Cloning, heterologous expression, and distinct substrate specificity of protein farnesyltransferase from Trypanosoma brucei

Abstract

Protein prenylation occurs in the protozoan that causes African sleeping sickness (Trypanosoma brucei), and the protein farnesyltransferase appears to be a good target for developing drugs. We have cloned the alpha- and beta-subunits of T. brucei protein farnesyltransferase (TB-PFT) using nucleic acid probes designed from partial amino acid sequences obtained from the enzyme purified from insect stage parasites. TB-PFT is expressed in both bloodstream and insect stage parasites. Enzymatically active TB-PFT was produced by heterologous expression in Escherichia coli. Compared with mammalian protein farnesyltransferases, TB-PFT contains a number of inserts of >25 residues in both subunits that reside on the surface of the enzyme in turns linking adjacent alpha-helices. Substrate specificity studies with a series of 20 peptides SSCALX (where X indicates a naturally occurring amino acid) show that the recombinant enzyme behaves identically to the native enzyme and displays distinct specificity compared with mammalian protein farnesyltransferase. TB-PFT prefers Gln and Met at the X position but not Ser, Thr, or Cys, which are good substrates for mammalian protein farnesyltransferase. A structural homology model of the active site of TB-PFT provides a basis for understanding structure-activity relations among substrates and CAAX mimetic inhibitors.

FIG. 1. Amino acid sequence of TB-PFT α-subunit (A) and β-subunit (B)

The alignment of TB-PFT and rat PFT sequences was carried out as described in the text. Identities are in bold type, and similarities are underlined. Amino acid segments that were found by Edman sequencing of tryptic peptides are shown with lowercase letters. Large inserts (>25 residues) present in TB-PFT are shown (αI–αV and βI–βIII).

FIG. 2. Northern blot analysis of bloodstream form (BSF) and procyclic (Pro) T. brucei

Total RNA was electrophoresed, blotted onto a nylon membrane, and hybridized with a ³²P-labeled probe to TB-PFT α-subunit (A). The blot was stripped and probed for TB-PFT β-subunit (B). The membrane was stripped again and probed for β-tubulin to demonstrate equivalent loading (C). Transcript sizes are marked in kilobases (Kb).

FIG. 3. Sequence comparison of rat PFT and TB-PFT α- (A) and β-subunits (B)

The thick bars indicate the full amino acid sequence. The α-helices are represented by the shaded boxes, with the paired helices underlying the tertiary structure combined for clarity. The starting and ending residue of each helix or pair of helices is shown in the box. The helices are numbered (1–15 α-subunit; 1–14 β-subunit) as described previously (14). Insertions greater than 25 residues in TB-PFT relative to rat PFT are numbered (αI–αV and βI–βIII).

FIG. 4. Structural comparison of rat PFT and TB-PFT

A ribbon diagram of rat PFT α-subunit (dark shading) and β-subunit (light shading) is shown. The active site is marked by the catalytic zinc in the center of the figure. The eight large TB-PFT insertions positioned relative to rat PFT are shown as spheres (larger diameter for larger inserts) and labeled according to Fig. 3.

FIG. 5. X-ray structure of rat PFT residues in the vicinity of the Met residue of bound CVIM (28)

Stereo diagram showing enzyme-bound CVIM (bold sticks) and rat PFT α- and β-subunit residues close to the Met of CVIM as well as Tyr-166α (see text for a discussion of these residues). Also shown is bound FPP (gray sticks, based on the x-ray structure of the ternary complex of rat PFT with CVIM and a hydroxyphosphonate FPP analog).

FIG. 6. Expression of TB-PFT in E. coli

E. coli BL21 pLysS was cultured in 5 ml of medium at 37 °C. Cells were collected before or after induction with 0.4 mm IPTG at room temperature for 2 h. Cell pellets were treated with 100 µl of Laemmli sample buffer, and sample aliquots (1.5 µl for lanes 1 and 2, and 4 µl for lane 3) were subjected to SDS-PAGE analysis using a 10% gel. Protein bands were visualized with Coomassie Blue staining. Lane 1, untransformed E. coli; lane 2, E. coli transformed with pRD578-TbPFT expressing T. brucei α- and β-subunits that was harvested after IPTG induction for 2 h; lane 3, same as lane 2 but without IPTG induction. Lane 4 was from a separate gel with different molecular mass markers and shows protein from the 120,000 × g pellet obtained from a lysate of E. coli expressing TB-PFT.

FIG. 7. CAAX specificity of TB-PFT

Purified TB-PFT (0.025 microunits) from procyclic parasites (A) or recombinant (about 3 µg of cytosolic protein from E. coli transformed with pRD578-TbPFT) (B) was incubated with 10 µm each of the 20 different SSCALX peptides and 0.75 µm (0.3 µCi) [³H]FPP for 30 min at 30 °C under standard assay conditions. Results are expressed as the radioactivity above the level measured with the no peptide control (6,000–10,000 cpm). For comparison, 0.05 µg of recombinant rat PFT (prepared by expression in the baculovirus/Sf9 cell system) (C) was also incubated with the peptides for 15 min at 30 °C. Based on replicate analyses, estimated errors are <15% for all cpm values.