A Calcium- and GTP-Dependent Transglutaminase in Leishmania infantum

Abstract

While human and animal leishmaniasis affect several millions of people worldwide, L. infantum is the species responsible for visceral leishmaniasis in Europe, Middle East, and America. Antileishmanial drugs present issues associated with drug toxicity and increasing parasite resistance. Therefore, the study of this parasite with a focus on new potential drug targets is extremely useful. Accordingly, we purified and characterized a transglutaminase (TGase) from L. infantum promastigotes. While Tgases are known to be involved in cell death and autophagy, it appears that these functions are very important for parasites' virulence. For the first time, we showed a Ca²⁺- and GTP-dependent TGase in Leishmania corresponding to a 54 kDa protein, which was purified by two chromatographic steps: DEAE-Sepharose and Heparin-Sepharose. Using polyclonal antibodies against a 50-amino-acid conserved region of the catalytic core of human TGase 2, we revealed two other bands of 66 and 75 kDa. The 54 kDa band appears to be different from the previously reported TGase, which was shown to be Ca²⁺- independent. Future research should address the identification of the purified enzyme sequence and, subsequently, its cloning to more comprehensively investigate its pathophysiological function and possible differences from mammal enzymes.

Keywords: GTP-dependent activity; Leishmania; calcium-dependent activity; protein cross-linking; transamidation; transglutaminase.

Figure 1

Detection of TGase activity in cultured promastigotes of L. infantum in vivo (A–C) and in vitro (D). (A) DAPI-stained nuclei of the parasites. (B) Fluorescein-cadaverine (FC) labelling of indigenous proteins via TGase activity. (C) Merged photo of A and B. Fluorescence was detected by Nikon Microphot FXA fluorescent microscope (×100 magnification). (D) 15% SDS-PAGE of L. infantum promastigotes lysate, labelled with FC. The green bands show excitation of the FC, and the red bands denote excited Cy5, which labels the molecular-mass marker. Molecular Imager System PHAROS Bio-Rad FX and software Quantity One 1-D were used for gel analysis. From left to right: lane M—molecular-mass marker; lane 1—parasite extract + FC + dimethyl casein (arrows); lane 2—parasite extract + FC; lane 3—parasite extract + FC + dimethyl casein+ putrescine; and lane 4—dimethyl casein alone (no fluorescence).

Figure 2

Ca²⁺ activation and GTP inhibition of L. infantum promastigotes’ activity. The percentages of the activities are represented. (A) Addition of EGTA to the reaction mix significantly reduced the enzyme’s activity (p < 0.001), and treatment of lysate with additional Ca²⁺ increased the enzyme’s activity. (B) Addition of GTP markedly reduced TGase activity.

Figure 3

10% SDS-PAGE (panel A) of the purified TGase and Western blot (panel B). Lane 1, fraction obtained from the affinity chromatography on Heparin-Sepharose; lane 2, molecular mass marker. (please find the WB full membrane in Figure S1).

Figure 4

Indirect immunofluorescent staining of L. infantum promastigotes smeared on glass slides with human TGase 2 rabbit polyclonal antibodies (orb2986) and FITC-tagged anti-rabbit antibodies. FITC fluorescence was visualized using a Nikon Microphot FXA fluorescent microscope (×40 magnification). (A) DAPI-stained nuclei of the parasites. (B) FITC labelling. (C) Merged image of A and B.

Figure 5

10% SDS-PAGE electrophoresis and immunoblotting of L. infantum promastigotes lysates using transglutaminase polyclonal antibodies (orb2986), HRP-conjugated Goat anti-Rabbit (H + L) secondary antibodies, and ECL detection system. Lane 1, purified guinea pig TGase; lane 2, IZSLER_MO1 L. infantum extract; and lane 3, MHOM/TN80/IPT1 L. infantum extract. (please find the WB full membrane in Figure S1).