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1.
Anal Chem ; 94(15): 5909-5917, 2022 04 19.
Article in English | MEDLINE | ID: covidwho-1882715

ABSTRACT

SARS-CoV-2 cellular infection is mediated by the heavily glycosylated spike protein. Recombinant versions of the spike protein and the receptor-binding domain (RBD) are necessary for seropositivity assays and can potentially serve as vaccines against viral infection. RBD plays key roles in the spike protein's structure and function, and thus, comprehensive characterization of recombinant RBD is critically important for biopharmaceutical applications. Liquid chromatography coupled to mass spectrometry has been widely used to characterize post-translational modifications in proteins, including glycosylation. Most studies of RBDs were performed at the proteolytic peptide (bottom-up proteomics) or released glycan level because of the technical challenges in resolving highly heterogeneous glycans at the intact protein level. Herein, we evaluated several online separation techniques: (1) C2 reverse-phase liquid chromatography (RPLC), (2) capillary zone electrophoresis (CZE), and (3) acrylamide-based monolithic hydrophilic interaction chromatography (HILIC) to separate intact recombinant RBDs with varying combinations of glycosylations (glycoforms) for top-down mass spectrometry (MS). Within the conditions we explored, the HILIC method was superior to RPLC and CZE at separating RBD glycoforms, which differ significantly in neutral glycan groups. In addition, our top-down analysis readily captured unexpected modifications (e.g., cysteinylation and N-terminal sequence variation) and low abundance, heavily glycosylated proteoforms that may be missed by using glycopeptide data alone. The HILIC top-down MS platform holds great potential in resolving heterogeneous glycoproteins for facile comparison of biosimilars in quality control applications.


Subject(s)
Biosimilar Pharmaceuticals , COVID-19 , Chromatography, Liquid , Chromatography, Reverse-Phase/methods , Glycoproteins/chemistry , Humans , Hydrophobic and Hydrophilic Interactions , Mass Spectrometry , Polysaccharides/analysis , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/chemistry
2.
Science ; 375(6578): eabl6251, 2022 01 21.
Article in English | MEDLINE | ID: covidwho-1650842

ABSTRACT

Many studies have examined the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants on neutralizing antibody activity after they have become dominant strains. Here, we evaluate the consequences of further viral evolution. We demonstrate mechanisms through which the SARS-CoV-2 receptor binding domain (RBD) can tolerate large numbers of simultaneous antibody escape mutations and show that pseudotypes containing up to seven mutations, as opposed to the one to three found in previously studied variants of concern, are more resistant to neutralization by therapeutic antibodies and serum from vaccine recipients. We identify an antibody that binds the RBD core to neutralize pseudotypes for all tested variants but show that the RBD can acquire an N-linked glycan to escape neutralization. Our findings portend continued emergence of escape variants as SARS-CoV-2 adapts to humans.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Immune Evasion , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , BNT162 Vaccine/immunology , Betacoronavirus/immunology , COVID-19/immunology , COVID-19/virology , Cross Reactions , Cryoelectron Microscopy , Crystallography, X-Ray , Epitopes , Evolution, Molecular , Humans , Models, Molecular , Mutation , Polysaccharides/analysis , Protein Binding , Protein Domains , Receptors, Coronavirus/chemistry , Receptors, Coronavirus/metabolism , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Viral Pseudotyping
3.
Carbohydr Polym ; 280: 119006, 2022 Mar 15.
Article in English | MEDLINE | ID: covidwho-1588175

ABSTRACT

Caulerpa lentillifera (Bryopsidophyceae, Chlorophyta) is an edible seaweed attracting great attention for its expansion of farming scale and increasing consumption in these years. In the present study, a sulfated polysaccharide (CLSP-2) was isolated and separated from C. lentillifera, and its chemical structure was elucidated by a series of chemical and spectroscopic methods. Among these methods, mild acid hydrolysis and photocatalytic degradation were applied to release mono- and oligo-saccharide fragments which were further identified by HPLC-MSn analysis, affording the information of the sugar sequences and the sulfate substitution in CLSP-2. Results indicated that the backbone of CLSP-2 was constructed of →6)-ß-Manp-(1→ with sulfated branches at C2, which were comprised of prevalent →3)-ß-Galp4S-(1→, →3)-ß-Galp2,4S-(1→, and minor Xyl. In addition, the virus neutralization assay revealed that CLSP-2 could effectively protect HeLa cells against SARS-CoV-2 infection with an IC50 of 48.48 µg/mL. Hence, the present study suggests CLSP-2 as a promising agent against SARS-CoV-2.


Subject(s)
COVID-19/virology , Caulerpa/chemistry , Polysaccharides/chemistry , Polysaccharides/pharmacology , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Chromatography, High Pressure Liquid/methods , HeLa Cells , Humans , Hydrolysis , Magnetic Resonance Spectroscopy/methods , Mass Spectrometry/methods , Molecular Weight , Polysaccharides/analysis , SARS-CoV-2 , Seaweed/chemistry , Spectroscopy, Fourier Transform Infrared/methods , Sulfates/chemistry
5.
Sci Rep ; 11(1): 20428, 2021 10 14.
Article in English | MEDLINE | ID: covidwho-1469984

ABSTRACT

Glycosylation is the most common post-translational modification and has myriad of biological functions. However, glycan analysis has always been a challenge. Here, we would like to present new techniques for glycan fingerprinting based on enzymatic fluorescent labeling and gel electrophoresis. The method is illustrated on SARS2 spike (S) glycoproteins. SARS2, a novel coronavirus and the causative agent of the COVID-19 pandemic, has had significant social and economic impacts since the end of 2019. To obtain the N-glycan fingerprint of an S protein, glycans released from the protein are first labeled through enzymatic incorporation of fluorophore-conjugated sialic acid or fucose, then separated by SDS-PAGE, and finally visualized with a fluorescent imager. To identify the labeled glycans of a fingerprint, glycan standards and glycan ladders are enzymatically generated and run alongside the samples as references. By comparing the mobility of a labeled glycan to that of a glycan standard, the identity of glycans maybe determined. O-glycans can also be fingerprinted. Due to the lack of an enzyme for broad O-glycan release, O-glycans on the S protein can be labeled with fluorescent sialic acid and digested with trypsin to obtain labeled glycan peptides that are then separated by gel electrophoresis. Glycan fingerprinting could serve as a quick method for globally assessing the glycosylation of a specific glycoprotein.


Subject(s)
COVID-19/virology , Polysaccharides/analysis , SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Carbocyanines/chemistry , Electrophoresis, Polyacrylamide Gel , Fluorescent Dyes/chemistry , Fucose/analogs & derivatives , Glycosylation , Humans , N-Acetylneuraminic Acid/analogs & derivatives , Optical Imaging
6.
Int J Nanomedicine ; 16: 4813-4830, 2021.
Article in English | MEDLINE | ID: covidwho-1406766

ABSTRACT

Human coronaviruses present a substantial global disease burden, causing damage to populations' health, economy, and social well-being. Glycans are one of the main structural components of all microbes and organismic structures, including viruses-playing multiple essential roles in virus infection and immunity. Studying and understanding virus glycans at the nanoscale provide new insights into the diagnosis and treatment of viruses. Glycan nanostructures are considered potential targets for molecular diagnosis, antiviral therapeutics, and the development of vaccines. This review article describes glycan nanostructures (eg, glycoproteins and glycolipids) that exist in cells, subcellular structures, and microbes. We detail the structure, characterization, synthesis, and functions of virus glycans. Furthermore, we describe the glycan nanostructures of different human coronaviruses, such as human coronavirus 229E (HCoV-229E), human coronavirus OC43 (HCoV-OC43), severe acute respiratory syndrome-associated coronavirus (SARS-CoV), human coronavirus NL63 (HCoV-NL63), human coronavirus HKU1 (HCoV-HKU1), the Middle East respiratory syndrome-associated coronavirus (MERS-CoV), and how glycan nanotechnology can be useful to prevent and combat human coronaviruses infections, along with possibilities that are not yet explored.


Subject(s)
Betacoronavirus/chemistry , Nanostructures/analysis , Nanostructures/chemistry , Polysaccharides/analysis , Polysaccharides/chemistry , Humans
7.
J Mol Biol ; 433(4): 166762, 2021 02 19.
Article in English | MEDLINE | ID: covidwho-1386060

ABSTRACT

The severity of SARS-CoV-2 infection is highly variable and yet the molecular basis for this effect remains elusive. One potential contribution are differences in the glycosylation of target human cells, particularly as SARS-CoV-2 has the capacity to bind sialic acid which is a common, and highly variable, terminal modification of glycans. The viral spike glycoprotein (S) of SARS-CoV-2 and the human cellular receptor, angiotensin-converting enzyme 2 (ACE2) are both densely glycosylated. We therefore sought to investigate whether the glycosylation state of ACE2 impacts the interaction with SARS-CoV-2 viral spike. We generated a panel of engineered ACE2 glycoforms which were analyzed by mass spectrometry to reveal the site-specific glycan modifications. We then probed the impact of ACE2 glycosylation on S binding and revealed a subtle sensitivity with hypersialylated or oligomannose-type glycans slightly impeding the interaction. In contrast, deglycosylation of ACE2 did not influence SARS-CoV-2 binding. Overall, ACE2 glycosylation does not significantly influence viral spike binding. We suggest that any role of glycosylation in the pathobiology of SARS-CoV-2 will lie beyond its immediate impact of receptor glycosylation on virus binding.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , COVID-19/metabolism , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/chemistry , Glycosylation , Host-Pathogen Interactions , Humans , Models, Molecular , Polysaccharides/analysis , Protein Binding
8.
Int J Mol Sci ; 22(15)2021 Jul 28.
Article in English | MEDLINE | ID: covidwho-1346499

ABSTRACT

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.


Subject(s)
Blood Group Antigens/metabolism , Erythrocytes/metabolism , Glycomics , Polysaccharides/analysis , Protein Processing, Post-Translational , Glycosylation , Humans , Proteomics , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
9.
J Am Chem Soc ; 143(31): 12014-12024, 2021 08 11.
Article in English | MEDLINE | ID: covidwho-1333882

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilizes an extensively glycosylated surface spike (S) protein to mediate host cell entry, and the S protein glycosylation plays key roles in altering the viral binding/function and infectivity. However, the molecular structures and glycan heterogeneity of the new O-glycans found on the S protein regional-binding domain (S-RBD) remain cryptic because of the challenges in intact glycoform analysis by conventional bottom-up glycoproteomic approaches. Here, we report the complete structural elucidation of intact O-glycan proteoforms through a hybrid native and denaturing top-down mass spectrometry (MS) approach employing both trapped ion mobility spectrometry (TIMS) quadrupole time-of-flight and ultrahigh-resolution Fourier transform ion cyclotron resonance (FTICR)-MS. Native top-down TIMS-MS/MS separates the protein conformers of the S-RBD to reveal their gas-phase structural heterogeneity, and top-down FTICR-MS/MS provides in-depth glycoform analysis for unambiguous identification of the glycan structures and their glycosites. A total of eight O-glycoforms and their relative molecular abundance are structurally elucidated for the first time. These findings demonstrate that this hybrid top-down MS approach can provide a high-resolution proteoform-resolved mapping of diverse O-glycoforms of the S glycoprotein, which lays a strong molecular foundation to uncover the functional roles of their O-glycans. This proteoform-resolved approach can be applied to reveal the structural O-glycoform heterogeneity of emergent SARS-CoV-2 S-RBD variants as well as other O-glycoproteins in general.


Subject(s)
Polysaccharides/analysis , SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Carbohydrate Sequence , Polysaccharides/chemistry , Protein Domains , Tandem Mass Spectrometry/methods
10.
Nat Methods ; 18(1): 28, 2021 01.
Article in English | MEDLINE | ID: covidwho-1065919
11.
Anal Chem ; 92(21): 14730-14739, 2020 11 03.
Article in English | MEDLINE | ID: covidwho-872627

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to a global pandemic of coronavirus disease 2019 (COVID-19). The spike protein expressed on the surface of this virus is highly glycosylated and plays an essential role during the process of infection. We conducted a comprehensive mass spectrometric analysis of the N-glycosylation profiles of the SARS-CoV-2 spike proteins using signature ions-triggered electron-transfer/higher-energy collision dissociation (EThcD) mass spectrometry. The patterns of N-glycosylation within the recombinant ectodomain and S1 subunit of the SARS-CoV-2 spike protein were characterized using this approach. Significant variations were observed in the distribution of glycan types as well as the specific individual glycans on the modification sites of the ectodomain and subunit proteins. The relative abundance of sialylated glycans in the S1 subunit compared to the full-length protein could indicate differences in the global structure and function of these two species. In addition, we compared N-glycan profiles of the recombinant spike proteins produced from different expression systems, including human embryonic kidney (HEK 293) cells and Spodoptera frugiperda (SF9) insect cells. These results provide useful information for the study of the interactions of SARS-CoV-2 viral proteins and for the development of effective vaccines and therapeutics.


Subject(s)
Betacoronavirus/chemistry , Polysaccharides/analysis , Spike Glycoprotein, Coronavirus/chemistry , Animals , Glycosylation , HEK293 Cells , Humans , Mass Spectrometry/methods , Polysaccharides/chemistry , SARS-CoV-2 , Spodoptera/chemistry
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