Modelling of the Human Major Prion Protein Three-dimensional Structure
Following assessment with the I-TASSER server [44], five 3D models of the huPrP were predicted, but only the best protein model (Fig. 2A) was selected based on the confidence score (C-score = -4.13), estimated template modelling score (TM-score = 0.28 ± 0.09), and estimated root mean square deviation (RMSD = 16.0 ± 3.1 Å) score. The Ramachandran plot analysis of huPrP by PDBsum server [46] showed 68.4% residues in most favoured region, 24.7% in allowed regions, 3.7% in generously allowed regions, and 3.2% residues in the disallowed regions for the predicted prion protein structure (Fig. 2B). The ProSA server [47] assesses the overall and local model quality of the predicted protein structure. The local model quality is shown by knowledge-based energy score, in which the positive values represent the erroneous or problematic regions of the input structure (Fig. 2C). The sequence position in the negative values of the knowledge-based energy confirms the good quality of our predicted protein structure. Moreover, ProSA also provides the z-score for overall model quality prediction and was − 5.74, indicating that it is located in the acceptable area [47] (Additional file 1: Figure S1A). The Verify 3D determine the compatibility of an atomic model (3D) with its own amino acid sequence (1D) by assigning a structural class based on its location and environment (alpha, beta, loop, polar, non-polar etc.) and compares the results to good structures [48]. For our protein structure, we found 80.63% of the residues have averaged 3D-1D score ≥ 0.2, well within the acceptable limit to be considered a good quality protein structure (Additional file 1: Figure S1B and Figure S1C).
Identification of Allergenic B Cell Epitopes in the Three-dimensional Structure of the Human Major Prion Protein
We predicted the linear B cell epitopes from the huPrP 3D structure and found 10 B cell linear epitopes ranging from 4 to 26 amino acids long with a protrusion index (PI) of 0.503 to 0.798 (Table 1) [66]. The full-length huPrP is divided into three major parts, including the flexible tail (FT) region (23–123), which comprises the octa-peptide repeats (OR) region (50–90) and the globular domain (GD) regions (124–230). We found four epitopes (epitope L7: 24–49, epitope L9: 56–66 and epitope L10: 76–86, epitope L2: 89–111) in the FT region, two of them located in the OR region. The GD contained three epitopes (epitope L4: 137–153, epitope L3: 167–174 and epitope L6: 189–204). On the contrary, the short epitope 1–4 (epitope L8) is located in the N-terminal region (signal peptide region) and comparatively two longer epitopes L1:222–238 (both in GD and non-structured region) and L5:245–253 (non-structured region) are located in the C-terminal region of the full length huPrP (Table 1). The position of the B cell linear epitopes on the protein structure are illustrated in (Fig. 3A & 3B). We also predicted 9 B cell conformational epitopes, shown in Table 2. The length of the B cell conformational epitopes ranged between 4 and 36 amino acid residues. The protrusion index value for the B cell conformational epitopes ranged between 0.55 and 0.883.
Table 1
Predicted linear B cell epitopes and their toxicity and allergenicity from the three-dimensional structure of the human major prion protein.
Epitope No.
|
Position
|
Peptide
|
Number of Residues
|
Score
|
Toxicity
|
Overall
|
Allergenicity
|
Overall
|
SVM Method
|
QM Method
|
AllergenFP
|
AllerTop 2.0
|
L1
|
222–238
|
SQAYYQRGSSMVLFSSP
|
17
|
0.798
|
Non-toxic
|
Non-toxic
|
|
Non-allergenic
|
Non-allergenic
|
|
L2
|
89–111
|
WGQGGGTHSQWNKPSKPKTNMKH
|
23
|
0.767
|
Non-toxic
|
Toxic
|
Toxic
|
Non-allergenic
|
Allergenic
|
Allergenic
|
L3
|
167–174
|
DEYSNQNN
|
8
|
0.751
|
Non-toxic
|
Non-toxic
|
|
Allergenic
|
Non-allergenic
|
Allergenic
|
L4
|
137–153
|
PIIHFGSDYEDRYYREN
|
17
|
0.737
|
Non-toxic
|
Toxic
|
Toxic
|
Allergenic
|
Non-allergenic
|
Allergenic
|
L5
|
245–253
|
SFLIFLIVG
|
9
|
0.701
|
Non-toxic
|
Non-toxic
|
|
Non-allergenic
|
Non-allergenic
|
|
L6
|
189–204
|
VTTTTKGENFTETDVK
|
16
|
0.685
|
Non-toxic
|
Non-toxic
|
|
Non-allergenic
|
Non-allergenic
|
|
L7
|
24–49
|
KRPKPGGWNTGGSRYPGQGSPGGNRY
|
26
|
0.676
|
Non-toxic
|
Toxic
|
Toxic
|
Non-allergenic
|
Non-allergenic
|
|
L8
|
1–4
|
MANL
|
4
|
0.559
|
Non-toxic
|
Non-toxic
|
|
Allergenic
|
Allergenic
|
Allergenic
|
L9
|
56–66
|
GWGQPHGGGWG
|
11
|
0.541
|
Non-toxic
|
Toxic
|
Toxic
|
Non-allergenic
|
Allergenic
|
Allergenic
|
L10
|
76–86
|
PHGGGWGQPHG
|
11
|
0.503
|
Non-toxic
|
Toxic
|
Toxic
|
Non-allergenic
|
Allergenic
|
Allergenic
|
Table 2
Predicted conformational B cell epitopes from the three-dimensional structure of the human major prion protein.
Epitope No.
|
Residues
|
Number of residues
|
Score
|
C1
|
Q227, R228, G229, S230, S231, M232, V233, L234, F235
|
9
|
0.883
|
C2
|
N100, K101, P102, S103, K104, P105, K106, T107, N108, M109, K110, H111, M112
|
13
|
0.811
|
C3
|
R25, P26, K27, P28, G29, G30, W31, N32, T33, G34, G35, S36, R37, Y38
|
14
|
0.747
|
C4
|
P137, I138, I139, H140, F141, G142, S143, D144, Y145, E146, D147, R148, Y149, R151, E152, N153, Y157, V189, T191, T192, T193, K194, G195, E196, N197, F198, T199, E200, T201, D202, V203, K204
|
32
|
0.717
|
C5
|
R164, E168, Y169, S170, N171, Q172, N173, N174, H177
|
9
|
0.708
|
C6
|
S222, Q223, A224, Y225
|
4
|
0.708
|
C7
|
S236, S237, P238, L242, S245, F246, L247, I248, F249, L250, I251, V252, G253
|
13
|
0.669
|
C8
|
P39, G40, Q41, G42, S43, P44, G45, G46, N47, R48, Y49
|
11
|
0.579
|
C9
|
G55, G56, W57, G58, Q59, P60, H61, G62, G63, G64, W65, G66, Q67, Q75, P76, H77, G78, G79, G80, W81, G82, Q83, P84, H85, G86, W89, G90, Q91, G92, G93, G94, T95, H96, S97, Q98, W99
|
36
|
0.55
|
The B cell linear epitope L2 overlapped with B cell conformational epitopes C2 and C9 (Tables 1 & 2). Further, linear epitope L4 also overlapped with conformational epitope C4 (Tables 1 & 2). On the other hand, rest of the other linear epitopes L1 overlapped with both C1 and C6; L3 overlapped with C5; L5 overlapped with C7; L6 overlapped with C4; L7 overlapped with both C3 and C8; and both L9 and L10 overlapped with C9 (Fig. 3C & 3D).
In this in silico analysis, the prediction of the toxicity for each linear epitope was achieved with ToxinPred server (http://crdd.osdd.net/raghava/toxinpred/) using Support Vector Machine (SVM) and Quantitative Matrix (QM) method [50]. The toxicity prediction results of the linear epitopes are shown in Table 1. The SVM method did not identify ‘toxic’ B cell linear epitopes from 3D structure however, the QM method identified epitopes L2 (89–111), L4 (137–153), L7 (24–49), L9 (56–66), and L10 (76–86) as being toxic. Of note, L2 and L4 epitope contain the binding sequences for the ‘neurotoxic’ anti-PrP antibodies ICSM35/POM3 antibodies [40, 43] and ICSM18/POM1 antibodies [40, 43], respectively. Interestingly, epitopes L9 (56–66) and L10 (76–86) are located in the octa-repeat region of the huPrP protein that contains the binding sequences for SAF 32 (59–89) [42] and POM2 (57–88) antibodies [43], respectively. We also predicted the allergenicity for the B cell linear epitopes using the AllergenFP [52] and AllerTop [51] allergenicity prediction server. L3 (167–174), L4 (137–153), and L8 (1–4) were predicted to be allergenic in AllergenFP, while AllerTop server identified epitopes L2 (89–111), L8 (1–4), L9 (56–66), and L10 (76–86) as allergenic. The allergenicity prediction results are shown in Table 1. Three linear epitopes were shown to be non-toxic and non-allergenic and included L1 (222–238) and L6 (189–204) on the globular domain and L5 (245–253) located on the non-structured region. We therefore investigated whether ICSM18, ICSM35, POM1, POM2, POM3, SAF32, and SAF70 antibodies trigger a neuronal allergenic reaction in vitro.
Anti-PrP Antibody Treatment of Mouse Neuroblastoma Cells Leads to Differential Expression of Fcγ receptors
Fcγ receptors (FcγRs) are subdivided into FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) according to their structural homology and binding affinity with IgG (reviewed in [67]). Moreover, FcγRs signal through immune tyrosine activating or inhibitory motifs to inhibit (FcγRIIb) or activate (FcγRI, FcγRIIa/c, FcγRIII) immune functions (reviewed in [67]). Here, we wanted to verify whether direct anti-PrP treatment of neurons or via co-culture of pre-treated microglia affects expression of both FcγRI and FcγRIII (FcγRIIa is not expressed in mice). Western blot analysis showed a ∼250 kDa CD64 (predicted MW: ~72 kDa) and CD16 (predicted MW: ~16 kDa) band associated with both SAF70 and POM1 when compared to untreated control in DAT (Fig. 4A). Interestingly, no such band was detected with ICSM 18 treatment following DMT, however, two bands ranging between 15–20 kDa were observed with the untreated control but significantly less intense with antibody treatment. Interestingly, a similar band pattern was also observed following probing with anti-phospho-serine and anti-phospho-tyrosine antibodies, signifying an inhibitory effect and/or dephosphorylation by anti-PrP antibodies (Fig. 4A). DMT with anti-PrP antibodies does not appear to affect expression of both CD64 and CD16 (Fig. 4B), however, DMT with ICSM18 showed one band pattern at ~ 150 kDa as compared with other antibodies that showed a two-band pattern at ~ 150 and 250 kDa following probing with anti-phospho-tyrosine antibody (Fig. 4B). Finally, IMT with anti-PrP antibodies led to increased CD64 and CD16 expression with ICSM18 and POM1, but lower expression with SAF70 (Fig. 4C). Similar band pattern was also observed when probing with anti-phospho-serine and anti-phospho-tyrosine antibodies (Fig. 4C). Of note, immunofluorescence studies have not been remarkable and did not show differences in the expression levels of FcγRs in N2a cells (data not shown).
Treatment of Mouse Neuroblastoma Cell lines with Anti-PrP Antibodies Leads to Neuronal Allergenicity
Treatment of N2a cells with anti-PrP antibodies ICSM18, ICSM35, POM1 and SAF70 led to the identification of 211 proteins (p < 0.05) after LC-MS analysis when compared with untreated N2a cells. Out of the 211 proteins, only the differentially expressed proteins were considered using maximum fold change ≥ 10, at least 2 identified unique peptides and a confidence score ≥ 40. The stringent parameters used here led to the identification of 26 proteins (Additional file 2: Table S1). Of note, the stringent parameters used to identify proteins associated with anti-PrP treatment are unusually high and would allow elimination of ‘false-negatives’ post LC-MS analysis. The 26 proteins were then assessed for allergenicity using AllergGAtlas database (http://biokb.ncpsb.org/AlleRGatlas/) [62] and 4 allergy related proteins, including beta-actin (ACTB), fatty acid-binding protein 5 (FABP5), protocadherin 11 (PCDH11X), and myomegalin (PDE4DIP). Among the 4 allergenic-related proteins, ACTB, PCDH11X and PDE4DIP were upregulated but FABP5 was found to be down regulated when compared to untreated control (Table 3). The functional annotation of the 4 allergenic-related proteins through DAVID bioinformatics resources [59, 60] showed that ACTB is associated with platelet aggregation and cellular response to electrical stimulus; PCDH11X is involved in the negative regulation of phosphatase activity; FABP5 was found to be associated with phosphatidylcholine biosynthetic process and transport while PDE4DIP was found to be involved in cellular protein complex assembly.
Table 3
Properties of the identified allergenic proteins following direct antibody treatment (DAT) of the neuroblastoma cell line. The properties were identified by Progenesis Software after the LC-MS analysis.
Accession
|
Gene Id
|
Similar Protein Family in AllerGAtlas Server
|
Protein Name
|
Anova (p)
|
Max Fold Change
|
Confidence Score
|
Peptides
|
Unique peptides
|
Highest Mean
|
Lowest Mean
|
Q05816
|
Fabp5
|
Fabp4
|
Fatty acid-binding protein 5
|
0.01718987
|
16
|
260
|
22
|
3
|
Control
|
DAT
|
B1AZR7
|
Pcdh11X
|
Pcdh1
|
Protocadherin 11
|
0.00666053
|
11.7
|
60.8
|
9
|
4
|
DAT
|
Control
|
Q3UBP6
|
Actb
|
-
|
Uncharacterized protein
|
0.01880576
|
12.3
|
802
|
58
|
3
|
DAT
|
Control
|
Q3UR03
|
Pde4dip
|
Pde4a
|
Myomegalin (Fragment)
|
0.02084224
|
29.9
|
49.1
|
7
|
3
|
DAT
|
Control
|
Protein-protein interaction of the identified 4 allergenic-related proteins with prion protein (PRNP) showed that PrP networks with ACTB via Cofilin-1 (CFL1), while no direct interaction was observed for FABP5, PCDH11X and PDE4DIP (Fig. 5A). Finally, analysis of individual anti-PrP antibody treatments revealed that PDE4DIP was present after DAT with ICSM18 and POM1 treatment, however, DAT with ICSM35 and SAF70 was not found to be associated with allergy related proteins (Table 4).
Table 4
Identification of antibody-specific allergenic proteins following direct antibody treatment (DAT). (√) Present and (-) Absent
Gene ID
|
Accession
|
ICSM Antibodies
|
SAF Antibody
|
POM Antibody
|
ICSM18
|
CTL
|
ICSM35
|
CTL
|
SAF70
|
CTL
|
POM1
|
CTL
|
Q05816
|
Fabp5
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
B1AZR7
|
Pcdh11X
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
Q3UBP6
|
Actb
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
Q3UR03
|
Pde4dip
|
√
|
-
|
-
|
-
|
-
|
-
|
√
|
-
|
Gene Ontology (GO) Analysis of Allergy Related Proteins Associated with Direct Antibody Treatment
We performed Gene Ontology (GO) analysis using the gene classification server, PANTHER classification system (v.14.0) [63], and the protein gene enrichment software, FunRich (Functional Enrichment analysis tool-Version 3.1.3) [64] (reference list “rodent database”) for the analysis of cellular components, molecular function, biological process, and the signalling pathway. PANTHER identified ACTB and PCDH11X allergenic- related genes involved in cellular components, biological processes and signalling pathways. The cellular component analysis of the 4 allergenic-related proteins showed that both ACTB and PCDH11X are associated with cell and membrane while ACTB is involved in membrane-enclosed lumen, organelle, protein-containing complex, and supramolecular complex (Fig. 5B). The molecular function of ACTB gene was found to be associated with binding and structural molecule activity (data not shown). The biological process of PCDH11X gene was found to be associated with biological adhesion, whereas ACTB was found to be involved in biogenesis, cellular process, developmental process, localization, locomotion, and multicellular organismal process (Fig. 5C). Signalling pathway analysis identified ACTB and PCDH11X association with cadherin signaling and Wnt signaling pathways. In addition, signaling pathway analysis of ACTB was found to be involved in Alzheimer disease-presenilin pathway, cytoskeletal regulation by Rho GTPase, Huntington disease, integrin signalling pathway, nicotinic acetylcholine receptor signaling pathway, and inflammation mediated by chemokine and cytokine signaling pathway (Fig. 5D).
FunRich analysis showed six cellular components such as postsynaptic density (ACTB, FABP5; p = 0.009), synapse (ACTB, FABP5; p = 0.065), postsynaptic actin cytoskeleton (ACTB; p = 0.027), NuA4 histone acetyltransferase complex (ACTB; p = 0.029), dense body (ACTB; p = 0.009), and tight junction (ACTB; p = 0.038) (Fig. 6A). FunRich analysis also identified different biological processes such as regulation of prostaglandin biosynthetic process (FABP5; p < 0.001), cellular response to cytochalasin B (ACTB; p = 0.004), regulation of norepinephrine uptake (ACTB; p = 0.01), regulation of retrograde trans-synaptic signaling by endocannabinoid (FABP5; p = 0.01), negative regulation of glucose transmembrane transport (FABP5; p = 0.025), protein localization to adherens junction (ACTB; p = 0.031), regulation of transepithelial transport (ACTB; p = 0.031), and positive regulation of microtubule nucleation (PDE4DIP; p = 0.038) (Fig. 6B). On the other hand, reactome pathway analysis through FunRich identified cell-extracellular matrix interactions (p = 0.014), interaction between L1 and ankyrins (p = 0.008), formation of annular gap junctions (p = 0.019), gap junction degradation (p = 0.021) for ACTB and triglyceride catabolism (p = 0.021) for FABP5 (Fig. 6C).
Co-Culture of Anti-PrP Antibody Treated-Microglia with Mouse Neuroblastoma Cell Line Leads to Neuronal Allergenicity
Co-culture of N2a cells with anti-PrP antibody-treated N11 cells led to the identification of 2346 proteins (only p < 0.05) after LC-MS analysis when compared with co-culture of N2a cells with untreated-N11 cells. Out of the 2346 proteins, only the differentially expressed proteins were considered using maximum fold change ≥ 10, at least 2 identified unique peptides and a confidence score ≥ 40. The stringent parameters used here led to the identification of 113 proteins (Additional file 2: Table S2). The 113 proteins were assessed for allergenicity using AllergGAtlas database and 8 proteins were confirmed to be allergenic (Table 5), including IF rod domain-containing protein (VIM), peroxiredoxin-1 (PRDX1), Legumain (LGMN), cytoskeletal beta-actin (ACTB), V(D)J recombination-activating protein 1 (RAG1), L-lactate dehydrogenase (LDHA), Receptor-type tyrosine-protein phosphatase C (PTPRC), and TIR domain-containing protein (TLR3). Among the identified 8 allergenic-related proteins, 7 (VIM, LGMN, ACTB, RAG1, LDHA, TLR3, PTPRC) showed the highest mean for DMT when compared with N2a cultured with untreated-N11 cells (Table 5). On the other hand, PRDX1 was found to be downregulated (Table 5).
Table 5
Properties of the identified allergenic proteins following direct microglia treatment (DMT) on the neuroblastoma cell line. The properties were identified by Progenesis Software after the LC-MS analysis.
Accession
|
Gene ID
|
Protein Name
|
Anova (p)
|
Max Fold Change
|
Confidence Score
|
Peptides
|
Unique Peptides
|
Highest Mean
|
Lowest Mean
|
Q3TWV0
|
Vim
|
IF rod domain-containing protein
|
0.000478
|
221
|
865
|
61
|
2
|
DMT
|
Control
|
B1AXW5
|
Prdx1
|
Peroxiredoxin-1 (Fragment)
|
0.010465
|
10.7
|
280
|
28
|
3
|
Control
|
DMT
|
A2RTI3
|
Lgmn
|
Legumain
|
3.27E-07
|
443
|
51.3
|
8
|
2
|
DMT
|
Control
|
O89054
|
Actb
|
Cytoskeletal beta-actin (Fragment)
|
2.63E-05
|
28.2
|
201
|
16
|
2
|
DMT
|
Control
|
Q78NA6
|
Rag1
|
V(D)J recombination-activating protein 1
|
2.91E-13
|
13.5
|
95.2
|
16
|
5
|
DMT
|
Control
|
Q3UDU4
|
Ldha
|
L-lactate dehydrogenase
|
7.9095E-05
|
13.5
|
474
|
41
|
6
|
DMT
|
Control
|
Q3TM31
|
Tlr3
|
TIR domain-containing protein
|
0.043612
|
14.8
|
43.2
|
9
|
2
|
DMT
|
Control
|
P06800
|
Ptprc
|
Receptor-type tyrosine-protein phosphatase C
|
6.11E-07
|
12.5
|
41.1
|
6
|
2
|
DMT
|
Control
|
Protein-protein interaction of the identified 8 allergenic-related proteins showed that PrP interacts with ACTB via Cofilin-1 (CFL1), while VIM, PTPRC, and LDHA directly interacts with ACTB (node 1, 2, 3, 4 in Fig. 7). It was previously shown that overexpression of PrPC itself activates the NADPH oxidase (NOS) for reactive oxygen species (ROS) production that initiates the cofilin activation and finally induce cofilin-actin rods in hippocampal neurons [68].
A study by Esue et al. demonstrated a direct interaction between actin and vimentin filaments mediated by the tail domain of vimentin [69]. Kristiansen et al. showed that mild proteasome impairment in prion-infected cells leads to the formation of aggresomes that contain VIM, HSP70, ubiquitin as well as proteasome subunits [70]. Of interest, PTPRC was found to be upregulated in mice brain following inoculation with prions [71]. A study by Ramljak et al. showed that there is a direct interaction between PrPC and lactate dehydrogenase (LDHA) and revealed that LDHA expression is increased under hypoxic conditions [72]. Protein-protein interaction also showed that both RAG1 (node 6 in Fig. 7) and TLR3 (node 5 in Fig. 7) indirectly interact with ACTB (node 1 in Fig. 7) via PTPRC (node 3 in Fig. 7) while PRDX1 (node 7 in Fig. 7) indirectly interacts with ACTB via LDHA (node 4 in Fig. 7) (Fig. 7). A study by Wagner and co-workers showed that PRDX6 was upregulated in scrapie-infected mice and neuronal cell lines [73]. However, LGMN (node 8 in Fig. 7) did not interact with any of the identified allergenic-related proteins as well as with prion protein (node 9 in Fig. 7).
Among the identified allergenic-related proteins, VIM was found to be involved in the progression of allergic diseases via inflammasome [74, 75] and VIM-P38MAPK complex facilitates mast cell activation via FcϵRI/CCR1 activation [76]. LDHA was identified as a potential marker in allergic alveolitis, airway inflammation, allergic encephalomyelitis, asthma disease [77–80]. PRDX1 was found as a negative regulator of inflammation [81], Th2-type airway inflammation, and allergen-related hyperresponsiveness [82]. PTPRC was found to be associated with asthma related phenotypes in a microarray analysis [83]. LGMN was found to be involved in allergic reaction by potentiating antigen processing [84]. A study by Sehra et al. showed that RAG1-deficient mice exhibited reduced mast cell infiltration when it was used as a chronic model of allergic inflammation [85].
TLR3 activation in an established experimental allergic asthma mice model increased the release of proinflammatory cytokines and mucus production which was also associated with the increased production of interleukin 17 (IL-17A) by natural killer (NK) cells [86].
The highest versus lowest mean of the allergenic-related protein expression by individual antibody treatments when compared to untreated control is shown in Table 6. Here, we show that ICSM18, ICSM35, and SAF70 share 62.5% allergy related proteins (5 proteins: VIM, LGMN, RAG1, LDHA, and PTPRC). On the other hand, both POM2 and SAF32 showed 75% effect with 6 proteins, but the proteins were found to be different for both POM2 (VIM, ACTB, LGMN, RAG1, LDHA, and TLR3) and SAF32 (VIM, ACTB, LGMN, RAG1, LDHA, and PTPRC). However, the lowest effect of antibody was observed for both POM1 (4 proteins; VIM, ACTB, RAG1, and TLR3) and POM3 (4 proteins; VIM, ACTB, LGMN, and LDHA) with 50% effect.
Table 6
Identification of antibody-specific allergenic proteins following direct microglia treatment (DMT). (√) Present and (-) Absent
Accession ID
|
Gene ID
|
ICSM Antibodies
|
POM Antibodies
|
SAF Antibodies
|
ICSM18
|
CTL
|
ICSM35
|
CTL
|
POM1
|
CTL
|
POM2
|
CTL
|
POM3
|
CTL
|
SAF32
|
CTL
|
SAF70
|
CTL
|
Q3TWV0
|
Vim
|
√
|
-
|
√
|
-
|
√
|
-
|
√
|
-
|
√
|
-
|
√
|
-
|
√
|
-
|
B1AXW5
|
Prdx1
|
-
|
√
|
-
|
√
|
-
|
-
|
-
|
√
|
-
|
-
|
-
|
√
|
-
|
√
|
A2RTI3
|
Lgmn
|
√
|
-
|
√
|
-
|
-
|
-
|
√
|
-
|
√
|
-
|
√
|
-
|
√
|
-
|
O89054
|
Actb
|
-
|
-
|
-
|
-
|
√
|
-
|
√
|
-
|
√
|
-
|
√
|
-
|
-
|
-
|
Q78NA6
|
Rag1
|
√
|
-
|
√
|
-
|
√
|
-
|
√
|
-
|
-
|
-
|
√
|
-
|
√
|
-
|
Q3UDU4
|
Ldha
|
√
|
-
|
√
|
-
|
-
|
-
|
√
|
-
|
√
|
-
|
√
|
-
|
√
|
-
|
Q3TM31
|
Tlr3
|
-
|
-
|
-
|
-
|
√
|
-
|
√
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
P06800
|
Ptprc
|
√
|
-
|
√
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
√
|
-
|
√
|
-
|
In order to verify whether the 8 allergenic-related proteins were specifically stimulated in neurons (and not in both neurons and microglia) following co-culture with antibody-treated microglia, we compared the proteome of the anti-PrP antibody-treated microglia without co-culture with neurons and found that anti-PrP antibody-treated microglia only did not display any common allergy-related proteins with DMT (Additional file 2: Table S3) indicating that our 8 identified allergy-related proteins were specifically activated in neurons.
Gene Ontology analysis of Allergy Related Proteins Associated with Direct Microglia Treatment
PANTHER analysis of the allergy related protein after DMT showed that the allergenic-related proteins are involved in cellular components, molecular functions, biological processes, signaling pathways, and protein classes (Fig. 8). Cellular components were classified into different groups in PANTHER analysis including cell (PRDX1, TLR3, ACTB) and membrane (TLR3, ACTB), membrane-enclosed lumen (ACTB), protein-containing complex (ACTB), and supramolecular complex (ACTB) (Fig. 8A). The molecular function was further classified into 3 different groups where it was observed that ACTB were shown to be involved in both structural molecule activity and binding activity and LGMN, PRDX1 and PTPRC are associated with catalytic activity (Fig. 8B). The biological process analysis showed that PRDX1 and TLR3 are involved in biological regulation, immune system process, and response to stimulus whereas PTPRC, LGMN, PRDX1, TLR3, and ACTB were found to be associated with cellular process (Fig. 8C). On the other hand, ACTB, was shown to be involved in biogenesis, developmental process, localization, locomotion, and multicellular organismal processes (Fig. 8C). The analysis also showed that PTPRC, LGMN, and PRDX1 are associated with metabolic processes while TLR3 is involved in signaling and multi-organism processes (Fig. 8C). The signaling pathway analysis is divided into 11 groups whereas PTPRC is involved in both B cell and T cell activation and JAK/STAT signaling pathway (Fig. 8D). On the other hand, ACTB is found to be associated with Alzheimer disease-presenilin pathway, cadherin signaling pathway, cytoskeletal regulation by Rho GTPase, Huntington disease, inflammation mediated by chemokine and cytokine signaling pathway, integrin signalling pathway, nicotinic acetylcholine receptor signaling pathway, and wnt signaling pathway (Fig. 8D).
Gene enrichment analysis of the 8 allergenic related proteins through FunRich showed 5 cellular components such as focal adhesion (ACTB, PTPRC; p = 0.02), ribonucleoprotein complex (VIM, ACTB; p = 0.018), bleb (PTPRC; p = 0.03), type III intermediate filament (VIM; p = 0.01), and membrane microdomain (PTPRC; p = 0.001) (Fig. 9A). FunRich analysis of the molecular function identified protein kinase binding (VIM, ACTB, PTPRC; p = 0.03), identical protein binding (VIM, PRDX1, ACTB, RAG1, TLR3; p = 0.001), and lactate dehydrogenase activity (LDHA; p = 0.009) (Fig. 9B).
Furthermore, the biological process analysis was found to be associated with cellular response to extracellular stimulus (LDHA, PTPRC; p < 0.001), cellular response to cytochalasin B (ACTB; p = 0.02), positive regulation of T cell differentiation (RAG1, PTPRC; p = 0.03), positive regulation of hematopoietic stem cell migration (PTPRC; p = 0.03), immunoglobulin biosynthetic process (PTPRC; p = 0.01), glucose catabolic process to lactate via pyruvate (LDHA; p = 0.004), negative regulation of ERBB signaling pathway (LGMN; p = 0.004), vacuolar protein processing (LGMN; p = 0.02), plasma membrane raft distribution (ACTB; p = 0.004), positive regulation of antigen receptor-mediated signaling pathway (ACTB; p = 0.004), positive regulation of protein tyrosine phosphatase activity (ACTB; p = 0.01), positive regulation of T cell mediated immunity (ACTB; p = 0.01), regulation of humoral immune response mediated by circulating immunoglobulin (ACTB; p = 0.004), regulation of interleukin-8 production (ACTB; p = 0.004), and regulation of Schwann cell migration (VIM; p = 0.004) (Fig. 9C).
Contactless Co-Culture of Anti-PrP Antibody Treated-Microglia and Mouse Neuroblastoma Cell Lines Leads to Neuronal Allergy.
Contactless co-culture of anti-PrP antibody treated-microglia N11 and N2a cells was designed to verify whether the allergenic effects caused by DMT were due to a direct cognate interaction of N2a and N11 or via indirect release of microglial factors which in turn might have led to allergenicity. N11 cells were initially treated with anti-PrP antibodies, including ICSM18, ICSM35, POM1, POM2, POM3, SAF32 or SAF70 on tissue culture inserts before placing the inserts containing antibody-treated microglia on tissue culture plate containing untreated N2a cells (IMT). IMT resulted in an initial dataset of 292 proteins (p < 0.05) after LC-MS analysis. Differentially expressed proteins (p < 0.05) were considered with a maximum fold change ≥ 10 and at least 2 identified unique peptides and a confidence score ≥ 10 and identified a total of 11 proteins (Additional file 2: Table S4). Out of the 11 proteins, AllergGAtlas database identified Integrin beta-4 (ITGB4) (upregulated, p = 0.034, maximum fold change 45, confidence score 33.6, peptide 6, unique peptide 3) as being allergenic.
The protein-protein interaction analysis showed that ITGB4 indirectly interacts with PRNP via ITGB6 and NCAM 1 (Additional file 1: Figure S2A). Santuccione and co-workers showed that activation of p59fyn was achieved via PrPC recruitment NCAM to lipid rafts [87]. Ghodrati et al. also showed that NCAM directly interacts with PrP and identified the transforming growth factor β and integrin signaling as prion interactors via gene ontology analysis [88].
Liu and co-workers revealed that ITGB4 is involved in airway hyper-responsiveness and lung inflammation in allergic asthma. ITGB4-deficient mice displayed increased infiltration of lymphocyte, neutrophil, and eosinophil as well as expression of IL-4, IL-13, and IL-13A in lung tissue [89]. Tang et al. showed that ITGB4-deficiency causes spontaneous exaggerated lung inflammation in early life [90]. A study by Yuan and co-workers showed that p53 pathway activation in ITGB4-deficiency prompts the senescence of airway epithelial cells [91]. Yuan and co-workers also demonstrated that the lack of ITGB4 is responsible for increased Th2 responses in allergic asthma by down-regulation of CCL17 and EGFR pathway in airway epithelial cells [92].
Gene Ontology analysis of Allergy Related Proteins Associated with indirect Microglia Treatment
The Gene classification through PANTHER showed that ITGB4 is associated with cellular components (cell junction, membrane, and protein-containing complex) (Additional file 1: Figure S2B), molecular function (binding) (Additional file 1: Figure S2C), biological process (biological adhesion, biological regulation, cellular process, localization, locomotion, response to stimulus, and signaling) (Additional file 1: Figure S2D), and signaling pathway (Integrin signalling pathway) (Additional file 1: Figure S2E). The Gene enrichment analysis ITGB4 through FunRich showed that the cellular components involved, included hemidesmosome (p < 0.001), integrin complex (p < 0.001), basal plasma membrane (p = 0.022), and cell leading edge (p = 0.032) (Additional file 1: Figure S3A). The biological process was found to be associated with trophoblast cell migration (p < 0.001), peripheral nervous system myelin formation (p < 0.001), hemidesmosome assembly (p = 0.002), cell adhesion mediated by integrin (p = 0.023), and cell motility (p = 0.036) (Additional file 1: Figure S3B). On the other hand, the reactome pathway analysis of ITGB4 identified type I hemidesmosome assembly (p = 0.002), syndecan interaction (p = 0.006), laminin interaction (p = 0.009), and assembly of collagen fibrils and other multimeric structures (p = 0.038) (Additional file 1: Figure S3C).