Cathepsin F of Teladorsagia circumcincta is a recently evolved cysteine protease

Abstract

Parasitic cysteine proteases are involved in parasite stage transition, invasion of host tissues, nutrient uptake, and immune evasion. The cysteine protease cathepsin F is the most abundant protein produced by fourth-stage larvae (L4) of the nematode Teladorsagia circumcincta, while its transcript is only detectable in L4 and adults. T. circumcincta cathepsin F is a recently evolved cysteine protease that does not fall clearly into either of the cathepsin L or F subfamilies. This protein exhibits characteristics of both cathepsins F and L, and its phylogenetic relationship to its closest homologs is distant, including proteins of closely related nematodes of the same subfamily.

Keywords: Cysteine protease; Teladorsagia circumcincta; bioinformatics; cathepsin; gastrointestinal nematode; homology modeling.

Figure 1.

Multiple sequence alignment of Teladorsagia circumcincta secreted cathepsin F (Tci-CF-1, GenBank accession no. ABA01328), the polymorphism discussed in Nisbet et al and variants 1, 2 and 3 identified in this study. Conserved residues indicated by a dot; stop codon by an asterisk; ERFNAQ, E/DxGTA, GxNxFxD and GCNGG motifs by square, cross, circle, and diamond, respectively; catalytic triad residues by a downward arrow; predicted N-glycosylation sites by a right-facing arrow; polymorphisms of interest in boxes.

Figure 2.

Teladorsagia circumcincta cathepsin F variant 3 gene exon/intron structure, constructed using PacBio and Illumina reads in SPAdes and CLC. Exons indicated by solid black arrows, introns indicated by white segments, gaps in contigs indicated by a break. The positions of intron-exon junctions and phase classes are denoted by diamonds.

Figure 3.

Phylogenetic tree of Tci-CF-1 and its 9 closest homologs. Tci-CF-1: Teladorsagia circumcincta secreted cathepsin F (GenBank accession no. ABA01328); Dp-HP-1: Diploscapter pachys hypothetical protein WR25_25536 (GenBank accession no. PAV60527); Hc-PI-1: Haemonchus contortus proteinase inhibitor I25 and proteinase inhibitor I29 and peptidase C1A domain-containing protein (GenBank accession no. CDJ88889); Hc-PI-2: H. contortus proteinase inhibitor I25 and proteinase inhibitor I29 and peptidase C1A domain-containing protein (GenBank accession no. CDJ92562); Dv-CF-1: Dictyocaulus viviparus cathepsin F1 (GenBank accession no. AFM37363); Ac-PFCP: Ancylostoma ceylanicum papain family cysteine protease (GenBank accession no. EPB70524); Aca-HP: Angiostrongylus cantonensis hypothetical protein Angca_010213 (GenBank accession no. KAE9418773); Sv-UP: Strongylus vulgaris unnamed protein product (GenBank accession no. VDM81154); Aco-UP: Angiostrongylus costaricensis unnamed protein product (GenBank accession no. VDM61191); Dp-HP-2: D. pachys hypothetical protein WR25_24125 (GenBank accession no. PAV67875). Scale-bar indicates branch lengths and bootstrap values are indicated following 1000 replications.

Figure 4.

Phylogenetic networks of Tci-CF-1 and its 9 closest homologues; Tci-CF-1: Teladorsagia circumcincta secreted cathepsin F (GenBank accession no. ABA01328); Dp-HP-1: Diploscapter pachys hypothetical protein WR25_25536 (GenBank accession no. PAV60527); Hc-PI-1: Haemonchus contortus proteinase inhibitor I25 and proteinase inhibitor I29 and peptidase C1A domain-containing protein (GenBank accession no. CDJ88889); Hc-PI-2: H. contortus proteinase inhibitor I25 and proteinase inhibitor I29 and peptidase C1A domain-containing protein (GenBank accession no. CDJ92562); Dv-CF-1: Dictyocaulus viviparus cathepsin F1 (GenBank accession no. AFM37363); Ac-PFCP: Ancylostoma ceylanicum papain family cysteine protease (GenBank accession no. EPB70524); Aca-HP: Angiostrongylus cantonensis hypothetical protein Angca_010213 (GenBank accession no. KAE9418773); Sv-UP: Strongylus vulgaris unnamed protein product (GenBank accession no. VDM81154); Aco-UP: Angiostrongylus costaricensis unnamed protein product (GenBank accession no. VDM61191); Dp-HP-2: D. pachys hypothetical protein WR25_24125 (GenBank accession no. PAV67875). (A) Neighbor-Net split network. (B) Reticulate network. Scale-bar indicates the distance of the edges.

Figure 5.

Annotated Teladorsagia circumcincta secreted cathepsin F sequence (GenBank accession no. DQ133568) with annotations. Signal sequence is underlined; predicted N-glycosylation sites marked with squiggle underline; N-terminal amino acid of mature protein indicated by black arrow; catalytic triad active site residues highlighted black; ERFNAQ, E/DxGTA, GxNxFxD and GCNGG motifs indicated by a square, cross, circle and diamond, respectively.

Figure 6.

Homology model of Teladorsagia circumcincta secreted cathepsin F (GenBank accession no. ABA01328). (A) Ribbon structure showing alpha helices, beta sheets, catalytic triad residues (C, H, N), predicted N-glycosylation sites, ERFNAQ, E/DxGTA, GCNGG, and GxNxFxD motifs. (B) Magnified ERFNAQ, E/DxGTA, GxNxFxD motifs and pro-region N-glycosylation site. (C) Magnified catalytic triad and GCNGG motif. (D) Magnified mature domain N-glycosylation site. Sidechains of residues of interest labeled and bold.

Figure 7.

Multiple sequence alignment of Tci-CF-1 with the 9 closest homologous sequences. Tci-CF-1: Teladorsagia circumcincta secreted cathepsin F (GenBank accession no. ABA01328); Dp-HP-1: Diploscapter pachys hypothetical protein WR25_25536 (GenBank accession no. PAV60527); Hc-PI-1: Haemonchus contortus proteinase inhibitor I25 and proteinase inhibitor I29 and peptidase C1A domain-containing protein (GenBank accession no. CDJ88889); Hc-PI-2: H. contortus proteinase inhibitor I25 and proteinase inhibitor I29 and peptidase C1A domain-containing protein (GenBank accession no. CDJ92562); Dv-CF-1: Dictyocaulus viviparus cathepsin F1 (GenBank accession no. AFM37363); Ac-PFCP: Ancylostoma ceylanicum papain family cysteine protease (GenBank accession no. EPB70524); Aca-HP: Angiostrongylus cantonensis hypothetical protein Angca_010213 (GenBank accession no. KAE9418773); Sv-UP: Strongylus vulgaris unnamed protein product (GenBank accession no. VDM81154); Aco-UP: Angiostrongylus costaricensis unnamed protein product (GenBank accession no. VDM61191); Dp-HP-2: D. pachys hypothetical protein WR25_24125 (GenBank accession no. PAV67875). Gaps indicated by a dash; ERFNAQ, E/DxGTA, GxNxFxD and GCNGG motif residues indicated by square, cross, circle and diamond, respectively; catalytic triad residues indicated by a downward arrow.

Figure 8.

Homology model of Teladorsagia circumcincta secreted cathepsin F (GenBank accession no. ABA01328). (A) Pro-region (line residues), mature domain (ribbon), locations of polymorphisms in variants 1, 2, and 3 (bold side-chain residues), and the catalytic triad (bold side-chain residues) which is exposed following cleavage of the pro-peptide. (B) Magnified view of the active site indicating bonds between the pro-region and catalytic triad residues.

Figure 9.

Structural comparison of human and Teladorsagia circumcincta cathepsin proteins. X-ray crystallography of mature domains of human cathepsins L and F, and homology modeling of T. circumcincta cathepsin F (GenBank accession no. ABA01328) and the pro-regions of human cathepsins L (UniProtKB accession no. P07711) and F (UniProtKB accession no. Q9UBX1). (A) Human cathepsin F; (B) T. circumcincta secreted cathepsin F; (C) Human cathepsin L; pro-region, mature domain and cystatin-like domain highlighted in different colors.