Serum N-glycomic profiling may provide potential signatures for surveillance of COVID-19.

Disease development and progression are often associated with aberrant glycosylation, indicating that changes in biological fluid glycome may potentially serve as disease signatures. The corona virus disease-2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) represents a significant threat to global human health. However, the effect of SARS-CoV-2 infection on the overall serum N-glycomic profile has been largely unexplored. Here, we extended our 96-well-plate-based high-throughput, high-sensitivity N-glycan profiling platform further with the aim of elucidating potential COVID-19-associated serum N-glycomic alterations. Use of this platform revealed both similarities and differences between the serum N-glycomic fingerprints of COVID-19 positive and control cohorts. Although there were no specific glycan peaks exclusively present or absent in COVID-19 positive cohort, this cohort showed significantly higher levels of glycans and variability. On the contrary, the overall N-glycomic profiles for healthy controls were well-contained within a narrow range. From the serum glycomic analysis, we were able to deduce changes in different glycan subclasses sharing certain structural features. Of significance was the hyperbranched and hypersialylated glycans and their derived glycan subclass traits. T-distributed stochastic neighbor embedding and hierarchical heatmap clustering analysis were performed to identify 13 serum glycomic variables that potentially distinguished the COVID-19 positive from healthy controls. Such serum N-glycomic changes described herein may indicate or correlate to the changes in serum glycoproteins upon COVID-19 infection. Furthermore, mapping the serum N-glycome following SARS-CoV-2 infection may help us better understand the disease and enable "Long-COVID" surveillance to capture the full spectrum of persistent symptoms.

35 years in plant lectin research: a journey from basic science to applications in agriculture and medicine.

Plants contain an extended group of lectins differing from each other in their molecular structures, biochemical properties and carbohydrate-binding specificities. The heterogeneous group of plant lectins can be classified in several families based on the primary structure of the lectin domain. All proteins composed of one or more lectin domains, or having a domain architecture including one or more lectin domains in combination with other protein domains can be defined as lectins. Plant lectins reside in different cell compartments, and depending on their location will encounter a large variety carbohydrate structures, allowing them to be involved in multiple biological functions. Over the years lectins have been studied intensively for their carbohydrate-binding properties and biological activities, which also resulted in diverse applications. The present overview on plant lectins especially focuses on the structural and functional characteristics of plant lectins and their applications for crop improvement, glycobiology and biomedical research.

Data-independent acquisition mass spectrometry for site-specific glycoproteomics characterization of SARS-CoV-2 spike protein.

The spike protein of SARS-CoV-2, the virus responsible for the global pandemic of COVID-19, is an abundant, heavily glycosylated surface protein that plays a key role in receptor binding and host cell fusion, and is the focus of all current vaccine development efforts. Variants of concern are now circulating worldwide that exhibit mutations in the spike protein. Protein sequence and glycosylation variations of the spike may affect viral fitness, antigenicity, and immune evasion. Global surveillance of the virus currently involves genome sequencing, but tracking emerging variants should include quantitative measurement of changes in site-specific glycosylation as well. In this work, we used data-dependent acquisition (DDA) and data-independent acquisition (DIA) mass spectrometry to quantitatively characterize the five N-linked glycosylation sites of the glycoprotein standard alpha-1-acid glycoprotein (AGP), as well as the 22 sites of the SARS-CoV-2 spike protein. We found that DIA compared favorably to DDA in sensitivity, resulting in more assignments of low-abundance glycopeptides. However, the reproducibility across replicates of DIA-identified glycopeptides was lower than that of DDA, possibly due to the difficulty of reliably assigning low-abundance glycopeptides confidently. The differences in the data acquired between the two methods suggest that DIA outperforms DDA in terms of glycoprotein coverage but that overall performance is a balance of sensitivity, selectivity, and statistical confidence in glycoproteomics. We assert that these analytical and bioinformatics methods for assigning and quantifying glycoforms would benefit the process of tracking viral variants as well as for vaccine development.

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).

N-Glycomics of Human Erythrocytes.

Glycosylation is a complex post-translational modification that conveys functional diversity to glycoconjugates. Cell surface glycosylation mediates several biological activities such as induction of the intracellular signaling pathway and pathogen recognition. Red blood cell (RBC) membrane N-glycans determine blood type and influence cell lifespan. Although several proteomic studies have been carried out, the glycosylation of RBC membrane proteins has not been systematically investigated. This work aims at exploring the human RBC N-glycome by high-sensitivity MALDI-MS techniques to outline a fingerprint of RBC N-glycans. To this purpose, the MALDI-TOF spectra of healthy subjects harboring different blood groups were acquired. Results showed the predominant occurrence of neutral and sialylated complex N-glycans with bisected N-acetylglucosamine and core- and/or antennary fucosylation. In the higher mass region, these species presented with multiple N-acetyllactosamine repeating units. Amongst the detected glycoforms, the presence of glycans bearing ABO(H) antigens allowed us to define a distinctive spectrum for each blood group. For the first time, advanced glycomic techniques have been applied to a comprehensive exploration of human RBC N-glycosylation, providing a new tool for the early detection of distinct glycome changes associated with disease conditions as well as for understanding the molecular recognition of pathogens.

Fostering "Education": Do Extracellular Vesicles Exploit Their Own Delivery Code?

Extracellular vesicles (EVs), comprising large microvesicles (MVs) and exosomes (EXs), play a key role in intercellular communication, both in physiological and in a wide variety of pathological conditions. However, the education of EV target cells has so far mainly been investigated as a function of EX cargo, while few studies have focused on the characterization of EV surface membrane molecules and the mechanisms that mediate the addressability of specific EVs to different cell types and tissues. Identifying these mechanisms will help fulfill the diagnostic, prognostic, and therapeutic promises fueled by our growing knowledge of EVs. In this review, we first discuss published studies on the presumed EV "delivery code" and on the combinations of the hypothesized EV surface membrane "sender" and "recipient" molecules that may mediate EV targeting in intercellular communication. Then we briefly review the main experimental approaches and techniques, and the bioinformatic tools that can be used to identify and characterize the structure and functional role of EV surface membrane molecules. In the final part, we present innovative techniques and directions for future research that would improve and deepen our understandings of EV-cell targeting.

Plasma Markers of Disrupted Gut Permeability in Severe COVID-19 Patients.

A disruption of the crosstalk between the gut and the lung has been implicated as a driver of severity during respiratory-related diseases. Lung injury causes systemic inflammation, which disrupts gut barrier integrity, increasing the permeability to gut microbes and their products. This exacerbates inflammation, resulting in positive feedback. We aimed to test whether severe Coronavirus disease 2019 (COVID-19) is associated with markers of disrupted gut permeability. We applied a multi-omic systems biology approach to analyze plasma samples from COVID-19 patients with varying disease severity and SARS-CoV-2 negative controls. We investigated the potential links between plasma markers of gut barrier integrity, microbial translocation, systemic inflammation, metabolome, lipidome, and glycome, and COVID-19 severity. We found that severe COVID-19 is associated with high levels of markers of tight junction permeability and translocation of bacterial and fungal products into the blood. These markers of disrupted intestinal barrier integrity and microbial translocation correlate strongly with higher levels of markers of systemic inflammation and immune activation, lower levels of markers of intestinal function, disrupted plasma metabolome and glycome, and higher mortality rate. Our study highlights an underappreciated factor with significant clinical implications, disruption in gut functions, as a potential force that may contribute to COVID-19 severity.

Site-specific characterization of SARS-CoV-2 spike glycoprotein receptor-binding domain.

The novel coronavirus SARS-CoV-2, the infective agent causing COVID-19, is having a global impact both in terms of human disease as well as socially and economically. Its heavily glycosylated spike glycoprotein is fundamental for the infection process, via its receptor-binding domains interaction with the glycoprotein angiotensin-converting enzyme 2 on human cell surfaces. We therefore utilized an integrated glycomic and glycoproteomic analytical strategy to characterize both N- and O- glycan site-specific glycosylation within the receptor-binding domain. We demonstrate the presence of complex-type N-glycans with unusual fucosylated LacdiNAc at both sites N331 and N343 and a single site of O-glycosylation on T323.

COVID-19 and Preparing Planetary Health for Future Ecological Crises: Hopes from Glycomics for Vaccine Innovation.

A key lesson emerging from COVID-19 is that pandemic proofing planetary health against future ecological crises calls for systems science and preventive medicine innovations. With greater proximity of the human and animal natural habitats in the 21st century, it is also noteworthy that zoonotic infections such as COVID-19 that jump from animals to humans are increasingly plausible in the coming decades. In this context, glycomics technologies and the third alphabet of life, the sugar code, offer veritable prospects to move omics systems science from discovery to diverse applications of relevance to global public health and preventive medicine. In this expert review, we discuss the science of glycomics, its importance in vaccine development, and the recent progress toward discoveries on the sugar code that can help prevent future infectious outbreaks that are looming on the horizon in the 21st century. Glycomics offers veritable prospects to boost planetary health, not to mention the global scientific capacity for vaccine innovation against novel and existing infectious agents.

How glycobiology can help us treat and beat the COVID-19 pandemic.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged during the last months of 2019, spreading throughout the world as a highly transmissible infectious illness designated as COVID-19. Vaccines have now appeared, but the challenges in producing sufficient material and distributing them around the world means that effective treatments to limit infection and improve recovery are still urgently needed. This review focuses on the relevance of different glycobiological molecules that could potentially serve as or inspire therapeutic tools during SARS-CoV-2 infection. As such, we highlight the glycobiology of the SARS-CoV-2 infection process, where glycans on viral proteins and on host glycosaminoglycans have critical roles in efficient infection. We also take notice of the glycan-binding proteins involved in the infective capacity of virus and in human defense. In addition, we critically evaluate the glycobiological contribution of candidate drugs for COVID-19 therapy such as glycans for vaccines, anti-glycan antibodies, recombinant lectins, lectin inhibitors, glycosidase inhibitors, polysaccharides, and numerous glycosides, emphasizing some opportunities to repurpose FDA-approved drugs. For the next-generation drugs suggested here, biotechnological engineering of new probes to block the SARS-CoV-2 infection might be based on the essential glycobiological insight on glycosyltransferases, glycans, glycan-binding proteins, and glycosidases related to this pathology.

Comprehensive characterization of N- and O- glycosylation of SARS-CoV-2 human receptor angiotensin converting enzyme 2.

The emergence of the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has created the need for development of new therapeutic strategies. Understanding the mode of viral attachment, entry and replication has become a key aspect of such interventions. The coronavirus surface features a trimeric spike (S) protein that is essential for viral attachment, entry and membrane fusion. The S protein of SARS-CoV-2 binds to human angiotensin converting enzyme 2 (hACE2) for entry. Herein, we describe glycomic and glycoproteomic analysis of hACE2 expressed in HEK293 cells. We observed high glycan occupancy (73.2 to 100%) at all seven possible N-glycosylation sites and surprisingly detected one novel O-glycosylation site. To deduce the detailed structure of glycan epitopes on hACE2 that may be involved in viral binding, we have characterized the terminal sialic acid linkages, the presence of bisecting GlcNAc and the pattern of N-glycan fucosylation. We have conducted extensive manual interpretation of each glycopeptide and glycan spectrum, in addition to using bioinformatics tools to validate the hACE2 glycosylation. Our elucidation of the site-specific glycosylation and its terminal orientations on the hACE2 receptor, along with the modeling of hACE2 glycosylation sites can aid in understanding the intriguing virus-receptor interactions and assist in the development of novel therapeutics to prevent viral entry. The relevance of studying the role of ACE2 is further increased due to some recent reports about the varying ACE2 dependent complications with regard to age, sex, race and pre-existing conditions of COVID-19 patients.

Mass Spectrometry Techniques in Emerging Pathogens Studies: COVID-19 Perspectives.

As corona virus disease 2019 (COVID-19) is a rapidly growing public health crisis across the world, our knowledge of meaningful diagnostic tests and treatment for severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) is still evolving. This novel coronavirus disease COVID-19 can be diagnosed using RT-PCR, but inadequate access to reagents, equipment, and a nonspecific target has slowed disease detection and management. Precision medicine, individualized patient care, requires suitable diagnostics approaches to tackle the challenging aspects of viral outbreaks where many tests are needed in a rapid and deployable approach. Mass spectrometry (MS)-based technologies such as proteomics, glycomics, lipidomics, and metabolomics have been applied in disease outbreaks for identification of infectious disease agents such as virus and bacteria and the molecular phenomena associated with pathogenesis. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS) is widely used in clinical diagnostics in the United States and Europe for bacterial pathogen identification. Paper spray ionization mass spectrometry (PSI-MS), a rapid ambient MS technique, has recently open a new opportunity for future clinical investigation to diagnose pathogens. Ultra-high-pressure liquid chromatography coupled high-resolution mass spectrometry (UHPLC-HRMS)-based metabolomics and lipidomics have been employed in large-scale biomedical research to discriminate infectious pathogens and uncover biomarkers associated with pathogenesis. PCR-MS has emerged as a new technology with the capability to directly identify known pathogens from the clinical specimens and the potential to identify genetic evidence of undiscovered pathogens. Moreover, miniaturized MS offers possible applications with relatively fast, highly sensitive, and potentially portable ways to analyze for viral compounds. However, beneficial aspects of these rapidly growing MS technologies in pandemics like COVID-19 outbreaks has been limited. Hence, this perspective gives a brief of the existing knowledge, current challenges, and opportunities for MS-based techniques as a promising avenue in studying emerging pathogen outbreaks such as COVID-19.