Evolutionary Exmpansion Of Nematode-specific Glycine-rich Secreted Peptides

Glycine-rich proteins can be classified into two classes according to the general structure:Class I,consisting of large glycine-rich proteins(GRPs,>200AA)characterized by a length of over 200 amino acids that typically function as structural components of cell walls;and Class II,consisting of small glycine-rich secreted peptides(GRSPs,<200AA)that contain a typical signal peptide followed by a mature peptide with a high glycine content.According to previous studies,genomes of almost all animals,plants and microorganisms were enriched for GRPs.The importance of the GRSPs in nematodes is strengthened by the fact that several members in GRSPs family have been shown to play important roles in C.elegans innate immunity.Objective:We were impressed by published works about family members of C.elegans GRSPs in immune responses and interested in knowing whether there were more GRSPs in nematodes and how GRSPs responded to C.elegans infections.Genome-wide identification of GRSPs would provide a global view of GRSPs evolution in the two nematodes.Integrated analysis of public transcriptome datasets of C.elegans innate immunity would gain insights into roles of C.elegans GRSPs to in innate immune.Method:1.Genome-wide protein sequences of the two nematodes were downloaded from the UCSC database and used to construct two local protein sequence databases.2.Local-Blastp and PSI-Blast programs from NCBI were carried out to identify C.elegans GRSPs with previously identified 6 members of GRSPs family(nlp-29,nlp-31,nlp-33,cnc-2,cnc-4 and cnc-6)as initial queries.3.C.elegans GRSPs were used as queries against the Gen Bank genome database(including H.sapiens,D.rerio,D.melanogaster,A.thaliana and D.discoideum).4.Peptides containing a signal peptide were confirmed by the signal P4.0 server5.Gene Expression Omnibus(GEO)datasets in NCBI and reads of the RNA sequencing project(PRJNA33023)in DRASearch were used to confirm transcriptional expression of C.elegans GRSPs and avoid false positives arising from genome annotation errors.6.Eight transcriptome datasets related to C.elegans infection quantified by microarray(GSE20053,E-MEXP-696,GSE27867,GSE54212,GSE53732,GSE41058,G SE37266 and GSE2740)were downloaded from the GEO database in NCBI.Differentially expressed genes were screened using the GEO2 R tool in the GEO database.7.Signal peptide sequences of the two nematode GRSPs were further analyzed by multi-sequence alignment using Muscle with manual adjustment in EBI database.8.Based on the signal peptide sequences of the two nematode GRSPs,a phylogenetic tree was constructed to analyze the evolution of GRSPs families by gene duplication using the program Molecular Evolutionary Genetics Analysis package version 6(MEGA6).9.Protein-coding sequences of the nematode GRSPs from subfamily ? to subfamily ?? were aligned according to their protein alignment using Clustal Omega with manual adjustment.10.Transition(Ti)/Transversion(Tv)ratios(R)among nucleotides,the number of synonymous(d S)and nonsynonymous(d N)substitutions per site,and the codon-based Z-test for purifying selection were estimated by MEGA 6.Results:1.Genome-wide comparisons across 10 species from algae Guillardia theta to mammal human indicated that Caenorhabditis elegans and Caenorhabditis briggsae were highly enriched for glycine-rich secreted peptides(GRSPs)(110 GRSPs in C.elegans and 93 in C.briggsae)in this study.2.Chromosomal mapping showed that most GRSPs were clustered on the two nematode genomes [103(93.64%)in C.elegans and 82(88.17%)in C.briggsae],which could be divided into 18 cluster units in C.elegans and 13 in C.briggsae,respevtively.Except for four C.elegans GRSPs clusters without matching clusters in C.briggsae,all other GRSPs clusters had paired synteny block between the two nematode genomes.3.Analyzing transcriptome datasets quantified by microarray indicated extensive genome-wide co-expression of GRSPs clusters after C.elegans infections.Highly homologous coding sequences and conserved exon-intron structures indicated that GRSPs tight clusters were likely derived from local DNA duplications.4.Purifying selection was carried out directly in sequence pairs and overall average by codon-based Z-test,which resulted in values equal to zero.The overall average difference of(d N-d S)was less than zero.Synonymous substitutions were clearly predominant on protein-coding sequences of the two nematode GRSPs,thus indicating occurrences of purifying selection.Conclusion:Highly homologous coding sequences of GRSPs with conserved exon-intron structure in tight clusters were likely derived from local DNA duplications.The phylogenetic conservation of synteny GRSPs clusters on their genomes,co-expression of GRSPs clusters and strong purifying selections may indicate evolutionary constraints acting on C.elegans to guarantee that C.elegans could rapidly respond to infections by genome-wide co-expression of GRSPs clusters.Significance:Phylogenetic conservation of synteny blocks between their genomes,co-expression of GRSPs clusters after C.elegans infections and strong purifying selections of coding sequences may indicate evolutionary constraints acting on C.elegans to guarantee that C.elegans could mount rapid systematic responses to infections by co-expression,co-regulation and co-functionality of GRSPs clusters.Glycine-rich secreted peptides(GRSPs),a family of proteins that are very critical for innate immune responses,are highly enriched in C.elegans and C.briggsae,compared with other species.Genome-wide identification of GRSPs would provide a global view of GRSPs evolutionary exmpansion in the two nematodes.Integrated analysis of public transcriptome datasets of C.elegans innate immunity would gain insights into roles of C.elegans GRSPs in innate immune.