1.11 Protozoan peptidases
1.11.2 Cysteine peptidases
1.11.2.1 Cathepsin L- like peptidase
Two peptidases from the C1 family of the CA clan of cysteine peptidases are expressed by kinetoplastid parasites and are similar to mammalian cathepsins B and L, and were named cathepsin B- and L- like peptidases (CATB and CATL) (Cazzulo et al., 1990; Caffrey et al., 2000). The CATL peptidases are key factors in the trypanosomal infectivity, cell differentiation, metabolism and elicit prominent immune responses and as such are considered to be potential chemotherapeutic and diagnostic targets (Lalmanach et al., 2002; Cortez et al., 2009). All kinetoplastid derived CATL and CATB peptidases share the same characteristic of other cysteine proteases, where they possess the conserved Cys, His catalytic dyad (Lecaille et al., 2002). In T. congolense infected cattle, it was found that TcCATB elicited a strong immune response and might be an appropriate antigen for the development of T. congolense specific diagnostic tests (Mendoza-Palomares et al., 2008). The kinetoplastid CATL peptidases possess an additional, highly immunogenic 11 to 13 kDa C-terminal extension (Caffrey et al., 2011). It has been recently shown that CATL is essential to the survival of the T. b. brucei parasite rather than CATB, and when completely inhibited the in vitro parasites died (Steverding et al., 2012). Thus CATL is an important chemotherapeutic and thus diagnostic target.
Congopain (EC 3.4.22.15), a CATL peptidase from T. congolense (TcCATL), has previously been shown to be a pathogenic factor in T. congolense infected cattle (Authié et al., 2001). It is a dominant antigen and as a result humoral and cellular responses are elicited against congopain (Lalmanach et al., 2002). Trypanotolerance in these cattle was correlated to anti-congopain antibodies (Authié et al., 1993a).
Detection of TcCATL and its associated proteolytic activity has been detected in the plasma of infected cattle (Authié et al., 1993a). Authié and co-workers (1993a; 1994) hypothesised that anti-TcCATL antibodies may alleviate disease symptoms and contribute to trypanotolerance in cattle.
Trypanopain (TbCATL) (Mackey et al., 2004), congopain (TcCATL) (Mendoza- Palomares et al., 2008) and rhodesian (TbrCATL) (Caffrey et al., 2001) are expressed throughout each of the four life cycle stages, but most significantly in the BSF stage
where it is localised in the lysosomes (Mbawa et al., 1991; Scory et al., 1999; Caffrey et al., 2001). The antigens used for serodiagnosis need to be expressed in the BSF and thus the CATL antigens are potential diagnostic antigens.
Many isoforms of congopain exist (Pillay et al., 2010) and the same trend is observed in the CATL peptidases from Leishmania mexicana and T. cruzi (Mottram et al., 1997;
Lima et al., 2001). Cruzipain is the major CATL lysosomal protease present in the T. cruzi parasite and is involved in the invasion process into mammalian smooth muscle and epithelial cells (Aparicio et al., 2004; Yoshida, 2006). It is thought that cruzipain might be involved in the mechanism of evasion from the immune system by its digestion of the hinge region of the human IgG subclasses (Berasain et al., 2003). It has been shown that along with TcrOPB and the metacyclic surface glycoprotein, gp82, that cruzipain plays a vital role in the mobilisation of Ca2+which results in host cell invasion by the T. cruzi parasite (Dorta et al., 1995; Burleigh et al., 1997; Caler et al., 1998).
The protozoan CATL enzymes have a similar 3D structure to that of papain-like cysteine peptidases and are comprised of an α-helical L-domain and an anti-parallel β-sheet R-domain with the propeptide settled in the substrate binding pocket (Lecaille et al., 2002). The 3D structures of only two kinetoplastid CATL have been solved to date in the presence of different substrates and inhibitors, namely those from T. cruzi and T. b. rhodesiense (Fig. 1.12).
Figure 1.12: The cathepsin L-like peptidase from T. b. rhodesiense in the presence of the K777 inhibtitor. TbrCATL (PDB ID: 2P7U) is a monomer with P1 symmetry and was resolved at 1.65 Å (Kerr et al., 2009).
The TDR targets database lists cruzipain and rhodesian as druggable targets (http://tdrtargets.org/targets/view?gene_id=48009, accessed 20/10/2103) and are thought to be potential diagnostic antigens. Rhodesian, the CATL from
T. b. rhodesiense, was thought to have diagnostic potential due to the fact that cruzipain was a diagnostic antigen and that it was highly expressed in BSF parasites, but was not recognised in infected human sera (Manful et al., 2010).
Vivapain (TvCATL, CCD21670.1), congopain (TcCATL, CAA81061.1) and cruzipain (TcrCATL, P25779.1) share a 78%, 72% and 71% sequence identity to rhodesian (TbrCATL, CAC67416.1) respectively and thus should possess similar diagnostic and chemotherapeutic potential (Fig. 1.13).
Figure 1.13: Sequence alignment of the cathepsin-L like peptidases from T. congolense, T. b. rhodesiense, T. vivax and T. cruzi. Multiple sequence alignment of the CA clan, C1 family of cysteine peptidases generated using ClustalX (Larkin et al., 2007).
Protein sequences were obtained from UniProtKB (http://www.uniprot.org/help/uniprotkb, accessed 25/08/2013): TcCATL from T. congolense, Q26895, TbrCATL from T. b. rhodesiense, Q95PM0, TvCATL from T. vivax, F9WRA9, and TcrCATL from T. cruzi, P25779. The amino acid residues involved in the signal peptide are highlighted in yellow, those in the propeptide in pink, the catalytic domain in blue, the catalytic site in grey, the C-terminal extension in green, and the 18-mer TcCATL N-terminal peptide in red and yellow. Alignment characters are annotated as follows: conserved residues (*), strongly similar properties (:) and weakly similar properties (.).
TcCATL MPRSEMTRTLRFSVGLLAVAACFVPVALGVLHAEQSLQQQFAAFKQKYSRSYKDATEEAF TbrCATL MPRTEMVRFVRLPVVLLAMAACLASVALGSLHVEESLEMRFAAFKKKYGKVYKDAKEEAF TvCATL ---MHAHALVTLLAAAVSVAPAAMAVLRADGPVEPLFAAFKQKYGRSYGTAAEEAF TcrCATL ---MSGWARALLLAAVLVVMACLVPAATASLHAEETLTSQFAEFKQKHGRVYESAAEEAF . :*. ...* . *:.: .: ** **:*:.: * * ****
TcCATL RFRVFKQNMERAKEEAAANPYATFGVTRFSDMSPEEFRATYHNGAEYYAAALKRPRKVVN TbrCATL RFRAFEENMEQAKIQAAANPYATFGVTPFSDMTREEFRARYRNGASYFAAAQKRLRKTVN TvCATL RLRVFEDNMRRSRMYAAANPHATFGVTPFSDLTPEEFRTRYHNGERHFEAARGRVRTLVQ TcrCATL RLSVFRENLFLARLHAAANPHATFGVTPFSDLTREEFRSRYHNGAAHFAAAQERARVPVK *: .*.:*: :: *****:****** ***:: ****: *:** :: ** * * *:
TcCATL VSTGKAPEAVDWRKKGAVTPVKDQGQCGSCWAFSAIGNIEGQWKVAGHELTSLSEQMLVS TbrCATL VTTGRAPAAVDWREKGAVTPVKDQGQCGSCWAFSTIGNIEGQWQVAGNPLVSLSEQMLVS TvCATL VPPGKAPAAVDWRRKGAVTPVKDQGTCGSCWSFSAIGNIEGQWAAAGNPLTSLSEQMLVS TcrCATL VEVVGAPAAVDWRARGAVTAVKDQGQCGSCWAFSAIGNVECQWFLAGHPLTNLSEQMLVS * ** ***** :****.***** *****:**:***:* ** **: *..********
TcCATL CDTNDFGCEGGLMDDAFKWIVSSNKGNVFTEQSYPYASGGGNVPTCDKSGKVVGAKIRDH TbrCATL CDTIDFGCGGGLMDNAFNWIVNSNGGNVFTEASYPYVSGNGEQPQCQMNGHEIGAAITDH TvCATL CDTKDNGCGGGLMDNAFEWIVKENSGKVYTEKSYPYVSGGGEEPPCKPRGHKVGATITGH TcrCATL CDKTDSGCSGGLMNNAFEWIVQENNGAVYTEDSYPYASGEGISPPCTTSGHTVGATITGH **. * ** ****::**:***..* * *:** ****.** * * * *: :** * .*
TcCATL VDLPEDENAIAEWLAKNGPVAIAVDATSFQSYTGGVLTSCISEHLDHGVLLVGYDDTSKP TbrCATL VDLPQDEDAIAAYLAENGPLAIAVDATSFMDYNGGILTSCTSEQLDHGVLLVGYNDSSNP TvCATL VDIPHDEDAIAKYLADNGPVAVAVDATTFMSYSGGVVTSCTSEALNHGVLLVGYNDSSKP TcrCATL VELPQDEAQIAAWLAVNGPVAVAVDASSWMTYTGGVMTSCVSEQLDHGVLLVGYNDSAAV *::*.** ** :** ***:*:****::: *.**::*** ** *:********:*::
TcCATL PYWIIKNSWSKGWGEEGYSALRR-HNQCLMKNLPSSAVVSG---PPPPPPTPTFTQEL TbrCATL PYWIIKNSWSNMWGEDGYIRIEKGTNQCLMNQAVSSAVVGGPTPPPPPPPPPSATFTQDF TvCATL PYWIIKNSWSSSWGEKGYIRIEKGTNQCLVAQLASSAVVGG---PGPTPTPTPTTN- TcrCATL PYWIIKNSWTTQWGEEGYIRIAKGSNQCLVKEEASSAVVGGP----GPTPEPTTTTTTSA *********:. ***.** : : ****: : *****.* * * *:.* * . TcCATL ---CEGAECQSKCTKATFPTGKCVQLSGAGSVIASCGSNNLTQIVYPLSSS TbrCATL ---CEGKGCTKGCSHATFPTGECVQTTGVGSVIATCGASNLTQIIYPLSRS TvCATL ---NNNNNDRTWP---IVKLHEDALQR--- TcrCATL PGPSPSYFVQMSCTDAACIVGCENVTLPTGQCLLTTSGVSAIVTCGAETLTEEVFLTSTH . * * *.. . * . TcCATL CSGFSVPLTVPLDKCLPIVIGSVMYECSDKAP---TESARLVRHE--- TbrCATL CSGLSVPITVPLDKCIPILIGSVEYHCSTNPP---TKAARLVPHQ--- TvCATL --- TcrCATL CSGPSVRSSVPLNKCNRLLRGSVEFFCGSSSSGRLADVDRQRRHQPYHSRHRRL