Human Proteinase 3, an important autoantigen of c-ANCA associated vasculitis, shares cross-reactive epitopes with serine protease allergens from mites: an in silico analysis [version 2; peer review: 1 approved, 2 approved with reservations]

Background: In autoimmune vasculitis, autoantibodies to Human Proteinase 3 (PR3), a human serine protease, seems to have a role on the inception of c-ANCA associated vasculitis. The origin of this autoreactive response remains unclear. However, for several autoreactive responses, molecular mimicry between environmental antigens and human proteins is key to trigger autoantibodies and finally autoimmunity manifestations. Considering that PR3 is a serine protease and house dust mite (HDM) group 3 allergens


Introduction
Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a life-threatening autoimmune disease affecting small vessels, compromising the respiratory mucosa, skin, lung, and the kidney 1 . This group of small vessel vasculitis includes various diseases: granulomatosis with polyangiitis, microscopic polyangiitis, kidney-limited vasculitis and Eosinophilic granulomatosis with polyangiitis, all of them having in common some degree of autoimmune response to the Human Proteinase 3 protein (PR3). Previous studies have shown that autoantibody binding to PR3 expressed on the neutrophil surface may activate its degranulation, eliciting tissue damage in small vessels and their irrigated organs. Also, while proinflammatory effector T cells have been implicated in vasculitis pathogenesis 2 , a specific PR3 T cell epitope has not been reported in AAV patients 3 . PR3 is a serine protease physiologically expressed in human neutrophils. Due to its enzymatic activity, it degrades various intercellular gap-junction proteins and collagen and may play a role in neutrophil transendothelial migration. In addition, this protein is an important autoantigen in AAV, and sera from patients with severe and relapsing forms of the disease can bind it in IgG ELISA assays [4][5][6] . Further, although a cause-effect relationship between PR3-autoantibodies and vasculitis is not clearly defined, animal models support a pathogenic role 7,8 , revealing that they may be involved in disease inception, progression and severity 1 .
Environmental exposures, specially to microbial components mimicking self-antigens have been proposed as triggers of autoimmunity 9,10 . Also, in AAV, it has been proposed that an endogenous immune response to a complementary protein to PR3 autoantigen could be implicated in disease inception, and this antisense protein harbors homology to various bacterial peptides 11 . PR3 crystal structure has been elucidated, and various epitopes are recognized by patients suffering AAV; however, its cross-reactivity with environmental antigens is poorly studied 12-14 .
Previous studies have shown that specific IgE to some self-proteins have been identified in autoimmune and allergic diseases like lupus, urticaria, dermatitis, allergic pulmonary aspergillosis and have a strong association with disease activity 15-18 . Some allergens can cross-react with human proteins and participates in autoimmunity inception in pemphigus vulgaris by a "hit-and-run" mechanism, opening the theoretical possibility for a similar mechanism to occur in another autoimmune disease such as AAV 19-22 .
In the tropics, house dust mites (HDM) are important ubiquitous allergen sources and exposure is perennial, increasing the possibilities of exposure in the general population 23 , and IgE sensitization to their components 24,25 . Sensitization to HDM group 3 allergens is common 26 , as they harbor serine protease activity and conserved structural homology 27 , making them potential PR3 cross reactive antigens; this has not been explored before. Here, we show in silico data suggesting cross-reactivity and epitope sharing between PR3 and HDM group 3 allergens.

Multiple alignment
Identity among all allergenic sequences homologous to PR3 was analyzed using the Jalview tool2.11.0 28 . First, all allergens and human PR3 codes were used as inputs in the Jalview tool. Second, the T coffee tool was chosen to assess alignment. Third, alignment was displayed as an identity percentage.

Construction of 3D model
The 3D model of Der p 3, a serine protease of Dermatophagoides pteronyssinus was generated by homology in the SWISS-MODEL server using the zymogen catalytic region of human MASP-2 (PDB: 1zjk f) as a template. The 3D model of Der p 3 was loaded into the ProSA-web server 29 , which was used to analyze its quality.
The model was refined in DeepView v4.1 (energy minimization and rotamer replacements). Its quality was evaluated by several tools, including Ramachandran graphs, WHATIF, QMEAN4 index, and energy values (GROMOS96 force field). For the validation of the Der p 3 structure we used the Minimize Structure option in the UCSF Chimera software, a procedure that adjust the energy and reduce the entropy of the model 30 .
Three-dimensional structure (PDB: 1FUJ) of the human PR3 serine protease was retrieved from the Protein Data Bank. A cartoon model was created using Pymol software v2.4. Root median square deviation (RMSD) value between Der p 3 and PR3 was calculated using Chimera software v1.0 30 .
B and T cell epitope prediction ElliPro v3.0 and BepiPred v2.0 tools were used to predict B and T cell epitopes on Der p 3 31,32 . With ElliPro, the 3D structure of Der p 3 was used to predict epitopes. Minimum score and maximum distance (Angstrom) were set to 0.5

Amendments from Version 1
The manuscript was revised and refined by a language style corrector. Some sentences in the methods and results section were improved giving more detail and explanations in accord to revisor suggestions.
Any further responses from the reviewers can be found at the end of the article REVISED and 6, respectively. Epitopes with high conserved rates were visualized in the 3D model. For prediction using BepiPred, an amino acid sequence of Der p 3 was used as input.

Conservation analysis
The 3D structure of Der p 3 was submitted to the ConSurf server to generate evolutionarily related conservation scores to help to identify functional regions in the proteins. HMMER algorithm, 1 iteration, E-value cutoff (0.0001) and UNIREF-90 database was set as default to generate multiple alignment, prior to evolutive analysis. All amino acid sequences in FASTA format were used.

Results
Human PR3 and HDM group 3 allergens exhibited identity and features of the serine protease family BLAST search identified various serine protease family members from HDM as homologous. The multiple sequence alignment analysis showed that Der p 3, Blo t 3, Gly d 3, Led p 3 and Tyr p 3 allergens shared 45% of identity in their aminoacid sequences with PR3. The most conserved region is located between residues 53 to 75, indicating the existence of molecular mimicry ( Figure 1). Among the members of HDM group 3 allergens, an identity until 41% was reported (Table 1), and a highly conserved region between residues 40 to 90 was found. When identity between PR3 and each allergen used in study was analyzed, a moderate level of identity was found (30%) ( Table 1).
A structural model of Der p 3 was obtained by homology modelling using the 3D structure of PR3 reported in the PDB database. According to modelling, the Der p 3 tertiary structure exhibited a typical fold of serine protease family, conformed by four α-helixes and fifteen β-strands with structural homology with PR3 (RMSD = 0.8) (Figure 2).
T and B cell cross-reactive epitopes were predicted between HDM group 3 allergens and PR3 Using ElliPro and BepiPred servers, a cross reactive B cell epitope was predicted on all serine protease used in this study. This epitope is formed by ten residues and is on the N-terminal region, spanning amino acids 29 and 39 with a surface area of 470 Å, not forming part of any domain within the protein. Conservative analysis indicated that the antigenic region predicted was highly conserved in the serine proteases ( Figure 3). According to ConSurf analysis, the region covering    the cross-reactive epitope is conserved among the serine protease family (Figure 4). T cell epitope prediction identified at least two epitopes with potential cross-reactivity among all sequences analyzed. Both epitopes are located on the first and second β strands: the first epitope spans the 45 to 59 region (ISLQSSSHFCGGTIL); and the second, the 63 to 77 region (WILTAAHCVAGQTAS) ( Figure 5; Table 2).

Discussion
In this study we found that PR3 and HDM group 3 serine protease allergens have conserved identity and homology. Also, for the first time, we predicted various T and B cell cross reactive epitopes between them through an in silico approach. PR3 is an important autoantigen in small vessel vasculitis and it seems to participate in disease inception, progression, and severity 1 . Our results have potential implications for the understanding of autoreactive response in AAV and open the possibility for a new environmental trigger of the autoreactive response in AAV.
In AAV, it has been proposed that autoantibodies directed to a complementary protein to PR3 autoantigen could be implicated in disease inception, and this antisense protein harbors homology to various bacterial peptides 11 -a theory named  In their seminal publication, Pendergraft et al. run a BLAST query to find homologues of PR3 protein in microbial or fungal microorganisms, and do not find matching sequences at that time 11 . However, they do not include Arachnida or other environmental sources of cross-reactivity. In our analysis we find matching PR3 protein sequences with various HDM group 3 serine protease allergens, and at least theoretically this finding could have many implications for the understanding of inception and even diagnosis of autoreactive response in AAV.
Recently, Qian et al. have shown that some allergens can crossreact with human proteins 19 and participate in autoimmunity inception in pemphigus vulgaris by a "hit-and-run" mechanism 22 , opening the theoretical possibility for a similar mechanism to occur in another autoimmune disease such as AAV. Similarly, in atopic dermatitis, Valenta and collaborators observe that some patients with severe complications from the disease, had IgE directed to the profilin of the Betula verrucosa, but also to the human homologue 40 .
In the tropics, HDM are important ubiquitous sources of protease allergens. Exposure is perennial, increasing the possibilities of exposure and IgE sensitization to their components in the general population 23-25 . Sensitization to HDM group 3 allergens is common 26 , and they harbor serine protease activity 27 , a characteristic that make them highly allergenic. Moreover, their conserved structural homology makes them highly immunogenic 41,42 and suitable for epitope spreading 43 . In this context, "hit-and-run" and epitope spreading establish framework mechanisms for environmental allergens with homology to autoantigens to potentially participate in the development of autoimmunity. We speculate that HDM group 3 allergens harbor two characteristics that make them suitable candidates for environmental triggering of AAV: their proteolytic activity that, as other protease allergens, set a tissue damaging microenvironment during antigen recognition 41 ; and molecular homology-epitope sharing with human PR3, that would elicit B cell autoantibody production and autoreactive T cell receptor generation. In conclusion, we observe that PR3 and HDM group 3 serine protease allergens have conserved identity, and for the first time we predict cross-reactive epitopes between them through an in silico approach.  Buendia et al. investigated in their manuscript possible link between human proteinase 3 (PR3) and house dust mite allergen protease that is denoted as allergen group 3. Authors suggest that similarity between PR3 and the group 3 mite allergens may be link to autoimmune vasculitis. Methodology and conclusions are based on structural homology based on in silico analysis. I believe that recommendations will improve the manuscript, which is of interest.

Data availability
Because methodology of the study is based on in silico tools, it is necessary to consider that the function is "possible" and needs to be confirmed in future.
Please note, that group 3 mite allergens become subject of debate in the domestic mites, that include both hose dust (HDMs) and stored product mite (SPMs) species. Immortally, authors should clarify that allergens of HDMs and SPMs were included in the study. In addition, these allergens are present in mite feces and, therefore, they are abundant in the environment. Thus, introduction should be improved.
It was found that occurrence of trypsin allergens in mites differs in species and strains. Comment to approach Authors used different sequences of group 3 mite allergens to predict similarity. It should be clear whether "pro" or mature form was used in analyses. It is recommended that authors will consider change "identity" to similarity. Usually, high identity is "only" to partial sequence. It is important to consider overall similarity.
In initial stage, authors used multiple Grp3 sequences, i.

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