key: cord-278169-elhz77ek
authors: Zhou, Dapeng; Tian, Xiaoxu; Qi, Ruibing; Peng, Chao; Zhang, Wen
title: Identification of 22 N-glycosites on spike glycoprotein of SARS-CoV-2 and accessible surface glycopeptide motifs: implications for vaccination and antibody therapeutics
date: 2020-06-10
journal: Glycobiology
DOI: 10.1093/glycob/cwaa052
sha: 
doc_id: 278169
cord_uid: elhz77ek

Coronaviruses hijack human enzymes to assemble the sugar coat on their spike glycoproteins. The mechanisms by which human antibodies may recognize the antigenic viral peptide epitopes hidden by the sugar coat are unknown. Glycosylation by insect cells differs from the native form produced in human cells, but insect cell–derived influenza vaccines have been approved by the US Food and Drug Administration. In this study, we analyzed recombinant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein secreted from BTI-Tn-5B1–4 insect cells, by trypsin and chymotrypsin digestion followed by mass spectrometry analysis. We acquired tandem mass spectrometry (MS/MS) spectrums for glycopeptides of all 22 predicted N-glycosylated sites. We further analyzed the surface accessibility of spike proteins according to cryogenic electron microscopy and homolog-modeled structures, and available antibodies that bind to SARS-CoV-1. All 22 N-glycosylated sites of SARS-CoV-2 are modified by high-mannose N-glycans. MS/MS fragmentation clearly established the glycopeptide identities. Electron densities of glycans cover most of the spike receptor-binding domain of SARS-CoV-2, except YQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQ, similar to a region FSPDGKPCTPPALNCYWPLNDYGFYTTTGIGYQ in SARS-CoV-1. Other surface-exposed domains include those located on central helix, connecting region, heptad repeats, and N-terminal domain. Because the majority of antibody paratopes bind to the peptide portion with or without sugar modification, we propose a snake-catching model for predicted paratopes: a minimal length of peptide is first clamped by a paratope, and sugar modifications close to the peptide either strengthen or do not hinder the binding.

Spike proteins are located on the surface of coronaviruses and serve as entry proteins for infection (Xu et al. 2004 ). The spike molecules form trimers, which must be cleaved by cellular proteases so that the fusion peptide can facilitate the fusion of virus membrane with the infected cells. The proteases generate S1 and S2 subunits from spike molecules, and the S1 subunit contains the critical receptor-binding domain to bind the ACE2 receptor on host cells. The receptor-binding motif of the receptor-binding domain, rich in tyrosine, forms direct contacts with ACE2. Fusion of the virus with host cells involves several other critical structures of the spike protein, including central helix and heptad repeat 1 and 2 domains.

Spike glycoproteins are major targets for vaccine design and antibody-based therapies for coronaviruses, including Middle East respiratory syndrome coronavirus; severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1), which caused a multi-country outbreak in 2002-2003; and SARS-CoV-2, which is responsible for a pandemic beginning in 2019. Several antibodies targeting spike proteins of SARS-CoV-1 have shown promising efficacy in preclinical trials (Berry et al. 2010 , Bian et al. 2009 , Greenough et al. 2005 , He et al. 2006 , He et al. 2005 , Hwang et al. 2006 , Ishii et al. 2009 , Miyoshi-Akiyama et al. 2011 , Pak et al. 2009 , Prabakaran et al. 2006 , Rockx et al. 2008 , Sui et al. 2014 , Sui et al. 2005 , ter Meulen et al. 2006 , Traggiai et al. 2004 , van den Brink et al. 2005 , Zhu et al. 2007 . Furthermore, structural studies suggest that domains other than the crucial receptor-binding domain are also potential targets for antibody binding; these include the fusion peptide, heptad repeat 1, and central helix domains (Yuan et al. 2017 ) (Table SI) . In all coronaviruses, spike glycoproteins are densely glycosylated, with more than 20 predicted sites for N-glycosylation. The function of these glycans in immune evasion by the virus remains unknown.

In this study, we analyzed a recombinant SARS-CoV-2 spike protein expressed by insect cells.

We acquired tandem mass spectrometry (MS/MS) spectrums for all glycopeptides generated by sequential digestion using trypsin and chymotrypsin. We further analyzed the cryogenic electron microscopy structures of the spike proteins to identify surface-exposed epitopes for antibody recognition and vaccine design.

A total of 22 N-glycosylation sites were found in the recombinant spike protein of SARS-CoV-2secreted from BTI-Tn-5B1-4 insect cells ( Figure 1 ). All 22 N-glycosites were confirmed by fragment ions of glycan moieties and characteristic b/y ions derived from peptide backbones (Supplemental Figure 1 and 2). Among them, eight are located in the N-terminal domain, two(N331 and N343) are located in the receptor-binding domain but are outside of the receptorbinding motif (limited to amino acids 438-506), and three are located in the rest of the S1 subunit. Nine are located in the S2 subunit. The glycosylation pattern of the spike protein is highly conserved in SARS-CoV-1, SARS-CoV-2, and Middle East respiratory syndrome coronavirus. The N-terminal and heptad repeat 2 domains are densely glycosylated. The fusion peptide domain is neighbored by N-glycosite N657. In contrast, the receptor-binding motif, central helix domain, and heptad repeat 1 domain are free of glycosylation. The majority of Nglycan moieties are a high-mannose type (Table SII) , which is consistent with the glycosylation pathway of the BTI-Tn-5B1-4 insect cell line used to produce the recombinant spike protein. To evaluate the efficiency of glycosylation at every N-glycosite, we calculated the relative ion

abundance of glycopeptide and non-glycosylated sequences generated by trypsin and chymotrypsin digestion. All 22 N-glycosylation sites were occupied by N-glycans (Table SIII) .

For 18 of the 22 N-glycosites, more than 90% of ions were glycosylated peptides. For Nglycosites 331, 1074, 1158, and 1173, more than 80% of ions were glycosylated peptides. We also searched for the O-glycosylated glycopeptides (Table SIV) . Preliminary analysis indicated that the ion abundance of O-glycopeptide sequences was less than 3% as compared with nonglycosylated sequences for all predicted O-glycosites (T323, T325, S678, S673, and S686).

By cryogenic electron microscopy structure modeling (Protein Data Bank [PDB]: 5X58) of the SARS-CoV-1 spike protein, 14 sites of N-glycosylation were observed. The Asn-GlcNAc groups were identified at the reducing end of the glycans at atomic resolution (PDB: 5X58, 3.2 Å), and the density maps of extending glycan chains were still visible although the density was relatively weak (Figure 2A , B, and C). The receptor-binding domain region of the SARS-CoV-1spike protein is densely covered by glycans except FSPDGKPCTPPALNCYWPLNDYGFYTTTGIGYQ, which overlaps with a previously identified "Achilles heel" (i.e., vulnerable spot) for antibody binding (Berry, et al. 2010) .

The spike protein of SARS-CoV-2 contains 22 N-glycosylation sites (in yellow in Figure 2D ).

When trimer structures of the S protein of SARS-CoV-1 and SARS-CoV-2 are aligned (rootmean-square deviation~1.32 for single chain), the structures are very similar except for a few loops, such as those at the N-terminal of the N-terminal domain (Supplemental Figure 3 ).

Sequence alignment and structure comparison revealed that the predicted glycosylation sites are highly conserved. Fourteen of 22 sites were observed by cryogenic electron microscopy for the

SARS-CoV-1 S protein, and most predicted sites of SARS-CoV-2 are located similarly to SARS-CoV-1 ( Figure 2E ). The receptor-binding domains were overall highly conserved with sequence identity (74.5%), structure (root-mean-square deviation~1.14Å), and two identical glycosylation sites near the N-terminal ( Figure 2F ), while the sequence specificity of epitopes remained unique in some regions (Tables I and II) . A similar surfaced-exposed region, or Achilles heel, YQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQ, was identified in the receptor-binding domain of SARS-CoV-2. Interestingly, the Achilles heel regions for both SARS-CoV-1 and SARS-CoV-2 were also free of glycosylation, whereas its neighboring fragments were covered or interacting with glycosylation. This region free of glycosylation is favorable for ACE2 and other protein binding ( Figure 2G ).

Accessible surface area profiling was used for predicting epitopes for monoclonal antibodies (MAbs) (Supplemental Figure 4) . Candidate epitopes are listed in Table I and Figure 3 . In addition to receptor-binding domains, multiple potential candidate epitopes were found from amino acid sequences at fusion peptide, heptad repeat 1, and central helix domains. Similar sites were found in receptor-binding domains and central helix domains of both viruses ( Figure 3 ).

However, unique sites were also found for each virus. For example, a unique epitope existing in SARS-CoV-2, but not in SARS-CoV-1, is the RARR (682-685) site for furin recognition (Supplemental Figure 5 ).

To evaluate the conservation of spike epitopes on a structural level, we further aligned the epitopes of SARS-CoV-1 and SARS-CoV-2 based on cryogenic electron microscopy structures.

Eleven predicted epitope pairs were found in receptor-binding domain, heptad repeat 1, and central helix (Table II, Figure 4 , and Supplemental Figure 6 ). Two structurally conserved epitope pairs (AH1/ah1 and AH2/ah2) were predicted at the Achilles heel region which interacts with ACE2 (Table II) . We also identified two conserved epitope pairs located on the surface of the receptor-binding domain but outside the ACE2-binding region (I/i and II/ii). Epitope pair II/ii has been proven to be a target for recognition by MAb S309 (Pinto et al. 2020 ), a potent neutralization antibody with half-maximal inhibitory concentration (IC50) at 69 ng/mL.

Neutralizing antibodies toward spike proteins are critical for protective immunity. Traggiai et al.

reported spike-specific MAbs isolated from a patient who recovered from SARS-CoV-1 infection, with in vitro neutralizing activity ranging from 10 -8 to 10 -11 M (Traggiai, et al. 2004) .

Several other groups have reported MAbs targeting spike (Berry, et al. 2010 , Bian, et al. 2009 , Greenough, et al. 2005 , He, et al. 2006 , He, et al. 2005 , Ishii, et al. 2009 , Miyoshi-Akiyama, et al. 2011 , Rockx, et al. 2008 , Sui, et al. 2014 , Sui, et al. 2005 , ter Meulen, et al. 2006 , van den Brink, et al. 2005 , Zhu, et al. 2007 . Spike protein has also been the focus for vaccine development. For example, mice vaccinated with DNA or subunit vaccines composed of spike proteins (or receptor-binding domain of spike proteins) and adjuvants had high titers of immunoglobulin G antibodies and were protected from SARS-CoV-1 or Middle East respiratory syndrome coronavirus infection (Du et al. 2010 , Du et al. 2007 , Honda-Okubo et al. 2015 , Iwata-Yoshikawa et al. 2014 , Li et al. 2013 , Lu et al. 2010 , Sekimukai et al. 2020 , Yang et al. 2004 , Zhao et al. 2014 ). Toll-like receptor ligands, delta inulin, and monophosphoryl lipid A were reported as effective adjuvants to be combined with subunit vaccines. However, to avoid the use

of adjuvant, inactivated SARS-CoV-1 or recombinant adeno-associated virus encoding the receptor-binding domain of the SARS-CoV-1 spike protein has also been studied; these induced potent protective antibody responses against infection (Du et al. 2008 , Okada et al. 2005 , See et al. 2006 , Spruth et al. 2006 . The safety and efficacy of antibody therapeutics and vaccines in human clinical trials remain to be studied, as well as the mechanisms for specific vaccine components and formulations. For example, pulmonary pathology was reported when alum was used as an adjuvant for a spike protein subunit vaccine (Tseng et al. 2012 ). Antibody-induced lung injury was also reported in a macaque model of SARS-CoV-1 infection (Liu et al. 2019) , which highlights the importance of avoiding antibody-mediated inflammation.

The receptor-binding domain has been a major focus for antibody and vaccine studies. Three antibodies,80R, m396, and F26G19, complexed with the receptor-binding domain of SARS-CoV-1 have been co-crystalized (Hwang, et al. 2006 , Pak, et al. 2009 , Prabakaran, et al. 2006 ).

All three antibodies recognize non-continuous, conformational epitopes (Table SI) . Several MAb clones that recognize linear continuous peptide sequences have also been reported (4D5, 17H9, F26G18, and 201), although co-crystal structures are not available yet.

In this study, we identified the accessible surface area profiling of the receptor-binding domain of SARS-CoV-2 and found a vulnerable region, YQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQ. Previously, the structural counterpart of this region was termed the Achilles heel of SARS-CoV-1 (Berry, et al. 2010) . It mostly overlaps with the interface between ACE2 and S protein ( Figure 2G ). For SARS-CoV-1, multiple MAbs targeting its Achilles heel have been generated, including F26G18,

4D5,CR3006,m396,FM39,CR3014,F26G19,and 80R (Table SI) . For antibody and vaccine development, ongoing studies are focusing on epitopes at the Achilles heel of SARS-CoV-2, especially ah1 and ah2 sites (listed in Table II and Figure 4) , which directly interact with ACE2.

However, neutralizing antibodies which do not directly compete with ACE2 binding also exist in recovered SARS-CoV-1 and SARS-CoV-2 patients; for example, the CR3022 MAb neutralizes SARS-CoV-1 but not SARS-CoV-2 (Yuan et al. 2020). The S309 MAb isolated from a SARS-CoV-1 patient neutralizes both SARS-CoV-1 and SARS-CoV-2, and structural analysis revealed its epitope to be a glycopeptide sequence located on the N343 glycosite. Notably, this is exactly in structurally conserved regions as we predicted for the II/ii epitope pair (Table II) . Clearly, other epitope pairs predicted in our study are candidate targets to isolate neutralizing antibodies as well.

It is well known that predicted epitopes of protein antigens may be masked by glycosylation.

Complex datasets and algorithms, such as spatial epitope prediction for protein antigens (SEPPA)

3.0, have been developed which are based on training parameters related to interactions of glycans and surrounding amino acids (Kong et al. 2015) . However, no experimental data are available on the effect of glycosylation sites on epitope surfaces. With the recent breakthrough by high-resolution cryogenic electron microscopy, many glycoproteins can be solved and modeled with glycosylation sites. Here we directly exploit experimental data of the SARS-CoV-1 spike protein from high-resolution cryogenic electron microscopy and screened epitopes for the SARS-CoV-2 spike protein by accessible surface area profiling based on homology-modeled structures. By this approach, we have identified an Achilles heel of SARS-CoV-2, as well as

11 multiple other surface-exposed epitopes within and outside the receptor-binding domain. For example, in the N-terminal domain (NTD) domain of the SARS-CoV-1spike protein, MAbs specific for linear epitopes have been reported (Table SI) (Greenough, et al. 2005) . MAbs specific to other regions of the S1 and S2 subunits of SARS-CoV spike proteins were also reported (Miyoshi-Akiyama, et al. 2011). As summarized in Table I , promising antibody binding sites within and outside the receptor-binding domain have been identified for SARS-CoV-2; our future investigations will focus on vaccination studies to validate their function as neutralizing epitopes with preventive and therapeutic effects in virus challenge experiments.

Dense glycosylation of glycoproteins is a well-known strategy used by viruses to conceal surface peptide epitopes that would otherwise elicit antibody responses, as exemplified by the Env protein of human immunodeficiency virus 1. However, after decades of effort, MAbs which bind to conformational epitopes on the surface of the Env protein have been identified (Garces et al. 2015 , Kong, et al. 2015 , Kong et al. 2013 . Most of these antibodies bind to the N-glycan portion neighboring the peptide epitopes, whereas some antibodies such as MAb 8ANC195 have evolved to recognize peptide epitopes with no dependence on glycan binding (Kong, et al. 2015) .

For antibodies specific to spike glycoproteins, there are no data available whether their recognition is hindered by the glycosylation of spike. However, antibodies that bind to both peptide and sugar portions of spike glycopeptides exist, such as MAb S309 which binds to a glycopeptide epitope on the N343 glycosylation site (Pinto, et al. 2020) . We propose a "snake catching" model: A snake-like epitope is elusive and difficult for an antibody to "catch" because of the highly mobile, "wiggly" sugar chains that hide the peptide portion. Therefore, to overcome the sugar barrier, a minimum length of peptide portion, either conformational or linear

continuous, must first be clamped by a paratope. This clamping effect may either be strengthened by sugars close to the peptide epitope or not hindered by sugar modification. Clearly, surfaceexposed glycopeptide motifs are critical for vaccine design.

In summary, our study clearly identified, by MS, all of the 22 N-glycosites of the SARS-CoV-2 spike protein. We have identified a list of linear surface-exposed candidate epitopes in the spike proteins of SARS-CoV-1 and SARS-CoV-2 and demonstrated the advantages of studying the effects of glycosylation with real cryogenic electron microscopy data. These candidate epitopes are critical for screening for MAb therapeutics to treat SARS-CoV-2, as well as mechanistic studies on vaccine development.

Spike proteins for SARS-CoV-2(GenBank Accession Number: MN908947),SARS-CoV-1 (AB263618),and Middle East respiratory syndrome virus (KM027290) were predicted by NetNGlyc.

The sequence identity of the spike proteins between SARS-CoV-2 and SARS-CoV-1 is as high as 84%, which is sufficient to build an accurate homolog model. The sequence of MN908947 was submitted to SWISS-MODEL, and the structural model was built against all available homolog structures as templates. One stable conformation of trimer structure models for SARS-CoV-2 is very close to the spike protein structure from SARS-CoV-1(PDB: 5X58), and their root-mean-square deviation of a single protein chain is approximately 1.32Å after the two structures were superimposed and compared in PyMOL( Figure 2D and E). 

S protein was precipitated with trichloroacetic acid solution (6.1N). The protein pellet was subsequently dissolved in 8 M urea in 100mM Tris-HCl, pH 8.5. Tris(2-carboxyethyl)phosphine (5 mM) was added and incubated for 20 minutes at room temperature to reduce the protein, and iodoacetamide (10mM) was subsequently added and incubated for 15 minutes to alkylate the protein. The protein mixture was digested with chymotrypsin (Wako, Richmond, VA) at a 1:100 ratio at 25°C, followed by trypsin (Promega) at 1:50 ratio (w/w) at 37°C. The reaction was terminated by adding formic acid, and the peptide mixture was desalted with a mono-Spin C18 column (GL Sciences).

The desalted peptide mixture was loaded onto a homemade 30-cm analytical column (ReproSil-Pur C18-AQ 1.9-μm resin, Dr. Maisch GmbH, 360μm OD× 75μm ID) connected to an Easy-nLC 

All acquired MS/MS and MS data were interpreted and analyzed as described (Liu et al. 2017) by using pGlyco 2.0 (version 2019.01.01, http://pfind.ict.ac.cn/software/pGlyco/index.html) glycopeptide identification and by using Byologic v3.5 for quantification. Parameters for our database search of intact glycopeptides were as follows: mass tolerance for precursors and fragment ions were set as ± 7 and ±20 ppm, respectively. The enzymes were trypsin and chymotrypsin. Maximal missed cleavage was 2. Fixed modification was carbamidomethylation on all Cys residues (C +57.022 Da). Variable modifications contained oxidation on Met (M +15.995 Da). The N-glycosylation sequon (N-X-S/T, X ≠ P) was modified by changing "N" to "J"

(the two shared the same mass). The glycan database was extracted from Glycome DB

(www.glycome-db.org). All identified spectra could be automatically annotated and displayed by the software tool gLabel embedded in pGlyco2.0, which facilitates manual verification.

Parameter settings in Byonic were the same as that in pGlyco2.0 except that the built-in Nglycan database (N-glycan 38 insect glycan) was used for database searching. The O-glycan database was homemade according to previously reported glycan structures by Gaunitz et al. (Lindberg et al. 2013) . The identified N-glycopeptides were further examined manually to verify the accuracy of identification. The glycopeptides were quantified by Byologic based on the extracted ion chromatogram area under the curve.

Glycosylation sites were solved and determined from high-resolution cryogenic electron microscopy density maps, and only N-Acetyl-D-glucosamine (Asn-GlcNAc) was determined to represent a whole glycan due to the glycan flexibility and disorder. TheSARS-CoV-1spike protein structure (PDB:5X58), together with the Asn-GlcNAc sites, were applied for molecular interface calculation with PISA (http://www.ccp4.ac.uk/pisa/). All the amino acids linking or interacting with Asn-GlcNAc were selected and excluded in epitope prediction. Besides the interaction between Asn-GlcNAc and amino acids, the effects of the larger structure of glycans extending from every Asn-GlcNAc may also need to be considered, as shown as in Figure 2C , although their electron densities are weak.

The aforementioned molecular interface calculation procedure was applied to calculate the accessible surface area and screen the corresponding antigen epitopes, except that the

glycosylation effect could not be measured because the structure is not yet available. Because most glycosylation sites are conserved due to the high similarity between these two spike proteins, we could predict the glycosylation site effects in the SARS-CoV-2 spike structure as well. When predicted epitopes coincided with the amino acid residues interacting with Asn-GlcNAc, they were removed from the candidates by cross-reference of the SARS-CoV-1 data. 

The authors declare no conflict of interest.

Dapeng Zhou, Chao Peng, and Wen Zhang designed this study. Dapeng Zhou, Xiaoxu Tian, Ruibing Qi, Chao Peng, and Wen Zhang contributed to the collection, analysis, and

interpretation of data. Dapeng Zhou and Wen Zhang wrote the manuscript. All authors read and approved the final manuscript. (Greenough, et al. 2005 , Hwang, et al. 2006 , Pak, et al. 2009 , Prabakaran, et al. 2006 

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