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

Deducing the N- and O-glycosylation profile of the spike protein of novel coronavirus SARS-CoV-2 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. Key words: COVID-19, SARS-CoV-2 glycosylation, spike protein, coronavirus vaccine, S1 S2 glycosylation Introduction To date, no specific medical treatments or vaccines for COVID-19 The current major health crisis is caused by the novel severe acute have been approved (Li and De Clercq 2020; WHO 2020a). There- respiratory syndrome coronavirus 2 (SARS-CoV-2) that rapidly fore, the scientific community is expending great effort in compiling spread globally within weeks in early 2020. This highly transmissible data regarding the virus, as well as the respiratory illness caused by infectious disease causes a respiratory illness named COVID-19 it, to find effective ways of dealing with this health crisis. (Huang et al. 2020; Wu et al. 2020). As of the 31 March 2020, The pathogenic SARS-CoV-2 enters human target cells via its 750,890 cases of COVID-19 and 36,405 COVID-19-related deaths viral transmembrane spike (S) glycoprotein. The spike protein is a have been confirmed globally by the World Health Organization trimeric class I fusion protein and consists of two subunits, namely (WHO 2020b). S1 and S2. The S1 subunit facilitates the attachment of the virus, © The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com 981 982 A Shajahan et al. Fig. 1. The SARS-CoV-2 spike proteins recombinantly expressed on HEK293 supernatant were fractionated through SDS-PAGE, subsequently digested by proteases and analyzed by nLC-NSI-MS/MS. The expression of SARS-CoV-2 spike protein subunits S1 and S2 on HEK 293 culture supernatant showed higher molecular weight upon SDS-PAGE than expected, because of glycosylation. Thus, the gel bands corresponding to the molecular weight of 200–100 kDa for S1 and 150–80 kDa for S2 were cut, proteins were lysed after reduction–alkylation and analyzed by LC-MS/MS (created with biorender.com). Purified S2 were processed after in-solution protease digestion. and subsequently the S2 subunit allows for the fusion of the viral Here, we report the site-specific quantitative N-linked and and human cellular membranes (Hoffmann et al. 2020; Walls et al. O-linked glycan profiling on SARS-CoV-2 subunit S1 and S2 2020; Zhou et al. 2020). The entry receptor for SARS-CoV-2 has been protein through glycoproteomics using high-resolution liquid identified as the human angiotensin-converting enzyme 2 (hACE2), chromatography-tandem mass spectrometry (LC-MS/MS). We used and recent studies determined a high binding affinity to hACE2 recombinant SARS-CoV-2 subunit S1 and S2 expressed in human (Hoffmann et al. 2020; Shang et al. 2020; Walls et al. 2020). Given cells, HEK 293, and observed partial N-glycan occupancy on 17 its literal key role, the S protein is one of the major targets for the out of 22 N-glycosylation sites. We found that the remaining development of specific medical treatments or vaccines: neutralizing five N-glycosylation sites were unoccupied. Remarkably, we have antibodies targeting the spike proteins of SARS-CoV-2 could prevent unambiguously identified two unexpected O-glycosylation sites at the virus from binding to the hACE2 entry receptor and therefore the receptor-binding domain (RBD) of subunit S1. O-glycosylation from entering the host cell (Shang et al. 2020). on the spike protein of SARS-CoV-2 is predicted in some recent Each monomer of the S protein is highly glycosylated with reports and most of these predictions are for sites in proximity 22 predicted N-linked glycosylation sites. Furthermore, three O- to furin cleavage site (S1/S2) as similar sites are O-glycosylated in glycosylation sites were also predicted (Andersen et al. 2020). Cryo- SARS-CoV-1 (Andersen et al. 2020; Uslupehlivan 2020). However, electron microscopy (Cryo-EM) provides evidence for the existence we observed O-glycosylation at two sites on the RBD of spike of 14–16 N-glycans on 22 potential sites in the SARS-CoV-2 S protein protein subunit S1, and this is the first report on the evidence for (Walls et al. 2020). The glycosylation pattern of the spike protein is such glycan modification at a crucial binding location of the spike a crucial characteristic to be considered regarding steric hindrance, protein. Site-specific analysis of N- and O-glycosylation information chemical properties and even as a potential target for mutation in the of SARS-CoV-2 spike protein provides basic understanding of the future. The N-glycans on S protein play important roles in proper viral structure, crucial for the identification of immunogens for protein folding and priming by host proteases (Walls et al. 2020). vaccine design. This in turn has the potential of leading to future As glycans can shield the amino acid residues and other epitopes therapeutic intervention or prevention of COVID-19. from cells and antibody recognition, glycosylation can enable the coronavirus to evade both the innate and adaptive immune responses Results (Walls et al. 2019, 2020). Elucidating the glycosylation of the viral S protein can aid in understanding viral binding with receptors, fusion, Studies over the past two decades have shown that glycosylation on entry, replication and also in designing suitable antigens for vaccine the protein antigens can play crucial roles in the adaptive immune development (Chakraborti et al. 2005; Zheng et al. 2018; Watanabe response. Thus, it is obvious that the glycosylation on the protein et al. 2019). Strategies for vaccine development aim to elicit such antigen is relevant for the development of vaccines, and it is widely adaptive immunity through an antibody response at the sites of viral accepted that the lack of information about the glycosylation sites entry (Afrough, Dowall, and Hewson 2019). hampers the design of such vaccines (Wolfert 2013). Deducing the N- and O-glycosylation profile of the spike protein of novel coronavirus SARS-CoV-2 983 Fig. 2. Glycosylation profile on coronavirus SARS-CoV-2 characterized by high-resolution LC-MS/MS. About 17 N-glycosylation sites were found occupied outof 22 potential sites along with two O-glycosylation sites bearing core-1 type O-glycans. Some N-glycosylation sites were partially glycosylated. Monosaccharide symbols follow the Symbol Nomenclature for Glycans (SNFG) system (Varki et al. 2015). Mapping N-glycosylation on SARS-CoV-2 spike protein and complex-type glycans across the N-glycosylation sites. We quantified the relative intensities of glycans at each site by comparing We have procured culture supernatants of HEK 293 cells expressing the area under the curve of each glycopeptide peak on the LC-MS SARS-CoV-2 subunit S1 and subunit S2 separately. The proteins were chromatogram. A recent preprint investigated the N-glycosylation expressed with a His tag with Val16 to Gln690 for subunit S1 and on SARS-CoV-2 spike protein expressed on FreeStyle293F human Met697 to Pro1213 for subunit S2. According to manufacturers, cells and reported prevalence of hybrid-type glycans (Watanabe et sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- al. 2020). In contrast, we observed a combination of high mannose PAGE) of the proteins showed a higher molecular weight than the and complex-type, but fewer hybrid-type glycans on most of the predicted 75 and 60 kDa, respectively, because of glycosylation. sites. We discovered predominantly highly processed sialylated Because the proteins were unpurified, we fractionated them through complex-type glycans on sites N165, N282, N801, N1074 and SDS-PAGE on separate lanes and cut the bands corresponding to N1098 (Figures 4 and 5). The highly sialylated glycans at N234 subunit S1 and subunit S2. The gels were stained with Coomassie dye, and N282 adjacent to RBD can act as determinant in viral binding and gel bands were cut into small pieces, destained, reduced, alkylated with hACE2 receptors (Tortorici et al. 2019; Hoffmann et al. and subjected to in-gel protease digestion. We employed trypsin, 2020; Walls et al. 2020). Similar to one recent report, we observed chymotrypsin and both trypsin and chymotrypsin in combination Man GlcNAc as a predominant structure across all S1 sites to generate glycopeptides that contain a single N-linked glycan 5 2 (Watanabe et al. 2020). However, we observed significantly site. Purified subunit S2 was also digested by trypsin–chymotrypsin unoccupied peptides on seven N-glycosylation sites (Figure 2). Sites combination through in-solution digestion. The glycopeptides N17, N603, N1134, N1158 and N1173 were completely unoccupied, were further analyzed by high-resolution LC-MS/MS, using a although further studies with higher concentration, alternative glycan oxonium ion product-dependent higher-energy collisional protease digestion strategies and purity of proteins are required to dissociation (HCD) triggered collision-induced dissociation (CID) validate this finding (Figures 1 and 2). On subunit S2, the assignments program. The LC-MS/MS data were analyzed using Byonic software, at sites N709 and N1134 was ambiguous as the quality of the MS/MS each detected spectrum was manually validated and false detections spectra was not satisfactory and we are currently evaluating the eliminated. possibilities of other posttranslational modifications adjacent to these We identified the glycan compositions at 17 out of the 22 sites. Although we detected unglycosylated peptide for site N1194 predicted N-glycosylation sites of the SARS-CoV-2 S1 and S2 proteins through high-quality MS/MS spectrum, glycosylation at this site is and found the remaining five sites unoccupied (Figures 2–4 and ambiguous. Supplementary Figures S1–S24). We observed high mannose, hybrid 984 A Shajahan et al. Identification of O-Glycosylation on SARS-CoV-2 spike protein We have evaluated O-glycosylation on the S1 and S2 subunits of SARS-CoV-2 spike protein by searching LC-MS/MS data for common O-glycosylation modifications. Interestingly, our O-glycoproteomic profiling indicated O-glycosylation at sites Thr323 and Ser325 on the S1 subunit of SARS-CoV-2 spike protein (Figures 3, 6 and 7). As O-glycosylation at Thr323 and Ser325 on the spike protein has not been reported before and is not indicated based on Cryo-EM data of SARS-CoV-2 S protein, we evaluated the detected O-glycopeptide manually (Walls et al. 2020). We observed very strong evidence for the presence of O-glycosylation at site Thr323 as b and y ions of 320 328 the peptide VQPTESIVR with high mass accuracy were present. Upon manual validation of the fragment ions of O-glycopeptide 320 328 VQPTESIVR , we observed that Thr323 is the predominantly occupied site. This conclusion was based on the b (b - m/z 228.13) and y (y - m/z 175.12, y - m/z 274.19, y - 474.30, y - 603.34 m/z 1 2 4 5 and y + glycan- 1748.77 m/z) ions we detected upon fragmentation of the glycopeptide (Figure 7a). In addition, neutral losses and the detection of oxonium ions also confirmed the presence of glycosyla- tion on these peptides. Core-1 mucin type O-glycans such as GalNAc, GalNAcGal and GalNAcGalNeuAc and Core-2 glycans GalNAc- GalNeuAc(GlcNAcGal) and GalNAcGalNeuAc(GlcNAcGalNeuAc) were observed on site Thr323 (Figure 7). Possibly, Ser325 is occupied with HexNAcHexNeuAc glycan together with T323 (Figure 6), but this could not be confirmed unambiguously as electron transfer dissociation fragmentation on this peptide was not successful because Fig. 3. 3D structure of SARS-CoV-2 spike glycoprotein showing the location of lower charge states of peptide (Supplementary Figures S3 and S4) of N- and O-glycosylation. Receptor-binding domain (RBD) is highlighted and (Shajahan et al. 2017). sites with predominant complex type N-glycans are represented with small squares. PBD ID: 6VXX: only one monomer is shown after removing the ligands. Discussion Two very recent preprints reporting N-glycosylation on spike protein Ser673, Thr678 and Ser686 are conserved O-glycosylation locations, of SARS-CoV-2 showed different glycosylation profiles, and both and SARS-CoV-2 S1 protein was suggested to be O-glycosylated at reports were different from our results although all three studies these locations (Andersen et al. 2020). Although it is unclear what utilized recombinant S protein from an HEK 293-based expres- function these predicted O-linked glycans perform, they have been sion system (Watanabe et al. 2020; Zhang et al. 2020). Although suggested to create a ‘mucin-like domain’, which could shield SARS- Watanabe et al. (2020) used the entire S protein extracellular domain CoV-2 spike protein epitopes or key residues (Bagdonaite 2018). for the glycoproteomics evaluation, we employed separate recom- Because some viruses can utilize mucin-like domains as glycan shields binant protein subunits S1 and S2 of the spike protein. However, for immunoevasion, researchers have highlighted the importance of they have made substitution at the furin cleavage sites and at proline experimental studies for the determination of predicted O-linked 986 and 987, presumably to stabilize the protein during expres- glycosylation sites (Bagdonaite 2018; Andersen et al. 2020). We sion and purification. Nevertheless, the authors indicated that this evaluated the O-glycosylation site prediction using the widely cited could influence the glycosylation at certain sites as the protein will tool Net-O-Gly server 4.0 (Steentoft et al. 2013). However, the tool undergo different glycosylation processing (Pritchard et al. 2015). did not find any strong prediction for the O-glycosylation except for The differences in the processing of the glycoprotein C-terminus by the sites Ser673, Thr678 and Ser686. oligosaccharyltransferase can also lead to differences in glycosylation Our analysis confirmed the presence of O-glycosylation at profile for the glycoprotein expressed as full length versus as subunits Thr323 and indicated possible glycosylation at Ser325 (Figures 3, 6 (Shrimal, Trueman and Gilmore 2013). This indicates the caution and 7). Intriguingly, the possible O-glycosylation at Thr323 of SARS- to be exercised in determining the glycosylation on viral antigens CoV-2 subunit S1 glycoprotein has been predicted by computational generated from various sources as the changes in glycosylation pat- analysis in a very recent preprint report (Uslupehlivan 2020). tern can influence the efficacy of potential vaccine candidates (Chang The accuracy of our observation of O-glycosylation at Thr323 2019). Study of glycosylation on the native virus isolated from clinical is further confirmed by the presence of proline at location 322, specimen would provide insights into how the virus actually utilizes considering the well-established fact that the frequency of occurrence host glycosylation machinery for the protein processing. of proline residues is higher adjacent to O-glycosylation sites (Thanka O-glycans, which are involved in protein stability and function, Christlet 2001). Cryo-EM studies on SARS-CoV-2 indicate that have been observed on some viral proteins and have been suggested the binding of S protein to the hACE2 receptor primarily involves to play roles in the biological activity of viral proteins (Bagdonaite extensive polar residue interactions between RBD and the peptidase 2018; Andersen et al. 2020). A comparative study of human SARS- domain of hACE2 (Hoffmann et al. 2020; Walls et al. 2020). The S CoV-2 S protein with other coronavirus S proteins has shown that protein RBD located in the S1 subunit of SARS-CoV-2 undergoes a Deducing the N- and O-glycosylation profile of the spike protein of novel coronavirus SARS-CoV-2 985 Fig. 4. Quantitative glycosylation profile of N-glycans on coronavirus SARS-CoV-2 spike protein characterized by high-resolution LC-MS/MS. (A)13sites on subunit S1; (B) 9 sites on subunit S2. RA: relative abundances. Monosaccharide symbols follow the SNFG system (Varki et al. 2015). hinge-like dynamic movement to enhance the capture of the receptor Our comprehensive N- and O-glycosylation characterization of RBD with hACE2, displaying 10–20-fold higher affinity for the SARS-CoV-2 expressed in a human cell system provides insights into human ACE2 receptor than SARS-CoV-1, which partially explains site-specific N- and O-glycan decoration on the trimeric spike protein. the higher transmissibility of the new virus (Wrapp et al. 2020; Yan We have employed an extensive manual interpretation strategy for the et al. 2020). The residues Thr323 and Ser325 are located at the RBD assignment of each glycopeptide structure to eliminate possibilities of of the S1 subunit of SARS-Cov-2, and thus the O-glycosylation at ambiguous software based annotation. We are currently working on this location could play a critical role in viral binding with hACE2 elucidating other potential posttranslational modifications on SARS- receptors (Figure 3)(Andersen et al. 2020; Hoffmann et al. 2020). CoV-2 spike protein as understanding the protein modifications in Our observation will pave the way for future studies to understand detail is important to guide future researches on disease interventions the implication of O-glycosylation at the RBD of S1 protein in viral involving spike protein. Detailed glycan analysis is important for the attachment with hACE2 receptors. development of glycoprotein-based vaccine candidates as a means 986 A Shajahan et al. Fig. 5. HCD and CID MS/MS spectra showing glycan neutral losses, oxonium ions and peptide fragments of (A) representative N-glycopeptide TQSLLIVN- NATNVVIK (site N122) of spike protein subunit S1; (B) representative N-glycopeptide TPPIKDFGGFNFSQILPDPSKPSKR (site N801) of spike protein subunit S2. Monosaccharide symbols follow the SNFG system (Varki et al. 2015). and chymotrypsin were purchased from Promega (Madison, WI). All other reagents were purchased from Sigma Aldrich unless indicated otherwise. Data analysis was performed using Byonic 3.5 software and manually using Xcalibur 4.2. The SARS-CoV-2 spike protein culture supernatant subunit S1 (Cat. No. 230-20407) and subunit S2 (Cat. No. 230-20408), and purified subunit S2 (Cat. No. 230-30163) were purchased from RayBiotech (Atlanta, GA). Protease digestion and extraction of peptides from SDS-PAGE The protein subunits S1 and S2 as HEK 293 culture supernatants Fig. 6. Quantitative glycosylation profile of O-glycans on sites T323 of coron- were fractionated on separate lanes using SDS-PAGE. The gel was avirus SARS-CoV-2 spike protein characterized by high-resolution LC-MS/MS. stained by Coomassie dye and the bands corresponding to subunit S1 Monosaccharide symbols follow the SNFG system (Varki et al. 2015). (200–100 kDa) and subunit S2 (150–80 kDa) were cut into smaller pieces (1 mm squares approx.) and transferred to clean tubes. The gel to correlate the structural variation with immunogenicity. Glycosy- pieces were destained by adding 100 μL acetonitrile (ACN): 50 mM lation can serve as a measure to evaluate antigen quality as various NH HCO (1:1) and incubated at room temperature (RT) for about 4 3 expression systems and production processes are employed in vaccine 30 min. Tubes were centrifuged, the supernatant was discarded and manufacture. The understanding of complex sialylated N-glycans and 100 μL ACN was added before incubation for 30 min. The proteins sialylated mucin type O-glycans, particularly in the RBD domain of on gel pieces were reduced by adding 350 μL 25 mM DTT and the spike protein of SARS-CoV-2, provides basic knowledge useful incubating at 60 C for 30 min. The tubes were cooled to RT and for elucidating the viral infection pathology in future therapeutic the supernatant removed. The gels were washed with 500 μL of possibilities, as well as in the design of suitable immunogens for ACN, 350 μL 90 mM IAA was added and the mixture was incubated vaccine development. at RT for 20 min in the dark. Proteins were digested by adding sequence-grade trypsin and/or chymotrypsin (we performed digests with both enzymes individually and as a cocktail) in digestion buffer Materials and methods (50 mM NH HCO ) for 18 h at 37 C separately. The peptides were 4 3 Dithiothreitol (DTT) and iodoacetamide (IAA) were purchased from extracted out from the gel by addition of 1:2 H O:ACN containing Sigma Aldrich (St. Louis, MO). Sequencing-grade modified trypsin 5% formic acid (500 μL), and the released peptides were speed-dried. Deducing the N- and O-glycosylation profile of the spike protein of novel coronavirus SARS-CoV-2 987 320 328 Fig. 7. HCD and CID MS/MS spectra showing glycan neutral losses, oxonium ions and peptide fragments of (A) representative O-Glycopeptide VQPTESIVR 320 328 with core 1 type GalNAcGalNeuAc glycan detected on site Thr323 of spike protein subunit S1; (B) representative O-Glycopeptide VQPTESIVR with core 2 type GalNAcGalNeuAc(GlcNAcGalNeuAc) glycan detected on site Thr323 of spike protein subunit S1. Monosaccharide symbols follow the SNFG system (Varki et al. 2015). The samples were reconstituted in aqueous 0.1% formic acid for Data analysis of glycoproteins LC-MS/MS experiments. Purified subunit S2 was reduced (25 mM The LC-MS/MS spectra of tryptic, chymotryptic and combined tryp- DTT and incubating at 60 C for 30 min), alkylated (90 mM IAA tic/chymotryptic digests of glycoproteins were searched against the and incubating at RT for 30 min in dark) and digested by trypsin– FASTA sequence of spike protein S1 and S2 subunit using the Byonic chymotrypsin combination (18 h at 37 C separately) through in- software by choosing appropriate peptide cleavage sites (semispecific solution digestion. cleavage option enabled). Oxidation of methionine, deamidation of asparagine and glutamine, possible common human N-glycans and O-glycan masses were used as variable modifications. The LC- Data acquisition of protein digest samples using MS/MS spectra were also analyzed manually for the glycopeptides nano-LC-MS/MS with the support of the Xcalibur software. The HCDpdCID MS The glycoprotein digests were analyzed on an Orbitrap Fusion Trib- spectra of glycopeptides were evaluated for the glycan neutral loss rid mass spectrometer equipped with a nanospray ion source and con- pattern, oxonium ions and glycopeptide fragmentations to assign the nected to a Dionex binary solvent system (Waltham, MA). Prepacked sequence and the presence of glycans in the glycopeptides. nano-LC columns of 15 cm length with 75 μm internal diameter (id), filled with 3 μm C18 material (reverse phase) were used for chromatographic separation of samples. The precursor ion scan Funding was acquired at 120,000 resolution in the Orbitrap analyzer and precursors at a time frame of 3 s were selected for subsequent MS/MS The US National Institutes of Health (S10OD018530); U.S. Depart- fragmentation in the Orbitrap analyzer at 15,000 resolution. The LC- ment of Energy, Office of Science, Basic Energy Sciences (under MS/MS runs of each digest were conducted for both 72 min and Award DE-SC0015662 to DOE—Center for Plant and Microbial 180 min in order to separate the glycopeptides. The threshold for Complex Carbohydrates at the Complex Carbohydrate Research triggering an MS/MS event was set to 1000 counts, and monoiso- Center, USA). topic precursor selection was enabled. MS/MS fragmentation was conducted with stepped HCD product triggered CID (HCDpdCID) program. Charge state screening was enabled, and precursors with Supplementary data unknown charge state or a charge state of +1 were excluded (positive ion mode). Dynamic exclusion was enabled (exclusion duration Supplementary data for this article are available online at http:// of 30 s). glycob.oxfordjournals.org/. 988 A Shajahan et al. Author contributions Shrimal S, Trueman SF, Gilmore R. 2013. 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Deducing the N- and O-glycosylation profile of the spike protein of novel coronavirus SARS-CoV-2

Glycobiology , Volume 30 (12) – May 4, 2020

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10.1093/glycob/cwaa042
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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. Key words: COVID-19, SARS-CoV-2 glycosylation, spike protein, coronavirus vaccine, S1 S2 glycosylation Introduction To date, no specific medical treatments or vaccines for COVID-19 The current major health crisis is caused by the novel severe acute have been approved (Li and De Clercq 2020; WHO 2020a). There- respiratory syndrome coronavirus 2 (SARS-CoV-2) that rapidly fore, the scientific community is expending great effort in compiling spread globally within weeks in early 2020. This highly transmissible data regarding the virus, as well as the respiratory illness caused by infectious disease causes a respiratory illness named COVID-19 it, to find effective ways of dealing with this health crisis. (Huang et al. 2020; Wu et al. 2020). As of the 31 March 2020, The pathogenic SARS-CoV-2 enters human target cells via its 750,890 cases of COVID-19 and 36,405 COVID-19-related deaths viral transmembrane spike (S) glycoprotein. The spike protein is a have been confirmed globally by the World Health Organization trimeric class I fusion protein and consists of two subunits, namely (WHO 2020b). S1 and S2. The S1 subunit facilitates the attachment of the virus, © The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com 981 982 A Shajahan et al. Fig. 1. The SARS-CoV-2 spike proteins recombinantly expressed on HEK293 supernatant were fractionated through SDS-PAGE, subsequently digested by proteases and analyzed by nLC-NSI-MS/MS. The expression of SARS-CoV-2 spike protein subunits S1 and S2 on HEK 293 culture supernatant showed higher molecular weight upon SDS-PAGE than expected, because of glycosylation. Thus, the gel bands corresponding to the molecular weight of 200–100 kDa for S1 and 150–80 kDa for S2 were cut, proteins were lysed after reduction–alkylation and analyzed by LC-MS/MS (created with biorender.com). Purified S2 were processed after in-solution protease digestion. and subsequently the S2 subunit allows for the fusion of the viral Here, we report the site-specific quantitative N-linked and and human cellular membranes (Hoffmann et al. 2020; Walls et al. O-linked glycan profiling on SARS-CoV-2 subunit S1 and S2 2020; Zhou et al. 2020). The entry receptor for SARS-CoV-2 has been protein through glycoproteomics using high-resolution liquid identified as the human angiotensin-converting enzyme 2 (hACE2), chromatography-tandem mass spectrometry (LC-MS/MS). We used and recent studies determined a high binding affinity to hACE2 recombinant SARS-CoV-2 subunit S1 and S2 expressed in human (Hoffmann et al. 2020; Shang et al. 2020; Walls et al. 2020). Given cells, HEK 293, and observed partial N-glycan occupancy on 17 its literal key role, the S protein is one of the major targets for the out of 22 N-glycosylation sites. We found that the remaining development of specific medical treatments or vaccines: neutralizing five N-glycosylation sites were unoccupied. Remarkably, we have antibodies targeting the spike proteins of SARS-CoV-2 could prevent unambiguously identified two unexpected O-glycosylation sites at the virus from binding to the hACE2 entry receptor and therefore the receptor-binding domain (RBD) of subunit S1. O-glycosylation from entering the host cell (Shang et al. 2020). on the spike protein of SARS-CoV-2 is predicted in some recent Each monomer of the S protein is highly glycosylated with reports and most of these predictions are for sites in proximity 22 predicted N-linked glycosylation sites. Furthermore, three O- to furin cleavage site (S1/S2) as similar sites are O-glycosylated in glycosylation sites were also predicted (Andersen et al. 2020). Cryo- SARS-CoV-1 (Andersen et al. 2020; Uslupehlivan 2020). However, electron microscopy (Cryo-EM) provides evidence for the existence we observed O-glycosylation at two sites on the RBD of spike of 14–16 N-glycans on 22 potential sites in the SARS-CoV-2 S protein protein subunit S1, and this is the first report on the evidence for (Walls et al. 2020). The glycosylation pattern of the spike protein is such glycan modification at a crucial binding location of the spike a crucial characteristic to be considered regarding steric hindrance, protein. Site-specific analysis of N- and O-glycosylation information chemical properties and even as a potential target for mutation in the of SARS-CoV-2 spike protein provides basic understanding of the future. The N-glycans on S protein play important roles in proper viral structure, crucial for the identification of immunogens for protein folding and priming by host proteases (Walls et al. 2020). vaccine design. This in turn has the potential of leading to future As glycans can shield the amino acid residues and other epitopes therapeutic intervention or prevention of COVID-19. from cells and antibody recognition, glycosylation can enable the coronavirus to evade both the innate and adaptive immune responses Results (Walls et al. 2019, 2020). Elucidating the glycosylation of the viral S protein can aid in understanding viral binding with receptors, fusion, Studies over the past two decades have shown that glycosylation on entry, replication and also in designing suitable antigens for vaccine the protein antigens can play crucial roles in the adaptive immune development (Chakraborti et al. 2005; Zheng et al. 2018; Watanabe response. Thus, it is obvious that the glycosylation on the protein et al. 2019). Strategies for vaccine development aim to elicit such antigen is relevant for the development of vaccines, and it is widely adaptive immunity through an antibody response at the sites of viral accepted that the lack of information about the glycosylation sites entry (Afrough, Dowall, and Hewson 2019). hampers the design of such vaccines (Wolfert 2013). Deducing the N- and O-glycosylation profile of the spike protein of novel coronavirus SARS-CoV-2 983 Fig. 2. Glycosylation profile on coronavirus SARS-CoV-2 characterized by high-resolution LC-MS/MS. About 17 N-glycosylation sites were found occupied outof 22 potential sites along with two O-glycosylation sites bearing core-1 type O-glycans. Some N-glycosylation sites were partially glycosylated. Monosaccharide symbols follow the Symbol Nomenclature for Glycans (SNFG) system (Varki et al. 2015). Mapping N-glycosylation on SARS-CoV-2 spike protein and complex-type glycans across the N-glycosylation sites. We quantified the relative intensities of glycans at each site by comparing We have procured culture supernatants of HEK 293 cells expressing the area under the curve of each glycopeptide peak on the LC-MS SARS-CoV-2 subunit S1 and subunit S2 separately. The proteins were chromatogram. A recent preprint investigated the N-glycosylation expressed with a His tag with Val16 to Gln690 for subunit S1 and on SARS-CoV-2 spike protein expressed on FreeStyle293F human Met697 to Pro1213 for subunit S2. According to manufacturers, cells and reported prevalence of hybrid-type glycans (Watanabe et sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- al. 2020). In contrast, we observed a combination of high mannose PAGE) of the proteins showed a higher molecular weight than the and complex-type, but fewer hybrid-type glycans on most of the predicted 75 and 60 kDa, respectively, because of glycosylation. sites. We discovered predominantly highly processed sialylated Because the proteins were unpurified, we fractionated them through complex-type glycans on sites N165, N282, N801, N1074 and SDS-PAGE on separate lanes and cut the bands corresponding to N1098 (Figures 4 and 5). The highly sialylated glycans at N234 subunit S1 and subunit S2. The gels were stained with Coomassie dye, and N282 adjacent to RBD can act as determinant in viral binding and gel bands were cut into small pieces, destained, reduced, alkylated with hACE2 receptors (Tortorici et al. 2019; Hoffmann et al. and subjected to in-gel protease digestion. We employed trypsin, 2020; Walls et al. 2020). Similar to one recent report, we observed chymotrypsin and both trypsin and chymotrypsin in combination Man GlcNAc as a predominant structure across all S1 sites to generate glycopeptides that contain a single N-linked glycan 5 2 (Watanabe et al. 2020). However, we observed significantly site. Purified subunit S2 was also digested by trypsin–chymotrypsin unoccupied peptides on seven N-glycosylation sites (Figure 2). Sites combination through in-solution digestion. The glycopeptides N17, N603, N1134, N1158 and N1173 were completely unoccupied, were further analyzed by high-resolution LC-MS/MS, using a although further studies with higher concentration, alternative glycan oxonium ion product-dependent higher-energy collisional protease digestion strategies and purity of proteins are required to dissociation (HCD) triggered collision-induced dissociation (CID) validate this finding (Figures 1 and 2). On subunit S2, the assignments program. The LC-MS/MS data were analyzed using Byonic software, at sites N709 and N1134 was ambiguous as the quality of the MS/MS each detected spectrum was manually validated and false detections spectra was not satisfactory and we are currently evaluating the eliminated. possibilities of other posttranslational modifications adjacent to these We identified the glycan compositions at 17 out of the 22 sites. Although we detected unglycosylated peptide for site N1194 predicted N-glycosylation sites of the SARS-CoV-2 S1 and S2 proteins through high-quality MS/MS spectrum, glycosylation at this site is and found the remaining five sites unoccupied (Figures 2–4 and ambiguous. Supplementary Figures S1–S24). We observed high mannose, hybrid 984 A Shajahan et al. Identification of O-Glycosylation on SARS-CoV-2 spike protein We have evaluated O-glycosylation on the S1 and S2 subunits of SARS-CoV-2 spike protein by searching LC-MS/MS data for common O-glycosylation modifications. Interestingly, our O-glycoproteomic profiling indicated O-glycosylation at sites Thr323 and Ser325 on the S1 subunit of SARS-CoV-2 spike protein (Figures 3, 6 and 7). As O-glycosylation at Thr323 and Ser325 on the spike protein has not been reported before and is not indicated based on Cryo-EM data of SARS-CoV-2 S protein, we evaluated the detected O-glycopeptide manually (Walls et al. 2020). We observed very strong evidence for the presence of O-glycosylation at site Thr323 as b and y ions of 320 328 the peptide VQPTESIVR with high mass accuracy were present. Upon manual validation of the fragment ions of O-glycopeptide 320 328 VQPTESIVR , we observed that Thr323 is the predominantly occupied site. This conclusion was based on the b (b - m/z 228.13) and y (y - m/z 175.12, y - m/z 274.19, y - 474.30, y - 603.34 m/z 1 2 4 5 and y + glycan- 1748.77 m/z) ions we detected upon fragmentation of the glycopeptide (Figure 7a). In addition, neutral losses and the detection of oxonium ions also confirmed the presence of glycosyla- tion on these peptides. Core-1 mucin type O-glycans such as GalNAc, GalNAcGal and GalNAcGalNeuAc and Core-2 glycans GalNAc- GalNeuAc(GlcNAcGal) and GalNAcGalNeuAc(GlcNAcGalNeuAc) were observed on site Thr323 (Figure 7). Possibly, Ser325 is occupied with HexNAcHexNeuAc glycan together with T323 (Figure 6), but this could not be confirmed unambiguously as electron transfer dissociation fragmentation on this peptide was not successful because Fig. 3. 3D structure of SARS-CoV-2 spike glycoprotein showing the location of lower charge states of peptide (Supplementary Figures S3 and S4) of N- and O-glycosylation. Receptor-binding domain (RBD) is highlighted and (Shajahan et al. 2017). sites with predominant complex type N-glycans are represented with small squares. PBD ID: 6VXX: only one monomer is shown after removing the ligands. Discussion Two very recent preprints reporting N-glycosylation on spike protein Ser673, Thr678 and Ser686 are conserved O-glycosylation locations, of SARS-CoV-2 showed different glycosylation profiles, and both and SARS-CoV-2 S1 protein was suggested to be O-glycosylated at reports were different from our results although all three studies these locations (Andersen et al. 2020). Although it is unclear what utilized recombinant S protein from an HEK 293-based expres- function these predicted O-linked glycans perform, they have been sion system (Watanabe et al. 2020; Zhang et al. 2020). Although suggested to create a ‘mucin-like domain’, which could shield SARS- Watanabe et al. (2020) used the entire S protein extracellular domain CoV-2 spike protein epitopes or key residues (Bagdonaite 2018). for the glycoproteomics evaluation, we employed separate recom- Because some viruses can utilize mucin-like domains as glycan shields binant protein subunits S1 and S2 of the spike protein. However, for immunoevasion, researchers have highlighted the importance of they have made substitution at the furin cleavage sites and at proline experimental studies for the determination of predicted O-linked 986 and 987, presumably to stabilize the protein during expres- glycosylation sites (Bagdonaite 2018; Andersen et al. 2020). We sion and purification. Nevertheless, the authors indicated that this evaluated the O-glycosylation site prediction using the widely cited could influence the glycosylation at certain sites as the protein will tool Net-O-Gly server 4.0 (Steentoft et al. 2013). However, the tool undergo different glycosylation processing (Pritchard et al. 2015). did not find any strong prediction for the O-glycosylation except for The differences in the processing of the glycoprotein C-terminus by the sites Ser673, Thr678 and Ser686. oligosaccharyltransferase can also lead to differences in glycosylation Our analysis confirmed the presence of O-glycosylation at profile for the glycoprotein expressed as full length versus as subunits Thr323 and indicated possible glycosylation at Ser325 (Figures 3, 6 (Shrimal, Trueman and Gilmore 2013). This indicates the caution and 7). Intriguingly, the possible O-glycosylation at Thr323 of SARS- to be exercised in determining the glycosylation on viral antigens CoV-2 subunit S1 glycoprotein has been predicted by computational generated from various sources as the changes in glycosylation pat- analysis in a very recent preprint report (Uslupehlivan 2020). tern can influence the efficacy of potential vaccine candidates (Chang The accuracy of our observation of O-glycosylation at Thr323 2019). Study of glycosylation on the native virus isolated from clinical is further confirmed by the presence of proline at location 322, specimen would provide insights into how the virus actually utilizes considering the well-established fact that the frequency of occurrence host glycosylation machinery for the protein processing. of proline residues is higher adjacent to O-glycosylation sites (Thanka O-glycans, which are involved in protein stability and function, Christlet 2001). Cryo-EM studies on SARS-CoV-2 indicate that have been observed on some viral proteins and have been suggested the binding of S protein to the hACE2 receptor primarily involves to play roles in the biological activity of viral proteins (Bagdonaite extensive polar residue interactions between RBD and the peptidase 2018; Andersen et al. 2020). A comparative study of human SARS- domain of hACE2 (Hoffmann et al. 2020; Walls et al. 2020). The S CoV-2 S protein with other coronavirus S proteins has shown that protein RBD located in the S1 subunit of SARS-CoV-2 undergoes a Deducing the N- and O-glycosylation profile of the spike protein of novel coronavirus SARS-CoV-2 985 Fig. 4. Quantitative glycosylation profile of N-glycans on coronavirus SARS-CoV-2 spike protein characterized by high-resolution LC-MS/MS. (A)13sites on subunit S1; (B) 9 sites on subunit S2. RA: relative abundances. Monosaccharide symbols follow the SNFG system (Varki et al. 2015). hinge-like dynamic movement to enhance the capture of the receptor Our comprehensive N- and O-glycosylation characterization of RBD with hACE2, displaying 10–20-fold higher affinity for the SARS-CoV-2 expressed in a human cell system provides insights into human ACE2 receptor than SARS-CoV-1, which partially explains site-specific N- and O-glycan decoration on the trimeric spike protein. the higher transmissibility of the new virus (Wrapp et al. 2020; Yan We have employed an extensive manual interpretation strategy for the et al. 2020). The residues Thr323 and Ser325 are located at the RBD assignment of each glycopeptide structure to eliminate possibilities of of the S1 subunit of SARS-Cov-2, and thus the O-glycosylation at ambiguous software based annotation. We are currently working on this location could play a critical role in viral binding with hACE2 elucidating other potential posttranslational modifications on SARS- receptors (Figure 3)(Andersen et al. 2020; Hoffmann et al. 2020). CoV-2 spike protein as understanding the protein modifications in Our observation will pave the way for future studies to understand detail is important to guide future researches on disease interventions the implication of O-glycosylation at the RBD of S1 protein in viral involving spike protein. Detailed glycan analysis is important for the attachment with hACE2 receptors. development of glycoprotein-based vaccine candidates as a means 986 A Shajahan et al. Fig. 5. HCD and CID MS/MS spectra showing glycan neutral losses, oxonium ions and peptide fragments of (A) representative N-glycopeptide TQSLLIVN- NATNVVIK (site N122) of spike protein subunit S1; (B) representative N-glycopeptide TPPIKDFGGFNFSQILPDPSKPSKR (site N801) of spike protein subunit S2. Monosaccharide symbols follow the SNFG system (Varki et al. 2015). and chymotrypsin were purchased from Promega (Madison, WI). All other reagents were purchased from Sigma Aldrich unless indicated otherwise. Data analysis was performed using Byonic 3.5 software and manually using Xcalibur 4.2. The SARS-CoV-2 spike protein culture supernatant subunit S1 (Cat. No. 230-20407) and subunit S2 (Cat. No. 230-20408), and purified subunit S2 (Cat. No. 230-30163) were purchased from RayBiotech (Atlanta, GA). Protease digestion and extraction of peptides from SDS-PAGE The protein subunits S1 and S2 as HEK 293 culture supernatants Fig. 6. Quantitative glycosylation profile of O-glycans on sites T323 of coron- were fractionated on separate lanes using SDS-PAGE. The gel was avirus SARS-CoV-2 spike protein characterized by high-resolution LC-MS/MS. stained by Coomassie dye and the bands corresponding to subunit S1 Monosaccharide symbols follow the SNFG system (Varki et al. 2015). (200–100 kDa) and subunit S2 (150–80 kDa) were cut into smaller pieces (1 mm squares approx.) and transferred to clean tubes. The gel to correlate the structural variation with immunogenicity. Glycosy- pieces were destained by adding 100 μL acetonitrile (ACN): 50 mM lation can serve as a measure to evaluate antigen quality as various NH HCO (1:1) and incubated at room temperature (RT) for about 4 3 expression systems and production processes are employed in vaccine 30 min. Tubes were centrifuged, the supernatant was discarded and manufacture. The understanding of complex sialylated N-glycans and 100 μL ACN was added before incubation for 30 min. The proteins sialylated mucin type O-glycans, particularly in the RBD domain of on gel pieces were reduced by adding 350 μL 25 mM DTT and the spike protein of SARS-CoV-2, provides basic knowledge useful incubating at 60 C for 30 min. The tubes were cooled to RT and for elucidating the viral infection pathology in future therapeutic the supernatant removed. The gels were washed with 500 μL of possibilities, as well as in the design of suitable immunogens for ACN, 350 μL 90 mM IAA was added and the mixture was incubated vaccine development. at RT for 20 min in the dark. Proteins were digested by adding sequence-grade trypsin and/or chymotrypsin (we performed digests with both enzymes individually and as a cocktail) in digestion buffer Materials and methods (50 mM NH HCO ) for 18 h at 37 C separately. The peptides were 4 3 Dithiothreitol (DTT) and iodoacetamide (IAA) were purchased from extracted out from the gel by addition of 1:2 H O:ACN containing Sigma Aldrich (St. Louis, MO). Sequencing-grade modified trypsin 5% formic acid (500 μL), and the released peptides were speed-dried. Deducing the N- and O-glycosylation profile of the spike protein of novel coronavirus SARS-CoV-2 987 320 328 Fig. 7. HCD and CID MS/MS spectra showing glycan neutral losses, oxonium ions and peptide fragments of (A) representative O-Glycopeptide VQPTESIVR 320 328 with core 1 type GalNAcGalNeuAc glycan detected on site Thr323 of spike protein subunit S1; (B) representative O-Glycopeptide VQPTESIVR with core 2 type GalNAcGalNeuAc(GlcNAcGalNeuAc) glycan detected on site Thr323 of spike protein subunit S1. Monosaccharide symbols follow the SNFG system (Varki et al. 2015). The samples were reconstituted in aqueous 0.1% formic acid for Data analysis of glycoproteins LC-MS/MS experiments. Purified subunit S2 was reduced (25 mM The LC-MS/MS spectra of tryptic, chymotryptic and combined tryp- DTT and incubating at 60 C for 30 min), alkylated (90 mM IAA tic/chymotryptic digests of glycoproteins were searched against the and incubating at RT for 30 min in dark) and digested by trypsin– FASTA sequence of spike protein S1 and S2 subunit using the Byonic chymotrypsin combination (18 h at 37 C separately) through in- software by choosing appropriate peptide cleavage sites (semispecific solution digestion. cleavage option enabled). Oxidation of methionine, deamidation of asparagine and glutamine, possible common human N-glycans and O-glycan masses were used as variable modifications. The LC- Data acquisition of protein digest samples using MS/MS spectra were also analyzed manually for the glycopeptides nano-LC-MS/MS with the support of the Xcalibur software. The HCDpdCID MS The glycoprotein digests were analyzed on an Orbitrap Fusion Trib- spectra of glycopeptides were evaluated for the glycan neutral loss rid mass spectrometer equipped with a nanospray ion source and con- pattern, oxonium ions and glycopeptide fragmentations to assign the nected to a Dionex binary solvent system (Waltham, MA). Prepacked sequence and the presence of glycans in the glycopeptides. nano-LC columns of 15 cm length with 75 μm internal diameter (id), filled with 3 μm C18 material (reverse phase) were used for chromatographic separation of samples. The precursor ion scan Funding was acquired at 120,000 resolution in the Orbitrap analyzer and precursors at a time frame of 3 s were selected for subsequent MS/MS The US National Institutes of Health (S10OD018530); U.S. Depart- fragmentation in the Orbitrap analyzer at 15,000 resolution. The LC- ment of Energy, Office of Science, Basic Energy Sciences (under MS/MS runs of each digest were conducted for both 72 min and Award DE-SC0015662 to DOE—Center for Plant and Microbial 180 min in order to separate the glycopeptides. The threshold for Complex Carbohydrates at the Complex Carbohydrate Research triggering an MS/MS event was set to 1000 counts, and monoiso- Center, USA). topic precursor selection was enabled. MS/MS fragmentation was conducted with stepped HCD product triggered CID (HCDpdCID) program. Charge state screening was enabled, and precursors with Supplementary data unknown charge state or a charge state of +1 were excluded (positive ion mode). Dynamic exclusion was enabled (exclusion duration Supplementary data for this article are available online at http:// of 30 s). glycob.oxfordjournals.org/. 988 A Shajahan et al. Author contributions Shrimal S, Trueman SF, Gilmore R. 2013. 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Journal

GlycobiologyPubmed Central

Published: May 4, 2020

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