Surface Glycoprotein Research Articles

TMIC-87. ICAM-1 REGULATE EXTRACELLULAR VESICLE-MEDIATED IMMUNOSUPPRESSIVE MONOCYTE IN HUMAN GLIOBLASTOMA CELL

Abstract Glioblastoma is the most common malignant primary brain tumor and is universally fatal. Treatment involves maximal safe resection with adjuvant chemoradiation. Poor prognosis in glioblastoma is multifactorial, and local and systemic immunosuppression is an important factor. Extracellular vesicles (EVs) are small, membrane-bound particles released by cells into the external environment. They play a key role in intercellular communication. In glioblastomas, EVs regulate immunosuppression through induction of myeloid-derived suppressor cells (MDSCs) in a process dependent on the PD-1/PD-L1 and IL-6 axes. Primary cilia are microtubule-based organelles that play crucial roles in various cellular processes and signal transduction cascades. We recently showed that primary cilia modulate IL-6 expression and consequently, MDSC induction. Comparative proteomic analysis of EVs derived from human glioblastoma cells devoid of cilia showed loss of ICAM-1 expression compared to control glioblastoma cells. ICAM-1 (Intercellular Adhesion Molecule-1) is a cell surface glycoprotein that mediates cell-cell adhesion and immune responses. In this study, we showed that depletion of primary cilia in human glioblastoma cells result in increased PD-L1 but loss of IL-6 and ICAM-1. RNAi-meidated loss of ICAM-1 was synergistic with primary cilia depletion in preventing MDSC inductionn normal human monocytes exposed to glioblastoma-derived EVs. Exogenous IFN-g was sufficient to restore IL-6 levels and MDSC induction despite loss of primary cilia and ICAM-1 suggesting that primary cilia act upstream of IFN-g, IL-6 and ICAM-1 in glioblastoma-mediated MDSC induction. This study is the first to connect primary cilia signal transduction to IL-6 and ICAM-1 expression and to MDSC induction. The evidence suggests a potential parallel primary cilia regulatory cascade independent of the PD-L1 axis that controls tumor-mediated immunosuppression. These results may inform novel therapeutic strategies aimed at increasing efficacy of immunotherapy in the glioblastoma and other neoplastic processes.

Tissue spaces are reservoirs of antigenic diversity for Trypanosoma brucei.

The protozoan parasite Trypanosoma brucei evades clearance by the host immune system through antigenic variation of its dense variant surface glycoprotein (VSG) coat, periodically 'switching' expression of the VSG using a large genomic repertoire of VSG-encoding genes1-6. Recent studies of antigenic variation in vivo have focused near exclusively on parasites in the bloodstream6-8, but research has shown that many, if not most, parasites reside in the interstitial spaces of tissues9-13. We sought to explore the dynamics of antigenic variation in extravascular parasite populations using VSG-seq7, a high-throughput sequencing approach for profiling VSGs expressed in populations of T. brucei. Here we show that tissues, not the blood, are the primary reservoir of antigenic diversity during both needle- and tsetse bite-initiated T. brucei infections, with more than 75% of VSGs found exclusively within extravascular spaces. We found that this increased diversity is correlated with slower parasite clearance in tissue spaces. Together, these data support a model in which the slower immune response in extravascular spaces provides more time to generate the antigenic diversity needed to maintain a chronic infection. Our findings reveal the important role that extravascular spaces can have in pathogen diversification.

Preclinical development of SGN-CD47M: Protease-activated antibody technology enables selective tumor targeting of the innate immune checkpoint receptor CD47.

CD47 is a cell surface glycoprotein that is expressed on normal human tissues and has a key role as a marker of self. Tumor cells have coopted CD47 overexpression to evade immune surveillance and thus blockade of CD47 is a highly active area of clinical exploration in oncology. However, clinical development of CD47-targeted agents has been complicated by its robust expression in normal tissues and the toxicities that arise from blocking this inhibitory signal. Further, pro-phagocytic signals are not uniformly expressed in tumors and antibody blockade alone is often not sufficient to drive antitumor activity. The inclusion of an IgG1 antibody backbone into therapeutic design has been shown to serve as an additional pro-phagocytic signal but also exacerbates toxicities in normal tissues. Therefore, a need persists for more selective therapeutic modalities targeting CD47. To address these challenges, we developed SGN-CD47M, a humanized anti-CD47 IgG1 monoclonal antibody linked to novel masking peptides through linkers designed to be cleaved by active proteases enriched in the tumor microenvironment. Masking technology has the potential to increase the amount of drug that reaches the tumor microenvironment, while concomitantly reducing systemic toxicities. We demonstrate that SGN-CD47M is well tolerated in cynomolgus monkeys and displays a 20-fold improvement in tolerability to hematologic toxicities when compared to the unmasked antibody. SGN-CD47M also displays preferential activation in the tumor microenvironment that leads to robust single-agent antitumor activity. For these reasons, SGN-CD47M may have enhanced antitumor activity and improved tolerability relative to existing therapies that target the CD47-SIRPα interaction.

Immunohistochemical Localization of Fibroblast Activation Protein in Coronary Arteries with Different Forms of Atherosclerosis

Background: Fibroblast activation protein (FAP) is a cell surface glycoprotein expressed by myofibroblasts in areas of active tissue remodeling. It plays a potentially important role in cardiac remodeling, atherosclerotic plaque formation, and plaque rupture. Given the distinct pathophysiology and morphology of different forms of atherosclerosis, we analyzed FAP expression in human coronary vessels with no coronary artery disease, atherosclerotic plaques at different levels of progression, and other distinct forms of coronary disease in post bypass vein grafting and cardiac allograft vasculopathy after a heart transplant. Methods: Immunohistochemical staining with monoclonal F19 mouse anti-human FAP antibody was performed to identify FAP in human atherosclerotic plaques, coronary bypass atherosclerosis, and post-transplant arteriosclerosis. The presence and distribution of FAP in different types and stages of human atherosclerosis were compared. Results: There was no FAP staining in patients with no significant coronary disease. All different types of human atherosclerotic lesioning lesions showed the presence of FAP expression, with different staining patterns in advanced atherosclerotic plaque, vein graft atherosclerosis lesions, and arteriosclerosis after a heart transplant. Conclusions: These data suggest that FAP may be a potential diagnostic marker and target for interventions, not only in coronary atherosclerotic plaque, but also in other forms of coronary disease, which have distinct pathophysiologies and currently limited treatment options.

Preparation and Construction of Chimeric Humanized Broadly Reactive Antibody 10H10 to Protein E of Tick-Borne Encephalitis Virus.

A full-length humanized chimeric antibody 10H10ch that specifically interacts with the surface glycoprotein E of flaviviruses was obtained. To construct it, we used variable fragments of the heavy and light chains of the monoclonal antibody 10H10 that form the active center of the antibody and a fragment of the constant part of the heavy chain of the human IgG1 antibody. The resulting full-length chimeric humanized antibody 10H10ch specifically interacted with the E protein of flaviviruses pathogenic to humans, such as tick-borne encephalitis, Zika, West Nile, and dengue viruses. An immunochemical assessment of the interaction constants of the 10H10ch antibody with a panel of native and recombinant flavivirus antigens by ELISA and biolayer interferometry showed that the dissociation constant (Kd) of the chimeric antibody is in the nanomolar region and is comparable to that of the high-affinity mouse monoclonal antibody 10H10. The possibility of using the resulting chimeric humanized antibody 10H10ch for the diagnosis, prevention, and treatment of various flavivirus infections is discussed.

Architectural organization and in situ fusion protein structure of lymphocytic choriomeningitis virus.

The impact of COVID-19 on public health has highlighted the importance of understanding zoonotic pathogens. Lymphocytic choriomeningitis virus (LCMV) is a rodent-borne human pathogen that causes hemorrhagic fever. Herein, we describe the in situ structure of LCMV proteins and their architectural organization on the viral envelope and around the nucleocapsid. The virion structure reveals the distribution of the surface glycoprotein complex (GPC) and the contact points between the viral envelope and the underlying matrix protein, as well as the association with the nucleocapsid. The morphology and sizes of virions, as well as the number of RNA polymerase L inside each virion vary greatly, highlighting the fast-changing nature of LCMV. A comparison between the in situ GPC trimeric structure and prior ectodomain structures identifies the transmembrane and endo domains of GPC and key interactions among its subunits. The work provides new insights into LCMV assembly and informs future structure-guided vaccine design.

Adenovirus type 5-expressing Gn induces better protective immunity than Gc against SFTSV infection in mice.

Severe fever with thrombocytopenia syndrome (SFTS) is caused by the SFTS virus (SFTSV) with high morbidity and mortality. The major immunodominant region of SFTSV surface glycoprotein (G) remains unclear. In this study, we constructed adenovirus type 5 (Ad5) vectored vaccine candidates expressing different regions of SFTSV G (Gn, Gc and Gn-Gc) and evaluated their immunogenicity and protective efficacy in mice. In wild-type mice, compared with Ad5-Gc or Ad5-Gn-Gc, Ad5-Gn recruited/activated more dendritic cells and B cells in lymph nodes or peripheral blood, causing Th1-/Th2-mediated responses in splenocytes and triggered a greater level of SFTSV-neutralizing antibodies. In IFNAR Ab-treated mice, immunization of Ad5-Gn exhibited better protection against SFTSV challenge than Ad5-Gc or Ad5-Gn-Gc. Furthermore, passive immunization revealed complete protective immunity of Gn-specific serum rather than Gc. Collectively, our data demonstrated that Gn is the immunodominant fragment of SFTSV G and could be a potential candidate for SFTSV vaccine development.

A Nitroreductase‐Activatable Metabolic Reporter for Covalent Labeling of Pathological Hypoxic Cells in Tumorigenesis

AbstractAberrant hypoxic stress will initiate a cascade of pathological consequence observed prominently in tumorigenesis. Understanding of hypoxia's role in tumorigenesis is highly essential for developing effective therapeutics, which necessitates reliable tools to specifically distinguish hypoxic tumor cells (or tissues) and correlate their dynamics with the status of disease in complex living settings for precise theranostics. So far, disparate hypoxia‐responsive probe molecules and prodrugs were designed via chemical or enzymatic reactions, yet their capability in real‐time reporting pathogenesis development is often compromised due to unrestricted diffusion and less selectivity towards the environmental responsiveness. Herein we present an oxygen‐insensitive nitroreductase (NTR)‐activatable glycan metabolic reporter (pNB‐ManNAz) capable of covalently labeling hypoxic tumor cells and tissues. Under pathophysiological hypoxic environments, the caged non‐metabolizable precursor pNB‐ManNAz exhibited unique responsiveness to cellular NTR, culminating in structural self‐immolation and the resultant ManNAz could incorporate onto cell surface glycoproteins, thereby facilitating fluorescence labeling via bioorthogonal chemistry. This NTR‐responsive metabolic reporter demonstrated broad applicability for multicellular hypoxia labeling, particularly in the dynamic monitoring of orthotopic tumorigenesis and targeted tumor phototherapy in vivo. We anticipate that this approach holds promise for investigating hypoxia‐related pathological progression, offering valuable insights for accurate diagnosis and treatment.

Some Glycoproteins Expressed on the Surface of Immune Cells and Cytokine Plasma Levels Can Be Used as Potential Biomarkers in Patients with Colorectal Cancer

Colorectal cancer (CRC) is a leading cause of mortality worldwide. Its incidence holds a major position among the most common life-threatening diseases. Hence, the early identification and precise characterization of disease activity based on proper biomarkers are of utmost importance for therapeutic strategy and patient survival. The identification of new biomarkers for colorectal cancer or disease-specific levels/combinations of biomarkers will significantly contribute to precise diagnosis and improved personalized treatment of patients. Therefore, the present study aims to identify colorectal cancer-specific immunological biomarkers. The plasma levels of several cytokines (interleukin-1β /IL-1β/, IL-2, IL-4, IL-10, IL-12, IL-15, TGFβ and IFNγ) of 20 patients with colorectal cancer and 21 healthy individuals were determined by ELISA. The expression of several types of glycoproteins on the surface of peripheral blood leukocytes isolated from CRC patients and healthy volunteers was evaluated by flow cytometry. Correlations between cytokine levels and cell surface glycoprotein expression were analyzed. The obtained results demonstrated significantly elevated levels of CD80, CD86, CD279 and CD274 expressing leukocyte populations in the cancer patient group, while the numbers of NK cells and CD8- and CD25-positive cells were decreased. Based on these data and the correlations with cytokine levels, it can be concluded that CD25, CD80, CD86, CD274 and CD279 glycoproteins combined with specific plasma levels of IL-1β, IL-2, IL-15 and TGFβ could represent potential biomarkers for colorectal cancer.

Exploring causal correlations between inflammatory cytokines and coronary heart disease: A Mendelian randomization study.

Coronary heart disease (CHD) is a global health concern, with inflammation significantly contributing to its pathogenesis. It is crucial to understand the relationship between inflammatory cytokines and CHD. This study investigates the causal correlations between circulating inflammatory cytokines and CHD using Mendelian randomization (MR), assessing both causative and resultant roles of these cytokines in CHD. In this bidirectional MR analysis, we used genetic data from a genome-wide association study (GWAS) of 60,801 CHD cases and 123,504 controls of European ancestry. We derived inflammatory cytokine data from a GWAS summary of 14,824 participants. The primary analytical approach was the inverse variance-weighted (IVW) method, supported by MR-Egger, weighted median, and weighted mode analyses. Heterogeneity was assessed using the Cochrane Q test, and horizontal pleiotropy was evaluated through the MR-Egger intercept and the MR-PRESSO global test, ensuring robustness against potential pleiotropic bias. This study pinpointed several cytokines as key upstream influencers on the risk of CHD, including eotaxin (CCL11) (odds ratio [OR]: 1.10, 95% confidence interval [CI]: 1.03-1.18, P = .003), C-C motif chemokine ligand 20 (CCL20) (OR: 1.15, 95% CI: 1.05-1.25, P = .002), macrophage colony-stimulating factor 1 (CSF1) (OR: 1.09, 95% CI: 1.01-1.17, P = .020), Fibroblast growth factor 21 (FGF21) (OR: 1.14, 95% CI: 1.01-1.29, P = .038), Fms-related tyrosine kinase 3 ligand (FLT3LG) (OR: 1.26, 95% CI: 1.09-1.44, P = .001), neurotrophin-3 (NT-3) (OR: 1.12, 95% CI: 1.01-1.24, P = .026), and leukemia inhibitory factor (LIF) (OR: 0.89, 95% CI: 0.80-0.99, P = .029). Conversely, T-cell surface glycoprotein CD5 (CD5) (beta: -0.15, 95% CI: -0.29 to -0.01, P = .042) were identified as downstream factors impacted by CHD. No evidence of heterogeneity or horizontal pleiotropy was detected across all results, and a leave-one-out analysis substantiated the robustness of these findings. These findings suggest that CCL11, CCL20, CSF1, FGF21, FLT3LG, NT-3, and LIF may play a crucial role in the pathogenesis of CHD. Additionally, CHD may impact the expression of CD5. Additional research is needed to explore the potential of these biomarkers in the prevention and treatment of CHD.

Graphene quantum dots disrupt the mitochondrial potential of Trypanosoma brucei by interacting with the p18 subunit of ATP synthase F1 after endocytosis via the VSG recycling pathway

HypothesisTrypanosomiasis is one of the main threats to human and animal health in African countries. Trypanosoma brucei can evade the host immune recognition by rapidly altering its variant surface glycoprotein (VSG). The ATP synthase F1 subunit of the parasite exhibits extremely low similarity to that of its mammalian hosts, hypothetically making it an ideal target for the development of novel therapeutics. ExperimentsGraphene quantum dots (GQDs) were synthesized, and their adhesion to T. brucei surface and internalization was observed microscopically. The activity of ATP synthase and mitochondrial membrane potential of T. brucei were measured after exposure to GQDs. Proteomics, biolayer interferometry, and molecular dynamic simulations were utilized to evaluate the interaction between GQDs with the target proteins. FindingsGQDs specifically adhered to the VSG of T. brucei and were conveyed inside the parasite via the VSG internalization pathway. The GQDs promoted intracellular ROS production, interacted with, and inhibited the activity of the p18 subunit of ATP synthase, disrupted parasite mitochondrial membrane potential. Additionally, the GQDs caused a decrease in aminoacyl – tRNA biosynthesis, and upregulated RNA and protein degradation pathways. The findings of this study offer a novel avenue for the target-oriented discovery of anti-trypanosome drugs.

Effects of selective serotonin reuptake inhibitors on platelet functions: a literature review.

Many epidemiological studies have evidenced an increased bleeding risk associated with selective serotonin reuptake inhibitors (SSRIs), yet the underlying mechanisms remain unclear. This review summarizes data on SSRIs' effects on platelet functions assessed with assays used in clinical practice and highlights the areas that deserve further investigation. Conflicting results of SSRI effects on platelet aggregation were observed irrespectively of the agonist used, the antidepressant drug or the study type. Alike, discrepant results were reported with flow-cytometry-based assays assessing either platelet surface glycoprotein levels, integrin activation, agonist-induced secretion of intraplatelet granule content or membrane anionic phospholipid exposure. Other tests may have detected a platelet function defect in SSRIs samples, however, results were largely inconsistent. Critical literature examination unveils very low certainty of evidence on potential SSRI effect on platelet functions. Findings are often inconsistent even when similar methods are used, most likely because of differences in study design, included patients (age, comorbid conditions), SSRIs' type and dose, uncontrolled confounding factors, and statistical analysis power. Further studies are needed to disentangle any intrinsic antiplatelet effect of SSRIs and the multiple confounding factors, mainly the depression control itself and the degree of platelet SERT inhibition.

Association between circulating inflammatory proteins and benign prostatic disease: a Mendelian randomization study

Previous research has suggested that circulating inflammatory proteins are associated with benign prostatic disease (BPD). This Mendelian randomization (MR) study was conducted to further investigate the causal relationship between 91 inflammatory proteins and BPD. Genome-wide association study (GWAS) summarized data of benign prostatic hyperplasia (BPH) and prostatitis were obtained from the FinnGen Biobank. The latest study offered the GWAS data on 91 proteins related to inflammation. We performed a bidirectional MR to investigate the causal association between inflammatory proteins and BPD. The outcomes of the IVW method indicated that decreased levels of circulating interleukin-17 C (IL-17 C) (OR = 0.92, 95%CI = 0.85–0.99, p-value = 0.0344) were suggestively associated with a higher risk of BPH and elevated levels of interleukin-10 receptor subunit alpha (IL-10RA) (OR = 1.24, 95%CI = 1.05–1.47, p-value = 0.0132) and urokinase-type plasminogen activator (uPA) (OR = 1.13, 95%CI = 1.00–1.28, p-value = 0.0421) were suggestively related to a higher risk of prostatitis. Furthermore, reverse MR revealed that BPH may promote the expression of circulating factors, including natural killer cell receptor 2B4 (CD244) (OR = 1.07, 95%CI = 1.01–1.13, p-value = 0.0192), T-cell surface glycoprotein CD6 isoform (CD6) (OR = 1.07, 95%CI = 1.01–1.13, p-value = 0.0192), and leukemia inhibitory factor receptor (LIF-R) (OR = 1.07, 95%CI = 1.01–1.15, p-value = 0.0163). Moreover, the results of sensitivity analyses indicate that heterogeneity and horizontal pleiotropy are unlikely to distort the findings. The results of this study indicate a potential association between circulating inflammatory proteins and BPD, which may become new diagnostic indicators or drug targets for clinical application in the prevention and treatment of BPD. However, further investigation is required.

Investigation on the influence of GPI-AP for the production of malic acid in Aspergillus niger

Based on the potential of microbial fermentation in the sustainable and environmentally friendly production of L-malic acid (L-MA), this study aims to investigate the effect of glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) on enhancing the stability of Aspergillus niger (A. niger) cell wall to increase L-MA production. Through experiments, we discovered and screened GPI2, a GPI-anchored cell surface glycoprotein, and confirmed for the first time that it has a promoting effect on L-MA, a metabolite of A. niger. This recombinant strain RG0098 that overexpressed gpi2 showed an increase in cell wall thickness and robustness, a decrease in intracellular reactive oxygen species (ROS) levels, which improved the antioxidant capacity. After 108 h of fermentation, the production of L-MA reached 114.7 ± 2.12 g/L, and the productivity was 1.06 ± 0.025 g/L/h. The strain RG0098 has enhanced the productivity of L-MA by a notable margin of 49.7 %. This study provides a sustainable and environmentally friendly pathway for the production of L-MA through microbial fermentation.

Prospects of universal influenza virus vaccine and the current challenges of new antiviral drugs

The profound impact of influenza viruses on human health persists as a significant burden, given their potent capacity to cause morbidity and mortality. Despite efforts to mitigate the annual burden of influenza, the effectiveness of current seasonal vaccines in providing substantial protection falls short, leaving undesirable pandemic of influenza viruses. The challenges posed by the influenza pandemic lies from the constant changes inherent in the virus itself, as well as the limitations of current immunization approaches in achieving sufficient immunogenicity. Influenza viruses exhibit antigenic drift and shift, undergoing antigenic evolution by altering the surface glycoproteins. These changes contribute to the persistent and dynamic nature of the influenza virus, posing formidable challenges to effective prevention and control strategies. Currently, there is growing recognition of unique viral targets that hold promise development of broad-protective vaccines against influenza. These targets are distinct from traditional vaccine targets and offer the potential for more comprehension protection against diverse strains of the virus. Here, we present a review about the novel drugs and vaccines that target the influenza virus would signify the unique immune correlates of protection that need to be initiated to accelerate the vaccine efficacy.

Inflammatory cytokines and their potential role in kidney stone disease: a Mendelian randomization study.

Previous studies have reported a complex relationship between inflammatory cytokines and kidney stone disease (KSD). The purpose of this paper is to investigate the potential causal impact of inflammatory cytokines on KSD by Mendelian randomization (MR) analysis. In our study, a thorough two-sample Mendelian randomization (MR) analysis was performed by us to determine the potential causal relationship between inflammatory cytokines and kidney stone disease. Utilizing GWAS summary data of inflammatory cytokines and KSD, we performed the first two-sample MR analysis. Genetic variants in GWASs related to inflammatory cytokines were employed as instrumental variables (IVs). The data on cytokines were derived from 14,824 participants and analyzed by utilizing the Olink Target-96 Inflammation Panel. GWAS summary data related to KSD (9713 cases and 366,693 controls) were obtained from the FinnGen consortium. The primary MR analysis method was Inverse variance weighted. Reverse MR analysis, Cochran's Q test, MR Egger, and MR-Pleiotropy RESidual Sum and Outlier (MR-PRESSO) were used to assess the stability of the results. 91 cytokines were enrolled in the MR analysis after strict quality control of IV. The IVW analysis revealed 2 cytokines as risk factors for KSD: Cystatin D (OR 1.06, 95% CI 1.01-1.11), Fibroblast growth factor 5 (OR 1.06, 95% CI 1.00-1.12), suggesting they are positively associated with the occurrence of kidney stones. We also found 3 protective associations between cytokines and KSD: Artemin (OR 0.86, 95% CI 0.78-0.96), T-cell surface glycoprotein CD6 isoform (OR 0.92, 95% CI 0.88-0.98), STAM-binding protein (OR 0.83, 95% CI 0.69-0.99). There was no horizontal pleiotropy or significant heterogeneity in our MR analysis, as determined by the p-value results of our MR Egger's intercept test, Cochrane Q-test, and MR-PRESSO, which were all > 0.05. Our study explored a variety of inflammatory cytokines related to KSD through MR analysis, which validated several previous findings and provided some new potential biomarkers for KSD. However, the findings require further investigation to validate their exact functions in the pathogenesis and evolution of KSD.

NCAM and attached polysialic acid affect behaviors of breast epithelial cells through differential signaling pathways.

Neural cell adhesion molecule (NCAM), a common mammalian cell surface glycoprotein, is the major substrate of polysialic acid (polySia). Polysialylated NCAM occurs in many types of cancer, but rarely in normal adult tissues. The functional role of NCAM hypersialylation in the epithelial-mesenchymal transition (EMT) process remains unclear. The present study indicates that NCAM and attached polysialic acid affect behaviors of breast epithelial cells through differential signaling pathways. NCAM and polysialylated NCAM are aberrantly regulated in breast cancer cells. They are both upregulated in normal breast epithelial cells undergoing EMT. Western blot analysis demonstrates that NCAM-140 overexpression induces EMT in breast epithelial cells and promotes cell proliferation and migration through activation of the β-catenin/slug signaling pathway. Modification of polySia attached to NCAM modulates cell adhesion and promotes cell motility through activation of the EGFR/STAT3 pathway. These observations contribute to clarifying the molecular mechanisms by which polysialic acid and its major substrate, NCAM, modulate cell behaviors, and highlight the significance of increased polysialylated expression on NCAM during EMT and tumor development.

Multi-omic analysis of a mucolipidosis II neuronal cell model uncovers involvement of pathways related to neurodegeneration and drug metabolism

Defining the molecular consequences of lysosomal dysfunction in neuronal cell types remains an area of investigation that is needed to understand many underappreciated phenotypes associated with lysosomal disorders. Here we characterize GNPTAB-knockout DAOY medulloblastoma cells using different genetic and proteomic approaches, with a focus on how altered gene expression and cell surface abundance of glycoproteins may explain emerging neurological issues in individuals with GNPTAB-related disorders, including mucolipidosis II (ML II) and mucolipidosis IIIα/β (ML IIIα/β). The two knockout clones characterized demonstrated all the biochemical hallmarks of this disease, including loss of intracellular glycosidase activity due to impaired mannose 6-phosphate-dependent lysosomal sorting, lysosomal cholesterol accumulation, and increased markers of autophagic dysfunction. RNA sequencing identified altered transcript abundance of several neuronal markers and genes involved in drug metabolism and transport, and neurodegeneration-related pathways. Using selective exo-enzymatic labeling (SEEL) coupled with proteomics to profile cell surface glycoproteins, we demonstrated altered abundance of several glycoproteins in the knockout cells. Most striking was increased abundance of the amyloid precursor protein and apolipoprotein B, indicating that loss of GNPTAB function in these cells corresponds with elevation in proteins associated with neurodegeneration. The implication of these findings on lysosomal disease pathogenesis and the emerging neurological manifestations of GNPTAB-related disorders is discussed.

A gain of function variant in RGS18 candidate for a familial mild bleeding syndrome

BackgroundInherited platelet diseases are bleeding disorders characterized by either defects in platelet count or platelet function, the latter being less common and very heterogeneous. Numerous gene variants associated with abnormal receptors, granules, and signaling pathways have been reported. Despite significant advancements in our understanding, many patients still lack a precise diagnosis. ObjectivesTo identify the genetic basis of a novel mild bleeding syndrome in a family exhibiting a selective defect of platelet aggregation. MethodsOur study included 6 family members across 3 generations who displayed reduced platelet aggregation in response to adenosine diphosphate, PAR1-AP, arachidonic acid, and epinephrine but not collagen. Platelet morphology, granule content, and expression of major surface glycoproteins were all found to be normal. Whole exome sequencing was performed for affected and nonaffected family members. ResultsWe identified RGS18, which encodes the regulator of G protein signaling (RGS) 18, as a candidate gene for the platelet function defect observed in this family. The RGS18 protein serves as a crucial negative regulator of G protein-coupled receptor signaling and coordinates the signaling pathways of natural platelet inhibitors. The heterozygous RGS18 c.643C>T, p.Arg215∗ variant was found to cosegregate among all 6 affected subjects. ConclusionTruncation at Arg215 removes the S216 and S218 phosphorylation sites, which are crucial regulatory domains for RGS18 activation. The impaired platelet function is thought to arise from excessive platelet downregulation due to constitutive activation of RGS18, resulting from a loss of association of the truncated form with the 14-3-3 protein.

N-glycoproteomic and proteomic alterations in SRD5A3-deficient fibroblasts.

SRD5A3-CDG is a congenital disorder of glycosylation (CDG) resulting from pathogenic variants in SRD5A3 and follows an autosomal recessive inheritance pattern. The enzyme encoded by SRD5A3, polyprenal reductase, plays a crucial role in synthesizing lipid precursors essential for N-linked glycosylation. Despite insights from functional studies into its enzymatic function, there remains a gap in understanding global changes in patient cells. We sought to identify N-glycoproteomic and proteomic signatures specific to SRD5A3-CDG, potentially aiding in biomarker discovery and advancing our understanding of disease mechanisms. Using tandem mass tag (TMT)-based relative quantitation, we analyzed fibroblasts derived from five patients along with control fibroblasts. N-glycoproteomics analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) identified 3,047 glycopeptides with 544 unique N-glycosylation sites from 276 glycoproteins. Of these, 418 glycopeptides showed statistically significant changes with 379 glycopeptides decreased (P<0.05) in SRD5A3-CDG patient-derived samples. These included high mannose, complex and hybrid glycan-bearing glycopeptides. High mannose glycopeptides from protocadherin Fat 4 and integrin alpha-11 and complex glycopeptides from CD55 were among the most significantly decreased glycopeptides. Proteomics analysis led to the identification of 5,933 proteins, of which 873 proteins showed statistically significant changes. Decreased proteins included cell surface glycoproteins, various mitochondrial protein populations and proteins involved in the N-glycosylation pathway. Lysosomal proteins such as N-acetylglucosamine-6-sulfatase and procathepsin-L also showed reduced levels of phosphorylated mannose-containing glycopeptides. Our findings point to disruptions in glycosylation pathways as well as energy metabolism and lysosomal functions in SRD5A3-CDG, providing clues to improved understanding and management of patients with this disorder.