A theory for the pathogenicity of COVID-19

1. A Theory for the Pathogenicity
of SARS-CoV-2
April 28/20
Updated January 19/22

2. Fcgbp = Fc of IgG binding protein
Fc of IgG binding domain
Von Willebrand Factor D domain
Cysteine rich
Trypsin Inhibitor – like domain
- 5405 amino acids
- 572 kDa

3. Biophysical Journal 111, July 26, 2016

4. VWFD Domains allow auto-oligomerization
of Fcgbp
1 2 3 4 5 6 7 8 9 10 11 12
13
1 2 3 4 5 6 7 8 9 10 11 12
13
1 2 3 4 5 6 7 8 9 10 11 12
13
VWF Factor VIII

5. Fcgbp Immunoprecipitation With Human
Serum

6. • It has a HMW with S-S linkages
• It is secreted with mucus from goblet cells into
the intestinal tract
• It may be glycosylated
• It contains cysteine-rich domains with the
conserved amino acid motif CGLCGN,
characteristic of thioredoxins, suggesting it may
function as an anti-oxidant.

7. Estimated Protein
Expression
FCGBP Genecard

8. Viral Infection:
1.
2.
Fcgbp
3.
4.
Virus breaches protective barrier,
infecting cells and eliciting an IgG
response if the body has seen the
invader before.
IgG binds virus with the antigen
binding domain and binds to Fcgbp
via the Fc domain, protecting the
tissue from further infection.
Fcgbp forms a scaffold within the
ECM overlaying the epithelium.

9. A comparison of the structures IgG and the Receptor for advanced glycation end
products (RAGE)
Taken from Wikipedia

10. December 2012, volume 7, issue 12
Disulfide bond-mediated RAGE dimerization in the ER is
the critical step of RAGE biogenesis. Without
formation of intermolecular disulfide bonds in the C2
region, RAGE fails to reach cell surface.

11. Fcgbp
sRAGE
Fc fragment of IgG aa sequence
Proposed mechanism for anti-inflammatory effect of sRAGE
HMGB1

12. 1 2 3 4 5 6
250 kDa
150 kDa
100 kDa
75 kDa
50 kDa
37 kDa
Lane 1: 75 ugs N1 total protein lysate
Lane 2: unbound RAGE IP
Lane 3: unbound Fcgbp IP
Lane 4: RAGE IP
Lane 5: Fcgbp IP
Lane 6: Precision Plus Protein Marker
Mouse Lung Protein

13. Immunoprecipitation of Fcgbp brings down RAGE
RAGE protein in mouse is ~44.2 kDa

14. Immunofluorescence: Airway in lung of control mouse.
Fcgbp shows up as red protein lining the lumen.
40x

15. Immunofluorescence: Fcgbp in the
airway of “house dust mite challenged”
mouse is down regulated or degraded.
20x

16. Immunofluorescence: sRAGE
presumably bound to Fcgbp in airway of
“house dust mite challenged” mouse.
20x

17. Fcgbp in human lung 20x

18. HL012 RAGE 40x-1

19. MaRAGE in human lung 20x

20. Receptor for advanced glycation end products
• RAGE is a multi-ligand binding member of the immunoglobulin superfamily.
• It is highly expressed as a membrane bound receptor on lung alveoli.
• It is also expressed in multiple cell types, including neurons of the central and
peripheral nervous system (COVID-19 patients report loss of sense of smell),
vascular and inflammatory cells (DCs and neutrophils. COVID-19 patients have
neutrophilia.)
• Its activation targets NFkB and CREB and AP-1 signaling which could influence
procalcitonin and Il-6 transcription, which have been observed to be
upregulated in patients with COVID-19.
• The soluble form of RAGE can act as a decoy to resolve inflammation. Evidence
suggests that it can bind to Fcgbp.

21. RAGE activation NFkB RAGE and Il-6
CREB Procalcitonin and Il-6, CCL3 and CCL4
AP-1 ADAM 10 and Il-6
Notes: ADAM 10 cleaves mRAGE to form sRAGE.
PCT is similar to the proteins belonging to the ADAM family.
CCL3 and CCL4 = Macrophage inflammatory protein 1 (MIP-1).
MIP-1 can cause rapid onset of fever. It also attracts neutrophils.
Transcriptome data from BALF of COVID-19 patients identified CCL3 and CCL4
as being highly expressed compared to controls.
Transcription activated by RAGE

22. FTGCVIAWNSNNLDSK VGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTP
KADKARYEREMKTYIPPKGET
SPIKE GP (429-479)
HMGB1 (65-85)
YGGC GGNRNNFDTEEYCMAVCG SAMSQSLLKTTQEPLARDPVKLPTTAASTP Amyloid precursor protein
(321-372)
WNNTAADDK QPJYEKKAAKLKEEKYEKDIAA YRAKGKP HMGB1 (133-168)
NCBI 2 Protein BLAST Results
Spike gp SARS CoV-2 QIC53213.1
HMGB1 CAG33144.1
Amyloid precursor protein PO5067.3
Ligands of RAGE
Can SARS-CoV-2 mimic
a ligand of RAGE?
The spike gp receptor-binding motif for ACE2 is amino acids 424-494. Amino acid residues 479 and 487
are important for virus progression. (PMID: 32338224)

23. Extracellular HMGB1: a therapeutic target in severe
pulmonary inflammation including COVID-19?
(Mol Med 2020, 26: 42)
• A hyperexcited HMGB1-RAGE axis can be expected in COVID-19 since necrotic respiratory
epithelial cells contribute with large amounts of extracellular HMGB1, and its cognate receptor
RAGE is constitutively abundantly expressed in the lungs.
• Arterial HMGB1 levels are considerably lower than those in venous blood and we propose that
this connects systemic HMGB1 biology to lung physiology.
• HMGB1 is a DNA- and RNA-binding molecule and HMGB1 has been demonstrated to function as
a gene delivery agent. There is a risk that HMGB1 might attach to viral RNA and bring it to the
cytosol via the RAGE-lysosomal pathway. This would implicate that there could be an additional
pathway apart from the angiotensin-converting enzyme 2 receptors enabling intracellular virus
replication.

24. • The autocrine/paracrine production of HMGB1 by maturing DCs leads to RAGE activation,
facilitating the maturation and mobilization of DCs.
• ….RAGE expression by maturing DCs is necessary for migration to the draining lymph node.
Maturing Dendritic Cells Depend on RAGE for
In Vivo Homing to Lymph Nodes
(J of Immuno, 2008, 180)
If SARS-CoV-2 mimics HMGB1, can this mechanism account for the lymphocytopenia and
necrosis of the spleen and lymph nodes observed in COVID-19 patients?

25. Hypothesis of COVID-19 Pathogenesis
• SARS-CoV-2 infects cells expressing the angiotensin-converting enzyme 2,
or ACE2. (A side effect of ACE2 inhibitors is a cough)
• ACE2 is expressed on lung alveolar epithelial cells, the same cell type
expressing RAGE.
• Infection of alveolar cells would cause cell death, releasing HMGB1, a
ligand of RAGE. This in turn would lead to ADAM 10 production, resulting in
membrane bound RAGE to be cleaved at the cell membrane surface,
generating sRAGE. Soluble RAGE can anchor itself with bound ligand to
Fcgbp (presumably to ameliorate local inflammation).
• If the SARS-CoV-2 virus is mimicking a RAGE ligand, it could be responsible
for the accumulation of extracellular protein observed in the histology of
alveolar tissue from these patients.

26. Polymorphisms of RAGE
• The SNP rs1899624 (or -374T/A polymorphism) is located in the promoter region of the AGER gene.
• It is associated with a three-fold increase in AGER expression in vitro, including sRAGE.
• In a paper published in 2013 (PMID 24284407), a group in Italy investigated the frequency of
rs1899624 in subjects over 90 years of age. (Apparently, Italy is home to the second oldest
population in the world.) They found an association between the A allele and genotype homozygous
for this allele (AA) and longer lifespan in the male population. They posit a possible role of -374/A
polymorphism in life longevity in their country. Intuitively it makes sense that the availability of
more sRAGE during episodes of inflammation could ameliorate symptoms and return the effected
tissue to homeostasis faster.
• If SARS-CoV-2 mimics a ligand of RAGE perhaps increased expression of the receptor becomes a
liability, causing more damage to the alveoli, and making it more difficult for the system to clear the
virus.
• I wonder if there is a genetic component to the extreme rates of mortality observed in the COVID-19
pandemic experience in Italy. Furthermore, I wonder if Italian ancestry (and greater frequency of
this polymorphism) is a factor in the similar trajectory and death rate observed in New York city.

27. Winnipeg Free Press April 9/20

28. Prevalence
N (%)
Median of sRAGE (pg/ml)
(Range)
P

29. • Rs2070600 located in exon 3, a putative site of ligand binding (V domain)
• G>A, Gly82Ser, 92.8 vs 7.2% in general population, 78.1 vs 21.9% in EAS (east Asian
pop.)
• Associated with an increased RAGE affinity for ligand AGEs
• Associated with a decrease in proteolysis of RAGE
• Minor allele is associated with a 50% decrease in sRAGE in American whites and
blacks
• 82Ser/Ser is rare and associated with higher risk for cardiovascular disease,
inflammation, oxidative stress, and insulin resistance
• Rs2071288 is located in intron 9, a splice site
• G>A, 94.6 vs 5.4% in general pop., 96.7 vs 3.3 in EAS
• Associated with 43% decrease in sRAGE in African Americans
• Minor allele frequency of 0.10 in African Americans

30. Could an increase in plasma soluble RAGE be responsible
for the additional damage from SARS-CoV-2?

31. Gene Expression Profile Protein Expression Profile
AGER Genecards

32. HDM-1 Chronic vs Fcgbp 40x-4 HDM-1 Chronic vs RAGE 40x-2
Lung From House Dust Mite Treated Mouse

33. • Findings suggest that among at-risk ICU patients, higher plasma sRAGE may identify
those who are more likely to develop Acute Respiratory Distress Syndrome.
• AGER SNP rs2070600 (Ser/Ser in LBD) was associated with increased ARDS risk and
higher plasma sRAGE in this cohort.

34. • Soluble RAGE correlated with sepsis severity (p=0.00001).
• Increased sRAGE was associated with 28-day mortality in patients with sepsis.

35. • Treatment with either anti-RAGE mAb or sRAGE improved lung injury, and
decreased alveolar inflammation in acid-injured animals.
• The balance of RAGE isoforms has been described as an important regulator of
RAGE activation. Generally, mRAGE may promote disease pathogenesis and
injury by activating the NFkB pathway, while sRAGE is protective by preventing
or reversing mRAGE signaling in disease.

36. • Treatment of the injured lung with rsRAGE significantly suppressed the lung
edema, the neutrophils infiltration, and the release of HMGB1.
• Supplementary therapy with sRAGE may be an effective therapeutic strategy for
ARDS.

37. Summary
SARS-CoV-2 infects type 1 and/or type 2 alveolar epithelial cells
Amplification and release of new virus particles
Cell death and release of HMGB1
HMGB1 binds to RAGE and activates NFkB, CREB, and AP-1 signaling, resulting in upregulation
of RAGE, Il-6, MIP-1, and procalcitonin. SARS-CoV-2 binds to RAGE (amplifying signal?)
mRAGE is cleaved to form sRAGE
sRAGE binds its ligands and anchors itself to Fcgbp Recovery
If there is not enough Fcgbp or sRAGE, complications arise (Cytokine storm?)
or
Death