Human brain glycoform coregulation network and glycan modification alterations in Alzheimer's disease.

Despite the importance of protein glycosylation to brain health, current knowledge of glycosylated proteoforms or glycoforms in human brain and their alterations in Alzheimer's disease (AD) is limited. Here, we report a proteome-wide glycoform profiling study of human AD and control brains using intact glycopeptide-based quantitative glycoproteomics coupled with systems biology. Our study identified more than 10,000 human brain N-glycoforms from nearly 1200 glycoproteins and uncovered disease signatures of altered glycoforms and glycan modifications, including reduced sialylation and N-glycan branching and elongation as well as elevated mannosylation and N-glycan truncation in AD. Network analyses revealed a higher-order organization of brain glycoproteome into networks of coregulated glycoforms and glycans and discovered glycoform and glycan modules associated with AD clinical phenotype, amyloid-ß accumulation, and tau pathology. Our findings provide valuable insights into disease pathogenesis and a rich resource of glycoform and glycan changes in AD and pave the way forward for developing glycosylation-based therapies and biomarkers for AD.

ELISA-based highly sensitive assay system for the detection of endogenous NGLY1 activity.

Cytosolic peptide:N-glycanase (NGLY1, PNGase) is an enzyme that cleaves N-glycans from misfolded glycoproteins. In 2012, a human genetic disorder, NGLY1 deficiency, was first reported to be caused by mutations of the NGLY1 gene. Since then, there has been rapid progresses on NGLY1 biology, and gene therapy has been proposed as a promising therapeutic option for NGLY1 deficiency. While a plasma/urine biomarker has also been developed for this disease, detection of NGLY1 activity could be another viable option for early diagnosis of NGLY1 deficiency. Thus far, several in vitro and in cellulo NGLY1 assays have been reported, but those assay systems have several issues that must be addressed in order to develop an assay system compatible for routine clinical examination. Here, we show a facile, highly sensitive in vitro assay system that could be used to detect NGLY1 activity by utilizing its sequence editing function, i.e. conversion of glycosylated Asn into Asp, followed by a detection of newly generated epitope (HA)-tag by anti-HA antibody. Using this ELISA-based assay, we detected endogenous NGLY1 activity in as little as 2 µg of crude extract, which is the equivalent of 5 × 103 cells. Our system also detects NGLY1 activity from cells with compromised NGLY1 activity, such as iPS cells from patient samples. This assay system could be applied in future clinical examinations to achieve an early diagnosis of NGLY1 deficiency.

Therapeutic administration of a cross-reactive mAb targeting the fusion glycoprotein of Nipah virus protects nonhuman primates.

No licensed vaccines or therapies exist for patients infected with Nipah virus (NiV), although an experimental human monoclonal antibody (mAb) cross-reactive to the NiV and Hendra virus (HeV) G glycoprotein, m102.4, has been tested in a phase 1 trial and has been provided under compassionate use for both HeV and NiV exposures. NiV is a highly pathogenic zoonotic paramyxovirus causing regular outbreaks in humans and animals in South and Southeast Asia. The mortality rate of NiV infection in humans ranges from 40% to more than 90%, making it a substantial public health concern. The NiV G glycoprotein mediates host cell attachment, and the F glycoprotein facilitates membrane fusion and infection. We hypothesized that a mAb against the prefusion conformation of the F glycoprotein may confer better protection than m102.4. To test this, two potent neutralizing mAbs against NiV F protein, hu1F5 and hu12B2, were compared in a hamster model. Hu1F5 provided superior protection to hu12B2 and was selected for comparison with m102.4 for the ability to protect African green monkeys (AGMs) from a stringent NiV challenge. AGMs were exposed intranasally to the Bangladesh strain of NiV and treated 5 days after exposure with either mAb (25 milligrams per kilogram). Whereas only one of six AGMs treated with m102.4 survived until the study end point, all six AGMs treated with hu1F5 were protected. Furthermore, a reduced 10 milligrams per kilogram dose of hu1F5 also provided complete protection against NiV challenge, supporting the upcoming clinical advancement of this mAb for postexposure prophylaxis and therapy.

Gn protein expressed in plants for diagnosis of severe fever with thrombocytopenia syndrome virus.

Severe fever with thrombocytopenia syndrome virus (SFTSV) causes the highly fatal disease in humans. To facilitate diagnosis, the native form of subunit glycoprotein (Gn), a prime target for potential vaccines and therapies, was produced in Nicotiana benthamiana using a Bamboo mosaic virus-based vector system. By fusion with secretory signal tags, SSExt, derived from the extension protein, and the (SP)10 motif, the yield of the recombinant Gn (rGn) was remarkably increased to approximately 7 mg/kg infiltrated leaves. Ultimately, an rGn-based ELISA was successfully established for the detection of SFTSV-specific antibodies in serum samples from naturally infected monkeys. As validated with the reference method, the specificity and sensitivity of rGn-ELISA were 94% and 96%, respectively. In conclusion, utilizing well-suited fusion tags facilitates rGn production and purification in substantial quantities while preserving its antigenic properties. The rGn-ELISA, characterized by its commendable sensitivity and specificity could serve as a viable alternative diagnostic method for assessing SFTSV seroprevalence. KEY POINTS: • SFTSV Gn, fused with secretory signal tags, was expressed by the BaMV-based vector. • The plant fusion tags increased expression levels and eased the purification of rGn. • The rGn-ELISA was established and validated; its specificity and sensitivity > 94%.

Exposing the molecular heterogeneity of glycosylated biotherapeutics.

The heterogeneity inherent in today's biotherapeutics, especially as a result of heavy glycosylation, can affect a molecule's safety and efficacy. Characterizing this heterogeneity is crucial for drug development and quality assessment, but existing methods are limited in their ability to analyze intact glycoproteins or other heterogeneous biotherapeutics. Here, we present an approach to the molecular assessment of biotherapeutics that uses proton-transfer charge-reduction with gas-phase fractionation to analyze intact heterogeneous and/or glycosylated proteins by mass spectrometry. The method provides a detailed landscape of the intact molecular weights present in biotherapeutic protein preparations in a single experiment. For glycoproteins in particular, the method may offer insights into glycan composition when coupled with a suitable bioinformatic strategy. We tested the approach on various biotherapeutic molecules, including Fc-fusion, VHH-fusion, and peptide-bound MHC class II complexes to demonstrate efficacy in measuring the proteoform-level diversity of biotherapeutics. Notably, we inferred the glycoform distribution for hundreds of molecular weights for the eight-times glycosylated fusion drug IL22-Fc, enabling correlations between glycoform sub-populations and the drug's pharmacological properties. Our method is broadly applicable and provides a powerful tool to assess the molecular heterogeneity of emerging biotherapeutics.

Restriction of Viral Glycoprotein Maturation by Cellular Protease Inhibitors.

The human genome is estimated to encode more than 500 proteases performing a wide range of important physiological functions. They digest proteins in our food, determine the activity of hormones, induce cell death and regulate blood clotting, for example. During viral infection, however, some proteases can switch sides and activate viral glycoproteins, allowing the entry of virions into new target cells and the spread of infection. To reduce unwanted effects, multiple protease inhibitors regulate the proteolytic processing of self and non-self proteins. This review summarizes our current knowledge of endogenous protease inhibitors, which are known to limit viral replication by interfering with the proteolytic activation of viral glycoproteins. We describe the underlying molecular mechanisms and highlight the diverse strategies by which protease inhibitors reduce virion infectivity. We also provide examples of how viruses evade the restriction imposed by protease inhibitors. Finally, we briefly outline how cellular protease inhibitors can be modified and exploited for therapeutic purposes. In summary, this review aims to summarize our current understanding of cellular protease inhibitors as components of our immune response to a variety of viral pathogens.

Role of LGL1 in cerebellar primordium of embryonic mice.

Lethal giant larvae 1 (LGL1) is originally recognized as a tumor suppressor, implicated in maintaining cell polarity in Drosophila and mammalian cells. Cell polarity plays a crucial role in tumorigenesis. We previously established Pax2-LGL1 -/- conditional knockout mice but did not focus on the tumorigenesis in cerebellar primordium. HE staining was used to detect the morphological structure of the cerebellar primordium during early embryonic development in Pax2-LGL1 -/- mice. Immunofluorescence assays were used to detect the expression of polar molecules. TUNEL staining assessed tissue apoptosis. Our findings reveal that deletion of LGL1 leads to the emergence of neuroblastoma-like tissues within the cerebellum primordium during early embryogenesis. This outcome can be attributed to alterations in expression patterns of polar molecules Cdc42 and ß-catenin following early deletion of LGL1, resulting in loss of cell polarity among neuroepithelial cells and subsequent formation of tumor-like tissues. However, further histological examination demonstrated that these tumor-like tissues disappear from embryonic day 15.5 onwards within the cerebellar primordium of Pax2-LGL1 -/- mice due to apoptosis-mediated cellular compensation. Our data emphasize the importance of LGL1 in maintaining neuroepithelial cell polarity and reveal a novel role for LGL1 in regulating tumorigenesis and ablation in the cerebellar primordium.

Mass Spectrometry Strategies for O-Glycoproteomics.

Glycoproteomics has accelerated in recent decades owing to numerous innovations in the analytical workflow. In particular, new mass spectrometry strategies have contributed to inroads in O-glycoproteomics, a field that lags behind N-glycoproteomics due to several unique challenges associated with the complexity of O-glycosylation. This review will focus on progress in sample preparation, enrichment strategies, and MS/MS techniques for the identification and characterization of O-glycoproteins.

Glycoproteome-Wide Discovery of Cortical Glycoproteins That May Provide Cognitive Resilience in Older Adults.

BACKGROUND AND OBJECTIVES: Molecular omics studies have identified proteins related to cognitive resilience but unrelated to Alzheimer disease and Alzheimer disease-related dementia (AD/ADRD) pathologies. Posttranslational modifications of proteins with glycans can modify protein function. In this study, we identified glycopeptiforms associated with cognitive resilience. METHODS: We studied brains from adults with annual cognitive testing with postmortem indices of 10 AD/ADRD pathologies and proteome-wide data from dorsal lateral prefrontal cortex (DLPFC). We quantified 11, 012 glycopeptiforms from DLPFC using liquid chromatography with tandem mass spectrometry. We used linear mixed-effects models to identify glycopeptiforms associated with cognitive decline correcting for multiple comparisons (p < 5 × 10-6). Then, we regressed out the effect of AD/ADRD pathologies to identify glycopeptiforms that may provide cognitive resilience. RESULTS: We studied 366 brains, average age at death 89 years, and 70% female with no cognitive impairment = 152, mild cognitive impairment = 93, and AD = 121 cognitive status at death. In models adjusting for age, sex and education, 11 glycopeptiforms were associated with cognitive decline. In further modeling, 8 of these glycopeptiforms remained associated with cognitive decline after adjusting for AD/ADRD pathologies: NPTX2a (Est., 0.030, SE, 0.005, p = 1 × 10-4); NPTX2b (Est.,0.019, SE, 0.005, p = 2 × 10-4) NECTIN1(Est., 0.029, SE, 0.009, p = 9 × 10-4), NPTX2c (Est., 0.015, SE, 0.004, p = 9 × 10-4), HSPB1 (Est., -0.021, SE, 0.006, p = 2 × 10-4), PLTP (Est., -0.027, SE, 0.009, p = 4.2 × 10-3), NAGK (Est., -0.027, SE, 0.008, p = 1.4 × 10-3), and VAT1 (Est., -0.020, SE, 0.006, p = 1.1 × 10-3). Higher levels of 4 resilience glycopeptiforms derived through glycosylation were associated with slower decline and higher levels of 4 derived through glycation were related to faster decline. Together, these 8 glycopeptiforms accounted for an additional 6% of cognitive decline over the 33% accounted for the 10 brain pathologies and demographics. All 8 resilience glycopeptiforms remained associated with cognitive decline after adjustments for the expression level of their corresponding protein. Exploratory gene ontology suggested that molecular mechanisms of glycopeptiforms associated with cognitive decline may involve metabolic pathways including pyruvate and NADH pathways and highlighted the importance of molecular mechanisms involved in glucose metabolism. DISCUSSION: Glycopeptiforms in aging brains may provide cognitive resilience. Targeting these glycopeptiforms may lead to therapies that maintain cognition through resilience.

Mechanistic insights into ligand dissociation from the SARS-CoV-2 spike glycoprotein.

The COVID-19 pandemic, driven by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spurred an urgent need for effective therapeutic interventions. The spike glycoprotein of the SARS-CoV-2 is crucial for infiltrating host cells, rendering it a key candidate for drug development. By interacting with the human angiotensin-converting enzyme 2 (ACE2) receptor, the spike initiates the infection of SARS-CoV-2. Linoleate is known to bind the spike glycoprotein, subsequently reducing its interaction with ACE2. However, the detailed mechanisms underlying the protein-ligand interaction remain unclear. In this study, we characterized the pathways of ligand dissociation and the conformational changes associated with the spike glycoprotein by using ligand Gaussian accelerated molecular dynamics (LiGaMD). Our simulations resulted in eight complete ligand dissociation trajectories, unveiling two distinct ligand unbinding pathways. The preference between these two pathways depends on the gate distance between two α-helices in the receptor binding domain (RBD) and the position of the N-linked glycan at N343. Our study also highlights the essential contributions of K417, N121 glycan, and N165 glycan in ligand unbinding, which are equally crucial in enhancing spike-ACE2 binding. We suggest that the presence of the ligand influences the motions of these residues and glycans, consequently reducing accessibility for spike-ACE2 binding. These findings enhance our understanding of ligand dissociation from the spike glycoprotein and offer significant implications for drug design strategies in the battle against COVID-19.

WNT2B activates macrophages via NF-κB signaling pathway in inflammatory bowel disease.

Inflammation is a significant pathological manifestation of inflammatory bowel disease (IBD), yet its mechanism has remained unclear. Although WNT2B is enriched in the intestinal inflammatory tissue of IBD patients, the specific mechanism of WNT2B in the formation of intestinal inflammation remains unclear. This study was aimed to investigate whether macrophages expressing WNT2B can aggravate intestinal tissue inflammation. Samples were collected from both normal individuals and patients with IBD at multiple colon sites. Macrophages were identified using tissue immunofluorescence. IκB kinase (IKK)-interacting protein (IKIP), which interacts with WNT2B, was found by protein cross-linking and protein mass spectrometry. The expression of WNT2B, IKIP, the NF-κB pathway, and downstream molecules were analyzed. An acute colitis model of C57BL/6J mice was established using an adeno-associated virus (AAV)-mediated WNT2B knockdown system and 3% dextran sulfate sodium (DSS). The degree of intestinal inflammation in mice was assessed upon WNT2B knockdown in macrophages. Macrophages expressing WNT2B were found to be enriched in the colitis tissues of IBD patients. WNT2B in macrophages activated the NF-κB pathway and enhanced the expression of downstream inflammatory cytokines. By competitively binding IKIP, WNT2B reduced the binding of IKIP to IKKß and promoted the activation of the NF-κB pathway. Using an AAV-mediated WNT2B knockdown system, WNT2B expression in intestinal macrophages was suppressed, leading to a reduction in intestinal inflammation. WNT2B activated the NF-κB pathway and enhanced the expression of downstream inflammatory cytokines by competitively binding to IKIP, potentially contributing to colon inflammatory injury in IBD.

Prognostic potency of plasma LRG1 measurement at multiple time points in acute ischemic stroke patients.

Objective: This study aimed to investigate the prognostic potency of LRG1 in acute ischemic stroke (AIS) patients. Methods: Plasma LRG1 levels were detected at admission and on days 3, 7 and 30 in 150 AIS patients. Results: LRG1 positively correlated with total cholesterol (p = 0.016), triglycerides (p = 0.046), C-reactive protein (p < 0.001), TNF-α (p = 0.001) and IL-6 (p = 0.004). After admission, LRG1 showed a decreasing trend (p < 0.001). Interestingly, LRG1 levels at admission (p = 0.014), day 3 (p = 0.027), day 7 (p = 0.008) and day 30 (p = 0.002) were higher in patients with modified Rankin scale score ≥2 versus those with scores <2. The LRG1 levels at day 7 (p = 0.032) and day 30 (p = 0.023) were higher in patients with recurrence versus no recurrence. Conclusion: LRG1 correlates with blood lipids, inflammation and short-term prognosis of AIS.

Decoding the glycoproteome: a new frontier for biomarker discovery in cancer.

Cancer early detection and treatment response prediction continue to pose significant challenges. Cancer liquid biopsies focusing on detecting circulating tumor cells (CTCs) and DNA (ctDNA) have shown enormous potential due to their non-invasive nature and the implications in precision cancer management. Recently, liquid biopsy has been further expanded to profile glycoproteins, which are the products of post-translational modifications of proteins and play key roles in both normal and pathological processes, including cancers. The advancements in chemical and mass spectrometry-based technologies and artificial intelligence-based platforms have enabled extensive studies of cancer and organ-specific changes in glycans and glycoproteins through glycomics and glycoproteomics. Glycoproteomic analysis has emerged as a promising tool for biomarker discovery and development in early detection of cancers and prediction of treatment efficacy including response to immunotherapies. These biomarkers could play a crucial role in aiding in early intervention and personalized therapy decisions. In this review, we summarize the significant advance in cancer glycoproteomic biomarker studies and the promise and challenges in integration into clinical practice to improve cancer patient care.

Modulation of autophagy affected tumorigenesis induced by the envelope glycoprotein of JSRV.

Ovine pulmonary adenocarcinoma (OPA), caused by the jaagsiekte sheep retrovirus (JSRV), is a chronic, progressive, and contagious lung tumor that seriously affects sheep production. It also represents a valuable animal model for several human lung adenocarcinomas. However, little is known about the role of autophagy in OPA tumorigenesis. Here, Western blotting combined with transmission electron microscopy examination and Cyto-ID dye staining was employed for evaluation of changes of autophagic levels. The results of the present study showed that expression of the autophagy marker proteins Beclin-1 and LC3 was decreased in OPA lung tissues, as well as in cells overexpressing the envelope glycoprotein of JSRV (JSRV Env). Reduced numbers of autophagosomes were also observed in cells overexpressing JSRV Env, although assessment of autophagic flux showed that JSRV Env overexpression did not block the formation of autophagosomes, suggesting increased degradation of autolysosomes. Last, mouse xenograft experiments indicated that inhibition of autophagy by 3-methyladenine suppressed both tumor growth and the epithelial-to-mesenchymal transition. In conclusion, JSRV, through JSRV Env, takes advantage of the autophagy process, leading to the development of OPA.

Epstein-Barr virus gp42 antibodies reveal sites of vulnerability for receptor binding and fusion to B cells.

Epstein-Barr virus (EBV) causes infectious mononucleosis and is associated with B cell lymphomas. EBV glycoprotein 42 (gp42) binds HLA class II and activates membrane fusion with B cells. We isolated gp42-specific monoclonal antibodies (mAbs), A10 and 4C12, which use distinct mechanisms to neutralize virus infection. mAb A10 was more potent than the only known neutralizing gp42 mAb, F-2-1, in neutralizing EBV infection and blocking binding to HLA class II. mAb 4C12 was similar to mAb A10 in inhibiting glycoprotein-mediated B cell fusion but did not block receptor binding, and it was less effective in neutralizing infection. Crystallographic structures of gH/gL/gp42/A10 and gp42/4C12 complexes revealed two distinct sites of vulnerability on gp42 for receptor binding and B cell fusion. Passive transfer of mAb A10 into humanized mice conferred nearly 100% protection from viremia and EBV lymphomas after EBV challenge. These findings identify vulnerable sites on EBV that may facilitate therapeutics and vaccines.

Afforestation enhances glomalin-related soil protein content but decreases its contribution to soil organic carbon in a subtropical karst area.

Afforestation on degraded croplands has been proposed as an effective measure to promote ecosystem functions including soil organic carbon (SOC) sequestration. Glomalin-related soil protein (GRSP) plays a crucial role in promoting the accumulation and stability of SOC. Nevertheless, mechanisms underlying the effects of afforestation on GRSP accumulation have not been well elucidated. In the present study, 14 pairs of maize fields and plantation forests were selected using a paired-site approach in a karst region of southwest China. By measuring soil GRSP and a variety of soil biotic and abiotic variables, the pattern of and controls on GRSP accumulation in response to afforestation were explored. The average content of total GRSP (T-GRSP) and its contribution to SOC in the maize field were 5.22 ± 0.29 mg g-1 and 42.33 ± 2.25%, and those in the plantation forest were 6.59 ± 0.32 mg g-1 and 25.77 ± 1.17%, respectively. T-GRSP content was increased by 26.4% on average, but its contribution to SOC was decreased by 39.1% following afforestation. T-GRSP content decreased as soil depth increased regardless of afforestation or not. Afforestation increased T-GRSP indirectly via its positive effects on arbuscular mycorrhizal fungi biomass, which was stimulated by afforestation through elevating fine root biomass or increasing the availability of labile C and N. The suppressed contribution of T-GRSP to SOC following afforestation was due to the relatively higher increase in other SOC components than T-GRSP and the significant increase of soil C:N ratio. Our study reveals the mechanisms underlying the effects of afforestation on T-GRSP accumulation, and is conducive to improving the mechanistic understanding of microbial control on SOC sequestration following afforestation.

Structural Studies of Henipavirus Glycoproteins.

Henipaviruses are a genus of emerging pathogens that includes the highly virulent Nipah and Hendra viruses that cause reoccurring outbreaks of disease. Henipaviruses rely on two surface glycoproteins, known as the attachment and fusion proteins, to facilitate entry into host cells. As new and divergent members of the genus have been discovered and structurally characterized, key differences and similarities have been noted. This review surveys the available structural information on Henipavirus glycoproteins, complementing this with information from related biophysical and structural studies of the broader Paramyxoviridae family of which Henipaviruses are members. The process of viral entry is a primary focus for vaccine and drug development, and this review aims to identify critical knowledge gaps in our understanding of the mechanisms that drive Henipavirus fusion.

Molecular-level architecture of Chlamydomonas reinhardtii's glycoprotein-rich cell wall.

Microalgae are a renewable and promising biomass for large-scale biofuel, food and nutrient production. However, their efficient exploitation depends on our knowledge of the cell wall composition and organization as it can limit access to high-value molecules. Here we provide an atomic-level model of the non-crystalline and water-insoluble glycoprotein-rich cell wall of Chlamydomonas reinhardtii. Using in situ solid-state and sensitivity-enhanced nuclear magnetic resonance, we reveal unprecedented details on the protein and carbohydrate composition and their nanoscale heterogeneity, as well as the presence of spatially segregated protein- and glycan-rich regions with different dynamics and hydration levels. We show that mannose-rich lower-molecular-weight proteins likely contribute to the cell wall cohesion by binding to high-molecular weight protein components, and that water provides plasticity to the cell-wall architecture. The structural insight exemplifies strategies used by nature to form cell walls devoid of cellulose or other glycan polymers.

Significance of Serum Leucine-rich Alpha-2 Glycoprotein as a Diagnostic Marker in Pediatric Inflammatory Bowel Disease.

Serum leucine-rich alpha-2 glycoprotein (LRG) has been utilized for adult inflammatory bowel disease (IBD); however, its efficacy in pediatric IBD remains unknown. The aim of this study was to compare the diagnostic accuracy of serum LRG for pediatric IBD with that of current inflammatory markers, erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP). This retrospective case-control study included pediatric patients, aged <16 years, who underwent colonoscopy and/or esophagogastroduodenoscopy between April 2017 and March 2022. All eligible patients were divided into two groups: patients with IBD, diagnosed with ulcerative colitis and Crohn's disease, and non-IBD controls. The optimal cut-off value of serum LRG for IBD diagnosis was determined from receiver operating characteristic analysis, and diagnostic accuracy of serum LRG was compared to serum ESR and CRP. A total of 53 patients (24 with IBD and 29 non-IBD controls) met the inclusion criteria. The cut-off value of serum LRG for IBD diagnosis was determined to be 19.5 µg/ml. At this cut-off value, serum LRG had a positive predictive value (PPV) of 0.80 and negative predictive value (NPV) of 0.88. In contrast, PPV and NPV were 0.78 and 0.70 for serum ESR and 0.82 and 0.72 for serum CRP, respectively. Serum LRG can be a potential diagnostic marker for pediatric IBD, with higher diagnostic accuracy than that of the conventional serum markers ESR and CRP.