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Sun Jul 19, 2020, 12:22 PM

Site-specific glycan analysis of the SARS-CoV-2 spike protein.

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.

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Reply Site-specific glycan analysis of the SARS-CoV-2 spike protein. (Original post)
NNadir Jul 2020 OP
CatLady78 Jul 2020 #1
NNadir Jul 2020 #2
CatLady78 Jul 2020 #3
NNadir Jul 2020 #4
CatLady78 Jul 2020 #5
NNadir Jul 2020 #6
CatLady78 Jul 2020 #7

Response to NNadir (Original post)

Mon Jul 20, 2020, 06:02 AM

1. Bookmarked

I keep bookmarking your cool, information dense posts. Not sure when I will get around to reading them however :-/..

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Response to CatLady78 (Reply #1)

Thu Jul 23, 2020, 09:02 AM

2. I'm glad you enjoy them. Frankly, I only get to read a small fraction of what I want to read, so..

...you're not alone in that.

It is a good thing, I think, to want to read more than one has time to do. It's a sign of freedom and a sign that your brain is working better than you may think it is.

Going through a recent issue of a journal I read quite a bit, ACS Sustainable and Chemical Engineering, Issue 23 (2020), I see I marked 16 papers for deeper reading, and thus far have only gotten into one at any level of depth, Energy-Saving CO2 Capture by H2 Gas Stripping for Integrating CO2 Separation and Conversion Processes, about sparging CO2 solutions with hydrogen for direct conversion to chemical fuels.

I haven't even marked the subsequent issues for papers of interest.

Over a long life though, I've managed to build a fairly large paper library and a far more massive electronic library, so whenever I have time, I can keep myself amused. If I wake up in the night with a new idea, I can usually go down to my office to find papers relevant to it.

Life is magnificent, and then you die.

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Response to NNadir (Reply #2)

Thu Jul 23, 2020, 10:00 AM

3. ...

I actually post science here to an extent to force myself to read more stuff and in more depth.
I made an embarassing error in an article I just posted on something I have a passing familiarity with-hair cell stereocilia. But it is making me read that paper properly.
Or I increasingly never do any reading outside a very narrow area in science and tied to specific problem solving.

As long as I don't leave anything inaccurate up here, it is an exercise that helps me. I'd hate to give people wrong information. So it forces me to check my work.

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Response to CatLady78 (Reply #3)

Thu Jul 23, 2020, 10:35 AM

4. The best scientists are those who are willing to be wrong about this or that.

I mostly write my posts here for exactly the same purposes, which is to force myself to clarify impressions in my head.

Our moderator here is very good at pointing out things that need refinement or correction.

Almost every issue of every journal has a corrections/errata paper at the end. It's OK to be wrong, as long as one can cheerfully admit to doing so.

We learn more deeply when we are corrected.

I could easily fill a notebook with things I believed at one point in my life that were wrong. I am best known on this website for my crusade to argue that nuclear energy, and only nuclear energy is sustainable in a world without poverty. This is certainly controversial on the left, regrettably, but as a lifelong leftist, when I was a stupid kid, I bought into the same idiotic rote anti-nuke rhetoric one can still hear on the left all the time. I was, until Chernobyl blew up, an anti-nuke.

It is extremely popular, but wrong, to argue that so called "renewable energy" is "renewable." It isn't, but millions upon millions of people, including very good scientists, argue otherwise. The journals are filled with papers with obeisances to this idea. I personally believed that so called "renewable energy" was a good thing for many years, up to perhaps, a few years back, until many of my critics with respect to nuclear energy forced me to look more deeply into it and I realized that the embrace of so called "renewable energy" was an extremely bad idea. (I am pleased to note that many papers are now written that are coming around on this point.)

It's more of a problem if you're wrong and insist you're infallible and right.

In science, a famous case would be Nobel Laureate H.C. Brown's campaign against the non-classical carbocation, but there are many others.

...exactly what everyone not named H. C. Brown had been saying it was for decades.

Even winning the Nobel Prize does not render one infallible.

Well before the crystal structure of the carbocation was resolved, NMR work had basically proved the point, this while Brown was alive, but in true Trumpian fashion, he denied it.

Charge/bond delocalization is now a central facet of chemistry, both inorganic and organic chemistry.

I appreciate your contributions here. You have no reason to be embarrassed or shy. The people who read and write in this forum do so because ideas matter more than absolute rectitude. Since you're relatively new in this forum, I for one, welcome you.

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Response to NNadir (Reply #4)

Thu Jul 23, 2020, 12:11 PM

5. Thanks a lot NNadir

I actually get a lot of encouragement from that. I appreciate your posts. I must confess a lot of it goes over my head but regardless I read them.
This forum helps me a lot.

I wish I could say that I do it in the interests of science-and there is a bit of that. But I do it more as an intellectual exercise to become comfortable with science communication - something I really struggle with.

Regarding your crusade, I am the sort of lazy environmentalist who backs green energy but certainly I do not know anything about it. I am struggling to keep up with my own field, so I don't get around to looking at stuff like that. I accept whatever is current dogma within those sciences. I hadn't really thought much about nuclear energy - I have the same generic orthodoxy on that most lefties do, but it isn't something I have strong views on. I will certainly check out your posts amd educate myself on that.

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Response to CatLady78 (Reply #5)

Thu Jul 23, 2020, 01:48 PM

6. This is a good place to practice communication.

Frankly I'm not great at science communication because I'm an old curmudgeon with a mean streak...

I have some advice for you though if you're not doing this already. You need to set a little bit of time each week to read through the advice in the Science and Nature Career sections. They're totally open sourced, no subscription or library access required.

Many of the struggles scientists go through are addressed in these little articles which are a quick read which can take your mind off things and make you recognize you're not alone.

I'm definitely too old to change my approach to my career, but I send links to notes in these career sections to my son all the time because, as an old man, I wish I had read them when I was younger. (Whether he actually reads them or not, I can't say, but he's always getting tiresome advice from his old man anyway. When I'm dead he may appreciate it.)

When you do science, you can feel very small, because the key to being a good scientist is knowing what you don't know and, frankly, being a little intimidated by what you don't know. When I was very young, the son of two parents who never finished high school and one who never started high school, I always thought everyone I met was smarter than I am. I wasted a lot of time worrying about that. Everyone struggles.

When my son was in elementary school, they had a little poster in the music department that referred to a quote from Albert Einstein which read, "If you think you have trouble with mathematics, I assure you mine are much worse." (He needed help from his friend Marcel Grossmann to get help around tensor algebra.)

I recently looked into the life of Donald Cram, who won the Nobel for Host/Guest chemistry, a key concept to this day. He said that his greatest satisfaction was recognizing at the end of a particular project how he should have started it, although invariably he didn't start it that way.

Life is like that, I think, which is why we old people say all the time, "I wish I knew what I know now when I was younger..." ...or, the other more bitter comment, "...youth is wasted on the young..."

I am vicariously young through my sons.

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Response to NNadir (Reply #6)

Fri Jul 24, 2020, 04:07 AM

7. I will keep that in mind

You are right about age. I am actually no spring chicken...but I do always wish I had the knowledge I have now 20 years ago....

I started in my field of choice relatively late. There are times when I feel that I am myself using the weird gliding motion of toxoplasma (and not as successfully-as it is chaotic rather than fast!) over the steady crawl I would prefer -silly joke..re: an article I posted earlier.

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