Molecular characterization of a cathepsin F-like protease in Trichinella spiralis

Abstract

Background: Trichinellosis is a re-emerging infectious disease, caused by Trichinella spp. Cathepsin F belongs to cysteine protease that is a major virulence factor for parasitic helminths, and it may be a potential anti-helminth drug target and vaccine candidate. The aim of this study was to clone, express and identify a cathepsin F-like protease in Trichinella spiralis and to investigate its biochemical characteristics.

Methods: The full-length cDNA encoding a putative cathepsin F-like protease in T. spiralis, TsCF1, was cloned and its biochemical characterization and expression profile were analyzed. Transcription of TsCF1 at different developmental stages of T. spiralis was observed by RT-PCR. The recombinant TsCF1 protein was expressed by prokaryotic expression system and recombinant TsCF1 (rTsCF1) was analyzed by western blotting. And expression of TsCF1 at muscle larvae stage was performed by immunofluorescent technique. Molecular modeling of TsCF1 and its binding mode with E-64 and K11777 were analyzed. Enzyme activity and inhibitory test with E-64 as inhibitor were investigated by using Z-Phe-Arg-AMC as specific substrate.

Results: Sequence analysis revealed that TsCF1 ORF encodes a protein of 366 aa with a theoretical molecular weight of 41.9 kDa and an isoelectric point of 7.46. The cysteine protease conserved active site of Cys173, His309 and Asn333 were identified and cathepsin F specific motif ERFNAQ like KLFNAQ sequence was revealed in the propeptide of TsCF1. Sequence alignment analysis revealed a higher than 40 % identity with other cathepsin F from parasitic helminth and phylogenetic analysis indicated TsCF1 located at the junction of nematode and trematode. RT-PCR revealed the gene was expressed in muscle larvae, newborn larvae and adult stages. SDS-PAGE revealed the recombinant protein was expressed with the molecular weight of 45 kDa. The purified rTsCF1 was used to immunize rabbit and the immune serum could recognize a band of about 46 kDa in soluble protein of adult, muscle larvae and ES product of muscle larvae. Immunolocalization analysis showed that TsCF1 located on the cuticle and stichosome of the muscle larvae. After renaturation rTsCF1 demonstrated substantial enzyme activity to Z-Phe-Arg-AMC substrate with the optimal pH 5.5 and this activity could be inhibited by cysteine protease inhibitor E-64. Further analysis showed the kinetic parameters of rTsCF1 to be Km = 0.5091 μM and Vmax = 6.12 RFU/s μM at pH 5.5, and the IC50 value of E64 was 135.50 ± 16.90 nM.

Conclusion: TsCF1 was expressed in all stages of T. spiralis and localized in the cuticle and stichosome. TsCF1 might play a role in the life cycle of T. spiralis and could be used as a potential vaccine candidate and drug target against T. spiralis infection.

Fig. 1

The nucleotide and deduced amino acid sequence of TsCF1 cDNA. The mature protease domain is shown lightly shaded; the conserved KLFNAQ, GNFN and GCNGG motifs are shown lightly shaded and in bold; and conserved papain family active site amino acids are indicated in the square frame of the sequence

Fig. 2

RT-PCR results of TsCF1 gene at different stages of T. spiralis. M: DL2000 DNA Marker; 1: muscle larvae; 2: newborn larvae; 3: 3 day old adult; 4: 5 day old adult

Fig. 3

Structural alignment of TsCF1 and 1M6D. Molecule of 1M6D is blue, residues of pocket are yellow, disulfide bonds are red. For TsCF1, molecule is shown in silver, residues of pocket is green, disulfide bonds is pink (the picture was created by VMD1.9.1)

Fig. 4

Stereo view of the interactions between TsCF1 (silver) and E64 (green), K11777 (yellow). Surface representation of the pocket of TsCF1, ligand displayed with sticks. a Binding mode of E64 and interactions between E64 and TsCF1. a and b represent the key interactions between TsCF1 and E64, surface representation of modeled TsCF1 active pocket, and inhibitor E64 are shown in green bonds. c Hydrophobic interactions between TsCF1 and E64. (a and b was created with VMD1.9.1, and c was created with LigPlot v.1.4.5) (color figure online). b Binding mode of K11777 and interactions between K11777 and TsCF1. a Key interactions between TsCF1 and K11777, surface representation of modeled TsCF1 active pocket, and K11777 are shown in yellow bonds. b Hydrophobic interactions between TsCF1 and K11777. (a was created with VMD1.9.1, and b was created with LigPlot v.1.4.5) (color figure online)

Fig. 5

Phylogenetic analysis of cathepsin F family proteins from different species. The GenBank accession number of each cathepsin F is : T. spiralis (EFV56510.1), (EFV55373.1), (XP_003378244.1), Loa loa (EFO21301.2), Brugia malayi (AAT07059), Teladorsagia circumcincta (ABA01328), Dictyocaulus viviparus (AFM37363.1), Caenorhabditis briggsae (CAP22767.1), Ascaris suum (ERG86355), Paragonimus westermani (AAF21461), (AAY81944), (AAW28151), (AAW28152), (AAW81947), (AAY81948), (AAY81942), (AAY81943), (AAY81946), (AAB93494), Schistosoma japonicum (AAW25775), Schistosoma mansoni (CCD77198), Clonorchis sinensis (AAD29130), (ABC69435), (AAP33049), (ABC69429), (AAP33050), Opisthorchis viverrini (AAV69023O) in GenBank and Caenorhabditis japonica (Wormbase ID: CJA01633) in wormbase. The tree was rooted using Homo sapiens (AAD41790)

Fig. 6

SDS-PAGE analysis of recombinant TsCF1 protein. M: Protein molecular weight marker; 1: purified recombinant protein by Ni-NTA

Fig. 7

Western blot analysis of antigens of T. spiralis. M: Protein molecular weight Marker; 1: rTsCF1; 2: crude extract of T. spiralis muscle larvae; 3: ES products of ML; 4: crude extract of T. spiralis adult

Fig. 8

Immunlocalization of TsCF1 in T. spiralis muscle larvae. a serum of healthy rabbit b anti-rTsCF1 serum

Fig. 9

Enzymatic activity of purified rTsCF1. a rTsCF1 activity. The concentration of substrate [Z-Phe-Arg-AMC] was plotted on the x-axis; the Z-Phe-Arg-AMC in relative fluorescence units (RFU)/s was plotted on the y-axis. b The curve of inhibitory activity of E64 with rTsCF1. The concentration of E64 was plotted on the x-axis; the relative activity of rTsCF1 with E64 is plotted on the y-axis