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Keywords = IgA1 glycosylation

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20 pages, 3857 KiB  
Article
Temporal and Sex-Dependent N-Glycosylation Dynamics in Rat Serum
by Hirokazu Yagi, Sachiko Kondo, Reiko Murakami, Rina Yogo, Saeko Yanaka, Fumiko Umezawa, Maho Yagi-Utsumi, Akihiro Fujita, Masako Okina, Yutaka Hashimoto, Yuji Hotta, Yoichi Kato, Kazuki Nakajima, Jun-ichi Furukawa and Koichi Kato
Int. J. Mol. Sci. 2025, 26(15), 7266; https://doi.org/10.3390/ijms26157266 - 27 Jul 2025
Viewed by 403
Abstract
We conducted systematic glycomic and glycoproteomic profiling to characterize the dynamic N-glycosylation landscape of rat serum, with particular focus on sex- and time-dependent variations. MALDI-TOF-MS analysis revealed that rat serum N-glycans are predominantly biantennary, disialylated complex-type structures with extensive O-acetylation [...] Read more.
We conducted systematic glycomic and glycoproteomic profiling to characterize the dynamic N-glycosylation landscape of rat serum, with particular focus on sex- and time-dependent variations. MALDI-TOF-MS analysis revealed that rat serum N-glycans are predominantly biantennary, disialylated complex-type structures with extensive O-acetylation of Neu5Ac residues, especially in females. LC-MS/MS-based glycoproteomic analysis of albumin/IgG-depleted serum identified 87 glycoproteins enriched in protease inhibitors (e.g., serine protease inhibitor A3K) and immune-related proteins such as complement C3. Temporal analyses revealed stable sialylation in males but pronounced daily fluctuations in females, suggesting hormonal influence. Neu5Gc-containing glycans were rare and mainly derived from residual IgG, as confirmed by glycomic analysis. In contrast to liver-derived glycoproteins, purified IgG exhibited Neu5Gc-only sialylation without O-acetylation, underscoring distinct sialylation profiles characteristic of B cell-derived glycoproteins. Region-specific glycosylation patterns were observed in IgG, with the Fab region carrying more disialylated structures than Fc. These findings highlight cell-type and sex-specific differences in sialylation patterns between hepatic and immune tissues, with implications for hormonal regulation and biomarker research. This study provides a valuable dataset on rat serum glycoproteins and underscores the distinctive glycosylation features of rats, reinforcing their utility as model organisms in glycobiology and disease research. Full article
(This article belongs to the Special Issue Glycobiology of Health and Diseases)
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11 pages, 1047 KiB  
Brief Report
Light Chain Isotype and Antibody-Specificity Impact on Virus Neutralization
by Lin Sun, Roman Palt, Georg Schütz, Esther Föderl-Höbenreich, Laura Brod, Antonia Hermle, Anja Lux, Herta Steinkellner and Somanath Kallolimath
Antibodies 2025, 14(2), 50; https://doi.org/10.3390/antib14020050 - 17 Jun 2025
Viewed by 459
Abstract
Therapeutic antibodies with lambda light chains (λ-Abs) are underrepresented compared to kappa light chains (κ-Abs). Here, we evaluated two SARS-CoV-2-specific monoclonal antibodies (mAbs) that exhibit high (P5C3) and low (H4) antigen binding as κ and λ variants. mAbs expressed in glycoengineered Nicotiana benthamiana [...] Read more.
Therapeutic antibodies with lambda light chains (λ-Abs) are underrepresented compared to kappa light chains (κ-Abs). Here, we evaluated two SARS-CoV-2-specific monoclonal antibodies (mAbs) that exhibit high (P5C3) and low (H4) antigen binding as κ and λ variants. mAbs expressed in glycoengineered Nicotiana benthamiana did not show differences in expression levels, glycosylation, and antigen binding, while κ-Abs exhibited slightly increased thermodynamic stability over λ-Abs. SARS-CoV-2 neutralization and IgG-FcγR immune complex studies revealed increased activities of H4 IgG1κ compared to H4 IgG1λ, with no differences observed between P5C3 variants. Our results indicate that constant light chain variability and Ab specificity contribute to Ab features, a fact that should be considered in engineering therapeutics. Full article
(This article belongs to the Section Antibody Discovery and Engineering)
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15 pages, 1218 KiB  
Article
Enhancing the Total Terminal Galactosylation of CHO Cell-Derived TNF-α Blocker-IgG1 Monoclonal Antibody Using Time-Dependent Galactose Supplementation
by Mallikarjuna Pulipeta, Pradeep Kumar Iyer, Rajendra Kumar Palakurthy, Narasimha Pullaguri, Rajasekhar Pinnamaneni and Srinivas Reddy Chilukuri
Biologics 2025, 5(2), 16; https://doi.org/10.3390/biologics5020016 - 11 Jun 2025
Viewed by 816
Abstract
Background: Recombinant monoclonal antibodies represent a vital category of biologics, constituting the largest class of molecules used to treat autoimmune disorders, cancers, rheumatoid arthritis, and other chronic conditions. The IgG1 subclass is the most potent among all the immunoglobulin gamma (IgG) antibodies, inducing [...] Read more.
Background: Recombinant monoclonal antibodies represent a vital category of biologics, constituting the largest class of molecules used to treat autoimmune disorders, cancers, rheumatoid arthritis, and other chronic conditions. The IgG1 subclass is the most potent among all the immunoglobulin gamma (IgG) antibodies, inducing Fc-related effector functions. N-linked glycan distribution of therapeutic IgG1s affects Fc-related effector functions such as CDC (complement-dependent cytotoxicity) and ADCC (antibody dependent cell-mediated cytotoxicity) biological activities and efficacy in vivo. Hence, as a critical quality attribute (CQA), the glycosylation profile of therapeutic IgG1s must be consistently preserved, which is primarily influenced by manufacturing process factors. In the era of biosimilars, it is challenging for biopharmaceutical manufacturers to not only obtain the desired glycan distribution consistently but also to meet the innovator molecule specifications as per the regulatory agencies. Methods: This study investigates the CHO fed-batch process parameters that affect the titer and terminal galactosylation of the TNF-α blocker-IgG1. It was hypothesized that galactose supplementation would enhance the galactosylation of TNF-α blocker-IgG1. Results: It was observed that such in-cultivation process shift does not affect cell culture parameters yet significantly enhances the galactosylation of TNF-α blocker-IgG1. Interestingly, the results indicate that supplementing D-galactose from the exponential phase of the CHO fed-batch process had the greatest effect on Fc galactosylation, increasing the amount of total galactosylated TNF-α blocker-IgG1 from 7.7% to 15.8%. Conclusions: Our results demonstrate a relatively easy and viable technique for cell culture engineering that is more appropriate for industrial production than costly in vitro glycoengineering. Full article
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16 pages, 2352 KiB  
Article
XBB.1.5 RBD-Based Bivalent Vaccines Induced Antibody Responses Against SARS-CoV-2 Variants in Mice
by Jiawen Liu, Tiantian Wang, Hongying Ren, Ruixi Liu, Qian Wang, Jun Wu and Bo Liu
Vaccines 2025, 13(5), 543; https://doi.org/10.3390/vaccines13050543 - 20 May 2025
Viewed by 699
Abstract
(1) Background: The currently circulating variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exhibits resistance to antibodies induced by vaccines. The World Health Organization recommended the use of monovalent XBB.1 sublineages (e.g., XBB.1.5) as an antigenic component in 2023. (2) Objective: In [...] Read more.
(1) Background: The currently circulating variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exhibits resistance to antibodies induced by vaccines. The World Health Organization recommended the use of monovalent XBB.1 sublineages (e.g., XBB.1.5) as an antigenic component in 2023. (2) Objective: In this study, we aimed to develop vaccines based on the XBB.1.5 receptor-binding domain (RBD) to combat the recently emerged SARS-CoV-2 XBB and JN.1 variants, as well as previously circulating variants. (3) Methods: Glycoengineered Pichia pastoris was utilized to produce a recombinant XBB.1.5 RBD protein with mammalian-like and fucose-free N-glycosylation. The XBB.1.5 RBD was mixed with Al(OH)3:CpG adjuvants to prepare monovalent vaccines. Thereafter, the XBB.1.5 RBD was mixed with the Beta (B.1.351), Delta (B.1.617.2), or Omicron (BA.2) RBDs (1:1 ratio), along with Al(OH)3:CpG, to prepare bivalent vaccines. BALB/c mice were immunized with the monovalent and bivalent vaccines. Neutralizing antibody titers were assessed via pseudovirus and authentic virus assays; humoral immune responses were analyzed by RBD-binding IgG subtypes. (4) Results: The monovalent vaccine induced higher neutralizing antibody titers against Delta, BA.2, XBB.1.5, and JN.1 compared to those in mice immunized solely with Al(OH)3:CpG, as demonstrated by pseudovirus virus assays. The XBB.1.5/Delta RBD and XBB.1.5/Beta RBD-based bivalent vaccines provided potent protection against the BA.2, XBB.1.5, JN.1, and KP.2 variants, as well as the previously circulating Delta and Beta variants. All monovalent and bivalent vaccines induced high levels of RBD-binding IgG (IgG1, IgG2a, IgG2b, and IgG3) antibodies in mice, suggesting that they elicited robust humoral immune responses. The serum samples from mice immunized with the XBB.1.5 RBD-based and XBB.1.5/Delta RBD-based vaccines could neutralize the authentic XBB.1.16 virus. (5) Conclusions: The XBB.1.5/Beta and XBB.1.5/Delta RBD-based bivalent vaccines are considered as potential candidates for broad-spectrum vaccines against SARS-CoV-2 variants. Full article
(This article belongs to the Section COVID-19 Vaccines and Vaccination)
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13 pages, 1776 KiB  
Article
Altered IgG N-Glycosylation at Onset of Type 1 Diabetes in Children Is Predominantly Driven by Changes in the Fab N-Glycans
by Branimir Plavša, Najda Rudman, Flemming Pociot and Olga Gornik
Biomedicines 2025, 13(5), 1206; https://doi.org/10.3390/biomedicines13051206 - 15 May 2025
Viewed by 426
Abstract
BackgroundN-glycosylation is a post-translational modification involving the attachment of oligosaccharides to proteins and is known to influence immunoglobulin G (IgG) effector functions and even antigen binding. IgG contains an evolutionarily conserved N-glycosylation site in its fragment crystallizable (Fc) region, [...] Read more.
BackgroundN-glycosylation is a post-translational modification involving the attachment of oligosaccharides to proteins and is known to influence immunoglobulin G (IgG) effector functions and even antigen binding. IgG contains an evolutionarily conserved N-glycosylation site in its fragment crystallizable (Fc) region, while during V-D-J recombination and somatic hypermutation processes it can also obtain N-glycosylation sites in its antigen binding fragment (Fab). Our previous study demonstrated altered IgG N-glycosylation in children at type 1 diabetes (T1D) onset, with the most prominent changes involving sialylated glycans, hypothesized to mainly come from the Fab region, however, the analytical method used could not distinguish between Fc and Fab. Methods: IgG was isolated from plasma from 118 children with T1D and 98 healthy controls from the Danish Registry of Childhood and Adolescent Diabetes. Isolated IgG was cleaved into Fc and Fab fragments using IdeS enzyme. N-glycans were enzymatically released from each fragment, fluorescently labelled with procainamide, and analyzed separately using the UPLC-MS method. Structural annotation of resulting chromatograms was performed using MS/MS. Results: T1D related N-glycosylation changes were more pronounced in the Fab glycans compared to Fc glycans, with five Fab glycans (Man5, Man7, FA2BG1S1, A2G2S2, FA2BG2S1) being significantly altered compared to only one in the Fc region (FA2[3]BG1). Comparing Fc and Fab glycosylation overall reveals stark differences in the types of glycans on each region, with a more diverse and complex repertoire being present in the Fab region. Conclusions: These findings suggest that N-glycosylation changes in early onset T1D predominantly originate from the Fab region, underscoring their potential role in modulating (auto)immunity and highlighting distinct glycosylation patterns between Fc and Fab. Full article
(This article belongs to the Special Issue Diabetes: Comorbidities, Therapeutics and Insights (2nd Edition))
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11 pages, 1659 KiB  
Perspective
Perspective on Immunoglobulin N-Glycosylation Status in Follicular Lymphoma: Uncovering BCR-Dependent and Independent Mechanisms Driving Subclonal Evolution
by Gloria Pokuaa Manu, Mariette Odabashian and Sergey Krysov
Cancers 2025, 17(7), 1219; https://doi.org/10.3390/cancers17071219 - 4 Apr 2025
Viewed by 606
Abstract
Follicular lymphoma (FL) is a heterogeneous and incurable disease. One of the hallmark features of FL cells is the introduction of N-glycosylation (N-gly) amino acid sequence motifs into the immunoglobulin variable (IgV) region through ongoing somatic hypermutation (SHM) in the early stages of [...] Read more.
Follicular lymphoma (FL) is a heterogeneous and incurable disease. One of the hallmark features of FL cells is the introduction of N-glycosylation (N-gly) amino acid sequence motifs into the immunoglobulin variable (IgV) region through ongoing somatic hypermutation (SHM) in the early stages of lymphoma development. These N-gly motifs, containing oligomannoses, are rarely found in healthy B cells but evidently play a crucial role in the clonal evolution and survival of FL cells in the hostile environment of germinal centers. The random nature of the ongoing SHM in FL occasionally results in the loss of productive immunoglobulin (Ig) genes or the elimination of N-gly motifs in productive genes. Such events typically lead to clonal deletion, as demonstrated by the longitudinal analysis of FL samples. However, rare N-gly-negative subclones demonstrate prolonged survival with evidence of ongoing SHM, giving rise to new N-gly-negative subclones before eventual deletion. This observation suggests the presence of specific mechanisms supporting their survival and proliferation. This perspective examines the current literature and explores whether a detailed transcriptomic and functional comparison of FL subclones characterized by different N-gly statuses, with a particular focus on N-gly-negative subclones, will lead to a comprehensive understanding of both N-gly-dependent and independent pro-survival and proliferative transcriptional signatures. Specifically, it aims to deepen our understanding of FL pathobiology and identify novel therapeutic targets for better disease management. Full article
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16 pages, 2258 KiB  
Article
Glycosylated Receptor-Binding-Domain-Targeting Mucosal Vaccines Protect Against SARS-CoV-2 Omicron and MERS-CoV
by Xiaoqing Guan, Abhishek K. Verma, Qian Liu, Melissa Palacios, Abby E. Odle, Stanley Perlman and Lanying Du
Vaccines 2025, 13(3), 293; https://doi.org/10.3390/vaccines13030293 - 10 Mar 2025
Viewed by 1002
Abstract
Background. The pathogenic coronaviruses (CoVs) MERS-CoV and SARS-CoV-2, which are responsible for the MERS outbreak and the COVID-19 pandemic, respectively, continue to infect humans, with significant adverse outcomes. There is a continuing need to develop mucosal vaccines against these respiratory viral pathogens to [...] Read more.
Background. The pathogenic coronaviruses (CoVs) MERS-CoV and SARS-CoV-2, which are responsible for the MERS outbreak and the COVID-19 pandemic, respectively, continue to infect humans, with significant adverse outcomes. There is a continuing need to develop mucosal vaccines against these respiratory viral pathogens to prevent entry and replication at mucosal sites. The receptor-binding domain (RBD) of the CoV spike (S) protein is a critical vaccine target, and glycan masking is a unique approach for designing subunit vaccines with improved neutralizing activity. Methods. We evaluated the efficacy of mucosal immunity, broad neutralizing activity, and cross-protection afforded by a combined glycosylated mucosal subunit vaccine encoding the RBDs of the original SARS-CoV-2 strain (SARS2-WT-RBD), the Omicron-XBB.1.5 variant (SARS2-Omi-RBD), and MERS-CoV (MERS-RBD). Results. Intranasal administration of the three-RBD protein cocktail induced effective, durable IgA and systemic IgG antibodies specific for the S protein of these CoVs, thereby neutralizing infection by pseudotyped SARS-CoV-2-WT, Omicron-XBB.1.5, and MERS-CoV. The mucosal vaccine cocktail protected immunized mice from challenge with SARS-CoV-2 Omicron-XBB.1.5 and MERS-CoV, leading to a significant reduction in the viral titers in the lungs. By contrast, the individual glycosylated RBD proteins only induced such immune responses and neutralizing antibodies against either SARS-CoV-2 or MERS-CoV, protecting against subsequent challenge with either SARS-CoV-2 or MERS-CoV; they did not provide simultaneous protection against both CoVs. Conclusions. This study describes a unique strategy for designing efficacious mucosal subunit vaccines that induce durable mucosal immunity, cross-neutralizing activity, and cross-protection against SARS-CoV-2 and MERS-CoV, highlighting the potential for the design of mucosal vaccines against other pathogens. Full article
(This article belongs to the Section COVID-19 Vaccines and Vaccination)
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18 pages, 5008 KiB  
Hypothesis
Hypothetical Pathogenetic Model of Membranous Nephropathy
by Irina Zdravkova, Eduard Tilkiyan, Desislava Bozhkova, Teodor Kuskunov, Yovko Ronchev and Boris Kirilov
Int. J. Mol. Sci. 2025, 26(5), 2206; https://doi.org/10.3390/ijms26052206 - 28 Feb 2025
Viewed by 810
Abstract
Membranous nephropathy (MN) is a disease with an etiology and pathogenesis that are still not fully understood, and it represents a great challenge. It is characterized by a variable course, spontaneous remissions and relapses. The inability to rely entirely on antibodies and the [...] Read more.
Membranous nephropathy (MN) is a disease with an etiology and pathogenesis that are still not fully understood, and it represents a great challenge. It is characterized by a variable course, spontaneous remissions and relapses. The inability to rely entirely on antibodies and the continuous threat of a malignant disease make the differentiation of MN types extremely difficult. Data of twelve patients with membranous nephropathy, ranging in age between 28 and 67 years, are presented; in total, seven men and five women were observed for a period of 2 to 10 years. In all patients, the diagnosis was confirmed through kidney biopsy and laboratory tests, including immunological, histopathological, and immunohistochemical tests. Histopathological and immunohistochemical tests were applied on available material from the thyroid gland in two patients and the gallbladder in two patients with MN. Data of 102 patients with MN and their comorbidities are evaluated in order to establish correlations. These and other data are used to build a hypothetical pathogenetic model that explains the etiology and the likely pattern of disease occurrence. We found a connection between chronic cholecystitis, thyroiditis, hepatitis, and other diseases in the occurrence of MN and disease course. From our practice and cases, we drew the conclusion that chronic inflammation in sites that express PLA2R leads to the formation of antibodies against PLA2R. These antibodies occur as a preformed immune complex or separately and are deposited in the subepithelial space, leading to MN appearance. Full article
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15 pages, 1500 KiB  
Article
Potential Glycobiomarkers in Maternal Obesity and Gestational Diabetes During Human Pregnancy
by Anna Farkas, Andrea Suranyi, Balint Kolcsar, Zita Gyurkovits, Zoltan Kozinszky, Sandor G. Vari and Andras Guttman
J. Clin. Med. 2025, 14(5), 1626; https://doi.org/10.3390/jcm14051626 - 27 Feb 2025
Viewed by 830
Abstract
Introduction: Obesity is a rapidly growing common health problem worldwide that can lead to the development of gestational diabetes mellitus (GDM). However, GDM not only affects women with obesity but can also develop at any time, even after the OGTT test; therefore, an [...] Read more.
Introduction: Obesity is a rapidly growing common health problem worldwide that can lead to the development of gestational diabetes mellitus (GDM). However, GDM not only affects women with obesity but can also develop at any time, even after the OGTT test; therefore, an increasing number of complications related to GDM can be seen in both mothers and their children. It is necessary to discover biomarkers capable of indicating the development of GDM or complications during/after pregnancy. Since the N-glycosylation motif of human IgG has been described to change under many physiological and pathological conditions, it is a promising target for biomarker research. In our study, the effects of obesity and GDM were investigated on human serum IgG N-linked glycosylation patterns during human pregnancy. Materials and Methods: The study participants were categorized into four groups according to their body mass index (BMI) and GDM status: normal weight as control, obese (BMI > 30 kg/m2), normal weight with GDM, and obese with GDM. The released N-glycan components of IgG were separated with capillary electrophoresis and detected using a laser-induced fluorescence detector. Results: The result revealed several differences between the N-glycosylation patterns of the four study groups. Of this, 17 of the 20 identified structures differed significantly between the groups. The ratios of sialylated to non-sialylated structures were not changed significantly, but the core fucosylation level showed a significant decrease in the GDM and obese GDM groups compared to the control subjects. The lowest degree of core fucosylation was observed in the GDM group. Conclusions: The findings indicate that obesity in isolation does not have a significant impact on the IgG N-glycosylation pattern in pregnancy. Conversely, alterations in the N-glycan profile of antibodies may serve as biomarkers for the diagnosis of GDM in mothers with a normal BMI, although more evidence is needed. By incorporating glycan-based biomarkers into clinical practice, healthcare providers can improve early detection, personalize management strategies, and potentially mitigate adverse pregnancy outcomes associated with obesity and GDM. Full article
(This article belongs to the Special Issue Gestational Diabetes: Cutting-Edge Research and Clinical Practice)
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16 pages, 4149 KiB  
Article
Hydrophilic Sulfonate Covalent Organic Frameworks for Serum Glycopeptide Profiling
by Shishu Yang, Yuheng Jiang, Shijie Jiang, Lihong Liu, Si Liu, Hua Zhang and Zhiyuan Gu
Int. J. Mol. Sci. 2025, 26(5), 1957; https://doi.org/10.3390/ijms26051957 - 24 Feb 2025
Viewed by 902
Abstract
Aberrant protein glycosylation is closely associated with a number of biological processes and diseases. However, characterizing the types of post-translational modifications (PTMs) from the complex biological samples is challenging for comprehensive glycoproteomic analysis. The development of high-performance enrichment materials and strategies during the [...] Read more.
Aberrant protein glycosylation is closely associated with a number of biological processes and diseases. However, characterizing the types of post-translational modifications (PTMs) from the complex biological samples is challenging for comprehensive glycoproteomic analysis. The development of high-performance enrichment materials and strategies during the sample pretreatment process is a prerequisite to glycoproteome research. Here in this work, a sulfonate-rich covalent organic framework (COF) called TpPa-(SO3H)2 (referred to as SCOF-2) was synthesized using the Schiff base reaction for the identification of glycopeptides. Benefiting from high hydrophilicity and abundant sulfonate affinity, a total of 28 and 16 glycopeptides could be efficiently detected from the standard glycoproteins of horseradish peroxidase (HRP) and immunoglobulin G (IgG) tryptic digest, respectively. Moreover, the as-prepared sulfonate-rich SCOF-2 has an ultralow detection limit (0.01 fmol μL−1), excellent enrichment selectivity (molar ratio HRP:BSA = 1:5000), satisfactory recovery rate (89.1%), high adsorption capacity (150 mg g−1) and good reusability in the individual enrichment. Meanwhile, by using the SCOF-2 adsorbent, 196 and 194 endogenous glycopeptides in the serum of ovarian cancer patients and healthy people among triplicates were successfully enriched and identified, respectively, using combined nanoLC–MS/MS technology. It demonstrated its great application potential in glycoproteomics research and provided a novel insight for the design of affinity materials. Full article
(This article belongs to the Special Issue Peptide Self-Assembly)
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8 pages, 1710 KiB  
Communication
The Glycosylation of Serum IgG Antibodies in Post-COVID-19 and Post-Vaccination Patients
by Csaba Váradi
Int. J. Mol. Sci. 2025, 26(2), 807; https://doi.org/10.3390/ijms26020807 - 18 Jan 2025
Viewed by 1390
Abstract
The signature of human serum IgG glycosylation is critical in the defense against pathogens. Alterations of IgG N-glycome were associated with COVID-19 (Coronavirus disease 2019) severity, although knowledge on the response to vaccination is limited. IgG N-glycome was analyzed in this study in [...] Read more.
The signature of human serum IgG glycosylation is critical in the defense against pathogens. Alterations of IgG N-glycome were associated with COVID-19 (Coronavirus disease 2019) severity, although knowledge on the response to vaccination is limited. IgG N-glycome was analyzed in this study in post-COVID-19 and post-vaccination patients to reveal potential glycosylation-based alterations using hydrophilic interaction liquid chromatography (HILIC-UPLC) with fluorescence (FLR) and mass-spectrometric (MS) detection. IgG antibodies were purified from serum samples through protein G affinity chromatography followed by PNGase F digestion-based deglycosylation. The released glycans were fluorescently derivatized by procainamide labeling and purified via solid-phase extraction. Higher levels of sialylation and afucosylation were identified in post-COVID-19 patients, which was further expanded by vaccination, but only in those who were previously SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2) infected. Full article
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20 pages, 7906 KiB  
Article
Developing a Label-Free Infrared Spectroscopic Analysis with Chemometrics and Computational Enhancement for Assessing Lupus Nephritis Activity
by Mei-Ching Yu, Xiang-Di Huang, Chin-Wei Kuo, Kai-Fu Zhang, Ping-Chung Liang, U-Ser Jeng, Pei-Yu Huang, Frederick Wai Keung Tam and Yao-Chang Lee
Biosensors 2025, 15(1), 39; https://doi.org/10.3390/bios15010039 - 11 Jan 2025
Viewed by 1224
Abstract
Patterns of disease and therapeutic responses vary widely among patients with autoimmune glomerulonephritis. This study introduces groundbreaking personalized infrared (IR)-based diagnostics for real-time monitoring of disease status and treatment responses in lupus nephritis (LN). We have established a relative absorption difference (RAD) equation [...] Read more.
Patterns of disease and therapeutic responses vary widely among patients with autoimmune glomerulonephritis. This study introduces groundbreaking personalized infrared (IR)-based diagnostics for real-time monitoring of disease status and treatment responses in lupus nephritis (LN). We have established a relative absorption difference (RAD) equation to assess characteristic spectral indices based on the temporal peak heights (PHs) of two characteristic serum absorption bands: ν1 as the target signal and ν2 as the PH reference for the ν1 absorption band, measured at each dehydration time (t) during dehydration. The RAD gap (Ψ), defined as the difference in the RAD values between the initial and final stages of serum dehydration, enables the measurement of serum levels of IgG glycosylation (ν1 (1030 cm−1), ν2 (1171 cm−1)), serum lactate (ν1 (1021 cm−1), ν2 (1171 cm−1)), serum hydrophobicity (ν1 (2930 cm−1), ν2 (2960 cm−1)), serum hydrophilicity (ν1 (1550 cm−1), ν2 (1650 cm−1)), and albumin (ν1 (1400 cm−1), ν2 (1450 cm−1)). Furthermore, this IR-based assay incorporates an innovative algorithm and our proprietary iPath software (ver. 1.0), which calculates the prognosis prediction function (PPF, Φ) from the RAD gaps of five spectral markers and correlates these with conventional clinical renal biomarkers. We propose that this algorithm-assisted, IR-based approach can augment the patient-centric care of LN patients, particularly by focusing on changes in serum IgG glycosylation. Full article
(This article belongs to the Section Optical and Photonic Biosensors)
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16 pages, 3400 KiB  
Article
Some Human Anti-Glycan Antibodies Lack the Ability to Activate the Complement System
by Nadezhda Shilova, Alexey Nokel, Alexander Lipatnikov, Nailya Khasbiullina, Yuri Knirel, Ludmila Baidakova, Alexander Tuzikov, Sergei Khaidukov, Polina Obukhova, Stephen Henry, Batozhab Shoibonov, Emin Salimov, Robert Rieben and Nicolai Bovin
Antibodies 2024, 13(4), 105; https://doi.org/10.3390/antib13040105 - 23 Dec 2024
Viewed by 2403
Abstract
Background. Naturally occurring human antibodies against glycans recognize and quickly eliminate infectious bacteria, viruses and aberrantly glycosylated neoplastic malignant cells, and they often initiate processes that involve the complement system. Methods. Using a printed glycan array (PGA) containing 605 glycoligands (oligo- and polysaccharides, [...] Read more.
Background. Naturally occurring human antibodies against glycans recognize and quickly eliminate infectious bacteria, viruses and aberrantly glycosylated neoplastic malignant cells, and they often initiate processes that involve the complement system. Methods. Using a printed glycan array (PGA) containing 605 glycoligands (oligo- and polysaccharides, glycopeptides), we examined which of the glycan-binding antibodies are able to activate the complement system. Using this PGA, the specificities of antibodies of the IgM and IgG classes were determined in the blood serum of healthy donors (suggested as mostly natural), and, then, using the same array, it was determined which types of the bound immunoglobulins were also showing C3 deposition. Results. It was found that about 30% of anti-glycan antibodies in human serum detected by the PGA did not activate the complement. They were mostly IgGs and directed to bacterial O-antigens; no apparent common structural motif within their target polysaccharides was found. Antibodies to blood group systems ABO and Forssman, xeno-antigens, a number of polysaccharides from various strains of S. enterica, E. coli and P. alcalifaciens, as well as small fragments of bacterial polysaccharides were recognized by complement-activating antibodies as expected. A complement-activating antibody was affinity-isolated on glycan-Sepharose from human serum, and, in the presence of the complement, it lysed red blood cells coated with the same glycan (kodecytes, where glycans expressed on biological membranes), while an isolated complement non-activating antibody did not, which confirms the validity of the solid-phase PGA results. Conclusions. Thus, ~30% of human anti-glycan antibodies lack the ability to activate the complement system. The function of the widely represented immunoglobulins that do not cause C3 deposition remains unclear. Full article
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16 pages, 8172 KiB  
Article
Amelioration of LPS-Induced Jejunum Injury and Mucus Barrier Damage in Mice by IgY Embedded in W/O/W Emulsion
by Zhaohui Wang, Ruihua Ye, Shidi Zhang, Chuanming Liu, Ke Chen, Kongdi Zhu, Pengjie Wang, Fuqing Wang and Jiaqiang Huang
Foods 2024, 13(24), 4138; https://doi.org/10.3390/foods13244138 - 20 Dec 2024
Viewed by 883
Abstract
Chicken yolk immunoglobulin (IgY) is a natural immunologically active antibody extracted from egg yolk and can be used as a natural dietary supplement for the treatment of inflammation and damage to the intestines. In our study, IgY was embedded in a double emulsion [...] Read more.
Chicken yolk immunoglobulin (IgY) is a natural immunologically active antibody extracted from egg yolk and can be used as a natural dietary supplement for the treatment of inflammation and damage to the intestines. In our study, IgY was embedded in a double emulsion (W/O/W; DE) to explore the therapeutic effect of the embedded IgY on Lipopolysaccharide (LPS)-induced jejunal injury in mice. The results showed that W/O/W-embedded IgY as a dietary supplement (IgY + DE) attenuated LPS-induced damage to mouse small intestinal structures and protected the integrity of the jejunal mucosal barrier. IgY + DE increased the amount of related transcription factors (Math1, Spdef, Elf3, and Klf4) and promoted thrush cell differentiation. IgY + DE ameliorated LPS-induced reduction in mucin quantity and markers. It promoted the expression of Muc1 and Muc2 and increased the mRNA expression levels of Muc1, Muc2, Muc3, Muc4, Muc13, and Agr2 (p < 0.05). IgY + DE increased the expression of several glycosyltransferases involved in mucin glycosylation. IgY + DE also neutralized the LPS attack on the expression of jejunal inflammatory factors IL-1β, IL-6, IL-4, and TNF-α. In conclusion, the IgY-embedded double emulsion can be used as a dietary supplement for immunotherapy to prevent LPS-induced jejunal injury in mice. Full article
(This article belongs to the Special Issue Bioavailability and Health Benefits of Bioactive Compounds in Foods)
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13 pages, 3871 KiB  
Article
LC-MS/MS-Based Site-Specific N-Glycosylation Analysis of VEGFR-IgG Fusion Protein for Sialylation Assessment Across IEF Fractions
by Kwang Hoe Kim, Eun Sun Ji, Ju Yeon Lee, Ju Hwan Song and Yeong Hee Ahn
Molecules 2024, 29(22), 5393; https://doi.org/10.3390/molecules29225393 - 15 Nov 2024
Viewed by 1660
Abstract
The glycosylation profile of therapeutic proteins significantly influences their efficacy, stability, and immunogenicity. Sialylation is crucial for the biological activity and pharmacokinetics of fusion proteins used in treating angiogenic disorders, making sialic acid levels a critical quality attribute in the development and production [...] Read more.
The glycosylation profile of therapeutic proteins significantly influences their efficacy, stability, and immunogenicity. Sialylation is crucial for the biological activity and pharmacokinetics of fusion proteins used in treating angiogenic disorders, making sialic acid levels a critical quality attribute in the development and production of biologics. In this study, we employed a mass spectrometry-based approach to assess sialylation levels through site-specific N-glycosylation analysis. To validate the method’s effectiveness, IEF fractions (acidic, main, and basic) obtained from the production media of the VEGFR-IgG fusion protein and anticipated to exhibit varying sialylation levels were analyzed. Our analytical method successfully evaluated the sialylation levels of each domain—IgG, VEGFR-1, and VEGFR-2—within the Fc-fusion protein. The results confirm that the overall sialylation level of the Fc-fusion protein correlated with the levels observed across the IEF fractions. This finding highlights the value of LC-MS/MS-based sialylation monitoring as a crucial tool for biosimilar development and quality control, particularly in optimizing target protein production. Additionally, glycopeptide-based LC-MS analysis enables site-specific sialylation evaluation, ensuring consistent profiles for robust quality assurance. Full article
(This article belongs to the Section Analytical Chemistry)
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