Angiostrongylus cantonensis an Atypical Presenilin: Epitope Mapping, Characterization, and Development of an ELISA Peptide Assay for Specific Diagnostic of Angiostrongyliasis

Background: Angiostrongyliasis, the leading cause universal of eosinophilic meningitis, is an emergent disease due to Angiostrongylus cantonensis (rat lungworm) larvae, transmitted accidentally to humans. The diagnosis of human angiostrongyliasis is based on epidemiologic characteristics, clinical symptoms, medical history, and laboratory findings, particularly hypereosinophilia in blood and cerebrospinal fluid. Thus, the diagnosis is difficult and often confused with those produced by other parasitic diseases. Therefore, the development of a fast and specific diagnostic test for angiostrongyliasis is a challenge mainly due to the lack of specificity of the described tests, and therefore, the characterization of a new target is required. Material and Methods: Using bioinformatics tools, the putative presenilin (PS) protein C7BVX5-1 was characterized structurally and phylogenetically. A peptide microarray approach was employed to identify single and specific epitopes, and tetrameric epitope peptides were synthesized to evaluate their performance in an ELISA-peptide assay. Results: The data showed that the A. cantonensis PS protein presents nine transmembrane domains, the catalytic aspartyl domain [(XD (aa 241) and GLGD (aa 332–335)], between TM6 and TM7 and the absence of the PALP and other characteristics domains of the class A22 and homologous presenilin (PSH). These individualities make it an atypical sub-branch of the PS family, located in a separate subgroup along with the enzyme Haemogonchus contournus and separated from other worm subclasses. Twelve B-linear epitopes were identified by microarray of peptides and validated by ELISA using infected rat sera. In addition, their diagnostic performance was demonstrated by an ELISA-MAP4 peptide. Conclusions: Our data show that the putative AgPS is an atypical multi-pass transmembrane protein and indicate that the protein is an excellent immunological target with two (PsAg3 and PsAg9) A. costarisencis cross-reactive epitopes and eight (PsAg1, PsAg2, PsAg6, PsAg7, PsAg8, PsAg10, PsAg11, PsAg12) apparent unique A. cantonensis epitopes. These epitopes could be used in engineered receptacle proteins to develop a specific immunological diagnostic assay for angiostrongyliasis caused by A. cantonensis.

strategic domains of these proteins through peptide microarray presents a faster and more efficient alternative to identify these determinants and then produce the desired antibodies.
Therefore, in this study, we describe the partial biochemical and immunological characterization of a putative presenilin protein of A. cantonensis and its epitopes in developing an ELISA test with high specificity and sensitivity for the diagnosis of the angiostrongyliasis.

Rat Infected Sera and Ethics Statement
Sera samples from 16 infected rats in the acute phase of angiostrongyliasis were obtained from the Laboratory of Biology and Parasitology of Wild Mammals Reservoirs (Oswaldo Cruz Institute, FIOCRUZ, Brazil). Another group included 25 uninfected rat sera.

Synthesis of the Cellulose-Membrane-Bound Peptide Array
A library of eight one, 15-mer peptides were designed to represent a consecutive overlapping coverage offset by nine amino acids across the entire coding region (415 aa) of the putative aspartyl protease (C7BVX5_ANGCA, UNIPROT) of A. cantonensis. The peptides were automatically prepared onto amino-PEG 500 -UC540 cellulose membranes (Intavis Bioanalytical Instruments, Köln, Germany) as previously described [44] using an Auto-Spot Robot ASP-222 (Intavis Bioanalytical Instruments AG, Köln, Germany) and the F-moc strategy. In brief, coupling reactions were followed by acetylation with acetic anhydride (4%, v/v) in N,N-dimethylformamide to render peptides unreactive during the subsequent steps. After acetylation, F-moc protective groups were removed by adding piperidine to render nascent peptides reactive. The remaining amino acids were added by this same process of coupling, blocking, and deprotection until the expected desired peptide was generated. After adding the last amino acid in the peptide, the amino acid side chains were deprotected using a solution of dichloromethane-trifluoracetic acid-tri isobutyl silane (1:1:0.05, v/v/v) and washed with methanol. Membranes containing the synthetic peptides were either probed immediately or stored at −20 • C until required. Negative controls [without peptide; IHLVNNESSEVIV HK (Clostridium tetani) precursor peptide] and positive controls were included in each assay.

Screening of SPOT Membranes
SPOT membranes were washed with TBS (50 mM Tris-buffer saline, pH 7.0) and blocked with TBS-CT (Tris-buffer saline, 3% casein, 0.1% Tween 20, pH 7.0) at room temperature under agitation or overnight at 4 • C. After extensive washing with TBS-T (Tris-buffer saline, 0.1% Tween 20, pH 7.0), the membrane presenting the peptide library was incubated for two hours with a pool of patients sera (1:100) in TBS-CT and then washed again with TBS-T. Afterward, the membrane was incubated with alkaline phosphatase-labeled goat anti-rat IgG (1:5000 in TBS-CT; KPL, Gaithersburg, MD, USA) for one hour, and then washed with TBS-T and CBS (50 mM citrate-buffer saline, pH 7.0). Chemiluminenscente CDP-Star ® Substrate (0.25 mM) with Nitro-Block-II™ Enhancer (Applied Biosystems, Waltham, MA, USA) was added to complete the reaction.

Scanning and Measurement of Spot Signal Intensities
As described previously, chemiluminescent signals were detected on an Odyssey FC (LI-COR Bioscience, Lincoln, NE, USA) [45]. Briefly, a digital image file was generated at a resolution of 5 MP, and the signal intensities were quantified using the TotalLab TL100 (v. 2009, Nonlinear Dynamics, Newcastle, Tyne, UK) software. The signal intensity (SI) used as a background was a set of negative controls spotted in each membrane.

Synthesis of Peptides and Preparation of the MAPs
Nine single A. cantonensis peptides (PSAg1-PSAg3, PSAg6-PSAg9, PSAg11, and PSAg12; Table 1) were synthesized by the F-moc strategy in a synthesizer machine (MultiPep-1 CEM, Corp, Charlotte, NC, USA). For the preparation of the dendrimer multi-antigen peptides (MAP4), the same synthesis protocol was used in the solid phase and the tetrameric F-moc-Lys2-Lys-β-Ala Wang resin, as described previously [46]. The constructs were prepared in the automated MultiPep-1 peptide synthesizer, and the side chains of tetrafunctional F-moc-amino acids were protected with TFA-labile protecting groups as required. Residues corresponding to the monovalent ('tail') part of the construct, up to the first (bis-Fmoc) Lys residue initiating the dendrimer structure, were incorporated via single couplings. Once sequence assembly was completed, the F-moc groups were removed, and the peptide-resin was cleaved and fully deprotected with TFA/H2O/EDT/TIS (94/2.5/2.5/1.0 v/v, 90 min). The peptide was precipitated by adding chilled diethyl ether, centrifuged for 3 × 10 min at 4 • C, and the pellet was taken up in aqueous AcOH (10% v/v), dried, and stored as a lyophilized powder. When necessary, the MAP was dissolved in water, centrifuged (10,000 g, 60 min, 15 • C) and the supernatant filtered on a centricon 10 filter. The single peptides were used without previous purification, but their identity was checked by MS (MALDI-TOF or electrospray).

ELISA-Peptide
The ELISA was realized, as described previously [47], with minor modifications. Briefly, ELISA plates (Immulon 4HB; Corning, NY, USA) were coated with 80 µL (50 µg) of each peptide prepared on coating buffer (Na 2 CO 3 -NaHCO 3 , pH 9.6) overnight at 4 • C. After each incubation step, the plates were washed three times using a PBS-T washing buffer (PBS with 0.1% Tween 20 adjusted to pH 7.2) and blocked (200 µL) with 2.5% BSA and incubated 2 h at 37 • C. The rat's sera were diluted (1:150) in coating buffer, and 100 µL were applied onto immunosorbent plates and incubated for 2 h at 37 • C. Following several washes with PBS-T, the plates were incubated with 100 µL goat anti-rat IgG conjugated to HRP

Database Searches, Computational, and Phylogeny Studies
To curate a family of 10 target sequences for C7BVX5-1, a set of aspartic protease sequences were retrieved from the Uniprot database (available online: http://www.uniprot. org/ accessed on 20 March 2021) using the following criteria: An EC designation of 3.4.23 for aspartic endopeptidases; a length between 300 to 800, and a reviewed annotation. Another set of sequences homologous to other proteins, which have been cited by Blast, was added after a delta-blast of the Refseq database with coverage greater than 80% of their sequence-issue and identity over 10-30%. From this set, an alignment was performed on the Mat server with blossum80 and a gap penalty of 2.5, followed by clustering to eliminate the excessive gaps with the method "minimum linkage." The potential transmembrane (TM) domains (TMD) of the A. cantonensis enzyme were analyzed by three different predictions programs; the TMpred [48], the TopCons [49], and MemConP [50]. The model most consistent with the identified epitopes and motifs were obtained using TopCons, which used an algorithm based on the statistical analysis of base. The prediction was made using a combination of several weight-matrices for scoring. Secondary structure predictions were obtained from PSIPRED (

Ethics Statement
The study was approved by the FIOCRUZ (IOC-CAAE: 52892216.8.0000.5248 and 1.896.362) study center ethics committee and conducted following all applicable Good Clinical Practice (GCP) regulatory requirements, including the Declaration of Helsinki.

Statistical Analysis
Statistical analysis was performed using GraphPad Prism version 5.0. The statistical differences using a t-test were considered if the p-value ≤ 0.05.

Identification and Mapping of Linear Epitopes Using Synthetic Peptides
The epitopes of the PS protein from A. cantonensis recognized by patients sera were mapped using the parallel Spot-synthesis strategy. The peptide library consisted of 69 peptide sequences of 15 amino acids that overlapped by 10 amino acids and covered the entire protein sequence. A representative experiment is presented in Figure 1. The list of synthesized peptides is shown in Supplementary Figure S1. Overall, each epitope displayed a relatively strong reactivity (containing 4-15 residues of extension). However, the most vigorous intensity was observed with the antigenic determinant in the peptide SIFWKGPMRLQQASL (B10) and PSSGRFIESFRMPSL (C2).
The analysis of spot signal intensity for the synthesized peptides from the PS sequence showed 12 epitopes ( Table 1). The secondary structure of the identified epitopes is also presented in Supplementary Figure S2 and was based on the results obtained by the I-Tasser prediction (accessed on 10 March 2021).  Figure S1. Each peptide was identified by the Spot-synthesis membrane position numbering. Spot intensities below 20% were considered as background. The positive peptides identified and assessed for overlap are gray and the resulting sequence within the rectangles.

Absence of Crucial Motifs for A22 Aspartyl Protease Family
The data available from studies on the γ-secretase complexes in mammalian cells have indicated that particular amino acid motifs are crucial for its proteolytic activity, substrate recognition, and complex assembly [52]. To prove that these critical motifs are also conserved in the putative A. cantonensis protein available in the protein database UniProtKB (http://www.uniprot.org/; accessed on 3 March 2021) with the number C7BVX5 belonging to the A22 family, multiple sequence alignments were performed. Potential homologs were identified by PSI-BLAST, and the level of similarity among the PSs from these species was aligned to the Homo sapiens sequence. Motif analysis and multiple sequence alignment of the A. cantonensis PS sequence (C7BVX5) with the H. sapiens PS1 protein (P49768) was performed and showed only one compatible conserved motif sequence. Those of the catalytic pocket (YD; 192-194 and GLGD; 287-290) located in TM7 aligned with XD (aa 241) and GLGD (aa 332-335) of A. cantonensis PS, in TMD6 and TDM7, respectively ( Figure 2).

Absence of Crucial Motifs for A22 Aspartyl Protease Family
The data available from studies on the γ-secretase complexes in mammalian cells have indicated that particular amino acid motifs are crucial for its proteolytic activity, substrate recognition, and complex assembly [52]. To prove that these critical motifs are also conserved in the putative A. cantonensis protein available in the protein database UniProtKB (http://www.uniprot.org/; accessed on 3 March 2021) with the number C7BVX5 belonging to the A22 family, multiple sequence alignments were performed. Potential homologs were identified by PSI-BLAST, and the level of similarity among the PSs from these species was aligned to the Homo sapiens sequence. Motif analysis and multiple sequence alignment of the A. cantonensis PS sequence (C7BVX5) with the H. sapiens PS1 protein (P49768) was performed and showed only one compatible conserved motif sequence. Those of the catalytic pocket (YD; 192-194 and GLGD; 287-290) located in TM7 aligned with XD (aa 241) and GLGD (aa 332-335) of A. cantonensis PS, in TMD6 and TDM7, respectively ( Figure 2).
Other motifs were also compared with PS proteins from other worms such as Caenorhabditis elegans (P52166), Angiostrongylus costaricensis (A0A0R3PEK8), and Schistosoma mansoni (C0SKM6). The human TMD8 has two motifs [GxxxG (GVKLG) and SxxxGxxxxA (SVLVGKASA)], which are localized close to the catalytic Asp. These motifs contain sites of FAD mutations, which form a portion of the catalytic core of the PSs and influence helix packing that may modulate enzymatic activity. In the A. cantonensis PS, these motifs were not identified. Another interesting difference is that, unlike humans and other nematodes, A. cantonensis PS lacks the conserved C-terminal PALP motif that determines the active site's conformation and characterizes the A22 family ( Figure 2). SxxxGxxxxA (SVLVGKASA)], which are localized close to the catalytic Asp. These motifs contain sites of FAD mutations, which form a portion of the catalytic core of the PSs and influence helix packing that may modulate enzymatic activity. In the A. cantonensis PS, these motifs were not identified. Another interesting difference is that, unlike humans and other nematodes, A. cantonensis PS lacks the conserved C-terminal PALP motif that determines the active site's conformation and characterizes the A22 family ( Figure 2).

Spatial Location of the Most Reactive Epitopes
To analyze the localization of the epitopes on the A. cantonensis protein, a 3D structure was obtained from the I-Tasser server ( Figure 3A), and the position of the 12 epitopes (Table 1) was identified by SPOT-Synthesis was depicted. In addition, transmembrane domains, hydropathy, and secondary structures were analyzed to pinpoint the epitope position in the presenilin antigen (PsAg) protein.
Four identified epitopes were exclusively in loop/coil (PsAg1, 2, 8, and 12) structures, while five (PsAg3, 6, 7, 9, and 11) shared amino acids in coil and helix, sometimes in the interface between membrane and cytosol/extracellular space. Three identified peptides were found in the alpha-helix (PsAg4, 5, and 10) (Table 1, Figure 3A). Although the epitope PsAg10 was predicted to stay partially embedded in the membrane helix, probably due to an unspecific reaction, this peptide was excluded from subsequent analysis. However, all other epitopes were present on the protein surface or adjacent to the membrane and intracellular/extracellular space, accessible to the solvent. The hydropathy plots of the protein and the amino acid accessibility, shown in Supplementary Figure S3, also suggested that residues in all of the epitopes were present on the surface of the protein. Figure 3B show a transmembrane model determined for the AgPS using the TopCons software and the distribution of the identified epitopes along with the primary structure. The predicted model depicted nine TM domains, which is consistent with the classical structure of the PS proteins. In addition, the expected model contemplates the catalytic domain consisting of the conserved motifs XD and GLGD, between TM6 and TM7.

Spatial Location of the Most Reactive Epitopes
To analyze the localization of the epitopes on the A. cantonensis protein, a 3D structure was obtained from the I-Tasser server ( Figure 3A), and the position of the 12 epitopes (Table 1) was identified by SPOT-Synthesis was depicted. In addition, transmembrane domains, hydropathy, and secondary structures were analyzed to pinpoint the epitope position in the presenilin antigen (PsAg) protein.
Four identified epitopes were exclusively in loop/coil (PsAg1, 2, 8, and 12) structures, while five (PsAg3, 6, 7, 9, and 11) shared amino acids in coil and helix, sometimes in the interface between membrane and cytosol/extracellular space. Three identified peptides were found in the alpha-helix (PsAg4, 5, and 10) (Table 1, Figure 3A). Although the epitope PsAg10 was predicted to stay partially embedded in the membrane helix, probably due to an unspecific reaction, this peptide was excluded from subsequent analysis. However, all other epitopes were present on the protein surface or adjacent to the membrane and intracellular/extracellular space, accessible to the solvent. The hydropathy plots of the protein and the amino acid accessibility, shown in Supplementary Figure S3, also suggested that residues in all of the epitopes were present on the surface of the protein. Figure 3B show a transmembrane model determined for the AgPS using the TopCons software and the distribution of the identified epitopes along with the primary structure. The predicted model depicted nine TM domains, which is consistent with the classical structure of the PS proteins. In addition, the expected model contemplates the catalytic domain consisting of the conserved motifs XD and GLGD, between TM6 and TM7. Membranes 2022, 11, x FOR PEER REVIEW 8 of 15  Table 1).

Phylogeny and Protein-Protein Interaction
A group of sequences for PSs was retrieved on the Uniprot server (Available online: http://www.uniprot.org accessed on 23 August 2020) through a search using the criteria name, "PS," and a length of 300 to 800. A phylogenetic tree was created with the sequences aligned by the neighbor-joining algorithm using a CLUSTALW program (Figure 4). The A. cantonensis aspartyl protease was localized in a separate subgroup along with the Haemonchus contortus enzyme detached from the other worm's subclasses of this protease family, such as Fasciola hepatica and Caenorhabditis elegans. Model of Ag/PS as a multi-pass transmembrane protein (TM), based on the predictive results obtained by bioinformatics (available online: https://topcons.net/pred accessed on 12 November 2020) and layout generated using Protter (available online: http://wlab.ethz.ch/protter/ accessed on 15 November 2020). The 9 TM domains reveal 12 identified epitopes and the catalytic pocket (orange) with the typical motifs XD (TMD6) and GLGD (TMD7) but the absence of the PALP domain (A). Three-dimensional structure and membrane-spanning model of A. cantonensis presenilin protein. 3D protein conformation and structure were determined using the I-Tasser, and epitopes were highlighted (purple), indicating the position of the 12 linear B epitopes identified by Spot-synthesis ((B), Table 1).

Phylogeny and Protein-Protein Interaction
A group of sequences for PSs was retrieved on the Uniprot server (Available online: http://www.uniprot.org accessed on 23 August 2020) through a search using the criteria name, "PS," and a length of 300 to 800. A phylogenetic tree was created with the sequences aligned by the neighbor-joining algorithm using a CLUSTALW program (Figure 4). The A. cantonensis aspartyl protease was localized in a separate subgroup along with the Haemonchus contortus enzyme detached from the other worm's subclasses of this protease family, such as Fasciola hepatica and Caenorhabditis elegans.

Selection of Putative Specific Epitopes
The first goal to eliminate cross-reactive epitopes was to search possible homology sequences of the epitopes in the Pir data bank. The peptide search database realized with the 12-epitope sequences returned, that 2 (PsAg3 and PsAg9) cross-react with A. costaricensis while 8 (PsAg1, PsAg2, PsAg6, PsAg7, PsAg8, PsAg10, PsAg11, PsAg12) are unique (Table 1; Supplementary Figure S4). PsAg4, PsAg5, and PsAg10 are defined in a helical well; they were not interpreted as B-linear epitopes.
Then, to confirm the immunogenicity of the epitopes, nine single peptides were synthesized (Table 1) and individually evaluated by ELISA assay using serum from infected rats. The results (media ± SD) are shown in Figure 5 and show that all the epitopes were highly reactive.
Thus, one antigenic determinant (PsAg2) was selected and synthesized as MAP4 containing 3 Gly in the N and 2 G in the C terminus (GGGVKRLYGPTDMGG) and analyzed by ELISA using a panel of rat infect sera. Based on a receiver operating characteristics (ROC) curve ( Figure 6B), the area under the curve (AUC) for PsAg2-MAP4 varied from 0.9817 to 0.9978 (p ≥ 0.0001) as detected by ELISA with an interval confidence of 95%, demonstrating high diagnostic accuracy for the PsAg2 epitope ( Figure 6).

Selection of Putative Specific Epitopes
The first goal to eliminate cross-reactive epitopes was to search possible homology sequences of the epitopes in the Pir data bank. The peptide search database realized with the 12-epitope sequences returned, that 2 (PsAg3 and PsAg9) cross-react with A. costaricensis while 8 (PsAg1, PsAg2, PsAg6, PsAg7, PsAg8, PsAg10, PsAg11, PsAg12) are unique (Table 1; Supplementary Figure S4). PsAg4, PsAg5, and PsAg10 are defined in a helical well; they were not interpreted as B-linear epitopes.
Then, to confirm the immunogenicity of the epitopes, nine single peptides were synthesized (Table 1) and individually evaluated by ELISA assay using serum from infected rats. The results (media ± SD) are shown in Figure 5 and show that all the epitopes were highly reactive.
Thus, one antigenic determinant (PsAg2) was selected and synthesized as MAP4 containing 3 Gly in the N and 2 G in the C terminus (GGGVKRLYGPTDMGG) and analyzed by ELISA using a panel of rat infect sera. Based on a receiver operating characteristics (ROC) curve ( Figure 6B), the area under the curve (AUC) for PsAg2-MAP4 varied from 0.9817 to 0.9978 (p ≥ 0.0001) as detected by ELISA with an interval confidence of 95%, demonstrating high diagnostic accuracy for the PsAg2 epitope ( Figure 6).  Table  1) with pool (n = 16) of serum from infected rat mice by IgG-ELISA-peptide. The solid and broken lines indicate the mean values of reactivity found with the set of peptides analyzed with positive sera (sera from infected rats) and negative sera (sera from healthy rats), respectively. Columns show the average of duplicates.

Discussion
Our group has been working for some years on the characterization of GSC proteins from lower eukaryotes [53,54] and we observed that this group of proteins has a very low homology (~20-30%) to each other, a fact that has made their identification difficult in genomics and proteomics [55,56] projects. However, on the other hand, this information points out that these proteins are more likely to have specific epitopes under the immunological point of view.
Therefore, to overcome the difficulty of the diagnostic specificity of angiostrongyliasis, we characterize a protein from A. cantonensis described in the database  Table 1) with pool (n = 16) of serum from infected rat mice by IgG-ELISA-peptide. The solid and broken lines indicate the mean values of reactivity found with the set of peptides analyzed with positive sera (sera from infected rats) and negative sera (sera from healthy rats), respectively. Columns show the average of duplicates.  Table  1) with pool (n = 16) of serum from infected rat mice by IgG-ELISA-peptide. The solid and broken lines indicate the mean values of reactivity found with the set of peptides analyzed with positive sera (sera from infected rats) and negative sera (sera from healthy rats), respectively. Columns show the average of duplicates.

Discussion
Our group has been working for some years on the characterization of GSC proteins from lower eukaryotes [53,54] and we observed that this group of proteins has a very low homology (~20-30%) to each other, a fact that has made their identification difficult in genomics and proteomics [55,56] projects. However, on the other hand, this information points out that these proteins are more likely to have specific epitopes under the immunological point of view.
Therefore, to overcome the difficulty of the diagnostic specificity of angiostrongyliasis, we characterize a protein from A. cantonensis described in the database Figure 6. Reactivity of rat infected sera (PS, n = 16) and healthy rat sera (HS, n = 25) with synthetic specific spanning epitope PsAg2-MAP4 in an ELISA. (A) The y-axis shows sera's mean reactivity (positive/mean negative) from infected rats. The ROC analysis (B) using PS showed that the sensitivity and specificity of the in-house ELISA-peptide was 100% for PsAg2. The dashed line shows the cut-off of the assay.

Discussion
Our group has been working for some years on the characterization of GSC proteins from lower eukaryotes [53,54] and we observed that this group of proteins has a very low homology (~20-30%) to each other, a fact that has made their identification difficult in genomics and proteomics [55,56] projects. However, on the other hand, this information points out that these proteins are more likely to have specific epitopes under the immunological point of view.
Therefore, to overcome the difficulty of the diagnostic specificity of angiostrongyliasis, we characterize a protein from A. cantonensis described in the database as a putative presenilin protein using bioinformatics, biochemical and immunological tools. This first characterization of an atypical transmembrane aspartyl protease from A. cantonensis.
Intramembrane proteases catalyze the unusual cleavage of peptide bonds in the plane of biological membranes. They are categorized according to their active site. For example, the MEROPS database (MEROPS database. Available online: https://www.ebi.ac.uk/ merops/ accessed on 18 November 2021) peptidase family, A22, contains membraneinserted endopeptidases and aspartates residues in the active area are characteristics of peptidases of family A22. The GxGD aspartyl proteases comprise the classical human PS1 and PS2, the signal peptide peptidase (SPP), and SPP-like (SPPL) proteases [57,58] and five homologous presenilin proteins, Presenilin is the catalytic portion of the gamma-secretase complex (GSC) along with other proteins, Anterior pharynx defective 1 (Aph1), Nicastrin, and presenilin enhancer 2 (Pen2), involved in complex stabilization and substrate binding. Most of the presenilins characterized until now are composed of 9-pass TM domains that undergo an endoproteolytic cleavage in the intracellular loop between TM6 and TM7, producing N-terminal and C-terminal fragments [59,60].
The predicted model for Ag-PS protein also presented 9 TM domains. Its topology is corroborated by the experiments of epitope identification (outside and inside) and contemplates the catalytic domain entailed of the conserved aspartates motifs XD (aa 241) and GLGD (aa 332-335), between TM6 and TM7, as occur in the human orthologue (YD; 192-194 and GLGD; 287-290) protein ( Figure 2). The presence of these critical motifs was also confirmed to be present in other worms such as Caenorhabditis elegans (P52166), Angiostrongylus costaricensis (A0A0R3PEK8), and Schistosoma mansoni (C0SKM6) (Figure 2).
However, the AgPS structure does not present other significant functional motifs such as the [GxxxG (GVKLG), SxxxGxxxxA (SVLVGKASA)] and the PALP. The first two motifs should be located at TMD8 of other eukaryotic proteins closely to the catalytic Asp and contribute to the helix packing. The PALP in the TMD9 stabilizes the GSC and characterizes the A22 family. This function has been demonstrated in studies performing mutations in the first Proline of PALP with leucine (P414L) which abrogated PS activity in Drosophila melanogaster and C. elegans [61].
Previously, a 46 kDa soluble aspartyl protease (GenBank accession no. NP 872129) from A. cantonensis was cloned and expressed and present in different infective levels larvae, young adults, and adult worms. Its primary function has been associated with exogenous hemoglobin degradation [62].
In this work, we describe, for the first time, the presence of an atypical presenilin-like aspartyl protease (AgPSat) in A. cantonensis. This nomenclature was attributed to proteins from different organisms that have high similarity with PSs but do not present certain domains characteristic of the PS family [62]. These non-catalytic activities may represent an ancestral PS function, described in non-mammalian animals, plants, and less complex eukaryotic organisms [63].
This sub-branch also possesses two conserved aspartic acid residues within adjacent predicted TM segments. However, the branch of presenilin homologous (PSH) proteins retains the conserved first Proline and the PALP motif but lacks several other motifs present in the A22 family [64,65]. Contrasting in the Ag-PS, the PALP motif is absent, but the protein has a proline located in the C-terminal fraction (aa 373). Interestingly, phylogenetic close related worms such as Haemonchus contortus (Figure 4) have the PALP motif. These collective data suggested that AgPS is an atypical aspartyl protease, with catalytic and non-catalytic functions essential for worm homeostasis [66][67][68][69][70][71].
The tissue and cellular location of this critical proteolytic enzyme and its cellular function need to be demonstrated.
To characterize this TM protein immunologically, a microarray of peptides was initially synthesized using the Spot-synthesis technique to identify linear B-cell epitopes in the putative PS of A. cantonesis that are recognized by infected rats IgG antibodies. Twelve epitopes were readily defined (Table 1), confirming the predicted AgPSat protein's likelihood to be expressed. In addition, the distribution of the peptides corroborates the topological model proposed for the AgPSat.
All peptides sequences were apparently specifically present in the A. cantonensis protein from the determinant profiles. Thus, initially, the immunogenicity of the 12 peptides was demonstrated by an ELISA-peptide assay using a pool of infected rats sera (Figure 4). An ELISA assay was developed using one of the peptides that highly showed sensitivity and specificity of MAP4, displaying its potential role to overcome the limitations of A. cantonensis diagnostics.

Conclusions
In this work, we have demonstrated that the putative aspartyl protease C7BVX5 (UNIPROT) is a 9-pass transmembrane atypical presenilin and proposed a structural model of membrane insertion. In addition, we have provided new insights describing, with more excellent coverage, the set of 12 IgG linear B-epitopes for this aspartyl protease from A. cantonensis and demonstrated the specificity and eligibility of some epitopes to enter phase IIB studies to develop new and fast diagnostic assays for angiostrongyliasis.

Supplementary Materials:
The following are available online at https://www.mdpi.com/article/ 10.3390/membranes12020108/s1, Figure S1: List of synthesized peptides covering the entire sequence of aspartyl protease (C7BVX5) from Angiostrongylus cantonensis. (F3, F4, F5, F11, F12, F13 positive controls and F9, F17 negative controls; Figure S2: Identity of the A. cantonensis aspartyl protease (C78VXS) used in our study with major other proteins deposited in data bank; Figure S3: Hydropathycyty (A) and amino acid accessibility (B) of aminoácids of the A. cantonensis aspartyl protease; Figure S4: Cross-reactivity of the six-presenilin nematode proteins deposited in the database with a higher degree of identity (45-80%) with A. cantonensis. BLAST was carried out in the UniProt (Universal Protein) database of the EBI (European Bioinformatics Institute), using the C7BVX5 protein from the organism "Angiostrongylus cantonensis" as input. Institutional Review Board Statement: The study was approved by the UNIGRANRIO (CAAE: 248 56610.0.0000.5283) study center ethics committee and conducted under good clinical practice and applicable regulatory requirements, including the Declaration of Helsinki.
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.