Evolutionary History of Cathepsin L (L-like) Family Genes in Vertebrates.

Cathepsin L family, an important cysteine protease found in lysosomes, is categorized into cathepsins B, F, H, K, L, S, and W in vertebrates. This categorization is based on their sequence alignment and traditional functional classification, but the evolutionary relationship of family members is unclear. This study determined the evolutionary relationship of cathepsin L family genes in vertebrates through phylogenetic construction. Results showed that cathepsins F, H, S and K, and L and V were chronologically diverged. Tandem-repeat duplication was found to occur in the evolutionary history of cathepsin L family. Cathepsin L in zebrafish, cathepsins S and K in xenopus, and cathepsin L in mice and rats underwent evident tandem-repeat events. Positive selection was detected in cathepsin L-like members in mice and rats, and amino acid sites under positive selection pressure were calculated. Most of these sites appeared at the connection of secondary structures, suggesting that the sites may slightly change spatial structure. Severe positive selection was also observed in cathepsin V (L2) of primates, indicating that this enzyme had some special functions. Our work provided a brief evolutionary history of cathepsin L family and differentiated cathepsins S and K from cathepsin L based on vertebrate appearance. Positive selection was the specific cause of differentiation of cathepsin L family genes, confirming that gene function variation after expansion events was related to interactions with the environment and adaptability.

[Cloning and expression analysis of Cathepsin L cDNA of Exopalaemon carinicauda].

Based on the EST sequence from a hemocyte cDNA library, the cathepsin L cDNA of Exopalaemon carinicauda (EcCatL) was cloned by rapid amplification of cDNA ends (RACE). The EcCatL cDNA was 1136 bp in length, which contains an open reading frame (ORF) of 960 bp, encoding a 319 amino-acid polypeptide. Homology analysis revealed that the amino acid sequence of EcCatL was highly conserved with its homologs in other crustaceans. The similarities of EcCatL with the CatL of Palaemonetes varians and Pandalus borealis were 92% and 76%, respectively. Phylogenetic analysis showed that EcCatL was in the same branch as that of Palaemonetes varians. The expression levels of EcCatL in different tissues were analyzed by quantitative real-time PCR. Expression of EcCatL was detected in all tested tissues of E. carinicauda, including hemocytes, gill, hepatopancreas, muscle, ovary, intestine, stomach and eyestalk, with the highest expression level in hepatopancreas. After challenged with Vibrio anguillarum or white spot syndrome virus, the expression of EcCatL were up-regulated in the hemocytes and hepatopancreas of E. carinicauda. Our results implied that EcCatL might play an important role in the prawn immune response.

Structural and functional conservation profiles of novel cathepsin L-like proteins identified in the Drosophila melanogaster genome.

Cathepsin L is a cysteine protease which degrades connective tissue proteins including collagen, elastin, and fibronectin. In this study, five well-characterized cathepsin L proteins from different arthropods were used as query sequences for the Drosophila genome database. The search yielded 10 cathepsin L-like sequences, of which eight putatively represent novel cathepsin L-like proteins. To understand the phylogenetic relationship among these cathepsin L-like proteins, a phylogenetic tree was constructed based on their sequences. In addition, models of the tertiary structures of cathepsin L were constructed using homology modeling methods and subjected to molecular dynamics simulations to obtain reasonable structure to understand its dynamical behavior. Our findings demonstrate that all of the potential Drosophila cathepsin L-like proteins contain at least one cathepsin propeptide inhibitor domain. Multiple sequence alignment and homology models clearly highlight the conservation of active site residues, disulfide bonds, and amino acid residues critical for inhibitor binding. Furthermore, comparative modeling indicates that the sequence/structure/function profiles and active site architectures are conserved.

Protection and antibody isotype responses against Fasciola hepatica with specific antibody to pIII-displayed peptide mimotopes of cathepsin L1 in sheep.

Fluke burdens, liver fluke size and biomass, fecal eggs counts, serum levels of hepatic enzymes and immune response were assessed in sheep immunized with peptide mimotopes of cathepsin L1 and challenged with Fasciola hepatica metacercariae. Twenty sheep were randomly allocated to four groups of five animals each; groups 1 and 2 were immunized at weeks 0 and 2 with mimotopes YVYRWVEAECVA and FSPAYLRDAALK, respectively; group 3 was immunized with wild-type M13KE phage and the control group received phosphate-buffered saline (PBS). All groups were challenged with 300 metacercariae at week 6 and slaughtered 16 weeks later. Groups 1 and 2 showed a reduction in fluke burden of 51.7% and 35.9%, respectively, when compared to the control group, but only the former was significant at the 5% level. Vaccinated animals showed a significant reduction in fluke length and width, wet weights and egg output. A significant diminution in the total biomass of parasites recovered was also observed in group 1. Levels of anti-phage total IgG increased rapidly within 2 weeks of the first immunization and were always significantly higher in groups 1 and 2 than in the infected control group. The fluke burden in group 1 was significantly correlated with IgG1 and total IgG. The vaccinated sheep with phage clones produced significantly high titres of IgG1 and IgG2 antibodies indicating a mixed Th1/Th2 response.