Researchers have developed a novel, simple and affordable method for N-glycan fingerprinting based on enzymatic fluorescent glycan labeling and electrophoresis. This new method may help us understand important changes in glycosylation in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein – something which could inform future therapeutic research.
Advances in genomics, proteomics and mass spectrometry have enabled scientists to identify specific glycans on proteins and their association with diseases. However, these methods are expensive, require highly trained experts to perform and are not easily available.
Researchers Zhengliang L. Wu and James M. Ertelt released the N-glycans present on a glycoprotein and labeled them enzymatically using fluorophore-conjugated sialic acid and fucose, and separated them using gel electrophoresis. The fingerprint of the glycan on the protein is identified using glycan standards and glycan ladders that are run along on the gel (to recognize the identities of individual bands). This novel method allows a quick look at the glycosylation pattern of any glycoprotein.
Glycans are important sugars on proteins that play various roles in immunity, such as modulating the T cell activation, leukocyte homing, and the immunogenicity of proteins. Aberration in the glycosylation pathway is believed to cause autoimmune disorders. Glycosylation is a common post-translational modification occurring on the membrane and secreted proteins. This increases the stability and solubility of these proteins.
SARS-CoV-2, the causative pathogen of coronavirus disease 2019 (COVID-19), enters human host cells using its membrane spike (S) glycoprotein. The spike protein contains 22 N-glycan sequons (N-X-S/T motifs, where X is any amino acid except proline).
The glycosylation on the spike protein camouflages the immunogenic epitopes of the protein, shielding from antibody neutralization – thereby enabling the virus to evade the host immune response. The vaccine development for SARS-CoV-2 is focused on proteins that initiate infection.
The spike protein is the principal target for vaccines and a critical component of serological assays. Therefore, it is important to understand the glycosylation of this protein, for which a new, novel method is proposed.
In this method, the glycans are first released by the peptide enzyme N-glycosidase F (PNGase F), and then labeled by a sialyltransferase or fucosyltransferase with a fluorophore-conjugated sialic acid or fucose, and O-glycans on a glycoprotein are labeled first followed by trypsin digestion, and finally, the freed labeled glycans or glycopeptides are separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
Fingerprinting N-glycans released from various SARS2 Spike proteins with ST6Gal1 (red) and FUT8 (green). N-glycans of various recombinant spike proteins released by PNGase F were labeled by ST6Gal1 and FUT8 with (+) or without (-) pretreatment of a neuraminidase (Neu). Labeled samples were separated on 17% SDS gel and imaged with regular protein imaging (upper panels) or fluorescent imaging (middle and lower panel in different contrasts). Bands in red are complex or hybrid glycans labeled by ST6Gal1 (via the incorporation of Cy5-conjugated sialic acid). Bands in green are oligo-mannose glycans labeled by FUT8 (via the incorporation of AlexaFluor 555-conjugated Fucose). For FUT8 labeling, MGAT1/UDP-GlcNAc were also included in the labeling mix to convert the oligo-mannose glycans to the substrates for FUT8. Labeled glycans from Ribonuclease B, S’1N1, S’1G1 and S’1G1f were run as references in (A) and a glycan ladder with 10 labeled standard glycans was run in (B). RS, RBD domain of SARS2 Spike protein expressed in Sf21 cells; RC, RBD domain of SARS2 Spike protein expressed in CHO cells; RH, RBD domain of SARS2 Spike protein expressed in HEK293 cells; SC, whole SARS2 Spike protein expressed in CHO cells; S1H, SARS2 S1 protein expressed in HEK293 cells; SH, SARS2 Spike protein expressed in HEK293 cells.
The researchers found that when a linkage of specific monosaccharide is added, the mobility of the glycan changes at a relatively constant rate. This further allows the researchers to deduce the identities of labeled bands.
For this glycan fingerprinting study, the researchers screened the labeling enzymes and optimized the substrate concentration for the labeling reaction using glycans released from the spike protein’s receptor-binding domain protein expressed in CHO cells as the substrates. The data from this study suggests that the RBD expressed in HEK293 cells mainly contains complex glycans.
Our study provides evidence to support that host cells determine the types of glycans attached to the spike proteins of SARS2, which further implies that the glycosylation pattern of the spike protein of SARS2 from COVID-19 patients could be different as well. ” Zhengliang L Wu, and James M Ertelt
In this study, they also show that the glycans of spike proteins expressed in insect cells and HEK293 cells are completely different.
Among the various sialyltransferases, they found ST6Gal1 and ST3Gal6 gave stronger signals. In their N-Glycan fingerprinting study of SARS-CoV-2 spike proteins with ST6Gal1, they found the existence of both sialylated and asialylated glycans on these proteins. When the same set of the SARS-CoV-2 spike protein samples were probed with ST3Gal6, they observed similar but distinctive glycan fingerprints. ST3Gal6 labeling also revealed some unique bands.
As a method of fingerprinting, the overall glycan patterning rather than individual glycan species is the focus,” the researchers say in their paper.
Even though the strategy does not allow site-specific and detailed structural glycan analysis, they discuss the advantages: simple, affordable, convenient, visually-informative, easy-to-interpret, multi-sampling possibility, efficient and quantitative.
The current widely used technique for glycan analysis is mass spectroscopy. Adding emphasis on the advantages of the new method, the researchers elaborate on the discrepancies found in mass spectroscopy data on glycans generated from different labs. For the NIST Monoclonal Antibody Reference Material 8671, analyzed by 66 labs around the world, the number of glycan compositions identified by each laboratory ranging from 4 to 48!
Likewise, the mass spectrometry analysis of the spike protein revealed it to be highly modified with complex, hybrid and oligomannose N-glycans. However, the glycan profiles reported by others varied greatly. The proposed method here provides an alternative for glycan research and analysis.
Glycan analysis is critical for researchers to better understand the biological functions of glycosylation on individual glycoproteins. Changes in glycosylation are often a hallmark of disease states. New therapeutic and diagnostic strategies that are based on the underlying glycobiology.
The two researchers from Bio-techne, R&D Systems, USA, have addressed an important methodology in glycobiology that could fast-track the science.
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