, which including matrix-remodeling-associated protein 5, prolow-density lipoprotein receptor-related protein 1, clusterin and lysosome-associated membrane glycoprotein 2. Gene Ontology analysis revealed the significantly differential glycoproteins mainly belonged to protein metabolic process, single-multicellular organism process, proteolysis, biological adhesion and cell adhesion. KEGG pathway and protein-protein interaction analysis suggested that the significantly differential glycoproteins were associated with PI3K-Akt signalling pathway, ECM-receptor interaction, protein processing in the endoplasmic reticulum and N-glycan biosynthesis. This information provides substantial insight into the role of protein glycosylation in the development of cartilage degeneration of ONFH patients.

Numerous studies on cancer, biopharmaceuticals, and clinical trials have necessitated comprehensive and precise analysis of protein O-glycosylation. However, the lack of updated and convenient databases deters the storage and utilization of emerging O-glycoprotein data. To resolve this issue, an O-glycoprotein repository named OGP was established in this work. It was constructed with a collection of O-glycoprotein data from different sources. OGP contains 9354 O-glycosylation sites and 11,633 site-specific O-glycans mapping to 2133 O-glycoproteins, and it is the largest O-glycoprotein repository thus far. Based on the recorded O-glycosites, an O-glycosylation site prediction tool was developed. Moreover, an OGP-backed website is already available (http://www.oglyp.org/). The website comprises four specially designed and user-friendly modules: Statistic Analysis, Database Search, Site Prediction, and Data Submit. The first version of OGP repository and the website allow users to obtain vast O-glycoprotein related information, such as protein accession numbers, glycopeptides, site-specific glycan structures, experimental methods, and potential glycosylation sites. O-glycosylation data mining can be performed efficiently on this website, which can greatly facilitates O-glycosylation studies.

Glycoproteins play crucial roles in many biological events such as protein folding, information transmission, nerve conduction, and molecular recognition. Some glycoproteins serve as disease biomarkers in clinical settings. However, selective detection of glycoprotein often faces great challenges, owing to its low abundance in complex biological samples. In this case, develop a highly sensitive and selective approach for glycoprotein detection is urgently needed. Molecularly imprinted polymers (MIPs) have proved to be an ideal absorbent material in detection and separation science. Herein, a novel biomass activated carbon–derived imprinted polymer (BAC@PEI/PBA/MIPs) was fabricated for selective recognition of glycoprotein. The as-prepared BAC@PEI/PBA/MIPs was synthesized using waste tea derived carbon as matrix, albumin chicken egg (OVA) as template, and dopamine as functional monomer. Branched polyethyleneimine (PEI) was covalently bonded on the BAC surface to increase the number of boronic acid moieties. Benefiting from the self-polymerization of dopamine and multi-boronic acid sites, a great number of recognition sites were presented under mild conditions. The static adsorption experiment showed that the BAC@PEI/PBA/MIPs exhibited a high binding capacity of 196.2 mg/g, rapid adsorption dynamics of 40 min, excellent selectivity and satisfactory reusability for OVA. Furthermore, the practicability of BAC@PEI/PBA/MIPs was verified by isolation of OVA from egg white. The good binding performance and facile preparation process make BAC@PEI/PBA/MIPs attractive for glycoprotein recognition, indicating its potential applications in biomedical research and clinical diagnostics.

Background and aims Inflammation is a pathophysiological mechanism of atherosclerosis, and several mediators have been proposed as biomarkers. Acute-phase serum glycoproteins are upregulated in the liver during systemic inflammation. Because of their unique biochemical characteristics, they can be measured by 1H-NMR, and their role as subclinical inflammation markers is under clinical evaluation. We aimed to assess the clinical value of plasma glycoproteins in familial hypercholesterolemia (FH) patients. Methods We recruited 295 FH patients (75.6% with FH-associated genetic variants). At baseline, a full glycoprotein profile, glycoprotein A and B (GlycA and B) concentrations and their height and width ratios (H/W) were analysed by 1H-NMR. A carotid artery ultrasound study was performed at baseline and prospectively at the 5-year follow-up in 144 FH patients. Results At baseline, the GlycA and GlycB concentrations and their H/W ratios were correlated with lipid profile and adiposity parameters, with the correlation between the GlycA and triglyceride concentrations (r = 0.780; p Conclusions The concentrations of serum glycoproteins as assessed by 1H-NMR are robust markers of subclinical inflammation. In FH patients, they are increased in the presence of subclinical vascular damage and could be considered atherosclerosis risk markers in the long term.

Salivary glycoproteins are known as an important barrier to inhibit influenza infection by presenting sialic acid (Sia) ligands that can bind with viral hemagglutination. Here, to further understand why pregnant women are more vulnerable to avian influenza virus (AIV), we investigated the alteration of protein sialylation in the saliva of women during pregnancy and postpartum, and its impact on the saliva binding affinity to AIV. Totally 1200 saliva samples were collected, the expression levels of terminal α2-3/6-linked Sia on salivary proteins were tested and validated, and the binding activities of salivary proteins were assessed against 3 strains of AIV and the H1N1 vaccine. Result showed that the expression of terminal α2-3-linked Sia in the saliva of women decreased dramatically during pregnancy compared to that of non-pregnancy control, especially for women in the second or third trimester (fold change = 0.53 and 0.37, p 0.001). And their salivary protein binding ability to AIV declined accordingly. The variation of terminal α2-3-linked Sia on salivary MUC5B and IgA was consistent with the above results. This study indicates that the decrease of terminal α2-3-linked Sia on salivary glycoproteins of pregnant women affects their binding ability to AIV, which may provide new insights into AIV prevention and control.

Hypothesis It has been recently shown that, in our organism, the secretions of Ca2+, Mg2+ and phosphate ions lead to the precipitation of amorphous magnesium-calcium phosphate nanoparticles (AMCPs) in the small intestine, where the glycoprotein mucin is one of the most abundant proteins, being the main component of the mucus hydrogel layer covering gut epithelium. Since AMCPs precipitate in vivo in a mucin-rich environment, we aim at studying the effect of this glycoprotein on the formation and features of endogenous-like AMCPs. Experiments AMCPs were synthesized from aqueous solution in the presence of different concentrations of mucin, and the obtained particles were characterised in terms of crystallinity, composition and morphology. Solid State NMR investigation was also performed in order to assess the interplay between mucin and AMCPs at a sub-nanometric level. Finding Results show that AMCPs form in the presence of mucin and the glycoprotein is efficiently incorporated in the amorphous particles. NMR indicates the existence of interactions between AMCPs and mucin, revealing how AMCPs in mucin-hybrid nanoparticles affect the features of both proteic and oligosaccharidic portions of the glycoprotein. Considering that the primary function of mucin is the protection of the intestine from pathogens, we speculate that the nature of the interaction between AMCPs and mucin described in the present work might be relevant to the immune system, suggesting a novel type of scenario which could be investigated by combining physico-chemical and biomedical approaches.

In order to achieve specific enrichment of target glycoproteins, double molecular recognition ligands functionalized surface solid-phase adsorption materials that can play a role under physiological pH conditions, have obvious advantages. In this work, a novel strategy was proposed for the synthesis of double molecular recognition functionalized magnetic graphene nanocomposites. Horseradish peroxidase was selected as the template proteins for evaluating the selectivity and binding capacity of the adsorption material. The bimolecular recognition was realized by introducing 4-carboxyphenylboronic acid (CPBA) and Suberic acid bis(3-sulfo-N-hydroxysuccinimide ester) sodium salt (Sulfo-DSS) as functional ligands. The abundant oxygen-containing groups in the complex (abbreviated as CPDS/CMCS/Fe3O4-rGO) were beneficial to reduce the pKa value of CPBA. Finally, CPDS/CMCS/Fe3O4-rGO realized the selective adsorption of glycoproteins in the neutral environment (pH 7.0). The resultant CPDS/CMCS/Fe3O4-rGO exhibited a high adsorption capacity of 1280.6 mg g−1 and excellent specificity toward glycoproteins compared to nonglycoproteins. Moreover, CPDS/CMCS/Fe3O4-rGO with strong magnetic responses (59.29 emu·g−1), could achieve rapid aggregation and separation under the external magnetic field. The results demonstrated the great potential of CPDS/CMCS/Fe3O4-rGO composites to separate and enrich glycoproteins from the complex biological sample for the glycoproteins analysis.

Human cytomegalovirus (HCMV) is currently a major cause of congenital disease in newborns and organ failure in transplant recipients. Despite decades of efforts, an effective vaccine against HCMV has yet to be developed. However, the discovery of pentameric gH complex on viral surface which contains potent neutralizing epitopes may help enable development of an effective vaccine. In our company ongoing Phase II clinical trial of whole-live virus HCMV vaccine (V160), the pentameric gH complex has been restored on the surface of live attenuated AD169 virus strain. The reconstructed HCMV virus contains a variety of surface glycoproteins including pentameric gH/gL/gUL128-131 complex, trimeric gH/gL/gO complex, gB glycoprotein, and gM/gN heterodimer complex. To further characterize this virus and enable the monitoring of multiple viral antigens during vaccine process development an effective and efficient analytical strategy was required to detect and quantify several viral surface proteins. In this paper, we present an innovative approach based on capillary western blot technology that allows fast and accurate quantitation of pentameric gH/gL/gUL128-131 complex, trimeric gH/gL/gO complex, and gB glycoprotein. This method is suitable for analyzing target proteins in multiple sample types including supernatants from infected cell culture, purification intermediates, concentration bulk, and the final vaccine product. In addition, the capillary western blot-based technology identified a previously unknown biochemical profile present in some HCMV viruses: triplet gH peaks of viral surface proteins in non-reducing environment, which could potentially present a new strategy for specificity and identity testing.

About half of all newly synthesized proteins have N-linked glycans. These glycans play pivotal roles in controlling the folding, sorting, and degradation of glycoproteins via several glycan-related proteins. The glycan-mediated protein quality control system is important for cellular homeostasis. In this review, we summarize recent advances in our understanding of the system and discuss structural insights from chemical and biological perspectives. In particular, we focus on the mechanisms by which these mediators respond to several folding states of glycoproteins.

Objective The study aimed to synthesize and characterize pyrimidine-linked benzimidazole hybrids, define their antimicrobial and antifungal activities in vitro, and determine their ability to inhibit the main protease and spike glycoprotein of SARS-CoV-2. Methods The ability of the synthesized compounds to inhibit the main protease and spike glycoprotein inhibitory of SARS-CoV-2 was investigated by assessing their mode of binding to the allosteric site of the enzyme using molecular docking. The structures of pyrimidine-linked benzimidazole derivatives synthesized with microwave assistance were confirmed by spectral analysis. Antibacterial and antifungal activities were determined by broth dilution. Results Gram-negative bateria (Escherichia coli and Pseudomonas aeruginosa) were more sensitive than gram-positive bateria (Staphylococcus aureus and Streptococcus pyogenes) to the derivatives. Candida albicans was sensitive to the derivatives at a minimal inhibitory concentration (MIC) of 250 μg/mL. The novel derivatives had better binding affinity (kcal/mol) than nelfinavir, lopinavir, ivermectin, remdesivir, and favipiravir, which are under investigation as treatment for SARS-CoV-2 infection. Compounds 2c, 2e, and 2g formed four hydrogen bonds with the active cavity of the main protease. Many derivatives had good binding affinity for the RBD of the of SARS-CoV-2 spike glycoprotein with the formation of up to four hydrogen bonds. Conclusion We synthesized novel pyrimidine-linked benzi-midazole derivatives that were potent antimicrobial agents with ability to inhibit the SARS-CoV-2 spike glycoprotein. Understanding the pharmacophore features of the main protease and spike glycoprotein offers much scope for the development of more potent agents. We plan to optimize the properties of the derivatives using models in vivo and in vitro so that they will serve as more effective therapeutic options against bacterial and SARS-CoV-2 infections.

Background and aims Glycoproteins play a key role in inflammatory and cardiometabolic processes. Their implication in atherosclerosis in type 1 diabetes (T1D) is unknown. We assessed the relationships between classic inflammatory markers, glycoproteins measured by nuclear magnetic resonance (1H-NMR), and preclinical atherosclerosis in these patients. Methods and results We selected patients with T1D, without cardiovascular disease (CVD), with: age ≥40 years, nephropathy (micro/macroalbuminuria), or ≥10 years of evolution with another risk factor. The presence of plaque (intima-media thickness >1.5 mm) was determined by ultrasonography. Concentrations of high-sensitive C-reactive protein (hsCRP), circulating leukocytes (classical inflammation markers) and 1H-NMR-glycoproteins (GlycA, GlycB, GlycF, and the height/width [H/W] ratios of GlycA and GlycB) were determined. We included 189 patients (58% male, age 47.0 [40.7–55.2] years). Thirty-five percent presented plaques (22%, ≥2 plaques). There was no association between hsCRP or leukocytes and atherosclerosis. However, in age- and sex-adjusted models, GlycA, GlycF, and the H/W ratios of GlycA and GlycB gradually increased with the number of plaques (0, 1, ≥2 plaques) only in patients without statins (p Conclusion In T1D individuals without lipid-lowering treatment, 1H-NMR-glycoproteins were independently associated with the presence and amount of carotid atherosclerosis, unlike other classical inflammatory markers. Further studies are needed to ascertain their utility as CVD biomarkers.

O-GalNAc type glycosylation is an abundant and complex protein modification. Recent developments in mass spectrometry resulted in significant success in quantitative analysis of O-GalNAc glycosylation. The analysis of released O-GalNAc type glycans expanded our horizons of understanding the glycome of various biological models. The site-specific analysis of glycosylation micro-heterogeneity of purified proteins opened perspectives for the improved design of glycoprotein therapeutics. Advanced gene editing and chemical technologies applied to O-glycoproteomics enabled to identify O-GalNAc glycosylation at unprecedented depth. Progress in the analysis of intact glycoproteins under native and reduced conditions enabled the monitoring of glycosylation proteoform variants. Despite of the astonishing results in quantitative O-GalNAc glycoproteomics, site-specific mapping of the full O-GalNAc structural repertoire in complex samples is yet a long way off. Here, we summarize the most common quantitative strategies in O-GalNAc glycoproteomics, review recent progress and discuss benefits and limitations of the various approaches in the field.

High Performance Size-Exclusion Chromatography coupled with Multi-Angle Light Scattering detection (HPSEC-MALS) is an important tool to provide a reliable molecular weight measurement for a large complex biomolecule. A recent HIV-1 soluble envelope trimer vaccine candidate, BG505 DS-SOSIP.664, is among the most glycosylated proteins to enter a clinical trial to date, and determination of its protein and glycan molecular weight is one of the key attributes in pre-clinical characterization. However, protein and glycans possess disparate dndcvalues making molecular weight measurement inaccurate in conventional SEC-MALS. To overcome these challenges, a simple mathematically guided experiment was explored, and a composite dndcvalue was established by utilizing protein and glycan mass contributions for the HIV-1 envelope trimer. This establishment was further verified by an orthogonal mass spectrometry analysis. This innovative, simple, and quick analytical approach can be applied broadly to measuring the molecular weight of various composite molecular structures, such as complex glycoconjugates.

Siglecs (sialic acid-binding immunoglobulin-like lectins) are a family of receptors that bind sialic acids in specific linkages on glycoproteins and glycolipids. Siglecs play roles in immune signalling and exhibit cell-type specific expression and endocytic properties. Recent studies suggest that Siglecs are likely to function as immune checkpoints that regulate responses in cancers and inflammatory diseases. In this review, we discuss strategies to target the Siglec-sialic acid axis in human diseases, particularly cancer, and the possibility of exploiting them for therapeutic intervention.

N-glycan imaging mass spectrometry (IMS) can rapidly and reproducibly identify changes in disease-associated N-linked glycosylation that are linked with histopathology features in standard formalin-fixed paraffin-embedded tissue samples. It can detect multiple N-glycans simultaneously and has been used to identify specific N-glycans and carbohydrate structural motifs as possible cancer biomarkers. Recent advancements in instrumentation and sample preparation are also discussed. The tissue N-glycan IMS workflow has been adapted to new glass slide-based assays for effective and rapid analysis of clinical biofluids, cultured cells, and immunoarray-captured glycoproteins for detection of changes in glycosylation associated with disease.

Although antiretroviral treatment lowers the burden of human immunodeficiency virus (HIV)-related disease, it does not always result in immunological recovery. This manifests as persistent chronic inflammation, immune activation or exhaustion that can promote the onset of co-morbidities. As the exact function of regulatory T (Treg) cells in HIV remains unclear, this cross-sectional study investigated three expression markers (Forkhead box protein P3 [FOXP3], glycoprotein A repetitions predominant [GARP], special AT-rich sequence binding protein 1 [SATB1]) and compared their expansion between CD4+CD25− and CD4+CD25++ T cells. Age-matched study subjects were recruited (Western Cape, South Africa) and sub-divided: HIV-negative subjects (n = 12), HIV-positive naive treated (n = 22), HIV-positive treated based on CD4 count cells/µL (CD4 > 500 and CD4 1000) (n = 34). Markers of immune activation (CD38) and coagulation (CD142) on T cells (CD8) were assessed by flow cytometry together with FOXP3, GARP and SATB1 expression on CD4+CD25− and CD4+CD25++ T cells. Plasma levels of interleukin-10 (IL-10; anti-inflammatory marker), IL-6 (inflammatory marker) and D-dimer (coagulation marker) were assessed. This study revealed three major findings in immuno-compromised patients with virological failure (CD4 1000): (1) the expansion of the unconventional Treg cell subset (CD4+CD25−FOXP3+) is linked with disease progression markers; (2) increased GARP expression in the CD4+CD25− and CD4+CD25++ subsets; and (3) the identification of a strong link between CD4+CD25−SATB1+ cells and markers of immune activation (CD8+CD38+) and coagulation (CD8+CD142+ and D-dimer).

Glycoprotein (GP)Ib that binds von Willebrand factor (vWF) and glycoprotein (GP)VI, that binds collagen play a significant role in platelet activation and aggregation, and are potential targets for antithrombotic treatment. They are targeted by snake venom proteinases. The effect of a such proteinase, mutalysin-II, on platelet aggregation was examined using washed human platelets and platelet-rich plasma. Its proteolytic activity on vWF, on its binding partner GPIbα, and on GPVI was analyzed by SDS-PAGE, and immunodetection with the corresponding antibodies after blotting. Dose- and time-dependently, mutalysin-II inhibits aggregation of washed platelets induced by vWF plus ristocetin and by convulxin, but with no significant effect on platelet-rich-plasma. Furthermore, mutalysin-II cleaves vWF into low molecular mass multimers of vWF and a rvWF-A1 domain to realease a ∼27-kDa fragment detectable by SDS-PAGE and blotting with mouse anti-rvWF-A1-domain IgG. Moreover, GPVI was cut by mutalysin-II into a soluble ∼55-kDa ectodomain and a fragment of ∼35-kDa. Thus, mutalysin-II inhibits vWF-induced platelet aggregation via cleavage of bound vWF-A1, and its receptor GPIbα. The additional cleavage of, GPVI, blocks collagen-induced platelets. Our data highlight mutalysin-II as an interesting platelet-directed tool targeting vWF-GPIbα binding and particularly GPVI. Thus, it might be suited for antithrombotic therapy as its combined inactivation of two receptors does not significantly compromise hemostasis, but shows high efficacy and safety. Studies are needed to further develop and demonstrate its potential benefits.

Made available in DSpace on 2021-06-25T10:53:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-05-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) EMC Dengue virus infection depends on its fusion with the host membrane, where the binding occurs through interaction between proteins on the virus cell surface and specific viral receptors on target membranes. This process is mediated by the fusion peptide located between residues 98 and 112 (DRGWGNGCGLFGKGG) that forms a loop in domain II of dengue E glycoprotein. In this study, we evaluated the role of fusion peptide surrounding regions (88–97 and 113–123) of the Dengue 2 subtype on its interaction with the membrane and fusion activity. These sequences are important to stabilize the fusion peptide loop and increase fusion activity. Three peptides, besides the fusion peptide, were synthesized by SPPS using the Fmoc chemical approach. The first contains the fusion peptide and the C-terminal region of the loop (sequence 98–123); another contains the N-terminal region (88–112) and the larger peptide contains both regions (88–123). The peptides were able to interact with a model membrane. Differences in morphology of the monolayer promoted by the peptides were assessed by Brewster Angle Microscopy (BAM). Our data indicated that the C-terminal region of fusion peptide loop is more efficient in promoting fusion and interacting with the membrane than the N-terminal sequence, which is responsible for the electrostatic initial interaction. We propose a 2-step mechanism for the interaction of the dengue virus fusion peptide with the host membrane, where the N-terminal sequence docks electrostatically on the headgroups and then the C-terminal interacts via hydrophobic forces in the acyl chains. Departamento de Bioquímica e Química Orgânica Instituto de Química UNESP -Univ Estadual Paulista Instituto de Física de São Carlos Universidade de São Paulo USP, São Carlos Department of Chemistry The Ohio State University Departamento de Bioquímica e Química Orgânica Instituto de Química UNESP -Univ Estadual Paulista

Alpha-1 acid glycoprotein (AGP) is a major acute-phase protein that is involved in drug/ligand binding and regulation of immune response. In response to inflammation, AGP secretion from the liver increases, resulting in elevated concentration of plasma AGP. AGP exhibits multiple N-glycosylation sites, and thus, is highly glycosylated. Although AGP glycosylation is considered to affect its functions, the significance of AGP glycosylation for its secretion is unclear. In this study, we investigated the effects of AGP glycosylation using glycosylation-deficient mouse AGP mutants lacking one, four, or all five N-glycosylation sites. Furthermore, we examined the effects of endoplasmic reticulum (ER) stress-inducing reagents, including tunicamycin and thapsigargin, which induce ER stress in an N-glycosylation-dependent and -independent manner, respectively. Here, we found that glycosylation deficiency and ER stress induce a little or no effect on AGP secretion. Conversely, thapsigargin significantly suppressed AGP secretion in glycosylation-independent manner. These findings indicate that AGP secretion is regulated via thapsigargin-sensitive pathway that might be further controlled by the intracellular calcium concentrations.

The heartland virus (HRTV) is a novel phlebovirus that causes severe infections in the USA and closely related to the severe fever thrombocytopenia syndrome virus (SFTSV), a causative agent for SFTS in Asia. The entry mechanisms of HRTV remain unclear. Here, we developed the pseudotyped vesicular stomatitis virus bearing the HRTV glycoprotein (GP) (HRTVpv), and the antigenicity and the entry mechanisms of HRTV were analyzed. HRTVpv was neutralized by anti-SFTSV Gc antibody, but not the anti-SFTSV Gn antibodies. Entry of HRTVpv to cells was inhibited by bafilomycin A1 and dynasore, and but it was enhanced in cells overexpressed with C-type lectins. Production of infectious HRTVpv and SFTSVpv was reduced by Nn-DNJ, α-glucosidase inhibitor. The entry of HRTV occurs via pH- and dynamin-dependent endocytosis. Furthermore, Nn-DNJ may be a possible therapeutic agent against HRTV and SFTSV.

A variety of opossum species are resistant to snake venoms due to the presence of antihemorrhagic and antimyotoxic acidic serum glycoproteins that inhibit several toxic venom components. Two virtually identical antihemorrhagic proteins isolated from either the North American opossum (D. virginiana) or the South American big-eared opossum (D. aurita), termed oprin or DM43 respectively, inhibit specific snake venom metalloproteinases (SVMPs). A better understanding of the structure of these proteins may provide useful insight to determine their mechanism of action and for the development of therapeutics against the global health concern of snake-bite envenomation. The aim of this work is to produce a recombinant snake venom metalloproteinase inhibitor (SVMPI) similar to the above opossum proteins in Escherichia coli and determine if this bacterially produced protein inhibits the proteolytic properties of Western Diamondback rattlesnake (C. atrox) venom. The resulting heterologous SVMPI was produced with either a 6-Histidine or maltose binding protein (MBP) affinity tag on either the C-terminus or N-terminus of the protein, respectively. The presence of the solubility enhancing MBP affinity tag resulted in significantly more soluble protein expression. The inhibitory activity was measured using two complementary assays and the MBP labeled SVMPI showed 7-fold less activity as compared to the 6-Histidine labeled SVMPI. Thus, the bacterially derived SVMPI with an unlabeled N-terminus showed high inhibitory activity (IC50 = 4.5 μM). The use of a solubility enhancing MBP fusion protein construct appears to be a productive way to express sufficient quantities of this mammalian protein in E. coli for further study.