Non-Enzymatic and Site-Specific Glycan Shedding: A Novel Protein

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Non-Enzymatic and Site-Specific Glycan Shedding: A Novel Protein Degradation Pathway Observed in a Stabilized Form of RSV Prefusion F Protein Jiang Qian, Eric Yearley, Sai Tian, Li Jing, Ankita Balsaraf, Paola Lo Surdo, Ying Huang, Sumana Chandramouli, Matthew J Bottomley, Nicolas Moniotte, and Zihao Wang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b02402 • Publication Date (Web): 28 Aug 2018 Downloaded from http://pubs.acs.org on September 3, 2018

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Analytical Chemistry

Non-Enzymatic and Site-Specific Glycan Shedding: A Novel Protein Degradation Pathway Observed in a Stabilized Form of RSV Prefusion F Protein Jiang Qian *, Eric Yearley *, Sai Tian §, Li Jing ¶, Ankita Balsaraf §, Paola Lo Surdo ¶, Ying Huang §, Sumana Chandramouli §, Matthew J. Bottomley §, Nicolas Moniotte *, and Zihao Wang*ǁ

*Analytical R&D, Slaoui Center for Vaccines Research, GSK Vaccines, 14200 Shady Grove RD. Rockville, MD 20850 § Preclinical R&D, Slaoui Center for Vaccines Research, GSK Vaccines, 14200 Shady Grove RD. Rockville, MD 20850 ¶ Drug Substance R&D , Slaoui Center for Vaccines Research, GSK Vaccines, 14200 Shady Grove RD. Rockville, MD 20850 ǁ Corresponding author. Tel.:+1 202 507 2777; fax: +1 919 567 6075; E-mail address: [email protected]

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ABSTRACT Stability is one of the critical attributes of a protein-based therapeutic or vaccine product, which is directly linked to product quality and efficacy. Elucidating protein degradation pathways is required to obtain thorough understanding of the product and ensure degradation products are properly monitored. We observed a unique protein degradation involving non-enzyme catalyzed loss of a complete N-linked glycan under stress condition from an engineered respiratory syncytial virus (RSV) pre-fusion F protein (RSVPreF3). Investigations involving mass spectrometry, molecular modeling, and mutagenesis revealed that the glycan shedding was site-specific, dependent on structural elements, and required a glycine residue immediately following the site of glycosylation. The glycan loss did not negatively affect the binding between the main immunogenic epitope Site Ø and the neutralizing antibody D25. Further study indicated that the glycan shedding followed a similar but different mechanism than that of conventional deamidation. Since glycosylation is an important attribute for many recombinant therapeutic proteins or vaccine antigens, the finding from this study suggests the need to monitor this new type of degradation, especially when glycosylation has an impact on efficacy or safety. INTRODUCTION Respiratory syncytial virus (RSV) infects almost all infants and young children up to 2 years of age and leads to acute lower respiratory tract infections 1. The RSV fusion protein (RSV-F), which is the target of most known neutralizing antibodies isolated from human sera, is a promising candidate antigen for developing vaccines against RSV infection 2,3. RSV-F is synthesized in vivo as a single-chain precursor and undergoes 2 ACS Paragon Plus Environment

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proteolytic processing resulting into two polypeptide chains termed F2 and F1, which are covalently linked together by multiple disulfide bonds 4,5. During entry into host cells, RSV-F transitions from an unstable pre-fusion conformation into a stable post-fusion conformation. Pre-fusion RSV-F is a better candidate antigen since it is the specific target of the most potent neutralizing antibodies 6. Recent studies have elucidated the crystal structures of both pre-fusion and post-fusion RSV-F, following which iterative structure-based designs have generated engineered molecules that remain relatively stable in the pre-fusion conformation and are capable of eliciting a strong protective immune response 6,7,8. RSVPreF3, which shares the same primary sequence as previously described 6, is stabilized in the pre-fusion state by an introduced extra disulfide bond, linking/connecting residues C155 (S155C) and C290 (S290C), and the cavity-filling mutations S190F and V207L 9. We discovered that RSVPreF3, when in liquid solution, non-enzymatically lost a whole N-glycan moiety in a site-specific manner under elevated temperature. It was also observed that the shedding of glycan was dependent on structural elements. Evidences suggested the shedding followed a similar but different mechanism from the typical protein deamidation mechanism. The impact of the glycan loss on the antigenicity of RSVPreF3 was also investigated. To the best of our knowledge, this unique pattern of protein degradation is the first reported for recombinant proteins, although there was speculation that deamidation may occur on glycosylated asparagine 10. This study highlights the needs to closely monitor this type of protein degradation and how it may affect the potency of protein drugs or vaccine products, in particular engineered recombinant proteins. 3 ACS Paragon Plus Environment


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EXPERIMENTAL SECTION Heat stress experiments and SDS-PAGE. Heat stressing was conducted by incubation at the temperatures of 25 °C, 40 °C or 55 °C for durations of 8 h, 52 h, 1 week, and 2 weeks. At the end of each incubation time, samples were analyzed by SDS-PAGE and stained by Coomassie Blue. Detection of shed N-glycans. Proteins from control and heat stressed materials were removed by precipitation with cold methanol. Then supernatants were collected, dried down completely, and re-suspended by 80% acetonitrile. The samples were analyzed by HILIC-LC-MS analysis on a Waters Xevo Q-TOF MS. Heat stress experiments of RSVPreF3 in H218O containing buffer. RSVPreF3 in the same phosphate-containing buffer as described in SI but containing 80% H218O was incubated at 55 °C for 2 weeks, after which cold acetone was added to precipitate the protein. Protein pellet was collected, re-solubilized in the 8 M GnCl (pH 8.5 in tris buffer), digested by Glu-C, and analyzed by LC-MS. For detailed experimental procedures about materials used, intact mass determination, peptide mapping, in silico modeling, expression of RSVPreF3 mutants, and immunoassay by biolayer interferometry, see Supplemental Information. RESULTS RSVPreF3 non-enzymatically loses N-glycan Under non-reducing condition, RSVPreF3 appeared as a single band with a molecular weight ~60 kDa on a SDS-PAGE gel. Under a boiled and reduced condition, RSVPreF3 4 ACS Paragon Plus Environment

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resolved as multiple bands. Two main bands included F1 polypeptide with gel mobility around 52 kDa and F2 polypeptide around 27 kDa. F2 and F1 contain two and one Nlinked glycosylation sites, respectively. Other bands corresponded to various productrelated variants, such as different glycoforms (Figure 1A). During a forced degradation study, it was observed that under elevated temperatures in liquid solution, the F2 band intensity dropped as early as 1 week and a new lower molecular weight band appeared (Figure 1A, right panel). The degradation band matched with a partially deglycosylated F2 band generated from brief PNGase F treatment (Figure 1B). Intact molecular weight analysis using LC-MS showed that the degraded material contained a species with ∆mass corresponding to an N-linked bi-antennary glycan (Table S1). To further confirm that a glycan was lost, supernatant from the heat stressed and control RSVPreF3 was analzyed by mass spectrometry. The results indicated that multiple N-glycan species were present in the supernatant of heat stressed RSVPreF3 but none in control material (Figure 1C). The identities of the glycans were in agreement with previous knowledge that F2 polypeptides were modified by complex type glycans. To investigate if there was clipping to F2 polypeptide in addition to glycan loss, the control and stressed materials were fully deglycosylated by PNGase F and then analyzed by LC-MS. The results showed that control material contained F2 species of 9190 Da, which matched the calculated theoretical mass; the stressed material contained only F2 species of 9191 Da. The additional 1 Da mass was presumably due to deamidation. Overall, the data suggested no clipping occurred to the F2 polypeptide backbone. (Figure 1D). F2 contains two N-linked glycans at 27N and 70N (numbering based on full length wild type RSV-F sequence)11, and SDS-PAGE suggested only one glycan was lost ( Figure 5 ACS Paragon Plus Environment


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1A, right panel and Figure 1B). A mass spectrometric analysis was used to pinpoint exact site of glycan loss. As shown in Figure 1E, multiple charge species corresponding to a peptide (NKC70DGTDAKVKLIKQE) were detected in stressed material but not in control material. Peptides containing unmodified 27N or 27D were not detected in neither control nor stressed materials. These data suggested that the glycan loss was strictly limited to 70N and the glycan loss was accompanied by conversion of Asn to Asp. A comprehensive proteomic study detected no presence of any endoglycosidase. In addition, an endoglycosidase inhibitor 12 was added to RSVPreF3 and showed no effect in preventing glycan loss (Figure 1F). Together, the data suggested that the glycan loss was not enzymatically catalyzed. RSVPreF3 N-glycan loss is structure-dependent The RSVPreF3, which is in pre-fusion conformation, shares largely the same primary amino acid sequence as post-fusion RSV-F but has dramatically different tertiary conformation 6,8. Post-fusion RSV-F protein was similarly stressed, but F2 degradant corresponding to loss of glycan at 70N was not observed (Figure S1A). More interestingly, when RSVPreF3 was denatured by sodium dodecyl sulfate (SDS) or surfactant RapiGest, the protein retained the glycan under heat stress (Figure S1B). These results indicated that structural elements were critical in the glycan loss. Effect of N-glycan loss on binding to neutralizing antibodies Site Ø is one of the main RSV-F epitopes targeted by neutralizing antibodies. Specifically, site Ø is present only in pre-fusion but not post-fusion RSV-F and is recognized by the monoclonal antibody (mAb) D25 6. The RSVPreF3 residue 70N is 6 ACS Paragon Plus Environment

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immediately adjacent to the stretch of amino acids (SNIKENKC70NGT, underscored) that is part of site Ø13. In the crystal structure of mAb 25 bound to the pre-fusion F protein, the glycans are not visible, perhaps due to their intrinsic flexibility and the relatively low resolution of the diffraction data 6. Therefore, we performed in silico analyses to evaluate possible positions of the glycans with respect to the protein. Indeed, computer modeling of the pre-fusion F structure with N-glycan present on 70N suggested the glycan might partially shield site Ø from being recognized (Figure 2A). We measured the equilibrium dissociation constant (KD) between D25 mAb and the control or stressed materials, and also evaluated KD changes in the mutants N70Q and T72D using biolayer interferometry immunoassay. In the heat stressed material, ~50% RSVPreF3 lost 70N glycan as estimated by SDS-PAGE, while in both mutants glycosylation on 70N position was completely absent because of either removing the Asparagine itself or removing the required N-linked glycosylation consensus motif (–NX-S/T-). As shown in Figures 2B and S2, neither partial loss of 70N glycan by heat stress nor complete loss by site-directed mutations negatively affected the binding affinity between RSVPreF3 and mAb D25. These data supported the in silico structural modeling result that absence of the glycan at 70N does not negatively affect or may even slightly favor the interaction because of better exposure of site Ø . Mechanism of non-enzymatic glycan loss Our next attempt was to understand the mechanism of the glycan loss. Asparagine was converted to an aspartate residue after the glycan moiety was lost (Figure 1E), same as observed in a typical protein deamidation. More interestingly, 70N is followed by a glycine residue. The -Asn-Gly-X- sequence is a known deamidation ‘hot spot’ 14,15. To 7 ACS Paragon Plus Environment


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test if the typical deamidation mechanism was followed in the glycan shedding observed here, RSVPreF3 was subjected to changes in pH and monitored for glycan loss. The RSVPreF3 protein was incubated at elevated temperature under different pH ranging from 7.0 to 9.0 and then separated by SDS-PAGE. The result showed that higher pH promoted the loss of glycan, consistent with the trend commonly observed for protein deamidation (Figure 3A). In addition, rates of glycan loss increased with higher temperature (Figure 3B). Following this observation, we generated three single amino acid mutants (G71A, G71I, G71S) that retained the 70N glycosylation site but removed the deamidation ‘hot spot’. The mutants were similarly stressed and analyzed for loss of glycans. The result showed that the 71G mutants did not lose 70N glycan under the same stress condition (Figure 3C). All these findings suggest that the glycan shedding may follow a similar mechanism as deamidation. To further investigate, an 18O labeling experiment was performed. ~ 80% of H2O in the original RSVPreF3 buffer was replaced with H218O, and the sample was then stressed by heating, after which the protein was digested and analyzed by LC-MS/MS. As expected, a mass increase of 2 Da was observed in the 70N-containing peptide in the heated sample indicating one 18O labeling. However, a mass increase of 4 Da was also observed, suggesting two 18O incorporation occurred (Figure 3D) as well. These complex MS spectra indicated a more complicated mechanism of glycan shedding than the typical protein deamidation. DISCUSSION Stability is one of the critical attributes of a drug or vaccine product, for which monitoring is required by regulatory agencies to ensure the product quality and efficacy. One way to gain understanding of product stability is to conduct forced degradation studies at 8 ACS Paragon Plus Environment

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elevated temperatures and extreme pH conditions. Known common protein degradations include higher order structure (HOS) changes, primary amino acid sequence clipping, amino acid side chain modifications, aggregation, etc.16. N-linked glycosylation is generally considered as a stable post-translational modification. Here we report a new type of protein degradation involving complete shedding of N-glycan. In contrast to known monosaccharide ‘peeling’, the shedding involved breakage of the Nglycosidic bond at the Asn side chain with the whole N-glycan being released into the solution. The shedding was limited to a single specific site (70N) as N-glycans at two other sites (27N and 500N) on the same molecule remained intact. More interestingly, the phenomenon was dependent on structural element(s), since denaturing of RSVPreF3 significantly suppressed glycan shedding. Post-fusion RSV, which shares largely the same primary amino acid sequence as RSVPreF3 but adopts a dramatically different tertiary structure 17, did not show glycan shedding under the same condition. RSVPreF3 was generated from iterative structure-based design, which stabilized the molecule in the most immunogenic pre-fusion conformation 6. D25, a potent neutralizing antibody isolated from RSV-infected patients, specifically recognizes site Ø, an epitope unique to pre-fusion RSV-F protein 6. The site of observed glycan shedding was immediately adjacent to one of the amino acid sequences that constitute site Ø 13. Klink et.al. showed that deletion of 70N glycan in bovine RSV resulted in higher immune response compared to fully glycosylated wild type 11. Zimmer et.al. found that loss of 70

N glycosylation led to significantly increased fusion activity18. Glycans are generally

considered as low in immunogenicity. It is also common that glycans are used by viruses as shield to evade immune recognition. We measured the KD of mAb D25 with 9 ACS Paragon Plus Environment


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stressed RSVPreF3 (partially lacking glycan at 70N) and mutant RSVPreF3 completely lacking 70N glycan, and found no impaired binding affinity. Thus, the glycan shedding did not appear to negatively affect the antigenicity of RSVPreF3 as a vaccine candidate. We also sought to understand the mechanism of the glycan shedding. The observation that glycan shedding was promoted by higher pH or temperature and that removing the deamidation ‘hot spot’ by mutating 71G abolished glycan shedding suggested that it might follow a mechanism similar to deamidation. On the other hand, an 18O incorporation study suggested it was not a canonical deamidation pathway. The resulting complex MS spectrum result (Figure 3D) suggested a sophisticated mechanism behind the shedding. It is known that during conversion from 70N to 70D, oxygen from H218O is incorporated into the hydroxyl group (-OH) of the side chain of 70D during both the deamidation and PNGase F mediated de-glycosylation process; and protein Asn deamidation generally proceeds through a succinimide intermediate before final 70D is formed. Based on the mass increase of 2 Da at 70N and known Asn deamidation mechanism, a mechanism similar to deamidation is proposed for the glycan shedding. As shown in Figure 3E (left), the amide bond between the 70N and glycan is broken as a result of a nucleophilic attack from the N atom of 71G. The succinimide intermediate is eventually hydrolyzed to 70D with one 18O being incorporated. The mass increase of 4 Da indicating a second 18O incorporation is not reported previously in neither deamidation nor deglycosylation studies. Moreover, as shown in Figure 3D, a ratio of ~1: 1 was observed between one 18O and two 18O incorporation. The result suggested that besides the Asn deamidation mechanism, a more complex glycan loss pathway might be involved. MS/MS fragmentation (data not 10 ACS Paragon Plus Environment

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shown) suggested that both mass shift (+2 and +4 Da) started from y13 ion containing (DGTDAKVKLIKQE), indicating both the first and second 18O incorporation occurred at 70

D. Based on the enol-keto equilibrium of the amide bond between the 70N and glycan

(Figure 3E, right), a mechanism featuring two 18O incorporation is proposed. As a result of the initial nucleophilic attack from the N atom of 71G, an unstable intermediate is formed with the glycan still attached. The intermediate will be eventually converted to the more stable intermediate succinimide as shown in Figure 3E (left). During the conversion, the glycan will be lost accompanied by the first 18O labeling. The succinimide containing 18O will then be hydrolyzed to become Asp, during which the second 18O will be incorporated. Since water molecules are involved in both pathways, solvent accessibility area (SAA) will be a key factor for the reaction to proceed. We speculate that 70N (but not 27N and 500N) has the ideal SAA only in the prefusion conformation. Further experiments are warranted to test these proposed mechanisms but are beyond the scope of this study. In summary, we observed and reported a new type of protein degradation in an engineered recombinant protein. Although the loss of glycan at 70N did not appear to affect antigenicity of RSVPreF3, proper glycosylation is critical for the stability, circulation half life, safety, and efficacy of many other recombinant therapeutic proteins19. Thus, it is of interest to further study the mechanism so to gain deeper understanding and so that preventive measures can be taken accordingly to preserve the consistency and efficacy of protein-based therapeutic or vaccine products.



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Figure Captions Figure 1. RSVPreF3 non-enzymatically loses the whole N-linked glycan at 70N position. (A) Non-reduced (left) and reduced (right) SDS-PAGE gels of RSVPreF3 in liquid or lyophilized (lyo) conditions treated at 5 °C or 40 °C for 1 or 2 weeks. RSVPreF3 freshly thawed from -80 °C was used as control. Red arrows point to the separated F1 and F2 polypeptides under reduced condition. A lower molecular weight band with MW smaller than 24 KDa was observed in 40 °C treated liquid RSVPreF3. (B) Partial deglycosylation of RSVPreF3 using PNGase F generated a band of similar gel mobility as the lower MW band generated by heat stress. The lowest MW band in PNGase F treated RSVPreF3 corresponded to complete removal of 2 N-glycans on F2. (C) Released N-glycans were detected in the heat stressed (lower panel) but not the control (upper panel) RSVPreF3. Inset shows identification of the LC peak by mass spectrometry. (D) Intact molecular weight analysis of PNGase F-deglycosylated control (upper panel) and heat stressed (lower panel) shows similar and expected MW. (E) A new peptide was detected in heat stressed RSVPreF3 (upper panel) and sequenced as NKC70DGTDAKVKLIKQE by MS/MS (lower panel), corresponding to loss of N-glycan at 70

N position. (F) Endoglycosidase inhibitor (Z-VAD-fmk) did not reduce glycan loss by

heat stress. Figure 2. Effects of 70N glycan shedding on RSVPreF3 antigenicity. (A) In silico modeling of glycosylated RSVPreF3 homotrimer showing partial shielding of Site Ø epitope by 70N glycan. Yellow: Site Ø epitope; Cyan: 70N glycan; Red: 71G residue; Green: 70N residue. (B) Comparison of D25 binding affinity among fresh, heat stressed RSVPreF3 that partially lost 70N glycan, and mutants that completely eliminated 70N 12 ACS Paragon Plus Environment

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glycan. Equilibrium dissociation constant KD (M) of mAb D25 targeting site Ø was measured through a kinetics experiment using biolayer interferometry (Octet) measurements. Kinetic parameters were derived by biolayer light interferometry using D25 as ligands immobilized on the sensor surface and RSVPreF3 as analytes in solution. Kon, association rate constant; Kdis, dissociation rate constant; control, nonheat stressed fresh RSVPreF3. For error bars, N=3. Figure 3. Proposed mechanism of non-enzymatic glycan shedding. (A) RSVPreF3 glycan loss increased with higher pH under same heat stress condition. (B) RSVPreF3 glycan loss increased with higher temperature. (C) Elimination of the deamidation ‘hotspot’ by mutating 71G to Ala, Ser, or Ile abolished the glycan shedding. (D) LCMS/MS analysis of heat stressed RSVPreF3 in the presence of ~80% H218O. m/z 616.328 corresponded to peptide NKC70DGTDAKVKLIKQE. ∆ mass of 2 Da or 4 Da indicated incorporation of 1 or 2 18O, respectively. (E) The glycan shedding follows a complex mechanism. ACKNOWLEDGEMENTS The authors would like to thank Drs Enrico Malito, Paolo Costantino, Feng Yan, Richard Buckholz, and Mr. Ratnesh Pandey for their intellectual inputs to this work.

Study Funding / Sponsorship: This work was sponsored by GlaxoSmithKline Biologicals SA, which was involved in all stages of the study conduct and analysis.

Author Contributions:



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ZW, JQ, EY, SC, and MJB were involved in the conception and design of the study. JQ, EY, ST, LJ, AB, YH, SC, and ZW acquired the data. ZW, JQ, EY, LJ, PLS, YH, SC, MJB and NM analyzed and interpreted the results. All authors were involved in drafting the manuscript or critically revising it for important intellectual content. All authors had full access to the data and approved the manuscript before it was submitted by the corresponding author.

Conflict of Interest: All authors have declared the following interests: All authors are employees of the GSK group of companies. PLS, YH, SC, MJB, NM and ZW report ownership of shares and/or restricted shares in the GSK group of companies.

Supporting Information Detailed additional information about materials and experimental procedures. Additional figures and table as referenced to in the text. REFERENCES (1)

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the human respiratory syncytial virus fusion protein at two distinct sites is required for activation of membrane fusion. Proc. Natl. Acad. Sci. USA. 2001, 98, 9859–9864. (5) Scheid, A.; Choppin, P.W. Two disulfide-linked polypeptide chains constitute the active F protein of paramyxoviruses.Virology 1977, 80, 54–66. (6) McLellan, J.S.; Chen, M.; Leung, S.; Graepel, K.W.; Du, X.; Yang, Y.; Zhou, T.; Baxa, U.; Yasuda, E.; Beaumont, T.; Kumar, A.; Modjarrad, K.; Zheng, Z.; Zhao, M.; Xia, N.; Kwong, P.D.; Graham, B.S. Structure of RSV Fusion Glycoprotein Trimer Bound to a Prefusion-Specific Neutralizing Antibody. Science 2013, 340, 1113–1117. (7) Joyce, M.G.; Zhang, B.; Ou, L.; Chen, M.; Chuang, G.Y.; Druz, A.; Kong, W.P.; Lai, Y.T.; Rundlet, E.J.; Tsybovsky, Y.; Yang, Y.; Georgiev, I.S.; Guttman, M.; Lees, C.R.; Pancera, M.; Sastry, M.; Soto, C.; Stewart-Jones, G.B.E.; Thomas, P.V.; Van Galen, J.G.; Baxa, U.; Lee, K.K.; Mascola, J.R.; Graham, B.S.; Kwong, P.D. Iterative structure-based improvement of a fusion-glycoprotein vaccine against RSV. Nat. Struct. Mol. Biol. 2016, 23, 811-820. (8) Swanson, K.A.; Settembre, E.C.; Shaw, C.A.; Dey, A.K.; Rappuoli, R.; Mandl, C.W.; Dormitzer, P.R.; Carfi, A. Structural basis for immunization with postfusion respiratory syncytial virus fusion F glycoprotein (RSV F) to elicit high neutralizing antibody titers. Proc. Natl. Acad. Sci. USA. 2011, 108, 96199624. (9) McLellan, J.S.; Chen,M.; Joyce,M.G.; Sastry, M.; Guillaume, B.E.; Stewart-Jones, G.B.E.; Yang, Y.; Zhang, B.; et al. Structure-based design of a fusion glycoprotein vaccine for respiratory syncytial virus. Science 2013, 342, 592-598. (10) Liu, S.; Moulton, K.R., Auclair, J.R., Zhou, S. Mildly acidic conditions eliminate deamidation artifact during proteolysis: digestion with endoprotease Glu-C at pH 4.5. Amino Acids 2016, 48, 1059-1067. (11) Klink, H.A.; Brady, R.P.; Topliff, C.L.; Eskridge, K.M.; Srikumaran, S.; Kelling, C.L. Influence of bovine respiratory syncytial virus F glycoprotein Nlinked glycans on in vitro expression and on antibody responses in BALB/c mice. Vaccine 2006, 24, 3388-3395. (12) Misaghi, S.; Pacold, M.E.; Blom, D.; Ploegh, H.L.; Korbel, G.A. Chem. Biol. Using a small molecule inhibitor of peptide: N-glycanase to probe its role in glycoprotein turnover. 2004, 11, 1677-1687. (13) Swanson, K.A.; Balabanis, K.; Xie, Y.; Aggarwal, Y.; Palomo, C.; Mas, V.; Metrick, C.; Yang, H.; Shaw, C.A.; Melero, J.A.; Dormitzer, P.R.; Carfi, A. J. Virol. A Monomeric Uncleaved Respiratory Syncytial Virus F Antigen Retains Prefusion-Specific Neutralizing Epitopes. 2014, 88, 11802-11810. (14) Robinson, N.E.; Robinson, A.B. Prediction of primary structure deamidation rates of asparaginyl and glutaminyl peptides through steric and catalytic effects. J. Pept. Res. 2004, 63, 437-448. (15) Robinson, N.E. Protein deamidation. Proc. Natl. Acad. Sci. USA. 2002, 99, 5283-5288.



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(16) Hawe, A.; Wiggenhorn, M.; van de Weert, M.; H O Garbe, J.; Mahler, H.; Jiskoot, W. Forced degradation of therapeutic proteins. J. Pharm. Sci. 2012, 101, 895-913. (17) McLellan, J.S.; Yang, Y.; Graham, B.S.; Kwong, P.D. Structure of Respiratory Syncytial Virus Fusion Glycoprotein in the Postfusion Conformation Reveals Preservation of Neutralizing Epitopes. J. Virol. 2011, 85, 7788–7796. (18) Zimmer, G.; Trotz, I.; Herrler, G. N-Glycans of F Protein Differentially Affect Fusion Activity of Human Respiratory Syncytial Virus. J. Virol. 2001, 75, 4744–4751. (19) Lalonde, M.E.; Durocher, Y. J. Biotech. Therapeutic glycoprotein production in mammalian cells. 2017, 251, 128-140


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Figure 1. A. C.

B.

Contro l

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D. F.

E.

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Control MW= 9190

1838.94 z=5 1683.95 z=1

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Stressed MW= 9191

1532.78 z=6

1141.7452 z=4

2627.04 2886.38 z=7 z=4 2500

3000

Figure 2. B. KD (M)

A.

Control

Heat stress

Control

N70Q

KD (M)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


T72D



Page 18 of 19

Figure 3. A.

D.

B.

C.

E.

Unstable intermediate

-Glycan loss nd 18

2

O incorporation

-Glycan loss

18

- O incorporation st 18

1

O incorporation


Page 19 of 19 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


For TOC Only


Non-Enzymatic and Site-Specific Glycan Shedding: A Novel Protein

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