Project description:PMM2-CDG is a rare inborn error of metabolism caused by deficiency of the phosphomannomutase (PMM2) enzyme, which leads to impaired protein glycosylation. While the disorder presents primarily with neurological symptoms, there is limited knowledge about the specific brain-related changes that result from PMM deficiency. We found aberrant neural activity in 2D neuronal networks from individuals with PMM2-CDG. Multi-omics datasets from 3D brain organoids derived from individuals with PMM2-CDG revealed widespread decrease in protein glycosylation, highlighting impaired glycosylation as a key pathological feature of PMM2-CDG. Further, we identified impaired mitochondrial structure and abnormal glucose metabolism in PMM2-CDG organoids indicating disturbances in energy metabolism. Correlation between PMM2 enzymatic activity in brain organoids and symptom severity suggests that the level of PMM2 enzyme function directly influences neurological manifestations. These findings enhance our understanding of specific brain-related perturbations associated with PMM2-CDG, offering insights into the underlying mechanisms and potential directions for therapeutic interventions.
Project description:Alterations of protein abundance and post-translational modifications in patients with Alzheimer’s disease (AD), such as glycosylation, phosphorylation, and ubiquitination, and their roles in disease progression and treatment outcome are areas of intense study. Little is known, however, about the overall N-glycosylation of proteins in human brains, and those from Alzheimer’s patients, particularly in regard to large-scale intact N-linked glycoproteomics analysis. To elucidate the glycoproteome landscape, we developed an approach based on multi-lectin affinity enrichment, hydrophilic interaction chromatography (HILIC), and LC-MS-based glycoproteomics analysis. We analyzed 10 normal, 10 asymptomatic, and 10 symptomatic AD brains, in which we detected >300 glycoproteins and >1,900 glycoforms across the samples. The majority of glycoproteins have N-glycans that are high-mannosidic and complex chains that are fucosylated and bisected. The Man5 N-glycan was found to occur most frequently at >20% of the total glycoforms. Unlike the glycoproteomes of other tissues, sialylation is a minor feature of the brain glycoproteome, occurring at <9% of N-glycans . We observed changes in the number of antennae, frequency of fucosylation, bisection, and other monosaccharides at individual glycosylation sites among normal, asymptomatic, and symptomatic AD samples. Further analysis revealed glycosylation differences in subcellular compartments. We did not observe a statistical difference between male and female patients. These results represent the first glycoproteomics landscape of brains from multiple AD individuals, which will facilitate a deeper understanding of AD and possible disease treatment options.
Project description:To understand the cellular functions of glycans and to exploit them as clinical targets it is essential to acquire information on their cell surface exposure. Here, we developed a sensitive and specific mass spectrometry method to globally study the N-glycoforms displayed at individual protein sites on the cell surface. This offers a valuable tool to discriminate between functionally relevant glycans and biosynthetic intermediates for future cellular glycoproteomic analyses.
Project description:Protein N-glycosylation is ubiquitous in brain and closely related to cognition and memory. Alzheimer's disease (AD) is a multifactorial disorder that lacks clear pathogenesis and treatment. Aberrant N-glycosylation has been suggested to be involved in AD pathology. While the systematic variations of protein N-glycosylation and their roles in AD have not been thoroughly investigated due to technically challenging. Here, we applied multilayered N-glycoproteomics to quantify the global protein expression, N-glycosylation sites, N-glycans, and site-specific N-glycopeptides in AD mice brains (APP/PS1 transgenic) versus wild type. The N-glycoproteome landscape exhibited the highly complex site-specific heterogeneity in AD brains. Quantitative analyses explored the generally dysregulated N-glycosylations in AD, involving proteins such as glutamate receptors, as well as fucosylated and oligo-mannose glycans. Furthermore, functional study revealed the crucial roles of N-glycosylation on proteins and neuron cells. Our work provided a robust multilayered N-glycoproteomics workflow for AD and can be applied to widespread biological systems.
Project description:this study discovered unique glycoprotein resources responsible for plant salt stress tolerance and suggested crucial roles of Nthis study discovered unique glycoprotein resources responsible for plant salt stress tolerance and suggested crucial roles of N-glycans in regulating salt responsive protein expression in Arabidopsis.-glycans in regulating salt responsive protein expression in Arabidopsis.
Project description:Sulfs represent a class of unconventional sulfatases, which differ from all other members of the sulfatase family by their structures, catalytic features and biological functions. Through their specific endosulfatase activity in extracellular milieu, Sulfs provide an original post-synthetic regulatory mechanism for heparan sulfate complex polysaccharides and have been involved in multiple physiopathological processes, including cancer. However, Sulfs remain poorly characterized enzymes, with major discrepancies regarding their in vivo functions. Here we show that human Sulf-2 (HSulf-2) features a unique polysaccharide post-translational modification. We identified a chondroitin/dermatan sulfate glycosaminoglycan (GAG) chain, attached to the enzyme substrate binding domain. We found that this GAG chain affects enzyme/substrate recognition and tunes HSulf-2 activity in vitro and in vivo using a mouse model of tumorigenesis and metastasis. In addition, we showed that mammalian hyaluronidase acted as a promoter of HSulf-2 activity by digesting its GAG chain. In conclusion, our results highlight HSulf-2 as a unique proteoglycan enzyme and its newly-identified GAG chain as a critical non-catalytic modulator of the enzyme activity. These findings contribute in clarifying the conflicting data on the activities of the Sulfs and introduce a new paradigm into the study of these enzymes.
Project description:Glycoproteins comprise more than half of current FDA-approved protein cancer markers but the development of new glycoproteins as disease biomarkers has been stagnant. Here we present a pipeline to develop glycoproteins from extracellular vesicles (EVs) through integrating quantitative glycoproteomics with a novel reverse phase glycoprotein array, and then apply it to identify novel biomarkers of breast cancer. EV glycoproteomics show promise in circumventing the problems plaguing current serum glycoproteomics and allowed us to identify hundreds of glycoproteins that have not been identified in serum. We identified 1,453 unique glycopeptides representing 556 glycoproteins in EVs, among which 20 are significantly higher in breast cancer patients. We further applied a novel glyco-specific reverse phase protein array to quantify a subset of the candidates. Together, this study demonstrates the great potential of this integrated pipeline for biomarker discovery.
Project description:Microalgae are a renewable and promising biomass for large-20 scale biofuel, food and nutrient production. However, their efficient exploitation depends on our knowledge of the cell wall composition and organization as it can limit access to high-value molecules. Here we provide an atomic-level model of the non-crystalline and water-insoluble glycoprotein-rich cell wall of Chlamydomonas reinhardtii. Using in situ solid-state and sensitivity-enhanced nuclear magnetic 25 resonance, we reveal unprecedented details on the protein and carbohydrate composition and their nanoscale heterogeneity, as well as the presence of spatially segregated protein- and glycan-rich regions with different dynamics and hydration levels. We show that mannose-rich lower molecular-weight proteins likely contribute to the cell wall cohesion by binding to high-molecular weight protein components, and that water provides plasticity to the cell-wall architecture. The 30 structural insight exemplifies strategies used by nature to form cell walls devoid of cellulose or other glycan polymers.
Project description:People with HIV (PWH) experience an increased vulnerability to premature aging and inflammation-associated comorbidities, even when HIV replication is suppressed by antiretroviral therapy (ART). However, the factors that contribute to or are associated with this vulnerability remain uncertain. In the general population, alterations in the glycomes of circulating IgGs trigger inflammation and precede the onset of aging-associated diseases. Here, we investigate the IgG glycomes of cross-sectional and longitudinal samples from 1,216 women and men, both living with virally suppressed HIV and those without HIV. Our glycan-based machine learning models indicate that living with chronic HIV significantly accelerates the accumulation of pro-aging associated glycomic alterations. Consistently, PWH exhibit heightened expression of senescence associated glycan-degrading enzymes compared to their controls. These glycomic alterations correlate with elevated markers of inflammatory aging and the severity of comorbidities, potentially preceding the development of such comorbidities. Mechanistically, HIV-specific antibodies glycoengineered with these alterations exhibit reduced anti-HIV IgG-mediated innate immune functions. These findings hold significant potential for the development of glycomic based biomarkers and tools to identify and prevent premature aging and comorbidities in people living with chronic viral infections.