Glycan Profiling Research Articles

Site specific N- and O-glycosylation mapping of the spike proteins of SARS-CoV-2 variants of concern

The glycosylation on the spike (S) protein of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, modulates the viral infection by altering conformational dynamics, receptor interaction and host immune responses. Several variants of concern (VOCs) of SARS-CoV-2 have evolved during the pandemic, and crucial mutations on the S protein of the virus have led to increased transmissibility and immune escape. In this study, we compare the site-specific glycosylation and overall glycomic profiles of the wild type Wuhan-Hu-1 strain (WT) S protein and five VOCs of SARS-CoV-2: Alpha, Beta, Gamma, Delta and Omicron. Interestingly, both N- and O-glycosylation sites on the S protein are highly conserved among the spike mutant variants, particularly at the sites on the receptor-binding domain (RBD). The conservation of glycosylation sites is noteworthy, as over 2 million SARS-CoV-2 S protein sequences have been reported with various amino acid mutations. Our detailed profiling of the glycosylation at each of the individual sites of the S protein across the variants revealed intriguing possible association of glycosylation pattern on the variants and their previously reported infectivity. While the sites are conserved, we observed changes in the N- and O-glycosylation profile across the variants. The newly emerged variants, which showed higher resistance to neutralizing antibodies and vaccines, displayed a decrease in the overall abundance of complex-type glycans with both fucosylation and sialylation and an increase in the oligomannose-type glycans across the sites. Among the variants, the glycosylation sites with significant changes in glycan profile were observed at both the N-terminal domain and RBD of S protein, with Omicron showing the highest deviation. The increase in oligomannose-type happens sequentially from Alpha through Delta. Interestingly, Omicron does not contain more oligomannose-type glycans compared to Delta but does contain more compared to the WT and other VOCs. O-glycosylation at the RBD showed lower occupancy in the VOCs in comparison to the WT. Our study on the sites and pattern of glycosylation on the SARS-CoV-2 S proteins across the VOCs may help to understand how the virus evolved to trick the host immune system. Our study also highlights how the SARS-CoV-2 virus has conserved both N- and O- glycosylation sites on the S protein of the most successful variants even after undergoing extensive mutations, suggesting a correlation between infectivity/ transmissibility and glycosylation.

Use of single analytic tool to quantify both absolute N-glycosylation and glycan distribution in monoclonal antibodies.

Recombinant proteins represent almost half of the top selling therapeutics-with over a hundred billion dollars in global sales-and their efficacy and safety strongly depend on glycosylation. In this study, we showcase a simple method to simultaneously analyze N-glycan micro- and macroheterogeneity of an immunoglobulin G (IgG) by quantifying glycan occupancy and distribution. Our approach is linear over a wide range of glycan and glycoprotein concentrations down to 25 ng/mL. Additionally, we present a case study demonstrating the effect of small molecule metabolic regulators on glycan heterogeneity using this approach. In particular, sodium oxamate (SOD) decreased Chinese hamster ovary (CHO) glucose metabolism and reduced IgG glycosylation by 40% through upregulating reactive oxygen species (ROS) and reducing the UDP-GlcNAc pool, while maintaining a similar glycan profile to control cultures. Here, we suggest glycan macroheterogeneity as an attribute should be included in bioprocess screening to identify process parameters that optimize culture performance without compromising antibody quality.

Human Milk Oligosaccharides And Site-Specific Glycosylation Heterogeneity Of Filipino Human Milk Glycoproteins Exhibit Dynamic Variations During Lactation As Influenced By Secretor Status And Coastal Environmental Condition – A Preliminary Study

Human Milk Oligosaccharides (HMOs) have a role in the immune system development of infants. HMOs and the N-glycans of human milk glycoproteins, especially highly fucosylated ones, function as prebiotics to the bifidogenic gut microbiota. This study reports the first longitudinal profile of HMOs and human milk glycoproteins from coastal, noncoastal, secretor and nonsecretor Filipino mothers analyzed and annotated using QTOF LC/MS and QqQ LC/MS - MRM, respectively. A milk glycan profile from coastal mothers revealed higher (P<0.05) presence of fucosylated branched HMOs, such as DFLNHa, LDFT, LNDFH I, and LNFP II. Moreover, secretor mothers expressed the highest 2’FL from colostrum (Day 5) until the end of the study (Month 6). Interestingly, the predominance of secretory IgA (64%) on the total milk glycoproteins from Filipino mothers supported their biological adaptation in conferring immune protection to their breastfeeding infant. Glycoform variations on milk glycoproteins, sIgA, IgG, Lactoferrin, and a1-Antitrypsin, in coastal mothers were observed. Moreover, lactoferrin was found to be highly glycosylated with 20 fucosylated and sialylated glycoforms. These results suggest that the coastal mother’s unique long-term seafood diet may have influenced their milk glycan profile and the potential incorporation of L-fucose derived from seafood in the biosynthesis of their milk glycans.

Screening Clones for Monoclonal Antibody Production Using Droplet Microfluidics Interfaced to Electrospray Ionization Mass Spectrometry.

As one of the most critical steps in process development for protein therapeutics, clone selection and cell culture optimization require a large number of samples to be screened for high titer and desirable molecular profiles. Typical analytical techniques, such as chromatographic approaches, often take minutes per sample which are inefficient for large-scale screenings. Droplet microfluidics coupled to mass spectrometry (MS) represents an attractive approach due to its low volume requirements, high-throughput capabilities, label-free nature, and ability to handle complex mixtures. In this work, we coupled a modified protein cleanup protocol with a droplet-MS workflow for mAb titer screening to guide clone selection. With this droplet approach we achieved a throughput of 0.04 samples/s with an LoD of 0.15 mg/mL and an LoQ of 0.45 mg/mL. To test its performance in a real-world setting, this workflow was applied to a 35-clone screen, where the top 20% producing clones were identified. In addition, we coupled our sample cleanup protocol to a high-resolution MS and compared the glycan profiles of the high titer clones. This work demonstrates that droplet-MS provides a rapid way of clone screening and cell culture optimization based on titer and molecular structure of the expressed proteins. Future work is aimed at increasing the throughput and automation of this droplet-MS technique.

Heightened Proinflammatory Glycosylation of Borrelia burgdorferi IgG Antibodies in Synovial Fluid in Patients With Antibiotic-Refractory Lyme Arthritis.

Terminal glycans on the Fc portion of IgG antibodies are critical for antibody-triggered, proinflammatory or antiinflammatory responses. We undertook this study to compare glycan profiles of total IgG1 and Borrelia burgdorferi (Bb)-specific IgG1 antibodies in patients with oral antibiotic-responsive or antibiotic-refractory Lyme arthritis (LA). Following affinity-column processing, glycan profiles of IgG antibodies were determined in serum and synovial fluid (SF) samples of 21 LA patients using glycoblotting with hydrazide glycan enrichment and determination of glycan structure by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. Correlations between glycan profiles and treatment outcomes were analyzed. Compared with patients with antibiotic-refractory LA, those with antibiotic-responsive LA had total and Bb-specific IgG1 antibody glycans with less intense inflammatory profiles, containing lower percentages of N-acetylglucosamine (GlcNAc) and bisecting GlcNAc and higher percentages of galactose and fucose. In contrast, patients with antibiotic-refractory LA prior to receiving IV antibiotic therapy had total IgG1 and Bb IgG1 antibodies with maximal, minimally opposed, proinflammatory glycan profiles, containing high percentages of GlcNAc and bisecting GlcNAc, intermediate percentages with galactose and fucose, and low percentages with N-acetylneuraminic acid (sialic acid). Patients with refractory LA who were first seen with synovitis after receiving IV antibiotic therapy still had Bb IgG1 antibodies with strongly inflammatory glycan profiles, but their inflammatory potential appeared to be waning. Patients with oral antibiotic-responsive LA had Bb IgG1 antibodies with more balanced proinflammatory/antiinflammatory glycan profiles, whereas patients with antibiotic-refractory LA had Bb IgG1 antibodies with maximal, minimally opposed, proinflammatory glycan profiles. Among patients with antibiotic-refractory LA, antibodies with this unbalanced inflammatory glycan profile may have a role in sustaining maladaptive joint inflammation.

Expression of placental glycans and its role in regulating peripheral blood NK cells during preeclampsia: a perspective.

Preeclampsia is a pregnancy-related multisystem disorder characterized by altered trophoblast invasion, oxidative stress, exacerbation of systemic inflammatory response, and endothelial damage. The pathogenesis includes hypertension and mild-to-severe microangiopathy in the kidney, liver, placenta, and brain. The main mechanisms involved in its pathogenesis have been proposed to limit trophoblast invasion and increase the release of extracellular vesicles from the syncytiotrophoblast into the maternal circulation, exacerbating the systemic inflammatory response. The placenta expresses glycans as part of its development and maternal immune tolerance during gestation. The expression profile of glycans at the maternal-fetal interface may play a fundamental role in physiological pregnancy changes and disorders such as preeclampsia. It is unclear whether glycans and their lectin-like receptors are involved in the mechanisms of maternal-fetal recognition by immune cells during pregnancy homeostasis. The expression profile of glycans appears to be altered in hypertensive disorders of pregnancy, which could lead to alterations in the placental microenvironment and vascular endothelium in pregnancy conditions such as preeclampsia. Glycans with immunomodulatory properties at the maternal-fetal interface are altered in early-onset severe preeclampsia, implying that innate immune system components, such as NK cells, exacerbate the systemic inflammatory response observed in preeclampsia. In this article, we discuss the evidence for the role of glycans in gestational physiology and the perspective of glycobiology on the pathophysiology of hypertensive disorders in gestation.

Glycans in spent embryo culture medium are related to the implantation ability of blastocysts

Research questionDoes glycan profile in spent blastocyst culture medium have the potential to be used as a biomarker to predict implantation outcome. DesignA nested case-control study was conducted in Northwest women’s and children’s Hospital, Xi’an, China. The patients underwent fresh IVF/ICSI cycles with single blastocyst transfer were included. Total 78 cases were included and separated into groups according to success (n = 39) and failure (n = 39) implantation outcomes. The glycosylation patterns in spent blastocyst culture medium were detected by lectin microarray containing 37 lectins using pooled samples and confirmed by reversed lectin microarray using individual sample. ResultsBinding signals of 10 lectins were found to be different between samples from successful and failed implantation. And 8 of them were confirmed that glycans binding to lectin NPA, UEA-I, MAL-I, LCA and GNA were significantly increased while DBA and BPL were decreased in the successful implantation compared to failed implantation. The glycan binding to lectin PHA-E + L had no difference between two groups. No significant differences in the glycan profile were found in spent culture medium of embryos with different morphological grades except the glycan binding to UEA-I between blastocysts of Poor and blastocysts of Medium. ConclusionDetection of glycan profile in spent culture medium may lead to a novel non-invasive assessment assay of embryo viability. In addition, these results may be helpful to further understanding molecular mechanisms in embryo implantation.

Role of IgG glycans in severe COVID-19 inflammatory cytokine responses

Abstract Severe COVID-19 and multisystem inflammatory syndrome in children (MIS-C) are characterized by hyperinflammation. Severe COVID in adults is associated with development of afucosylated SARS-CoV-2 IgG that induce monocytes to secrete inflammatory cytokines. This study aimed to analyze the relationship between spike IgG Fc glycosylation and inflammatory markers during acute pediatric COVID or MIS-C or following COVID vaccination in adults. We assessed cross-sectional and longitudinal samples from 195 participants: Adult vaccinees, acute COVID patients, and healthy controls, as well as pediatric acute COVID patients, MIS-C patients, and healthy controls. We developed capillary electrophoresis (CE) methods to analyze bulk and spike IgG Fc glycans and measured ten relevant cytokines/chemokines by multiplexed assay. Analysis of healthy control bulk IgG by CE found 95.3–99.6% fucosylated, 2.7–6.9% bisected, 55.8–66.8% galactosylated, and 4.6–15.6% sialylated Fc glycans, compared to 99.8%, 6.7%, 45.4%, and 8.2% by mass spectrometry, respectively. IFN-γ, IL-10, CXCL10, and CCL3 were significantly increased in MIS-C compared to pediatric healthy controls; IL-10, CCL2, and TNF were only increased in severe adult COVID compared to healthy controls or less severe COVID. Initial results confirmed significantly more afucosylated spike IgG in adult severe COVID than afucosylated bulk IgG in controls. Our high-throughput methods for IgG Fc glycan profiling generated comparable results to mass spectrometry. Continued assessment will determine if COVID- or vaccine-induced spike IgG glycoforms correlate with inflammatory markers. Future mechanistic work will examine how modified glycosylation induces inflammatory cytokines. Supported by an IDCRC New Investigator Pilot Award to E.M.S. (NIH UM1AI148684), by the Centers for Disease Control and Prevention through a cooperative agreement with the Georgia Emerging Infection Program (grant no. U50CK000485), and by grant from NIH to P.A. (R24GM137782).

Effect of Xenogeneic Substances on the Glycan Profiles and Electrophysiological Properties of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Human induced pluripotent stem cell (hiPSC)-derived cardiomyocyte (CM) hold great promise as a cellular source of CM for cardiac function restoration in ischemic heart disease. However, the use of animal-derived xenogeneic substances during the biomanufacturing of hiPSC-CM can induce inadvertent immune responses or chronic inflammation, followed by tumorigenicity. In this study, we aimed to reveal the effects of xenogeneic substances on the functional properties and potential immunogenicity of hiPSC-CM during differentiation, demonstrating the quality and safety of hiPSC-based cell therapy. We successfully generated hiPSC-CM in the presence and absence of xenogeneic substances (xeno-containing (XC) and xeno-free (XF) conditions, respectively), and compared their characteristics, including the contractile functions and glycan profiles. Compared to XC-hiPSC-CM, XF-hiPSC-CM showed early onset of myocyte contractile beating and maturation, with a high expression of cardiac lineage-specific genes (ACTC1, TNNT2, and RYR2) by using MEA and RT-qPCR. We quantified N-glycolylneuraminic acid (Neu5Gc), a xenogeneic sialic acid, in hiPSC-CM using an indirect enzyme-linked immunosorbent assay and liquid chromatography-multiple reaction monitoring- mass spectrometry. Neu5Gc was incorporated into the glycans of hiPSC-CM during xeno-containing differentiation, whereas it was barely detected in XF-hiPSC-CM. To the best of our knowledge, this is the first study to show that the electrophysiological function and glycan profiles of hiPSC-CM can be affected by the presence of xenogeneic substances during their differentiation and maturation. To ensure quality control and safety in hiPSC-based cell therapy, xenogeneic substances should be excluded from the biomanufacturing process.

Real time on-line amino acid analysis to explore amino acid trends and link to glycosylation outcomes of a model monoclonal antibody.

Bioreactor parameters can have significant effects on the quantity and quality of biotherapeutics. Monoclonal antibody products have one particularly important critical quality attribute being the distribution of product glycoforms. N-linked glycosylation affects the therapeutic properties of the antibody including effector function, immunogenicity, stability, and clearance rate. Our past work revealed that feeding different amino acids to bioreactors altered the productivity and glycan profiles. To facilitate real-time analysis of bioreactor parameters and the glycosylation of antibody products, we developed an on-line system to pull cell-free samples directly from the bioreactors, chemically process them, and deliver them to a chromatography-mass spectroscopy system for rapid identification and quantification. We were able to successfully monitor amino acid concentration on-line within multiple reactors, evaluate glycans off-line, and extract four principal components to assess the amino acid concentration and glycosylation profile relationship. We found that about a third of the variability in the glycosylation data can be predicted from the amino acid concentration. Additionally, we determined that the third and fourth principal component accounts for 72% of our model's predictive power, with the third component indicated to be positively correlated with latent metabolic processes related to galactosylation. Here we present our work on rapid online spent media amino acid analysis and use the determined trends to collate with glycan time progression, further elucidating the correlation between bioreactor parameters such as amino acid nutrient profiles, and product quality. We believe such approaches may be useful for maximizing efficiency and reducing production costs for biotherapeutics.

Surface Glycan Profiling of Extracellular Vesicles by Lectin Microarray and Glycoengineering for Control of Cellular Interactions.

Extracellular vesicles (EVs)are a group of cell-derived membrane vesicles that carry a variety of cargo such as protein, nucleic acids, andlipids, and are secreted by almost all cell types. Functionally, EVs play important roles in physiological andpathological processes such as immune responses and tumor growth through intercellular communication bytransferring this molecular information between cells. Therefore, they have potential versatile clinical applicationsas disease biomarkers and drug delivery carriers. Notably, subpopulations of EVsexhibit distinct characteristics depending on their cell of origin, including the expression of surface glycans, whichhave been implicated in a variety of cellular processes such as field cancerization, cell recognition, and signaltransduction. However, these are features have not been fully exploited because of the difficulty in analyzing theseproteins. In this paper, we summarize the advancements in glycoengineering andhigh-performance lectin microarray for high-throughput analysis of EV glycans to generate an index ofheterogeneity to identify disease biomarkers, and describe how understanding the function of EVs in disease canenhance their potential application in the clinic.

Insights into the Shape-Selective Retention Properties of Porous Graphitic Carbon Stationary Phases and Applicability for Polar Compounds

In this article, the ability of porous graphitic carbon (PGC) to discriminate between structurally similar compounds is described. After a brief overview of the particle technology and its use in liquid chromatography (LC), two case studies are presented using small-molecule probes to illustrate the importance on how molecular shape and the ability to interact with the graphitic surface lead to the resolution of structural isomers. Finally, an application of this technology in the area of glycan analysis is showcased where released N-glycans from human immunoglobulin G (IgG) were resolved without derivatization, enabling the glycan profile of a biotherapeutic to be determined.

The role of glycan characterisation in ATMP development and evaluation

AbstractAdvanced therapeutic medicinal products (ATMP) aim to transform treatment strategies for unmet clinical needs. ATMP development requires product standardisation, high‐quality consistent starting materials, and safety and efficacy, while reducing adverse reactions. Glycosylation is a common post‐translational modification of glycans to specific peptide sites. Changes in glycosylation in starting materials or culture can potentially affect the bioactivity, safety and efficacy of glycosylated products. Glycan analysis is applied to few biological products, most commonly monoclonal antibodies and glycoconjugate vaccines. Regulatory authorities are starting to scrutinise the glycan profiles of biomedical products more carefully since glycosylation‐mediated activity has been recognised to lead to adverse effects in this class of therapies, including allergic/hypersensitivity reactions, anti‐drug antibodies and product heterogeneity in biologic and advanced treatments. This review highlights the impact of glycosylation in cell and tissue substrates for use in advanced therapies, identifying the need for glycan analysis to design safer, consistent products.

Identification of tagged glycans with a protein nanopore

Structural complexity of glycans derived from the diversities in composition, linage, configuration, and branching considerably complicates structural analysis. Nanopore-based single-molecule sensing offers the potential to elucidate glycan structure and even sequence glycan. However, the small molecular size and low charge density of glycans have restricted direct nanopore detection of glycan. Here we show that glycan sensing can be achieved using a wild-type aerolysin nanopore by introducing a facile glycan derivatization strategy. The glycan molecule can induce impressive current blockages when moving through the nanopore after being connected with an aromatic group-containing tag (plus a carrier group for the neutral glycan). The obtained nanopore data permit the identification of glycan regio- and stereoisomers, glycans with variable monosaccharide numbers, and distinct branched glycans, either independently or with the use of machine learning methods. The presented nanopore sensing strategy for glycans paves the way towards nanopore glycan profiling and potentially sequencing.

Life stage-specific glycosylation of extracellular vesicles from Schistosoma mansoni schistosomula and adult worms drives differential interaction with C-type lectin receptors DC-SIGN and MGL.

Schistosomes can survive in mammalian hosts for many years, and this is facilitated by released parasite products that modulate the host's immune system. Many of these products are glycosylated and interact with host cells via C-type lectin receptors (CLRs). We previously reported on specific fucose-containing glycans present on extracellular vesicles (EVs) released by schistosomula, the early juvenile life stage of the schistosome, and the interaction of these EVs with the C-type lectin receptor Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN or CD209). EVs are membrane vesicles with a size range between 30-1,000nm that play a role in intercellular and interspecies communication. Here, we studied the glycosylation of EVs released by the adult schistosome worms. Mass spectrometric analysis showed that GalNAcβ1-4GlcNAc (LacDiNAc or LDN) containing N-glycans were the dominant glycan type present on adult worm EVs. Using glycan-specific antibodies, we confirmed that EVs from adult worms were predominantly associated with LDN, while schistosomula EVs displayed a highly fucosylated glycan profile. In contrast to schistosomula EV that bind to DC-SIGN, adult worm EVs are recognized by macrophage galactose-type lectin (MGL or CD301), and not by DC-SIGN, on CLR expressing cell lines. The different glycosylation profiles of adult worm- and schistosomula-derived EVs match with the characteristic glycan profiles of the corresponding life stages and support their distinct roles in schistosome life-stage specific interactions with the host.

Relevance of glyco-biomakers and glycan profiles in cancer stem cells.

Altered and aberrant glycosylation signatures have been linked to being a hallmark in a variety of human disorders including cancer. Cancer stem cells (CSCs), capable of self-renewal and differentiation, have recently been credited with a unique notion of disease genesis and implicated as the cause for initiation and recurrence of the disease in a new regime of neoplastic transformations hypothesis. Many biomarkers relating to diagnostic and prognostic intents have been discovered using the ubiquitous and abundant surface glycan patterns on CSCs. Various technological advancements have been developed to identify and determine concerns with glycosylation structure. However, the nature and purpose of the glycan moiety on these glycosylation pattern have not yet been thoroughly investigated. This review, thus, summarizes the process of glycosylation in CSCs, variations in glycosylation patterns in various stem cells, aberrant glycosylation patterns in cancer, the role of glycosylation in tumor cell adhesion, cell-matrix interactions, and signaling, as well as cancer detection and treatment. The function of carbohydrates as prospective serum biomarkers, some clinically authorized biomarkers, and potential novel biomarkers relating to cancer disease diagnosis and prognosis are also discussed in the review.

Spatiotemporal changes of tissue glycans depending on localization in cardiac aging.

We investigated glycan localization in the cardiac tissue of normal mice and their alterations during aging, using evanescent-field fluorescence-assisted lectin microarray, a technique based on lectin-glycan interaction, and lectin staining. The glycan profiles in the left ventricle showed differences between the luminal side (medial) and wall side (lateral) regions. The medial region was characterized by the presence of sialic acid residues. Moreover, age-related changes in glycan profiles were observed at a younger age in the medial region. The difference in the age-related decrease in the level of α-galactose stained with Griffonia simplicifolia lectin-IB4 in different regions of the left ventricle suggests spatiotemporal changes in the number of microvessels. The glycan profile, which retains diverse glycan structures, is supported by many cell populations, and maintains cardiac function. With further research, glycan localization and changes have the potential to be developed as a marker of the signs of heart failure.

Retrospective screening of serum IgG glycosylation biomarker for primary Sjögren's syndrome using lectin microarray.

Primary Sjögren's syndrome (PSS) is a systemic autoimmune disease resulting in significant loss of systemic gland secretory function. IgG glycosylation abnormalities had been found to play important roles in autoimmune diseases. Here, we aim to explore the specific changes of IgG glycosylation in PSS patient serum that could serve as potential biomarkers for disease diagnosis and differential diagnosis. From 2012 to 2018, patients diagnosed with PSS or primary biliary cholangitis (PBC) admitted consecutively to the department of Rheumatology at Peking Union Medical College Hospital were retrospectively included in this study. Glycan profiles of serum IgG from 40 PSS patients, 50 PBC patients, and 38 healthy controls were detected with lectin microarray containing 56 lectins. Lectins with significantly different signal intensity among groups were selected and validated by lectin blot assay. Lectin microarray analysis revealed that binding levels of Amaranthus Caudatus Lectin (ACL, prefers glycan Galβ3GalNAc, P = 0.011), Morniga M Lectin (MNA-M, prefers glycan mannose. P = 0.013), and Lens Culinaris Agglutinin (LCA, prefers glycan fucose) were significantly increased, while Salvia sclarea Agglutinin (SSA, prefers glycan sialylation, P = 0.001) was significantly decreased in PSS patients compared to PBC group. Compared to healthy controls, MNA-M (P = 0.001) and LCA (P = 0.028) were also significantly increased, while Phaseolus Vulgaris Erythroagglutinin and Phaseolus Vulgaris Leucoagglutinin (PHA-E and PHA-L, prefer glycan galactose, P = 0.004 and 0.006) were significantly decreased in PSS patients. The results of LCA and MNA-M were further confirmed using lectin blot assay. Changes in serum IgG glycosylation in PSS increased binding levels of LCA and MNA-M lectins using microarray techniques compared to PBC patients and healthy controls, which could provide potential diagnostic value. Increased core fucose and mannose alteration of IgG may play important roles in PSS disease.

Changes of serum IgG glycosylation patterns in rheumatoid arthritis

BackgroundRA is a common chronic and systemic autoimmune disease, and the diagnosis is based significantly on autoantibody detection. This study aims to investigate the glycosylation profile of serum IgG in RA patients using high-throughput lectin microarray technology.MethodLectin microarray containing 56 lectins was applied to detect and analyze the expression profile of serum IgG glycosylation in 214 RA patients, 150 disease controls (DC), and 100 healthy controls (HC). Significant differential glycan profiles between the groups of RA and DC/HC as well as RA subgroups were explored and verified by lectin blot technique. The prediction models were created to evaluate the feasibility of those candidate biomarkers.ResultsAs a comprehensive analysis of lectin microarray and lectin blot, results showed that compare with HC or DC groups, serum IgG from RA patients had a higher affinity to the SBA lectin (recognizing glycan GalNAc). For RA subgroups, RA-seropositive group had higher affinities to the lectins of MNA-M (recognizing glycan mannose) and AAL (recognizing glycan fucose), and RA-ILD group had higher affinities to the lectins of ConA (recognizing glycan mannose) and MNA-M while a lower affinity to the PHA-E (recognizing glycan Galβ4GlcNAc) lectin. The predicted models indicated corresponding feasibility of those biomarkers.ConclusionLectin microarray is an effective and reliable technique for analyzing multiple lectin–glycan interactions. RA, RA-seropositive, and RA-ILD patients exhibit distinct glycan profiles, respectively. Altered levels of glycosylation may be related to the pathogenesis of the disease, which could provide a direction for new biomarkers identification.

Golgi fragmentation - One of the earliest organelle phenotypes in Alzheimer's disease neurons.

Alzheimer's disease (AD) is the most common cause of dementia, with no current cure. Consequently, alternative approaches focusing on early pathological events in specific neuronal populations, besides targeting the well-studied amyloid beta (Aβ) accumulations and Tau tangles, are needed. In this study, we have investigated disease phenotypes specific to glutamatergic forebrain neurons and mapped the timeline of their occurrence, by implementing familial and sporadic human induced pluripotent stem cell models as well as the 5xFAD mouse model. We recapitulated characteristic late AD phenotypes, such as increased Aβ secretion and Tau hyperphosphorylation, as well as previously well documented mitochondrial and synaptic deficits. Intriguingly, we identified Golgi fragmentation as one of the earliest AD phenotypes, indicating potential impairments in protein processing and post-translational modifications. Computational analysis of RNA sequencing data revealed differentially expressed genes involved in glycosylation and glycan patterns, whilst total glycan profiling revealed minor glycosylation differences. This indicates general robustness of glycosylation besides the observed fragmented morphology. Importantly, we identified that genetic variants in Sortilin-related receptor 1 (SORL1) associated with AD could aggravate the Golgi fragmentation and subsequent glycosylation changes. In summary, we identified Golgi fragmentation as one of the earliest disease phenotypes in AD neurons in various in vivo and in vitro complementary disease models, which can be exacerbated via additional risk variants in SORL1.