O-Glycosylation patterns in Post-viral Fatigue Syndrome: Sialic Acid-preserving Chemical Release, 2025, de Otazo Hernández

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O-Glycosylation patterns in Post-viral Fatigue Syndrome: Sialic Acid-preserving Chemical Release

Daniel García de Otazo Hernándeza,b, Gianluigi Sabatinoa, Eva Untersmayr b, Selina Kepplerc, Davide Reta,b

a Research Unit Macromolecular Chemistry, Institute of Applied Synthetic Chemistry, TU Wien, 1060 Vienna, Austria daniel.garcia@tuwien.ac.at

b Institute of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, 1090 Vienna, Austria c Division of rheumatology and Immunology Medical University of Graz, 8010 Graz, Austria

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a complex inflammatory condition characterized by chronic fatigue, post-exertional malaise, and immune dysregulation whose underlying mechanisms remain poorly understood. Glycosylation, the process of attaching glycans to proteins and lipids, plays a crucial role in immune cell communication and inflammation [1]. As sialic acid has great importance in autoimmune and inflammatory diseases, the focus was directed towards a controlled release and labelling reaction with all conditions avoiding acidic hydrolysis of sialic acid [Figure 1.].

O-glycan from blood sera and purified antibodies where methyl amidated to stabilize sialic acid [2]. The release reaction proceeds via non-reductive β-elimination and subsequent labelling with a fluorescent compound in conditions able to conserve sialic acid in antennary position. The O-glycan profiles are analyzed by HPLC with fluorescence and MALDI mass spectrometry.

O-glycosylation profiles with intact sialylation of ME/CFS patients and healthy controls reveal an altered O-Glycan pattern which may contribute to the chronic inflammatory state observed in ME/CFS.

Figure 1. Graphical abstract.

O-Glycans from blood serum and antibodies were analyzed from patients and healthy controls using a novel release and labelling method. Image created with BioRender.

References:

1. Rohrhofer, J., et al., Immunological Patient Stratification in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome. Journal of Clinical Medicine, 2024. 13(1): p. 275.

2. Ret, D., et al., DMTMM-mediated methylamidation for MALDI mass spectrometry analysis of N-glycans with structurally conserved sialic acid residues in biological fluids "via direttissima". Talanta, 2022. 242: p. 123326.

The 22nd European Carbohydrate Symposium, 6-10 July 2025 GDAŃSK, POLAND
 
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I've shared this paper before, but one possible reason for changes in glycosylation is endoplasmic reticulum stress. ER stress causes a homeostatic response called the 'unfolded protein response' which has many effects, including, the paper says, changing the way glycans get added to proteins.

And when those glyosylated proteins are expelled from the cell, they can act as a signal of cellular stress or infection.

Protein Glycosylation Patterns Shaped By the IRE1-XBP1s Arm of the Unfolded Protein Response
Kenny Chen 1 , Matthew D Shoulders 1 2
Affiliations

Abstract
The unfolded protein response (UPR) is a sensing and signaling pathway that surveys the endoplasmic reticulum (ER) for protein folding challenges and responds whenever issues are detected. UPR activation leads to upregulation of secretory pathway chaperones and quality control factors, as well as reduces the nascent protein load on the ER, thereby restoring and maintaining proteostasis. This paradigm-defining view of the role of the UPR is accurate, but it elides additional key functions of the UPR in cell biology. In particular, recent work has revealed that the UPR can shape the structure and function of N- and O-glycans installed on ER client proteins. This crosstalk between the UPR's response to protein misfolding and the regulation of glycosylation remains insufficiently understood. Still, emerging evidence makes it clear that the UPR, and particularly the IRE1-XBP1s arm of the UPR, may be a central regulator of protein glycosylation with important biological consequences. In this review, we discuss the crosstalk between proteostasis, the UPR, and glycosylation, present progress towards understanding biological functions of this crosstalk, and examine potential roles in diseases such as cancer.


This all ties back to Hwang's study where he found signs of endoplasmic reticulum stress in mecfs,that was causing wasf3 to get into mitochondria. If there is chronic ER stress it could be having effects on glycosylation too.
 
I have been anxiously awaiting the results of Davide Ret's project on glycosylation!

In the meantime, I am wondering about the O-glycosylation pattern that they are interested in.

In reading about sialic acid, I am wondering if desialylation is a cause of issues in pwME/CFS.

I found this in a study:
Besides to function as recognition sites, or act as biological mask to shield recognition sites, Sialic acid residues have also been reported to serve as antioxidant to scavenge free radicals. In addition, more and more emerging evidence suggests that under some pathological conditions, cell-surface sialic acids also can be desialylated by reactive oxygen species (ROS)/reactive nitrogen species (RNS) via a nonenzymatic, chemical desialylation process, which potentially provides a new dimension for the...
(I don't have access to the study that is needed to finish the sentence.)

Edit in: Desialylation is a pivotal part of sialic acid metabolism, which initiates the catabolism of glycans by removing the terminal sialic acid residues on glycans.
 
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Related editorial:

Editorial: CD24 in the regulation of cellular development and disease​


CD24 (heat stable antigen or nectadrin) is a GPI-linked, highly glycosylated, dynamically expressed molecule present on cells of the immune system, myeloid cells (including granulocytes), keratinocytes, adipocytes, and also on astrocytes, microglia and neurons (1).

Moreover, Mensah et al. previously showed a clear increase in CD24 expression in both naïve and memory IgD+B cell populations in the circulation of individuals with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS)

B cell experiments explore the role of CD24, CD38, and energy metabolism in ME/CFS​

www.frontiersin.org

Frontiers | In vitro B cell experiments explore the role of CD24, CD38, and energy metabolism in ME/CFS

Disturbances of energy metabolism contribute to clinical manifestations of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS). Previously we found t...
www.frontiersin.org www.frontiersin.org

Lastly, Armstrong et al. found that upon in vitro activation with T-dependent or Toll-like-receptor 9 (TLR)-dependent agonists CD24 expression on B cells from ME/CFS patients had a delayed decrease, consistent with in vivo results. Additional metabolomic studies further confirmed an association of CD24 with B cell metabolism, namely a correlation with glucose usage and lactate production. B cells from ME/CFS patients also had significantly higher uptake of amino acids, which has been associated with an increased degradation of nucleotides to AMP perhaps showing a possible link with fatigue in patients with ME/CFS. Thus, alterations in CD24 expression may potentially be useful in exploring B cell immunometabolism.

Much more in there.
 
I don't know if the SIGlecs are what Davide Ret is researching, but looking for links to sialic acid and glycosylation led to the information about SIGlec CD24.



Sorry, CD22 is n-linked glycosylated. I am going to edit that out.
 
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arnoble said:
er, what "chronic inflammatory state" is that?
Click to expand...
I wonder if that would be autoimmunity.
@manruipa at X dot com does have information about how ME/CFS can be related to autoimmune diseases.
This study doesn't specifically mention ME/CFS, but many people with ME/CFS do have symptoms of autoimmunity.

Abnormal B cell glycosylation in autoimmunity: A new potential treatment strategy​


www.frontiersin.org

Frontiers | Abnormal B cell glycosylation in autoimmunity: A new potential treatment strategy

Systemic lupus erythematosus (SLE) and primary Sjögren’s syndrome (pSS) are two autoimmune diseases characterized by the production of pathogenic autoreactiv...
www.frontiersin.org www.frontiersin.org

Systemic lupus erythematosus (SLE) and primary Sjögren’s syndrome (pSS) are two autoimmune diseases characterised by the production of pathogenic autoreactive antibodies.

They likely involve a physiological process known as glycosylation. Both SLE T cell markers and pSS-associated autoantibodies exhibit abnormal glycosylation. Such dysregulation suggests that defective glycosylation may also occur in B cells, thereby modifying their behaviour and reactivity.

This study aimed to investigate B cell subset glycosylation in SLE, pSS and healthy donors and to extend the glycan profile to serum proteins and immunoglobulins. We used optimised lectin-based tests to demonstrate specific glycosylation profiles on B cell subsets that were specifically altered in both diseases.

Compared to the healthy donor B cells, the SLE B cells exhibited hypofucosylation, whereas only the pSS B cells exhibited hyposialylation. Additionally, the SLE B lymphocytes had more galactose linked to N-acetylglucosamine or N-acetylgalactosamine (Gal-GlcNAc/Gal-GalNAc) residues on their cell surface markers.

Interestingly, some similar alterations were observed in serum proteins, including immunoglobulins. These findings indicate that any perturbation of the natural glycosylation process in B cells could result in the development of pathogenic autoantibodies. The B cell glycoprofile can be established as a preferred biomarker for characterising pathologies and adapted therapeutics can be used for patients if there is a correlation between the extent of these alterations and the severity of the autoimmune diseases.
 
Violeta said:
I wonder if that would be autoimmunity.
@manruipa at X dot com does have information about how ME/CFS can be related to autoimmune diseases.
This study doesn't specifically mention ME/CFS, but many people with ME/CFS do have symptoms of autoimmunity.
Click to expand...

The consensus amongst members here is that there is no real evidence for autoimmunity in ME/CFS. There is no relation to other autoimmune disease that I know of, other than some papers suggesting a higher rate of thyroid disease, which may be partly due to overlapping symtoms causing diagnositc ascertainment issues.

There aren't any symptoms of autoimmunity per se, as I understand it. People with ME/CFS will have symptom overlap with lots of other conditions but that in itself tells us nothing much.

The basic question is what they meant by 'chronic inflammatory state'. There isn't any good evidence for inflammation to date. (And autoimmunity isn't necessarily inflammatory.)

Glycosylation changes in disease were trendy in the 1980s and people like Tom Rademacher and John Axford tried to show that the changes were diagnostic or involved in pathogenesis. But it all pretty much fizzled out because nothing clear came out of it. I think ths may be people re-inventing a flat tyre.
 
Jonathan Edwards said:
The basic question is what they meant by 'chronic inflammatory state'. There isn't any good evidence for inflammation to date.
Click to expand...
Oh, maybe the study is referring to inflammation in the brain (myalgic encephalomyelitis).

Molecular Mechanisms of Neuroinflammation in ME/CFS and Long COVID to Sustain Disease and Promote Relapses​

Molecular Mechanisms of Neuroinflammation in ME/CFS and Long COVID to Sustain Disease and Promote Relapses - PMC

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a disease now well-documented as having arisen commonly from a viral infection, but also from other external stressors, like exposure to agricultural chemicals, other types of infection, ...
pmc.ncbi.nlm.nih.gov pmc.ncbi.nlm.nih.gov

Brain and muscle chemistry in myalgic encephalitis/chronic fatigue syndrome (ME/CFS) and long COVID: a 7T magnetic resonance spectroscopy study​


www.nature.com

Brain and muscle chemistry in myalgic encephalitis/chronic fatigue syndrome (ME/CFS) and long COVID: a 7T magnetic resonance spectroscopy study - Molecular Psychiatry

Myalgic encephalitis/chronic fatigue syndrome (ME/CFS) is a common debilitating medical condition, whose main symptoms - fatigue, post-exertional malaise and cognitive dysfunction – are also present in many cases of long COVID. Magnetic resonance spectroscopy (MRS) allows the insight into their...
www.nature.com www.nature.com
 
Violeta said:
Oh, maybe the study is referring to inflammation in the brain (myalgic encephalomyelitis).
Click to expand...

Myalgic encephalomyelitis is an anachronistic name referring to apparent localising neurological signs suggesting encephalitis in an acute epidemic illness (at the Royal Free Hospital and maybe in Iceland before) that was thought to precede what we now call ME/CFS. In the end no evidence for. any specific illness with encephalitis was established. Putative neuroinflammation in terms of this myalgic encephalomyelitis has nothing to do with our current concept of ME/CFS.

Unfortunately, there seem to be a number of researchers who have not quite grasped this!

The question is whether or not there is brain inflammation in ME/CFS (a term with a different meaning). The Tate study is speculative. The other study is one of many that have produced conflicting results. The overall body of evidence points to no inflammation but maybe there are some changes in that direction in ME/CFS that we have missed.
 
Violeta said:
What does the ME in ME/CFS stand for?
Click to expand...

It came from 'myalgic encephalomyelitis' but as part of a confusion between myalgic encephalomyelitis as an acute infective epidemic disease with signs of brain injury and the 'chronic ME' that Ramsay described as following it, which was ME/CFS but Ramsay probably thought it was a later phase of the other illness that never materialised.

Names in disease nomenclature are often illogical and borrowed by mistake. Rheumatoid arthritis is called that because it seemed a bit like rheumatic fever but very likely what people thought was chronic rheumatic fever was rheumatoid arthritis in the first place. Polymyalgia rheumatica used to be called senile rheumatic gout but it has nothing to do with gout, and so on.

The great thing about 'ME/CFS' to my mind is that like 'SLE' or 'MS' it allows you to forget where the letters come from and focus on the modern definition of the clinical problem.
 
«ME/CFS» just means the syndrome that’s roughly what’s described by the Canada Consensus Criteria etc.

It doesn’t mean «ME or CFS» or anything like that.
 
I just googled ME/CFS, and there are pages of sites that say it is myalgic encephalomyelitis.
I don't know what's going on here, but I'm out of here.
This is crazy.
 
Violeta said:
I just googled ME/CFS, and there are pages of sites that say it is myalgic encephalomyelitis.
Click to expand...
The ME in ME/CFS does stand for myalgic encephalomyelitis, it is confusing. But the point people are making is that despite "encephalomyelitis" being in the name, no actual brain inflammation has been proven. It was initially suspected in the historical cases of ME, and despite not being supported by evidence, the terminology ended up getting rolled into the name "ME/CFS" by a different group
 
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