scholarly journals O-Glycosylation Landscapes of SARS-CoV-2 Spike Proteins

2021 ◽  
Vol 9 ◽  
Author(s):  
Yong Zhang ◽  
Wanjun Zhao ◽  
Yonghong Mao ◽  
Yaohui Chen ◽  
Shanshan Zheng ◽  
...  
Keyword(s):  
High Resolution Mass Spectrometry ◽  
Collision Induced Dissociation ◽  
Energy Collision ◽  
Cell Type ◽  
Glycosylation Pattern ◽  
S Protein ◽  
Viral Attachment ◽  
Viral Binding ◽  
Pngase F ◽  
S Proteins

The densely glycosylated spike (S) proteins that are highly exposed on the surface of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) facilitate viral attachment, entry, and membrane fusion. We have previously reported all the 22 N-glycosites and site-specific N-glycans in the S protein protomer. Herein, we report the O-glycosylation landscapes of SARS-CoV-2 S proteins, which were characterized through high-resolution mass spectrometry. Following digestion with trypsin and trypsin/Glu-C, and de-N-glycosylation using PNGase F, we determined the GalNAc-type O-glycosylation pattern of S proteins, including O-glycosites and the six most common O-glycans occupying them, via Byonic identification and manual validation. Finally, 255 intact O-glycopeptides composed of 50 peptides sequences and 43 O-glycosites were discovered by higher energy collision-induced dissociation (HCD), and three O-glycosites were confidently identified by electron transfer/higher energy collision-induced dissociation (EThcD) in the insect cell-expressed S protein. Most glycosites were modified by non-sialylated O-glycans such as HexNAc(1) and HexNAc(1)Hex (1). In contrast, in the human cell-expressed S protein S1 subunit, 407 intact O-glycopeptides composed of 34 peptides sequences and 30 O-glycosites were discovered by HCD, and 11 O-glycosites were unambiguously assigned by EThcD. However, the measurement of O-glycosylation occupancy hasn’t been made. Most glycosites were modified by sialylated O-glycans such as HexNAc(1)Hex (1)NeuAc (1) and HexNAc(1)Hex (1)NeuAc (2). Our results reveal that the SARS-CoV-2 S protein is an O-glycoprotein; the O-glycosites and O-glycan compositions vary with the host cell type. These comprehensive O-glycosylation landscapes of the S protein are expected to provide novel insights into the viral binding mechanism and present a strategy for the development of vaccines and targeted drugs.

scholarly journals Mucin-type O-glycosylation Landscapes of SARS-CoV-2 Spike Proteins

2020 ◽  
Author(s):  
Yong Zhang ◽  
Wanjun Zhao ◽  
Yonghong Mao ◽  
Yaohui Chen ◽  
Jingqiang Zhu ◽  
...  
Keyword(s):  
High Resolution Mass Spectrometry ◽  
Targeted Drugs ◽  
Cell Type ◽  
Glycosylation Pattern ◽  
Site Specific ◽  
S Protein ◽  
Viral Attachment ◽  
Viral Binding ◽  
Pngase F ◽  
S Proteins

ABSTRACTThe densely glycosylated spike (S) proteins that are highly exposed on the surface of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) facilitate viral attachment, entry, and membrane fusion. We have previously reported all the 22 N-glycosites and site-specific N-glycans in the S protein protomer. Herein, we report the comprehensive and precise site-specific O-glycosylation landscapes of SARS-CoV-2 S proteins, which were characterized using high-resolution mass spectrometry. Following digestion using trypsin and trypsin/Glu-C, and de-N-glycosylation using PNGase F, we determined the mucin-type (GalNAc-type) O-glycosylation pattern of S proteins, including unambiguous O-glycosites and the 6 most common O-glycans occupying them, via Byonic identification and manual validation. Finally, 43 O-glycosites were identified in the insect cell-expressed S protein. Most glycosites were modified by non-sialylated O-glycans such as HexNAc(1) and HexNAc(1)Hex(1). In contrast, 30 O-glycosites were identified in the human cell-expressed S protein S1 subunit. Most glycosites were modified by sialylated O-glycans such as HexNAc(1)Hex(1)NeuAc(1) and HexNAc(1)Hex(1)NeuAc(2). Our results are the first to reveal that the SARS-CoV-2 S protein is a mucin-type glycoprotein; clustered O-glycans often occur in the N- and the C-termini of the S protein, and the O-glycosite and O-glycan compositions vary with the host cell type. These site-specific O-glycosylation landscapes of the SARS-CoV-2 S protein are expected to provide novel insights into the viral binding mechanism and present a strategy for the development of vaccines and targeted drugs.

scholarly journals Deducing the N- and O-glycosylation profile of the spike protein of novel coronavirus SARS-CoV-2

Glycobiology ◽  
2020 ◽  
Vol 30 (12) ◽  
pp. 981-988 ◽  
Author(s):  
Asif Shajahan ◽  
Nitin T Supekar ◽  
Anne S Gleinich ◽  
Parastoo Azadi
Keyword(s):  
High Resolution Mass Spectrometry ◽  
Critical Role ◽  
Spike Protein ◽  
Protein Subunit ◽  
Binding Studies ◽  
Viral Attachment ◽  
Protein Subunits ◽  
Glycosylation Profile ◽  
Viral Binding ◽  
Novel Coronavirus

Abstract The current emergence of the novel coronavirus pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demands the development of new therapeutic strategies to prevent rapid progress of mortalities. The coronavirus spike (S) protein, which facilitates viral attachment, entry and membrane fusion is heavily glycosylated and plays a critical role in the elicitation of the host immune response. The spike protein is comprised of two protein subunits (S1 and S2), which together possess 22 potential N-glycosylation sites. Herein, we report the glycosylation mapping on spike protein subunits S1 and S2 expressed on human cells through high-resolution mass spectrometry. We have characterized the quantitative N-glycosylation profile on spike protein and interestingly, observed unexpected O-glycosylation modifications on the receptor-binding domain of spike protein subunit S1. Even though O-glycosylation has been predicted on the spike protein of SARS-CoV-2, this is the first report of experimental data for both the site of O-glycosylation and identity of the O-glycans attached on the subunit S1. Our data on the N- and O-glycosylation are strengthened by extensive manual interpretation of each glycopeptide spectra in addition to using bioinformatics tools to confirm the complexity of glycosylation in the spike protein. The elucidation of the glycan repertoire on the spike protein provides insights into the viral binding studies and more importantly, propels research toward the development of a suitable vaccine candidate.

scholarly journals Deducing the N- and O- glycosylation profile of the spike protein of novel coronavirus SARS-CoV-2

2020 ◽  
Author(s):  
Asif Shajahan ◽  
Nitin T. Supekar ◽  
Anne S. Gleinich ◽  
Parastoo Azadi
Keyword(s):  
High Resolution Mass Spectrometry ◽  
Critical Role ◽  
Spike Protein ◽  
Protein Subunit ◽  
Binding Studies ◽  
Viral Attachment ◽  
Protein Subunits ◽  
Glycosylation Profile ◽  
Viral Binding ◽  
Novel Coronavirus

AbstractThe current emergence of the novel coronavirus pandemic caused by SARS-CoV-2 demands the development of new therapeutic strategies to prevent rapid progress of mortalities. The coronavirus spike (S) protein, which facilitates viral attachment, entry and membrane fusion is heavily glycosylated and plays a critical role in the elicitation of the host immune response. The spike protein is comprised of two protein subunits (S1 and S2), which together possess 22 potential N-glycosylation sites. Herein, we report the glycosylation mapping on spike protein subunits S1 and S2 expressed on human cells through high resolution mass spectrometry. We have characterized the quantitative N-glycosylation profile on spike protein and interestingly, observed unexpected O-glycosylation modifications on the receptor binding domain (RBD) of spike protein subunit S1. Even though O-glycosylation has been predicted on the spike protein of SARS-CoV-2, this is the first report of experimental data for both the site of O-glycosylation and identity of the O-glycans attached on the subunit S1. Our data on the N- and O-glycosylation is strengthened by extensive manual interpretation of each glycopeptide spectra in addition to using bioinformatics tools to confirm the complexity of glycosylation in the spike protein. The elucidation of the glycan repertoire on the spike protein provides insights into the viral binding studies and more importantly, propels research towards the development of a suitable vaccine candidate.

scholarly journals Modeling Coronavirus Spike Protein Dynamics: Implications for Immunogenicity and Immune Escape

2021 ◽  
Author(s):  
Genevieve Kunkel ◽  
Mohammad Madani ◽  
S. J. White ◽  
Paulo Verardi ◽  
Anna Tarakanova
Keyword(s):  
Protein Dynamics ◽  
Antibody Binding ◽  
Phase Iii ◽  
Coarse Grained ◽  
Mode Analysis ◽  
S Protein ◽  
Viral Attachment ◽  
Binding Behavior ◽  
The Impact ◽  
S Proteins

The ongoing COVID-19 pandemic is a global public health emergency requiring urgent development of efficacious vaccines. While concentrated research efforts are underway to develop antibody-based vaccines that would neutralize SARS-CoV-2, and several first-generation vaccine candidates are currently in Phase III clinical trials or have received emergency use authorization, it is forecasted that COVID-19 will become an endemic disease requiring second-generation vaccines. The SARS-CoV-2 surface Spike (S) glycoprotein represents a prime target for vaccine development because antibodies that block viral attachment and entry, i.e. neutralizing antibodies, bind almost exclusively to the receptor binding domain (RBD). Here, we develop computational models for a large subset of S proteins associated with SARS-CoV-2, implemented through coarse-grained elastic network models and normal mode analysis. We then analyze local protein domain dynamics of the S protein systems and their thermal stability to characterize structural and dynamical variability among them. These results are compared against existing experimental data, and used to elucidate the impact and mechanisms of SARS-CoV-2 S protein mutations and their associated antibody binding behavior. We construct a SARS-CoV-2 antigenic map and offer predictions about the neutralization capabilities of antibody and S mutant combinations based on protein dynamic signatures. We then compare SARS-CoV-2 S protein dynamics to SARS-CoV and MERS-CoV S proteins to investigate differing antibody binding and cellular fusion mechanisms that may explain the high transmissibility of SARS-CoV-2. The outbreaks associated with SARS-CoV, MERS-CoV, and SARS-CoV-2 over the last two decades suggest that the threat presented by coronaviruses is ever-changing and long-term. Our results provide insights into the dynamics-driven mechanisms of immunogenicity associated with coronavirus S proteins, and present a new approach to characterize and screen potential mutant candidates for immunogen design, as well as to characterize emerging natural variants that may escape vaccine-induced antibody responses.

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

2020 ◽  
Author(s):  
Asif Shajahan ◽  
Stephanie Archer-Hartmann ◽  
Nitin T. Supekar ◽  
Anne S. Gleinich ◽  
Christian Heiss ◽  
...  
Keyword(s):  
Angiotensin Converting Enzyme ◽  
Viral Entry ◽  
Glycosylation Site ◽  
Converting Enzyme ◽  
S Protein ◽  
Angiotensin Converting Enzyme 2 ◽  
Viral Attachment ◽  
Viral Binding ◽  
Glycosylation Sites

AbstractThe emergence of the COVID-19 pandemic caused by 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 human 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.

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

Glycobiology ◽  
2020 ◽  
Author(s):  
Asif Shajahan ◽  
Stephanie Archer-Hartmann ◽  
Nitin T Supekar ◽  
Anne S Gleinich ◽  
Christian Heiss ◽  
...  
Keyword(s):  
Angiotensin Converting Enzyme ◽  
Viral Entry ◽  
Glycosylation Site ◽  
Hek293 Cells ◽  
Converting Enzyme ◽  
S Protein ◽  
Angiotensin Converting Enzyme 2 ◽  
Viral Attachment ◽  
Viral Binding ◽  
Glycosylation Sites

Abstract 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.

From the discovery to molecular understanding of cellular iron-sulfur protein biogenesis

2020 ◽  
Vol 401 (6-7) ◽  
pp. 855-876 ◽  
Author(s):  
Roland Lill
Keyword(s):  
De Novo ◽  
Current Knowledge ◽  
Protein Stabilization ◽  
S Protein ◽  
Cellular Iron ◽  
Iron Sulfur ◽  
Protein Biogenesis ◽  
Sulfur Protein ◽  
Initial Discovery ◽  
S Proteins

AbstractProtein cofactors often are the business ends of proteins, and are either synthesized inside cells or are taken up from the nutrition. A cofactor that strictly needs to be synthesized by cells is the iron-sulfur (Fe/S) cluster. This evolutionary ancient compound performs numerous biochemical functions including electron transfer, catalysis, sulfur mobilization, regulation and protein stabilization. Since the discovery of eukaryotic Fe/S protein biogenesis two decades ago, more than 30 biogenesis factors have been identified in mitochondria and cytosol. They support the synthesis, trafficking and target-specific insertion of Fe/S clusters. In this review, I first summarize what led to the initial discovery of Fe/S protein biogenesis in yeast. I then discuss the function and localization of Fe/S proteins in (non-green) eukaryotes. The major part of the review provides a detailed synopsis of the three major steps of mitochondrial Fe/S protein biogenesis, i.e. the de novo synthesis of a [2Fe-2S] cluster on a scaffold protein, the Hsp70 chaperone-mediated transfer of the cluster and integration into [2Fe-2S] recipient apoproteins, and the reductive fusion of [2Fe-2S] to [4Fe-4S] clusters and their subsequent assembly into target apoproteins. Finally, I summarize the current knowledge of the mechanisms underlying the maturation of cytosolic and nuclear Fe/S proteins.

scholarly journals The Yeast Scaffold Proteins Isu1p and Isu2p Are Required inside Mitochondria for Maturation of Cytosolic Fe/S Proteins

2004 ◽  
Vol 24 (11) ◽  
pp. 4848-4857 ◽  
Author(s):  
Jana Gerber ◽  
Karina Neumann ◽  
Corinna Prohl ◽  
Ulrich Mühlenhoff ◽  
Roland Lill
Keyword(s):  
Iron Homeostasis ◽  
De Novo ◽  
Mitochondrial Targeting ◽  
Protein Maturation ◽  
Iron Sulfur Cluster ◽  
Subcellular Compartment ◽  
S Protein ◽  
Iron Sulfur ◽  
Protein Biogenesis ◽  
S Proteins

ABSTRACT Iron-sulfur (Fe/S) proteins are located in mitochondria, cytosol, and nucleus. Mitochondrial Fe/S proteins are matured by the iron-sulfur cluster (ISC) assembly machinery. Little is known about the formation of Fe/S proteins in the cytosol and nucleus. A function of mitochondria in cytosolic Fe/S protein maturation has been noted, but small amounts of some ISC components have been detected outside mitochondria. Here, we studied the highly conserved yeast proteins Isu1p and Isu2p, which provide a scaffold for Fe/S cluster synthesis. We asked whether the Isu proteins are needed for biosynthesis of cytosolic Fe/S clusters and in which subcellular compartment the Isu proteins are required. The Isu proteins were found to be essential for de novo biosynthesis of both mitochondrial and cytosolic Fe/S proteins. Several lines of evidence indicate that Isu1p and Isu2p have to be located inside mitochondria in order to perform their function in cytosolic Fe/S protein maturation. We were unable to mislocalize Isu1p to the cytosol due to the presence of multiple, independent mitochondrial targeting signals in this protein. Further, the bacterial homologue IscU and the human Isu proteins (partially) complemented the defects of yeast Isu protein-depleted cells in growth rate, Fe/S protein biogenesis, and iron homeostasis, yet only after targeting to mitochondria. Together, our data suggest that the Isu proteins need to be localized in mitochondria to fulfill their functional requirement in Fe/S protein maturation in the cytosol.

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