The major leucyl aminopeptidase of Trypanosoma cruzi (LAPTc) assembles into a homohexamer and belongs to the M17 family of metallopeptidases

Abstract

BackgroundPathogens depend on peptidase activities to accomplish many physiological processes, including interaction with their hosts, highlighting parasitic peptidases as potential drug targets. In this study, a major leucyl aminopeptidolytic activity was identified in Trypanosoma cruzi, the aetiological agent of Chagas disease.ResultsThe enzyme was isolated from epimastigote forms of the parasite by a two-step chromatographic procedure and associated with a single 330-kDa homohexameric protein as determined by sedimentation velocity and light scattering experiments. Peptide mass fingerprinting identified the enzyme as the predicted T. cruzi aminopeptidase EAN97960. Molecular and enzymatic analysis indicated that this leucyl aminopeptidase of T. cruzi (LAPTc) belongs to the peptidase family M17 or leucyl aminopeptidase family. LAPTc has a strong dependence on neutral pH, is mesophilic and retains its oligomeric form up to 80°C. Conversely, its recombinant form is thermophilic and requires alkaline pH.ConclusionsLAPTc is a 330-kDa homohexameric metalloaminopeptidase expressed by all T. cruzi forms and mediates the major parasite leucyl aminopeptidolytic activity. Since biosynthetic pathways for essential amino acids, including leucine, are lacking in T. cruzi, LAPTc could have a function in nutritional supply.

The Activity of a Hexameric M17 Metallo-Aminopeptidase Is Associated With Survival of Mycobacterium tuberculosis

“…Substrate hydrolysis was determined by AMC release measured fluorometrically using SpectraMax M5 Microplate Reader with SoftMax Pro Data Acquisition and Analysis Software (Molecular Devices) as described previously (Cadavid-Restrepo et al, 2011). Enzyme cation preference was performed using different concentrations of CaCl 2 , CoCl 2 , CuSO 4 , FeCl 2 , MgCl 2 , MnCl 2 , NiSO 4 , or ZnSO 4 in Tris-HCl pH 7.5 at 25°C containing Leu-AMC as substrate.…”

Purification and characterization of a Cys-Gly hydrolase from the gastropod mollusk,Patella caerulea

“…Recently, metallopeptidases belonging to the M17 family have been reported to be involved in parasitic infections in humans [37][38][39][40] ; they help parasites to survive by acting in the preliminary stages of their development, as, for example, eggs hatch in Schistosoma 41 or the maturation of immature trophozoites in P. falciparum 42 . Thus, the search for inhibitors of this enzyme activity has been proposed as a promising strategy for the treatment of various parasitic diseases.…”

Applications of Zymography (Substrate-SDS-PAGE) for Peptidase Screening in a Post-Genomic Era

“…Alternatively, an overlay with fluorogenic or chromogenic peptide substrates can be done (Cadavid-Restrepo et al 2011). The acrylamide concentration in gels varies more commonly from 7 to 15%, which impact on protein separation; low molecular mass proteins usually require higher acrylamide concentration for better protein resolution.…”