1.11 Protozoan peptidases
1.11.1 Serine peptidases
Serine peptidases play a vital role in various physiological functions such as digestion, immune responses, fibrinolysis to name but a few (Polgár, 2005). These peptidases are characterised by a Ser, His, Asp catalytic triad (Polgár, 2005). The mechanism by which classical serine peptidases act is detailed in Fig. 1.8.
Figure 1.8: The reaction mechanism and amino acid orientation during serine peptidase mediated proteolysis. The catalytic triad consists of Asp, His and Ser residues whereby the serine nucleophile attacks the substrate carbonyl carbon (Erez et al., 2009).
The serine hydroxyl nucleophile attacks the carbonyl carbon of the substrate whereby a tetrahedral intermediate and an imidazolium ion is formed (Polgár, 2005). The tetrahedral intermediate is stabilised by an oxyanion hole in the enzyme, which provides the hydrogen bonds to the oxygen atom, and is formed by the backbone amide of the Gly193 and Ser195 residues (Polgár, 1987; Hedstrom, 2002). The formation of the acyl enzyme and the amine product, from the intermediate, is achieved by acid catalysis (Polgár, 2005). The acyl enzyme is subsequently deacylated by the attack of a water molecule rather than the serine nucleophile (Polgár, 2005).
Serine peptidases are classed in 12 clans, namely SB, SC, SE, SF, SH, SJ, SK, SO, SP, SR, SS and ST along with 7 families which have not yet been assigned. Prolyl oligopeptidase (POP) and oligopeptidase B (OPB), present in trypanosomes, are members of the S9 family within the SC clan of serine peptidases (Coetzer et al., 2008;
Rawlings et al., 2008). The SC clan possesses the same α/β-hydrolase fold containing the catalytic triad common to the classical serine peptidases with the exception in the
reverse of handedness (Fülöp et al., 1998). Whereas POP cleaves peptides, no bigger than 30 residues, after a proline residue (Rawlings et al., 1991; Barrett and Rawlings, 1992), OPB belongs to a small subgroup of peptidases from the S9 family which are able to cleave peptides C-terminal to basic residues (Burleigh et al., 1997; Polgár, 2002).
1.11.1.1 Oligopeptidase B
African trypanosomes possess an OPB enzyme which has been implicated in trypanosomiasis since the serine peptidase inhibitors (serpins) found in the host bloodstream are unable to inhibit their activity such as the degradation of host peptide hormones (Troeberg et al., 1996; Morty et al., 2005; Coetzer et al., 2008). This enables the trypanosomal parasite to disrupt the hormone metabolism of the host during infection, as is illustrated by the decrease in hormone levels during the course of trypanosomal infection (Tizard et al., 1978; Tetaert et al., 1993; Brandenberger et al., 1996). The OPB enzyme (EC 3.4.21.83) has a trypsin-like specificity for small peptide substrates and cleaves peptides after basic Arg or Lys residues (Troeberg et al., 1996;
Fülöp et al., 1998; Morty et al., 2005; Coetzer et al., 2008). The peptidase‟s specificity for small peptides is due to the N-terminal β-propeller domain which restricts access to the catalytic triad (Fülöp et al., 1998; Gérczei et al., 2000). The invasion of the host cell by T. cruzi requires calcium signalling which is generated by the proteolytic activity of OPB (Burleigh et al., 1997). Research has shown that OPB is able to hydrolyse and thus inactivate atrial natriuretic factor in the plasma of infected rats (Morty et al., 2001;
Morty et al., 2005) which is a contributing factor for the characteristic hypervolemia symptoms in AT infections (Anosa and Isoun, 1976). The OPB from T. b. brucei has been shown to retain its catalytic activity in the bloodstream of the host and is responsible for the degradation of the host‟s peptide hormones whereby pathogenesis is promoted (Morty et al., 1999; Morty et al., 2001). Trypanocidal drugs, pentamidine, diminazene, and suramin, have been found to target the OPB enzyme (Morty et al., 1998). Since there are no mammalian OPB orthologues, it is considered to be an attractive chemotherapeutic target (Coetzer et al., 2008; Bastos et al., 2013). The application of OPB as a diagnostic antigen, however, has yet to be investigated.
The OPB from T. congolense (TcOPB, CCD12722.1), T. vivax (TvOPB, CCC53885.1) and T. cruzi (TcrOPB, XP_809967.1), and Leishmania major OPB (LmjOPB, XP_001681251.1) have a 81, 75, 72 and 61% sequence identity to T. b. brucei OPB (TbOPB, AAC80459.1) respectively (Fig. 1.9). The 3D structure of kinetoplastid OPB has only been solved for Leishmania major (McLuskey et al., 2010) and T. b. brucei
(Canning et al., 2013) in an attempt to understand the catalytic mechanism and regulation of the peptidase activity (Fig. 1.10).
Figure 1.9: A sequence comparison between the oligopeptidase B enzymes of the various animal and human infective Trypanosoma and a human infective Leishmania species. Multiple sequence alignment of the SC clan, S9A family of serine oligopeptidases generated using ClustalX (Larkin et al., 2007). Protein sequences were obtained from UniProtKB (http://www.uniprot.org/help/uniprotkb, accessed 25/08/2013):
T. b. brucei, O76728, T. congolense, F9W6B7, T. vivax, G0U8G0, T. cruzi, Q4D6H1, and Leishmania major, Q4QHU7. The amino acid residues involved in the catalytic site are highlighted in yellow. Alignment characters are annotated as follows: conserved residues (*), strongly similar properties (:) and weakly similar properties (.).
Figure 1.10: Structure of oligopeptidase B from T. b. brucei. The open state, non-ligand bound TbOPB is a dimer with a P3 symmetry which was resolved at a 2.4Å resolution (Canning et al., 2013). The blue, green and yellow β sheets form the N-terminal β-propeller domain whilst the blue, red and orange α helices form the catalytic domain.
TbOPB GKYLTKRNTFMDFIACAEHLISSGLTTPAQLSCEGRSAGGLLVGAVLNMRPDLFHVALAG TcOPB GKYLTKRNTFMDFISCAEHLISSGVTTPPQLACEGRSAGGLLVGAVLNMRPDLFRVAVAG TvOPB AKYLTKRNTFMDFIACAEHLISSGLTTPNQLACEGRSAGGLLIGAVLNMRPDLFHVALAG TcrOPB AKYLTKRNTFSDFIACAEYLIEIGLTTPSQLACEGRSAGGLLIGAVLNMRPDLFRVALAG LmjOPB AKYLTKRNTFSDFIAAAEFLVNAKLTTPSQLACEGRSAGGLLMGAVLNMRPDLFKVALAG .********* ***:.**.*:. :*** **:**********:***********:**:**
TbOPB VPFVDVMTTMCDPSIPLTTGEWEEWGNPNEYKFFDYMNSYSPIDNVRAQDYPHLMIQAGL TcOPB VPFVDVMTTMCDPSIPLTTGEWEEWGNPNEYKFFDYMNSYSPIDNVRPQDYPNLIIQAGL TvOPB VPFVDVMTTMCDPTIPLTTFEWEEWGNPNEYKYFDYMNSYSPIDNVRAQAYPHLMIQAGL TcrOPB VPFVDVMTTMCDPSIPLTTGEWEEWGNPNEYKFFDYMNSYSPVDNVRAQDYPHLMIQAGL LmjOPB VPFVDVMTTMCDPSIPLTTGEWEEWGNPNEYKYYDYMLSYSPMDNVRAQEYPNIMVQCGL *************:***** ************::*** ****:****.* **::::*.**
TbOPB HDPRVAYWEPAKWASKLRELKTDSNEVLLKMDLESGHFSASDRYKYLRENAIQQAFVLKH TcOPB HDPRVAYWEPAKWASKLRELKTDNNEVLLKMDLDSGHFSASDRYKYLREHAIQQAFVLKH TvOPB HDPRVAYWEPVKWASRLRQLKTDGNEVLVKMDLDSGHFSSADRYKYWREMAIQQAFVLKH TcrOPB HDPRVAYWEPAKWASKLRALKTDSNEVLLKMDLESGHFSASDRYRYWREMSFQQAFVLKH LmjOPB HDPRVAYWEPAKWVSKLRECKTDNNEILLNIDMESGHFSAKDRYKFWKESAIQQAFVCKH **********.**.*:** ***.**:*:::*::*****: ***:: :* ::***** **
TbOPB LN--VRQLLRK TcOPB LG--VRRLLRH TvOPB LN--VRCLLRR TcrOPB LN--ARTLLRR LmjOPB LKSTVRLLVRR * .* *:*: