Identification and characterization of epicuticular proteins of nematodes sharing motifs with cuticular proteins of arthropods

Specific collagens and insoluble proteins called cuticlins are major constituents of the nematode cuticles. The epicuticle, which forms the outermost electron-dense layer of the cuticle, is composed of another category of insoluble proteins called epicuticlins. It is distinct from the insoluble cuticlins localized in the cortical layer and the fibrous ribbon underneath lateral alae. Our objective was to identify and characterize genes and their encoded proteins forming the epicuticle. The combination between previously obtained laboratory results and recently made available data through the whole-genome shotgun contigs (WGS) and the transcriptome Shotgun Assembly (TSA) sequencing projects of Ascaris suum allowed us to identify the first epicuticlin gene, Asu-epic-1, on the chromosome VI. This gene is formed of exon1 (55 bp) and exon2 (1067 bp), separated by an intron of 1593 bp. Exon 2 is formed of tandem repeats (TR) whose number varies in different cDNA and genomic clones of Asu-epic-1. These variations could be due to slippage of the polymerases during DNA replication and RNA transcription leading to insertions and deletions (Indels). The deduced protein, Asu-EPIC-1, consists of a signal peptide of 20 amino acids followed by 353 amino acids composed of seven TR of 49 or 51 amino acids each. Three highly conserved tyrosine motifs characterize each repeat. The GYR motif is the Pfam motif PF02756 present in several cuticular proteins of arthropods. Asu-EPIC-1 is an intrinsically disordered protein (IDP) containing seven predicted molecular recognition features (MoRFs). This type of protein undergoes a disorder-to-order transition upon binding protein partners. Three epicuticular sequences have been identified in A. suum, Ascaris lumbricoides, and Toxocara canis. Homologous epicuticular proteins were identified in over 50 other nematode species. The potential of this new category of proteins in forming the nematode cuticle through covalent interactions with other cuticular components, particularly with collagens, is discussed. Their localization in the outermost layer of the nematode body and their unique structure render them crucial candidates for biochemical and molecular interaction studies and targets for new biotechnological and biomedical applications.Funding provided by: Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen ForschungCrossref Funder Registry ID: http://dx.doi.org/10.13039/501100001711Award Number: Funding provided by: Université de NeuchâtelCrossref Funder Registry ID: http://dx.doi.org/10.13039/501100005353Award Number: Funding provided by: Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen ForschungAward Number: 008439Funding provided by: Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen ForschungAward Number: 26764Funding provided by: Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen ForschungAward Number: 33961Funding provided by: Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen ForschungAward Number: 047194The public accessible cDNA (X92101.2, 31B1A; AJ408885.1, C1; AJ408886.1, C2; AJ408887.1, C3) and genomic clones (AJ408888.1, G1; AJ408889.1, G2; AJ408890.1, G3), coding for A.suum epicuticlins, were used for a comparison of their common properties through alignments using Jalview. Based on the conserved common characteristics (aagaggaa), nucleotide Blasts were carried out on the following databases: NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi) nucleotide collection, whole-genome shotgun contigs (wgs) of A. suum; WormbaseParasite and the EMBL-EBI services. Protein Blasts were carried out again on the same platforms and also using the protein knowledge platform UniProt. Multiple or pairwise nucleotide or protein sequence alignments were carried out using the EMBL-EBI services: Clustal Omega for multiple alignments and Needle or Matcher for pairwise alignments. Radar was used for the automatic alignment of protein repeats and Phobius to identify signal peptides. For the analysis of the C. elegans genes, Wormbase was the authoritative data source. Following Expasy services (https://www.expasy.org/) were applied for the in-depth study of the epicuticlins: STRING, ProtParam and Compute pI/MW