Site-specific glycan analysis of the SARS-CoV-2 spike protein. [View all]

[NNadir]

The paper I'll discuss in this post is this one: Site-specific glycan analysis of the SARS-CoV-2 spike (Yasunori Watanabe*, Joel D. Allen*, Daniel Wrapp, Jason S. McLellan, Max Crispin, Science, 17 Jul 20, Vol. 369, Issue 6501, pp. 330-333)

In recent years, I've been dragging myself, when I have time, into a consideration of the 4th, and clearly the most challenging, structural motif in molecular biology, following on amino acid based structures (proteins and peptides), nucleic acid derived structures, obviously DNA and RNA, but also including biological energetics, lipids and the complex structural and signalling pathways, especially in the signalling of inflammation, and finally, the difficult one, about which I am working to learn, the sugars. All of these classes of molecules play in the dance of metabolism, the signalling and transformations that make living things be, well, living. In what little spare time I have for it, I have been working to understand glycobiology as well as the structures of the "simple" sugars (which are not necessarily simple), modified sugars, glycosides, ordinary and modifed glycopolymers and their derivatives.

It is worth noting that the largest fraction of the total biomass on this planet is a glycopolymer, cellulose.

Many important molecules in physiology are hybrid molecules containing the core subunits of basic biological motifs. A hybrid molecule containing a sugar bonded in a specific way, via it's oxidized carbon (acetal form) is called a "glycoside." Here is an example of a glycoside that is bonded to a fat, a "GPI anchor":



Lipid Web

A simplified version of this diagram, from the same website, is here:



"GPI anchors" = Glycosylphosphatidylinositol-Anchors

These molecules have three biological motifs represented, four sugars, including an amino sugar, a sugar derived molecule important in physiology, inositol, a diacyl fat, and a protein, which may or may not have other glycosides (glycans) attached. If one looks carefully at the second diagram, where the sugars are designated "Man" (for mannose) and the aminosugar is designated "GlcN" (for glucosamine, one can see that each of the mannoses can be bonded to the other mannose at any of four different positions (since stereochemistry robs mannose of its symmetry) - actually there are five different ways, because one bond would be through an oxygen than can have either of two spacial orientations.

I had the pleasure of working briefly on a GPI anchored protein, alkaline phosphatase, found in the alimentary canal; in the shown example, this GPI anchor works to anchor proteins to the membranes of red blood cells.

Reflection on this point should give an immediate feel for the complexity of systems involving sugars, which should give an appreciation of the sophistication of the paper being discussed.

Glycans on proteins themselves be quite complex; the paper under discussion is about a very complex molecule of this type, a special type of glycoside, " glycan" which is a glycoside of a protein of a specific type that is a key to the understanding of SARS-CoV-2.

Glycans come in two forms, the first and most extensively studied being the N-glycans, which are bonded to proteins at very specific residues, asparagine residues, at the β amide nitrogen. Historically and currently these have been studied by releasing them from the protein using an set enzymes (PGNase) and studying their structure instrumentally in isolation from the parent protein. The other form of glycans, has been somewhat more challenging to release. Nevertheless, the release of glycans for study eliminates the most important information in connection with their function, which is their location on the protein (or peptide chain).

Modern advances in software have gone a long way to address this problem. An example of such software is that of Protein Metrics, the Byonic software. Here is a presentation (on N-Glycans) from that company: Byonic™: N-Linked Glycopeptide Analysis. I recently had the pleasure of watching a scientific webinar by scientists in the groups out which the Science paper comes, the McClellan group and the Crispin group.

There are, of course, other approaches to addressing glycan analysis involving both software and chemistry. I had the pleasure of attending a lecture by Dr. Hui Zhang of Johns Hopkins when she spoke at a conference in New Jersey. Here is an open sourced paper from her group on the subject of O and N glycan analysis: Classification of Tandem Mass Spectra for Identification of N- and O-linked Glycopeptides (Zhang et al., Scientific Reports volume 6, Article number: 37189 (2016))

Anyway, to return to the subject of glycosylation of SARS-Cov-2 S (Spike) Protein.

From the introduction to the paper:

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative pathogen of coronavirus 2019 (COVID-19) (1, 2), induces fever, severe respiratory illness, and pneumonia. SARS-CoV-2 uses an extensively glycosylated spike (S) protein that protrudes from the viral surface to bind to angiotensin-converting enzyme 2 (ACE2) to mediate host-cell entry (3). The S protein is a trimeric class I fusion protein, composed of two functional subunits, responsible for receptor binding (S1 subunit) and membrane fusion (S2 subunit) (4, 5). The surface of the envelope spike is dominated by host-derived glycans, with each trimer displaying 66 N-linked glycosylation sites. The S protein is a key target in vaccine design efforts (6), and understanding the glycosylation of recombinant viral spikes can reveal fundamental features of viral biology and guide vaccine design strategies (7, 8).

Viral glycosylation has wide-ranging roles in viral pathobiology, including mediating protein folding and stability and shaping viral tropism (9). Glycosylation sites are under selective pressure as they facilitate immune evasion by shielding specific epitopes from antibody neutralization. However, we note the low mutation rate of SARS-CoV-2 and that as yet, there have been no observed mutations to N-linked glycosylation sites (10). Surfaces with an unusually high density of glycans can also enable immune recognition (9, 11, 12). The role of glycosylation in camouflaging immunogenic protein epitopes has been studied for other coronaviruses (10, 13, 14). Coronaviruses form virions by budding into the lumen of endoplasmic reticulum–Golgi intermediate compartments (15, 16). However, observations of complex-type glycans on virally derived material suggests that the viral glycoproteins are subjected to Golgi-resident processing enzymes (13, 17).

The type of analysis performed here, and implied in the discussions above is what we call "bottom up" analysis, which involves the enzymatic digestion (usually trypsin is the enzyme most widely used, although there are others) of a protein into smaller peptide fragments.

Some pictures from the text:



The caption:

Fig. 1 Expression and validation of the SARS-CoV-2 S glycoprotein.
(A) Schematic representation of the SARS-CoV-2 S glycoprotein. The positions of N-linked glycosylation sequons (N-X-S/T, where X ≠ P) are shown as branches (N, Asn; X, any residue; S, Ser; T, Thr; P, Pro). Protein domains are illustrated: N-terminal domain (NTD), receptor binding domain (RBD), fusion peptide (FP), heptad repeat 1 (HR1), central helix (CH), connector domain (CD), and transmembrane domain (TM). (B) SDS–polyacrylamide gel electrophoresis analysis of the SARS-CoV-2 S protein (indicated by the arrowhead) expressed in human embryonic kidney (HEK) 293F cells. Lane 1: filtered supernatant from transfected cells; lane 2: flow-through from StrepTactin resin; lane 3: wash from StrepTactin resin; lane 4: elution from StrepTactin resin. (C) Negative-stain EM 2D class averages of the SARS-CoV-2 S protein. 2D class averages of the SARS-CoV-2 S protein are shown, confirming that the protein adopts the trimeric prefusion conformation matching the material used to determine the structure (4).

Mapping of glycans on the SARS-CoV-2 protein:

Fig. 2 Site-specific N-linked glycosylation of the SARS-CoV-2 S glycoprotein.
The schematic illustrates the color code for the principal glycan types that can arise along the maturation pathway from oligomannose- to hybrid- to complex-type glycans. The graphs summarize quantitative mass spectrometric analysis of the glycan population present at individual N-linked glycosylation sites simplified into categories of glycans. The oligomannose-type glycan series (M9 to M5; Man9GlcNAc2 to Man5GlcNAc2) is colored green, afucosylated and fucosylated hybrid-type glycans (hybrid and F hybrid) are dashed pink, and complex glycans are grouped according to the number of antennae and presence of core fucosylation (A1 to FA4) and are colored pink. Unoccupancy of an N-linked glycan site is represented in gray. The pie charts summarize the quantification of these glycans. Glycan sites are colored according to oligomannose-type glycan content, with the glycan sites labeled in green (80 to 100%), orange (30 to 79%), and pink (0 to 29%). An extended version of the site-specific analysis showing the heterogeneity within each category can be found in table S1 and fig. S2. The bar graphs represent the mean quantities of three biological replicates, with error bars representing the standard error of the mean.

Figure 3 invites some commentary after the caption.



The caption:

Fig. 3 Structure-based mapping of SARS-CoV-2 S N-linked glycans.
Representative glycans are modeled onto the prefusion structure of the trimeric SARS-CoV-2 S glycoprotein (PDB ID 6VSB) (4), with one RBD in the “up” conformation and the other two RBDs in the “down” conformation. The glycans are colored according to oligomannose content as defined by the key. ACE2 receptor binding sites are highlighted in light blue. The S1 and S2 subunits are rendered with translucent surface representation, colored light and dark gray, respectively. The flexible loops on which the N74 and N149 glycan sites reside are represented as gray dashed lines, with glycan sites on the loops mapped at their approximate regions.

It is notable that there are many regions in this protein that are not covered by glycans. In virology there is something known as a "glycan shield." We have been working for decades to produce an HIV vaccine. The difficulty in doing that has been informed by the very large glycan shield that covers the HIV virus, which prevents the binding of antibodies to the peptide sequence of the HIV viral proteins. The less extensive glycan shield in SAR-CoV-2, as shown in this cartoon offers hopes for a vaccine.

The authors note as much:

Highly dense glycan shields, such as those observed on LASV GPC and HIV-1 Env, feature so-called mannose clusters (22, 24) on the protein surface (Fig. 4). Whereas small mannose-type clusters have been characterized on the S1 subunit of Middle East respiratory syndrome (MERS)–CoV S (10), no such phenomenon has been observed for the SARS-CoV-1 or SARS-CoV-2 S proteins. The site-specific glycosylation analysis reported here suggests that the glycan shield of SARS-CoV-2 S is consistent with other coronaviruses and similarly exhibits numerous vulnerabilities throughout the glycan shield (10). Last, we detected trace levels of O-linked glycosylation at Thr323/Ser325 (T323/S325), with over 99% of these sites unmodified (fig. S4), suggesting that O-linked glycosylation of this region is minimal when the structure is native-like.

This is a marvelous paper in my opinion, and it shows that we are not technologically disarmed, yet, in the battle against this terrible disease, even if we are temporarily led by an ignorant, obviously emotionally and cognitively impaired anti-science moron.

Our scientific instructure, though damaged, is still intact and there will be time under Joe Biden, to repair it.

I trust you're having a pleasant afternoon despite this being the days of Covid. We are locked inside here in New Jersey because of extreme heat, driven by climate change, an artifact of anti-science on the right, and regrettably on the left as well. I trust you will behave safely, and thus remain safe and well.