Pyroglutamyl peptidase type I from Trypanosoma brucei: a new virulence factor from African trypanosomes that de-blocks regulatory peptides in the plasma of infected hosts

Abstract

Peptidases of parasitic protozoans are emerging as novel virulence factors and therapeutic targets in parasitic infections. A trypanosome-derived aminopeptidase that exclusively hydrolysed substrates with Glp (pyroglutamic acid) in P1 was purified 9248-fold from the plasma of rats infected with Trypanosoma brucei brucei. The enzyme responsible was cloned from a T. brucei brucei genomic DNA library and identified as type I PGP (pyroglutamyl peptidase), belonging to the C15 family of cysteine peptidases. We showed that PGP is expressed in all life cycle stages of T. brucei brucei and is expressed in four other blood-stream-form African trypanosomes. Trypanosome PGP was optimally active and stable at bloodstream pH, and was insensitive to host plasma cysteine peptidase inhibitors. Native purified and recombinant hyper-expressed trypanosome PGP removed the N-terminal Glp blocking groups from TRH (thyrotrophin-releasing hormone) and GnRH (gonadotropin-releasing hormone) with a k(cat)/K(m) value of 0.5 and 0.1 s(-1) x microM(-1) respectively. The half-life of TRH and GnRH was dramatically reduced in the plasma of trypanosome-infected rats, both in vitro and in vivo. Employing an activity-neutralizing anti-trypanosome PGP antibody, and pyroglutamyl diazomethyl ketone, a specific inhibitor of type I PGP, we demonstrated that trypanosome PGP is entirely responsible for the reduced plasma half-life of TRH, and partially responsible for the reduced plasma half-life of GnRH in a rodent model of African trypanosomiasis. The abnormal degradation of TRH and GnRH, and perhaps other neuropeptides N-terminally blocked with a pyroglutamyl moiety, by trypanosome PGP, may contribute to some of the endocrine lesions observed in African trypanosomiasis.

Figure 1. Identification of type I PGP activity in the plasma of trypanosome-infected rats

(A) PGP activity in infected rat plasma is attributable to a type I PGP. Glp-AMC-hydrolysing activity in 100 μl aliquots of healthy rat plasma (closed bars) or infected rat plasma (open bars), preincubated (15 min, 37 °C) with 1,10-phenanthroline (Phen; 1 mM), Glp-DMK (100 μM), or preimmune (p.i.; 100 μg·ml⁻¹), anti-rat PGP (rPGP; 100 μg·ml⁻¹) or anti-trypanosome PGP (tPGP; 100 μg·ml⁻¹) antibodies are shown. Results shown are the means±S.D. (n=3). Significance probabilities are indicated for infected plasma versus healthy plasma (open versus closed bars, within the same group). (B) Time course for the appearance of type I PGP activity in infected rat plasma and Glp-AMC-hydrolysing activity of plasma drawn from the tail vein of rats at 2 day intervals, starting 4 days prior to infection and ending at 12 days post-infection are shown. The bars represent the data range, while the boxes represent lower and upper quartiles. The line within the quartile box indicates the median (n=5). (C) PGP activity-neutralizing activity of week 9 rat anti-trypanosome PGP antibodies. Recombinant trypanosome PGP (1 nM active concentration, by mass) was preincubated with preimmune rat (open squares) or week 9 anti-trypanosome PGP (closed squares) antibodies (1–1000 μg·ml⁻¹) for 15 min at 37 °C prior to assessment of activity against Glp-AMC. Results shown are the means±S.D. (n=3). AFU, arbitrary fluorescence units; NS, not significant; **P<0.05; ****P<0.001.

Figure 2. Type I PGP from T. brucei brucei

(A) Purity of PGP preparations. Native PGP purified from infected rat plasma, and recombinant catalytically active wild-type (WT) or a catalytically inactive C167A variant (C167A) were resolved on a reducing SDS/15% polyacrylamide gel, and proteins were visualized by Coomassie Blue staining. (B) An unrooted dendrogram was prepared by comparing the full-length PGP amino acid sequences using the ClustalW alignment software of the MEGALIGN program (DNASTAR) with a PAM250 weight table set with: ktuple=1, gap penalty=3 and gap window=5. The scale at the bottom measures the evolutionary distance between sequences. The units indicate the number of substitution events. Sequences were obtained from the GenBank®/EBI database under the following accession numbers: Q6GDB4 (Staphylococcus aureus), P65678 (Streptococcus pneumoniae), P46107 (B. amyloliquefaciens), Q8D4N5 (Vibrio vulnificus), AAK44557 (Mycobacterium tuberculosis), AAB63524 (My. bovis), NP_624759 (Streptomyces coelicolor), CAC03615 (Mus musculus), BAD01533 (Rattus norvegicus), CAC03610 (Homo sapiens), AAH75524 (Xenopus tropicalis), CAC33026 (Takifugu rubripes) and AAY40294 (T. brucei). (C) Multiple sequence alignment of PGP from T. brucei (T.b.), Mus musculus (M.m.), H. sapiens (H.s.), B. amyloliquefaciens (B.a.) and Staph. aureus (S.a.). Sequence sources are as in (B). Substrate-binding residues are indicated by an ‘S’; catalytic residues are indicated with an asterisk (*). Overlined residues represent peptides generated by endoproteinase Lys-C digests.

Figure 3. pH activity and stability optima for trypanosome PGP

The activity (open circles) and stability (closed circles) of T. brucei brucei PGP was assessed in AMT buffer over the pH range 4–10.5 as detailed in the Experimental section. Values represent the means±S.D. (n=3).

Figure 4. Trypanosome PGP is ubiquitously expressed

(A) Expression of the pgp gene in different life cycle stages of T. brucei brucei. The pgp gene expression was demonstrated by RT–PCR (+RT) from bloodstream-form trypomastigotes (bT) and cultured insect-form promastigotes (cP) mRNA. An RT reaction mixture to which no RT had been added (−RT) and plasmid pRM228 (containing the full-length T. brucei brucei pgp gene) served as negative and positive control templates respectively. RNA equivalence was demonstrated by amplifying a 1500 bp fragment of the opdb gene from the RT reaction mixture. CT, control. (B) Expression of the pgp gene in different African trypanosomes. Extracts (30 μg) of T. brucei brucei (T.b.b.), T. brucei rhodesiense (T.b.r.), T. brucei gambiense (T.b.g.), T. evansi (T.e.) and T. congolense (T.c.) were resolved on a reducing SDS/15% polyacrylamide gel, transferred on to a PVDF membrane and probed with anti-trypanosome PGP antibodies.

Figure 5. Trypanosome PGP is a cytosolic enzyme that is released into the plasma of infected rats

(A–C) Trypanosome PGP is a cytosolic enzyme. T. brucei brucei cell bodies were fractionated into crude nuclear (N), large granule (LG), small granule (SG), crude microsomal (M) and high-speed soluble supernatant (S) fractions. The specific activities of tyrosine aminotransferase (A), α-mannosidase (B) and PGP (C), in subcellular fractions, are expressed relative to the specific activity (RSA) of the homogenate (primary y-axis). Similarly, specific activities of these three enzymes in T. brucei brucei cell-culture supernatants (SNT) are expressed relative to the specific activity (RSA) in naïve media (secondary y-axis). (D) Immunoreactive PGP is present in the plasma of infected rats. Partially fractionated plasma, from three healthy rats (lanes 1–3) or three trypanosome-infected rats (lanes 4–6), was resolved by reducing Tris/Tricine SDS/PAGE on an SDS/15% polyacrylamide gel, transfer on to a PVDF membrane and probing with anti-trypanosome PGP antibodies.

Figure 6. Trypanosome PGP reduces the plasma half-life TRH and GnRH in vitro

A single representative experiment is illustrated in a scatter plot that shows the loss of radioactive tracer from the plasma, while the insets give the averaged values of three independently determined half-lives for each experimental group. Half-life of [L-His-4-³H(n),L-Pro-3,4-³H(n)]TRH in (A) untreated plasma or (B) plasma treated with Glp-DMK (100 μM; 37 °C, 15 min), from healthy (Ctrl; ■) or infected rats (Inf; □). (C) Half-life of [L-His-4-³H(n),L-Pro-3,4-³H(n)]TRH in plasma from infected rats preincubated with rat antitrypanosome PGP antibodies (PGP; ■) or preimmune rat antibodies (p.i.; □) (100 μg·ml⁻¹; 37 °C, 15 min). (D) Half-life of ¹²⁵I-Tyr⁵-GnRH in (D) untreated plasma or (E) plasma treated with Glp-DMK (100 μM; 37 °C, 15 min), from healthy (Ctrl; ■) or infected rats (Inf; □). (F) Half-life of ¹²⁵I-Tyr⁵-GnRH in plasma from infected rats preincubated with rat anti-trypanosome PGP antibodies (PGP; ■) or preimmune rat antibodies (p.i.; □) (100 μg·ml⁻¹; 37 °C, 15 min). In the scatter plots, solid lines indicate the regression analysis of the plots, while dashed lines indicate the 95% confidence intervals. In the insets, bars represent the means±S.D. (n=3). NS, not significant; ***P<0.05; ****P<0.001.

Figure 7. Trypanosome PGP reduces the in vivo plasma half-life of TRH and GnRH

(A) Plasma half-life of [L-His-4-³H(n),L-Pro-3,4-³H(n)]TRH injected into the jugular vein of infected rats preinfused for 2 h (25 μg·min⁻¹) with rat anti-trypanosome PGP (PGP; □) or preimmune rat antibodies (p.i.; ■). The inset also describes the t_1/2 for [L-His-4-³H(n),L-Pro-3,4-³H(n)]TRH in healthy rats (Ctrl; original results not shown). (B) Plasma half-life of ¹²⁵I-Tyr⁵-GnRH injected into the jugular vein of infected rats preinfused for 2 h (25 μg·min⁻¹) with rat anti-trypanosome PGP (PGP; □) or preimmune rat antibodies (p.i.; ■). The inset also describes the t_1/2 for ¹²⁵I-Tyr⁵-GnRH in healthy rats (Ctrl; original results not shown). In the scatter plots, solid lines indicate the regression analysis of the plots, while broken lines indicate 95% confidence intervals. In the insets, the bars reflect the means±S.D. (n=3). *P<0.5.