Glycosylation Patterns Research Articles

Specificity of widely used lectins as probed with oligosaccharide and plant polysaccharide arrays.

Glycan-binding specificity was studied for Jacalin, RCA 120, SBA, PHA-L, PHA-E, WGA, UEA, AAL, LTL, LEL, SNA, DSA, LCA, MAH and Con A, lectins widely used in histochemistry. Oligosaccharide- and polysaccharide-based glycan arrays were applied. Expected specificity was confirmed for only 6 of the 15 lectins and the glycan binding profiles of some lectins were dramatically broader than generally accepted. WGA, LEL and DSA known as chitooligosaccharide-specific, were unexpectedly polyreactive, binding to other glycans with the same affinity as to chitobiose, ABH antigens and oligolactosamines (unsubstituted and sialylated). SBA, in addition to expected binding to glycans with terminal GalNAcα, also had high affinity for the GM1 ganglioside. MAH demonstrated much higher affinity to a variety of sulfated glycans compared to Neu5Acα2-3Galβ1-3GalNAcα. Contrary to the common view, LCA demonstrated the maximum binding to (GlcNAcβ1-2Manα1)2-3,6-Manβ1-4GlcNAcβ1-4GlcNAc N-glycan, while it had no interaction with corresponding Gal or Neu5Ac terminated versions. This observed polyreactivity of some lectins casts doubt on their use in accurately determining the presence of a specific glycan structure by histochemical studies. However, comparisons of sera from healthy and diseased individuals with help of a lectin array can easily establish differences in glycosylation patterns and presumptive glycan identities, which can later be clarified using more accurate methods of structural analysis.

The evolution of hemagglutinin-158 and neuraminidase-88 glycosylation sites modulates antigenicity and pathogenicity of clade 2.3.2.1 H5N1 avian influenza viruses

Clade 2.3.2.1 of the H5N1 avian influenza virus (AIV) evolved into several subclades. However, the effect of glycosylation on the biological characteristics of hemagglutinin (HA) and/or neuraminidase (NA) from H5N1 AIVs remains unclear. Here, we determined that the global prevalence of clade 2.3.2.1 H5N1 AIVs with deglycosylated residue 158 on HA (HA158-) and glycosylated residue 88 on NA (NA88+) were predominant via multiple sequence analysis. The deglycosylation of residue on NA 88 (NA88-) was observed in clade 2.3.2.1a (new) and clade 2.3.2.1e H5N1 AIVs. Interestingly, NA88- was coupled with the acquisition of 158 glycosylation sites on HA (HA158+) in clade 2.3.2.1e H5N1 AIVs from China, and clade 2.3.2.1a (new) H5N1 AIVs exhibiting the HA158-NA88- pattern were predominant in Bangladesh. Meanwhile, the temporal distribution of strain HA158+ NA88- was highly consistent with the implementation of Re-6 vaccine in China. The recombinant H5N1 AIVs constructed using a reverse genetic system showed that the acquisition of the HA158 glycosylation site facilitated viral evasion form Re-6 antisera, and the virus lacking glycosylation sites at HA158 and NA88 resulted in reduced NA activity, replication in mammalian cells, and pathogenicity in both chickens and mice compared to that of the viruses with alternative glycosylation patterns. Therefore, the acquisition of HA158+ in clade 2.3.2.1e H5N1 AIVs enables evasion of Re-6 vaccination pressure, and the virulence of clade 2.3.2.1 H5N1 AIVs is modulated by the absence of glycosylation sites at HA158 and NA88. Our finding highlighting the importance of epidemiological surveillance and timely updating vaccines of H5 AIVs.

Synthetic Retinoid Sulfarotene Selectively Inhibits Tumor-Repopulating Cells of Intrahepatic Cholangiocarcinoma via Disrupting Cytoskeleton by P-Selectin/PSGL1 N-Glycosylation Blockage.

Intrahepatic cholangiocarcinoma (ICC) is a highly lethal malignancy that currently lacks effective clinical treatments. Eliminating stem cell-like cancer cells is an extremely promising but challenging strategy for treating ICC. A recently developed synthetic retinoid, sulfarotene, abrogates proliferation, and induces apoptosis of tumor-repopulating cells (TRCs) that exhibit stem cell-like properties, yet its effect and underlying mechanisms remain elusive in ICC. It is found that although 5-fluorouracil, cisplatin, pemigatinib, and gemcitabine all inhibit ICC-TRCs, sulfarotene demonstrates superior efficacy. Sulfarotene induces retinoic acid receptor alpha (RARɑ) translocation from the cytoplasm to the nucleus, suppressing P-selectin expression at the transcriptional level. Moreover, it directly interacts with fucosyltransferase 8 (FUT8), inhibiting the core fucosylation of P-selectin glycoprotein ligand 1 (PSGL1). These actions collectively inhibit ICC-TRCs via destroying PSGL1-regulated cytoskeleton. The findings provide a strategy of inhibiting P-selectin/PSGL1 interaction and altering PSGL1 glycosylation pattern to compromise the cytoskeletal integrity and eliminate ICC-TRCs.

Autism-Linked Mutations in α2δ-1 and α2δ-3 Reduce Protein Membrane Expression but Affect Neither Calcium Channels nor Trans-Synaptic Signaling

Background: α2δ proteins regulate membrane trafficking and biophysical properties of voltage-gated calcium channels. Moreover, they modulate axonal wiring, synapse formation, and trans-synaptic signaling. Several rare missense variants in CACNA2D1 (coding for α2δ-1) and CACNA2D3 (coding for α2δ-3) genes were identified in patients with autism spectrum disorder (ASD). However, the pathogenicity of these variants is not known, and the molecular mechanism by which α2δ proteins may contribute to the pathophysiology of autism is, as of today, not understood. Therefore, in this study we functionally characterized two heterozygous missense variants in α2δ-1 (p.R351T) and α2δ-3 (p.A275T), previously identified in patients with ASD. Methods: Electrophysiological recordings in transfected tsA201 cells were used to study specific channel-dependent functions of mutated α2δ proteins. Membrane expression, presynaptic targeting, and trans-synaptic signaling of mutated α2δ proteins were studied upon expression in murine cultured hippocampal neurons. Results: Homologous expression of both mutated α2δ proteins revealed a strongly reduced membrane expression and synaptic localization compared to the corresponding wild type α2δ proteins. Moreover, the A275T mutation in α2δ-3 resulted in an altered glycosylation pattern upon heterologous expression. However, neither of the mutations compromised the biophysical properties of postsynaptic L-type (CaV1.2 and CaV1.3) and presynaptic P/Q-type (CaV2.1) channels when co-expressed in tsA201 cells. Furthermore, presynaptic expression of p.R351T in the α2δ-1 splice variant lacking exon 23 did not affect trans-synaptic signaling to postsynaptic GABAA receptors. Conclusions: Our data provide evidence that the pathophysiological mechanisms of ASD-causing mutations of α2δ proteins may not involve their classical channel-dependent and trans-synaptic functions. Alternatively, these mutations may induce subtle changes in synapse formation or neuronal network function, highlighting the need for future α2δ protein-linked disease models.

Identification of Hepatic-like Erythropoietin with Enhanced Activity As a New Cause of Hereditary and Acquired Erythrocytosis

Background:Hereditary and acquired erythrocytosis are characterized by increased red cell mass. They can develop secondary to disorders that cause tissue hypoxia or to an inappropriate increase in erythropoietin (EPO) production by interstitial kidney cells. EPO is the primary regulator of erythropoiesis during early fetal development and throughout adult life. Although EPO is produced in the fetal liver before birth, the major site of EPO production becomes the kidney shortly after birth.In this work, we identified six unrelated families with hereditary erythrocytosis associated with circulating EPO levels within the normal range, and characterized this condition as a novel molecular and functional entity.Methods:Luciferase reporter constructs were placed under the control of the mutant EPO regulatory elements to study the impact on EPO gene expression. Induced pluripotent stem cells (iPSC) were derived from patients' blood, which were differentiated into hepatocyte-like EPO-producing cells. EPO proteins from affected family members and healthy newborns were isolated from blood and analyzed by isoelectric focusing (IEF), then the activity of the purified EPO was quantified.Results:Using next-generation sequencing and segregation analysis, we identified three novel non-coding heterozygous mutations in the promoter and intron 1 of the EPO gene in the six families. Experiments with reporter assays and iPSC-derived hepatocyte-like cells showed that mutations target previously uncharacterized regulatory elements of the EPO gene that exhibit high responsiveness to HIF-2α. The circulating EPO protein from all patients with hereditary erythrocytosis examined in this study exhibited a unique and distinctive IEF profile compared to EPO produced by the kidneys of healthy adults. An identical IEF profile was also found in premature neonates, in whom EPO is produced by the liver, as well as in patients with acquired erythrocytosis associated with liver diseases. Interestingly, this profile, which shows a shift of the main EPO isoforms to a more basic region, was related to glycosylation modifications. This suggests lower contents of sialic acids and other modifications, indicating a liver-type glycosylation pattern, which differs from the classic kidney-type glycosylation pattern observed in adulthood. Finally, our functional assays of EPO protein purified from patients' plasma and umbilical cord blood revealed a statistically significant enhanced activity, as reflected by increased EPO receptor signaling in a human cell line, pointing to a potential gain-of-function for the liver-type glycosylation of EPO.Conclusions:We report a new entity of secondary erythrocytosis named Hepatic-like Erythropoietin Polycythemia (HEP), characterized by the production of liver-like EPO with an atypical glycosylation pattern and increased activity. This new form of EPO has been observed in all patients with hereditary EPO-mutated erythrocytosis, in cases of erythrocytosis associated with liver disease, and in neonates.Interestingly, the shift from a more active liver-type EPO to kidney-type EPO in newborns may explain the significant decrease in hemoglobin levels typically observed in the first weeks of life.Thus, it is essential to consider quantitative and qualitative EPO screening as part of the biological tests for the diagnosis of idiopathic and hereditary erythrocytosis.

Glycosylation changes of vWF in circulating extracellular vesicles to predict depression

The clinical diagnosis of major depressive disorder (MDD) still depends on subjective information in terms of various symptoms regarding mood. Detecting the characterization of extracellular vesicles (EVs) in blood may result in finding a diagnostic biomarker that reflects the depressive stage of patients with MDD. Here, we report the results on the glycosylation pattern of enriched plasma EVs from patients with MDD. We compared glycosylation patterns by lectin blotting expressed in EVs isolated from the plasma of both patients with MDD and age-matched healthy control participants (HCs) using size-exclusion chromatography. The levels of Wheat germ agglutinin (WGA), N-acetyl glucosamine (GlcNAc), and N-Acetylneuraminic acid (Neu5Ac, sialic acid) - binding lectin, were significantly decreased in patients with MDD in the depressive state compared to HCs and in remission state. Furthermore, proteome analysis revealed that the von Willebrand factor (vWF) was a significant factor recognized by WGA. WGA-binding vWF antigen differentiated patients with MDD versus HCs and the same patients with MDD in a depressive versus remission state. In this study, the change patterns in the glycoproteins contained in plasma EVs support the usability of testing to identify patients who are at increased risk of depression during antidepressant treatment.

Glycomic profiling of parathyroid neoplasms via lectin microarray analysis.

Parathyroid carcinoma (PC) is a rare malignancy with a poor prognosis. Diagnosis of PC is often difficult in clinical practice and efficient diagnostic markers are still needed for differential diagnosis. Aberrant glycosylations of glycoproteins were identified with lectin microarray in various cancers, while relevant information is lacking in PC. In this study, 8 PC and 6 parathyroid adenoma (PA) tissues were assessed using a microarray consisting of 70 lectins. Overall lectin-specific glycosylation patterns were compared between PA and PC tissues. Lectins with significant differential response between PC and PA were further validated by lectin histochemistry. The difference in signal intensities was found in 71.4% (50/70) of the lectins between the two groups (P < 0.05). The vast majority of PCs had higher intensity signals than PAs (PCs vs. PAs, ratio >1) and amaranthus caudatus (ACL) showed the most significantly different response between them (ratio = 2.45). Lectin histochemistry further confirmed higher ACL intensity in PCs than in PAs. The differentially expressed glycans in PC tissues were primarily glucose, mannose, and galactose-based. PC presented unique glycomic features and ACL may serve as a candidate diagnostic marker for PC.

Non-targeted N-glycome profiling reveals multiple layers of organ-specific diversity in mice

N-glycosylation is one of the most common protein modifications in eukaryotes, with immense importance at the molecular, cellular, and organismal level. Accurate and reliable N-glycan analysis is essential to obtain a systems-wide understanding of fundamental biological processes. Due to the structural complexity of glycans, their analysis is still highly challenging. Here we make publicly available a consistent N-glycome dataset of 20 different mouse tissues and demonstrate a multimodal data analysis workflow that allows for unprecedented depth and coverage of N-glycome features. This highly scalable, LC-MS/MS data-driven method integrates the automated identification of N-glycan spectra, the application of non-targeted N-glycome profiling strategies and the isomer-sensitive analysis of glycan structures. Our delineation of critical sub-structural determinants and glycan isomers across the mouse N-glycome uncovered tissue-specific glycosylation patterns, the expression of non-canonical N-glycan structures and highlights multiple layers of N-glycome complexity that derive from organ-specific regulations of glycobiological pathways.

Modifying the Glycocalyx of Melanoma Cells via Metabolic Glycoengineering Using N-Acetyl-d-glucosamine Analogues.

Tumor cells are decorated with aberrant glycan structures on cell surfaces. It is well known that the glycocalyx serves as a main cellular regulator, although its role in cancer is still not completely understood. Over recent decades, several non-natural monosaccharides carrying clickable groups have been introduced in melanoma cells. This technique, called Metabolic Glycoengineering (MGE), opens up the possibility of altering the cell's glycocalyx via click chemistry using a two-step approach. This study expands the field of MGE by showing the successful metabolic incorporation of novel alternative artificial glucosamine derivatives. The latter were either deoxygenated or blocked by methyl ether in position 4 to generate deficient glycosylation patterns, while being extended by an alkyne to enable click chemistry as a one-step approach. As a result, we observed a reduced proliferation rate of melanoma cells. Furthermore, using a lectin array, the decrease in high mannose epitopes was observed. In summary, the successful use of alternative artificial glucosamine derivatives enabled a significant alteration in the glycocalyx, consequently influencing cell behavior.

Differential CD38 Glycosylation Patterns in Acute Myeloid Leukemia and T-Cell Acute Lymphoblastic Leukemia: Implications for Targeted Immunotherapy

Differential CD38 Glycosylation Patterns in Acute Myeloid Leukemia and T-Cell Acute Lymphoblastic Leukemia: Implications for Targeted Immunotherapy

Mining and Engineering the Di-O-glycosylation Pattern of UGT72B1 for the Highly Efficient O-Glycosylation of Endogenous Quercetin.

Compared with mono-O-glycosylation, di-O-glycosylation endows the precursor with better performance. However, the mining and engineering of di-O-glycosylation patterns of glycosyltransferases are limited, hindering their synthetic applications. Here, an Arabidopsis xenobiotic-transforming glycosyltransferase, UGT72B1, was found to catalyze the glycosylation of endogenous quercetin and its monoglycosides, generating di-O-glucosides. Mutating M17/G18/Y315 into L/T/Q in UGT72B1 altered its regioselectivity toward quercetin 7-O-glucoside, enzymatically generating another 3,7-di-O-glycoside with up to a 100% conversion rate, and increased the sugar donor preference. Altering the regiospecificity of glycosyltransferases likely required coordination between the entrance and the active site, where the orientations of the sugar acceptors and donors shift to adopt a lower binding energy state. Moreover, quercetin 3,4'-di-O-β-d-glucoside and quercetin 3,7-di-O-β-d-glucoside synthesized were found to have the highest anti-inflammatory activities. Overall, this work presents an efficient strategy to engineer glycosylation patterns for the synthesis of quercetin di-O-β-d-glucosides to be used as food additives, therapeutics, and nutraceuticals.

Investigation of Fibrillar Aggregates Formed by Pathogenic Pre-Pro-Vasopressin Mutants that Cause ADNDI.

Aggregations of unfolded or misfolded proteins, both inside and outside cells, are implicated in numerous diseases, collectively known as amyloidosis. Particularly, Autosomal dominant neurohypophyseal diabetes insipidus (ADNDI) is a rare disease caused by mutations in the AVP-NPII gene, leading to the inability to secrete arginine vasopressin (AVP). These misfolded proteins accumulate within the endoplasmic reticulum (ER), causing cellular dysfunction. This study aimed to investigate the formation of amyloid-like aggregates within the cell resulting from misfolded mutant precursor proteins, which induce disulfide-linked oligomers due to the G45C, 207_209delGGC, G88V, C98X, C104F, E108D-1, E108D-2 and R122H mutations identified by our group in the AVP-NPII gene of ADNDI patients. De-glycosylation studies were performed to analyze the glycosylation patterns of mutant protein precursors. The involvement of these precursors in the ER-related degradation pathway was studied by conducting protease inhibition experiments. Disulfide-linked oligomer analysis determined the oligomerization status of the mutant precursors. Immunofluorescence and electron microscopy studies provided evidence of aggregate structures in the ER lumen. In vitro studies involving bacterial expression and fibril formation in E. coli. Our findings demonstrated that the N-glycan structure of mutant precursors remains intact within the ER. Protease inhibition experiments indicated the involvement of these precursors in the ER-related degradation pathway. Disulfide-linked oligomer analysis revealed homo-oligomer structures in mutations. Immunofluorescence and electron microscopy studies confirmed the presence of aggregate structures in the ER lumen. In vitro studies showed that mutant precursors could form fibril structures in E. coli. Our study may support the idea that ADNDI belongs to the group of neurodegenerative diseases due to the formation of fibrillar amyloid aggregates in the cell.

Giardia spp.-induced microbiota dysbiosis disrupts intestinal mucin glycosylation

ABSTRACT Infection with the protozoan parasite Giardia duodenalis (syn. intestinalis, lamblia) has been associated with intestinal mucus disruptions and microbiota dysbiosis. The mechanisms remain incompletely understood. Mucus consists primarily of densely glycosylated mucin glycoproteins. Mucin O-glycans influence mucus barrier properties and mucin–microbe interactions and are frequently altered during disease. In this study, we observed time-dependent and regiospecific alterations to intestinal mucin glycosylation patterns and the expression of mucin-associated glycosyltransferase genes during Giardia infection. Glycosylation alterations were observed in Giardia-infected mice in the upper small intestine, the site of parasite colonization, and in the distal colon, where active trophozoites were absent. Alterations occurred as early as day 2 post-infection and persisted in mice after parasite clearance. We also observed small intestinal goblet cell hyperplasia and thinning of the distal colon mucus barrier during early infection, and microbiota alterations and altered production of cecal SCFAs. Giardia-induced alterations to mucin glycosylation were at least in part dependent on microbiota dysbiosis, as transplantation of a dysbiotic mucosal microbiota collected from Giardia-infected mice recapitulated some alterations. This study describes a novel mechanism by which Giardia alters intestinal mucin glycosylation, and implicates the small intestinal microbiota in regulation of mucin glycosylation patterns throughout the gastrointestinal tract.

6644 Glycosylation Patterns of the IgG Variable Region in Thyroglobulin Antibodies and Their Impact on Antigen Binding Affinity

Abstract Disclosure: S. Wang: None. Y. Cao: None. C. Zhao: None. C. Huang: None. K. Zhao: None. Y. Li: None. Y. Huang: None. Z. Ying: None. Y. Zhang: None. Y. Gao: None. Objective: Thyroglobulin antibodies (TgAb), key serological markers in Hashimoto's thyroiditis (HT) patients, are predominantly in immunoglobulin G (IgG) form. Prior research has highlighted the importance of increased glycosylation in TgAb IgG from HT patients. This study investigates the unique glycosylation patterns of F(ab')₂ fragments in TgAb IgG in HT patients, compared to healthy individuals. It focuses on how these patterns differ and their impact on binding to thyroglobulin (Tg). Exploring the role of N-glycosylation in the F(ab')₂ segment of TgAb IgG is crucial for understanding its effect on immune responses in HT, potentially unveiling key mechanisms of the disease. Methods: To assess serum TgAb levels in HT patients (n≥28), we used an electrochemiluminescence immunoassay, dividing patients into medium-level (mHT, n≥14) and high-level (hHT, n≥14) groups. TgAb IgG was isolated from serum of both HT and control groups (n = 14) using affinity chromatography. TgAb IgG was then cleaved with Ides enzyme to yield TgAb IgG F(ab’)2 and Fc fragments. The F(ab')2 fragment of TgAb IgG underwent MALDI-QIT-TOF-MS mass spectrometry for glycosylation profiling. Additionally, mixed TgAb IgG was separated using columns that recognize mannose and terminal sialic acid, yielding high mannose and high terminal sialic acid TgAb IgG. Different TgAb IgG glycoforms were prepared through enzymatic treatment with peptide-N-glycosidase F, β1-4 galactosidase, and α2-3,6,8 neuraminidase. The binding affinity of these TgAb IgG to Tg antigen was measured using Tg specific ELISA and surface plasmon resonance (SPR). Results: Using MALDI-TOF MS, we identified 22 IgG glycan types in both HT and control (Con) groups. In the hHT group, six glycan types (G0F, G1F, G2F, M5A1, A3G1F, A3F) were upregulated compared to Con, with statistical significance (p &amp;lt; 0.05). The mHT group showed increased levels of G1F and G2F compared to Con (p &amp;lt; 0.05). Between hHT and mHT, only A3F was significantly elevated (p &amp;lt; 0.05). Removal of N-glycosylation from IgG TgAb F(ab)2 reduced binding to Tg antigen. Presence of both mannose and terminal sialic acid in TgAb enhanced the binding to Tg. Conclusions: Most TgAb IgG in the variable region of HT patients contain N-glycans. The glycosylation patterns of TgAb IgG F(ab)2 in HT differ significantly from healthy controls, affecting antibody binding affinity. Our findings reveal the complex role of glycosylation patterns in TgAb IgG, providing new insights into HT pathogenesis and potential research directions. Presentation: 6/1/2024

Effects of testosterone and metformin on the GlycanAge index of biological age and the composition of the IgG glycome.

With aging, the body's ability to maintain regular functions declines, increasing susceptibility to age-related diseases. Therapeutic interventions targeting the underlying biological changes of aging hold promise for preventing or delaying multiple age-related diseases. Metformin, a drug commonly used for diabetes treatment, has emerged as a potential gerotherapeutic agent due to its established safety record and preclinical and clinical data on its anti-aging effects. Glycosylation, one of the most common and complex co- and post-translational protein modifications, plays a crucial role in regulating protein function and has been linked to aging and various diseases. Changes in immunoglobulin G (IgG) glycosylation patterns have been observed with age, and these alterations may serve as valuable biomarkers for disease predisposition, diagnosis, treatment monitoring, and overall health assessment. In this study, we analyzed the IgG glycosylation patterns of white men from Europe, aged 29-45years, under treatment with metformin, testosterone, metformin plus testosterone, and placebo (trial registration number NCT02514629, 2013/07/04), and investigated the longitudinal changes in glycosylation over time. We observed statistically significant differences in the IgG glycome composition between participants on testosterone therapy and placebo, with decreased agalactosylation and increased galactosylation and sialylation. However, metformin therapy did not result in statistically significant changes in glycosylation patterns. These findings contribute to our understanding of the impact of therapeutic interventions on IgG glycosylation and confirm the value of IgG glycosylation as a significant biomarker, capable of assessing biological age using the GlycanAge index and providing insight into overall health compared to chronological age.

Glycoprofile Comparison of the SARS-CoV-2 Spike Proteins Expressed in CHO and HEK Cell Lines.

Coronavirus SARS-CoV-2 spike protein remains a key focus of research due to a continued need for diagnostic and therapeutic tools to monitor and respond to new variants. Glycosylation of the spike protein is critical for the protein's functions in viral attachment and host cell entry. For scalable and cost-effective production of the spike protein, expression system-driven divergence in glycosylation patterns on recombinant spike proteins needs to be fully understood. This study assessed the N-glycosylation profiles of a full-length trimeric spike protein expressed in either Human Embryonic Kidney (HEK Expi293F) or Chinese Hamster Ovary (CHO-S) cells. Glycopeptide analysis was performed using a tandem mass spectrometry workflow and BioPharma incorporating HEK and CHO glycan databases for protein characterisation. The results outline important differences in the variety and types of N-glycan generated by the two cell lines across the 22 known N-glycosylation sites of the spike protein. A notable increase in terminal sialylation, as well as the presence of the potentially immunogenic N-glycolylneuraminic acid at a functionally key N-glycosylation site, was observed in the CHO-S derived spike protein. With the potential for the relatively vast and more complex CHO glycan repertoire (182 glycans relative to 39 human glycans) to produce functional implications with CHO-S expressed spike protein, this study adds valuable knowledge to aid Quality by Design approaches and enable Multi Attribute Monitoring of specific N-glycosylation sites for proteoform analyses. This can further inform antigen development with future variants in order to devise updated diagnostic tests and therapeutic vaccine designs.

Efficient purification of soluble receptor for advanced glycation end-products from Sus scrofa lung tissue and synthesis of its binding ligand, glycated bovine serum albumin

A receptor for advanced glycation end products (RAGE) has emerged as a crucial player in various pathological conditions due to its involvement in inflammation and cellular dysfunction. Its soluble isoform, sRAGE, has garnered significant attention for its competitive inhibitory effects and potential therapeutic applications. However, obtaining sRAGE with appropriate glycosylation patterns for binding to glycated proteins has been challenging, often requiring costly expression systems. Here, we present a novel approach for producing and purifying sRAGE from Sus scrofa lungs, bypassing the need for expensive expression systems. Previous protocols for sRAGE extraction faced reproducibility issues due to high viscosity and haemoglobin content of the solution. To address this, we developed a method for selective haemoglobin precipitation using a zinc-containing buffer, enabling purification via various chromatographic methods. Through a combination of chromatographic techniques, we obtained sRAGE in suitable purity, identified using HPLC-MS/MS. Additionally, producing glycated proteins for RAGE receptor activation often involved lengthy protocols or inadequate separation from reactants. Thus, we devised a rapid method for producing and purifying pure BSA glycated with ribose, addressing a critical gap in the field. Functional studies, conducted using Native PAGE, demonstrated the capability of purified proteins to bind to each other.

Multiplex Determination of Glycan Profiles on Urinary Prostate-Specific Antigen by Quartz-Crystal Microbalance Combined with Surface-Enhanced Raman Scattering.

Prostate cancer remains a major health concern, with prostate-specific antigen (PSA) being a key biomarker for its detection and monitoring. However, PSA levels often fall into a "gray zone", where PSA levels are not clearly indicative of cancer, thus complicating early diagnosis and treatment decisions. Glycosylation profiles, which often differ between healthy and diseased cells, have emerged as potential biomarkers to enhance the specificity and sensitivity of cancer diagnosis in these ambiguous cases. We propose the integration of two complementary techniques, namely quartz-crystal microbalance with dissipation (QCM-D) and surface-enhanced Raman scattering (SERS) to study PSA glycan profiles. QCM-D offers real-time operation, PSA mass quantification, and label-free detection with high sensitivity, as well as enhanced specificity and reduced cross-reactivity when using nucleic acid aptamers as capture ligands. Complementary SERS sensing enables the determination of the glycosylation pattern on PSA, at low concentrations and without the drawbacks of photobleaching, thereby facilitating multiplexed glycosylation pattern analysis. This integrated setup could retrieve a data set comprising analyte concentrations and associated glycan profiles in relevant biological samples, which may eventually improve early disease detection and monitoring. Prostate-specific antigen (PSA), a glycoprotein secreted by prostate epithelial cells, serves as our proof-of-concept analyte. Our platform allows multiplex targeting of PSA multiplex glycosylation profiles of PSA at "gray zone" concentrations for prostate cancer diagnosis. We additionally show the use of SERS for glycan analysis in PSA secreted from prostate cancer cell lines after androgen-based treatment. Differences in PSA glycan profiles from resistant cell lines after androgen-based treatment may eventually improve cancer treatment.

Serum levels of prostate specific antigen, free PSA, [-2]proPSA, fPSA/tPSA ratio, Prostate Health Index, and glycosylation patterns of free PSA in patients with benign prostatic hyperplasia pharmacotherapy.

The medication used to treat benign prostate hyperplasia (BPH), a common condition in men over 50 years of age, can alter the levels of biomarkers used in prostate cancer detection. Commonly used medications for BPH include alpha-blockers, 5-alpha reductase inhibitors (5-ARIs), and muscarinic antagonists. We studied the impact of these drugs on total prostate-specific antigen (tPSA), free PSA (fPSA), [-2]proPSA, fPSA/tPSA ratio, and the Prostate Health Index (PHI), as well as novel potential biomarkers in the form of glycan composition of fPSA. Serum samples were collected from 564 males with BPH, with a mean age of 68.5 years. The samples were used to measure levels of tPSA, fPSA, and [-2]proPSA. The fPSA/tPSA and PHI were then calculated. The glycan composition of fPSA was analyzed using lectin-based glycoprofiling. Pharmacotherapy data was collected from the patients' medical records. Alpha-blocker monotherapy was associated with higher fPSA and fPSA/tPSA ratio, and decreased PHI. Levels of tPSA were not impacted. Alpha-blocker and 5-ARI dual therapy was associated with reduced levels of fPSA, [-2]proPSA, and PHI. Therapy combining alpha-blockers and antimuscarinic agents did not significantly influence biomarker levels apart from an increase in a Maackia amurensis lectin-recognized glycan originating in fPSA. BPH pharmacotherapy notably affects prostate cancer biomarkers. Recognizing the impact of pharmacotherapy is crucial for achieving an accurate diagnosis of prostate cancer and for planning treatment.

Integrated proteomic and glycoproteomic analysis reveals heterogeneity and molecular signatures of brain metastases from lung adenocarcinomas

Brain metastasis is a major cause of poor prognosis and death in lung adenocarcinoma (LUAD); however, the understanding of therapeutic strategies and mechanisms for brain metastases from LUAD (BM-LUAD) remains notably limited, especially at the proteomics levels. To address this issue, we conducted integrated proteomic and glycoproteomic analyses on 49 BM-LUAD tumors, revealing two distinct subtypes of the disease: BM-S1 and BM-S2. Whole exome sequencing analysis revealed that somatic mutations in STK11 and KEAP1, as well as copy number deletions on chr19p13.3, such as STK11, UQCR11, and SLC25A23, were more frequently detected in BM-S2. In BM-S1 tumors, we observed significant infiltration of GFAP + astrocytes, as evidenced by elevated levels of GFAP, GABRA2, GABRG1 and GAP43 proteins and an enrichment of astrocytic signatures in both our proteomic data and external spatial transcriptomic data. Conversely, BM-S2 tumors demonstrated higher levels of PD-1 immune cell infiltration, supported by the upregulation of PD-1 and LAG-3 genes. These findings suggest distinct microenvironmental adaptations required by the different BM-LUAD subtypes. Additionally, we observed unique glycosylation patterns between the subtypes, with increased fucosylation in BM-S1 and enhanced sialylation in BM-S2, primarily affected by glycosylation enzymes such as FUT9, B4GALT1, and ST6GAL1. Specifically, in BM-S2, these sialylation modifications are predominantly localized to the lysosomes, underscoring the critical role of N-glycosylation in the tumor progression of BM-LUAD. Overall, our study not only provides a comprehensive multi-omic data resource but also offers valuable biological insights into BM-LUAD, highlighting potential mechanisms and therapeutic targets for further investigation.