Characterization of the human platelet N- and O-glycome upon storage using tandem mass spectrometry.

Changes in surface glycan determinants, specifically sialic acid loss, determine platelet life span. The gradual loss of stored platelet quality is a complex process that fundamentally involves carbohydrate structures. Here, we applied lipophilic extraction and glycan release protocols to sequentially profile N- and O-linked glycans in freshly isolated and 7-day room temperature-stored platelet concentrates. Analytical methods including matrix assisted laser desorption/ionization time-of-flight mass spectrometry, tandem mass spectrometry, and liquid chromatography were used to obtain structural details of selected glycans and terminal epitopes. The fresh platelet repertoire of surface structures revealed diverse N-glycans, including high mannose structures, complex glycans with polylactosamine repeats, and glycans presenting blood group epitopes. The O-glycan repertoire largely comprised sialylated and fucosylated core-1 and core-2 structures. For both N- and O-linked glycans, we observed a loss in sialylated epitopes with a reciprocal increase in neutral structures as well as increased neuraminidase activity after platelet storage at room temperature. The data indicate that loss of sialylated glycans is associated with diminished platelet quality and untimely removal of platelets after storage.

Chronic exposure to low doses of ionizing radiation impacts the processing of glycoprotein N-linked glycans in Medaka (Oryzias latipes).

PURPOSE: Ionizing radiation is found naturally in the environment. Low doses of IR may have beneficial applications, yet there is also potential for detrimental long-term health effects. Impacts following exposure to low levels of IR have been refractory to identification and quantification. Glycoprotein glycosylation is vital to cell-cell communication and organismal function, and sensitive to changes in an organism's macro- and cellular environment. We investigated whether accumulated low doses of IR (LoDIR) affect the N-linked glycoprotein glycans using Medaka fish (Oryzias latipes). MATERIALS AND METHODS: State-of-the-art methods in radiation exposure and glycan analysis were applied to study N-glycan changes after 190 day exposure at three different rates of gamma irradiation (2.25, 21.01, and 204.3 mGy/day) in wild-type adult Medaka. Tissue N-glycans were analyzed following enzymatic release from extracted proteins. RESULTS: N-linked glycan profiles are dominated by complex type N-glycans modified with terminal sialic acid and core fucose. Fucosylation and sialylation of N-linked glycoprotein glycans are affected by LoDIR and a subset of N-glycans are involved in the organismal radio-response. CONCLUSION: This is the first indication that the glycome can be interrogated for biomarkers that report the impact of chronic exposure to environmental stressors, such as low-level IR.

Glycomics-informed glycoproteomic analysis of site-specific glycosylation for SARS-CoV-2 spike protein.

This protocol describes an integrated approach for analyzing site-specific N- and O-linked glycosylation of SARS-CoV-2 spike protein by mass spectrometry. Glycoproteomics analyzes intact glycopeptides to examine site-specific microheterogeneity of glycoproteins. Glycomics provides structural characterization on any glycan assignments by glycoproteomics. This procedure can be modified and applied to a variety of N- and/or O-linked glycoproteins. Combined with bioinformatics, the glycomics-informed glycoproteomics may be useful in generating 3D molecular dynamics simulations of certain glycoproteins alone or interacting with one another. For complete details on the use and execution of this protocol, please refer to Zhao et al. (2020).

Virus-Receptor Interactions of Glycosylated SARS-CoV-2 Spike and Human ACE2 Receptor.

The current COVID-19 pandemic is caused by the SARS-CoV-2 betacoronavirus, which utilizes its highly glycosylated trimeric Spike protein to bind to the cell surface receptor ACE2 glycoprotein and facilitate host cell entry. We utilized glycomics-informed glycoproteomics to characterize site-specific microheterogeneity of glycosylation for a recombinant trimer Spike mimetic immunogen and for a soluble version of human ACE2. We combined this information with bioinformatic analyses of natural variants and with existing 3D-structures of both glycoproteins to generate molecular dynamics simulations of each glycoprotein alone and interacting with one another. Our results highlight roles for glycans in sterically masking polypeptide epitopes and directly modulating Spike-ACE2 interactions. Furthermore, our results illustrate the impact of viral evolution and divergence on Spike glycosylation, as well as the influence of natural variants on ACE2 receptor glycosylation that, taken together, can facilitate immunogen design to achieve antibody neutralization and inform therapeutic strategies to inhibit viral infection.

Virus-Receptor Interactions of Glycosylated SARS-CoV-2 Spike and Human ACE2 Receptor.

The SARS-CoV-2 betacoronavirus uses its highly glycosylated trimeric Spike protein to bind to the cell surface receptor angiotensin converting enzyme 2 (ACE2) glycoprotein and facilitate host cell entry. We utilized glycomics-informed glycoproteomics to characterize site-specific microheterogeneity of glycosylation for a recombinant trimer Spike mimetic immunogen and for a soluble version of human ACE2. We combined this information with bioinformatics analyses of natural variants and with existing 3D structures of both glycoproteins to generate molecular dynamics simulations of each glycoprotein both alone and interacting with one another. Our results highlight roles for glycans in sterically masking polypeptide epitopes and directly modulating Spike-ACE2 interactions. Furthermore, our results illustrate the impact of viral evolution and divergence on Spike glycosylation, as well as the influence of natural variants on ACE2 receptor glycosylation. Taken together, these data can facilitate immunogen design to achieve antibody neutralization and inform therapeutic strategies to inhibit viral infection.

Prediction of Response to Therapy for Autoimmune/Inflammatory Diseases Using an Activated Macrophage-Targeted Radioimaging Agent.

The ability to select patients who will respond to therapy is especially acute for autoimmune/inflammatory diseases, where the costs of therapies can be high and the progressive damage associated with ineffective treatments can be irreversible. In this article we describe a clinical test that will rapidly predict the response of patients with an autoimmune/inflammatory disease to many commonly employed therapies. This test involves quantitative assessment of uptake of a folate receptor-targeted radioimaging agent ((99m)Tc-EC20) by a subset of inflammatory macrophages that accumulate at sites of inflammation. Murine models of four representative inflammatory diseases (rheumatoid arthritis, inflammatory bowel disease, pulmonary fibrosis, and atherosclerosis) show markedly decreased uptake of (99m)Tc-EC20 in inflamed lesions upon initiation of successful therapies, but no decrease in uptake upon administration of ineffective therapies, in both cases long before changes in clinical symptoms can be detected. This predictive capability should reduce costs and minimize morbidities associated with failed autoimmune/inflammatory disease therapies.