mAb CZP-315.D9: An Antirecombinant Cruzipain Monoclonal Antibody That Specifically Labels the Reservosomes ofTrypanosoma cruziEpimastigotes

Abstract

Reservosomes are large round vesicles located at the posterior end of epimastigote forms of the protozoan Trypanosoma cruzi, the etiological agent of Chagas disease. They are the specific end organelles of the endocytosis pathway of T. cruzi, and they play key roles in nutrient uptake and cell differentiation. These lysosome-like organelles accumulate ingested macromolecules and contain large amounts of a major cysteine proteinase (cruzipain or GP57/51 protein). Aim of this study was to produce a monoclonal antibody (mAb) against a recombinant T. cruzi cruzipain (TcCruzipain) that specifically labels the reservosomes. BALB/c mice were immunized with purified recombinant TcCruzipain to obtain the mAb. After fusion of isolated splenocytes with myeloma cells and screening, a mAb was obtained by limiting dilution and characterized by capture ELISA. We report here the production of a kappa-positive monoclonal IgG antibody (mAb CZP-315.D9) that recognizes recombinant TcCruzipain. This mAb binds preferentially to a protein with a molecular weight of about 50 kDa on western blots and specifically labels reservosomes by immunofluorescence and transmission electron microscopy. The monoclonal CZP-315.D9 constitutes a potentially powerful marker for use in studies on the function of reservosomes of T. cruzi.

Knockout of the gamma subunit of the AP-1 adaptor complex in the human parasite Trypanosoma cruzi impairs infectivity and differentiation and prevents the maturation and targeting of the major protease cruzipain

“…This hydrolase is synthesized as a zymogen of 57 kDa, from which the N-terminal pro-domain is removed autocatalytically, to generate the active enzyme (51 kDa) [ 57 ]. Once cruzipain is processed in the endoplasmic reticulum/Golgi complex, it is delivered to reservosomes, which only contain mature cruzipain [ 40 ]. Given the potential importance of TcAP1-γ in vesicular traffic from the TGN—including reservosome-bound traffic in T .…”

“…cruzi –we investigated whether the transport of cruzipain to reservosomes was affected in TcγKO parasites. First we analyzed the expression of cruzipain in TcγKO cells using an anti-cruzipain antiserum that recognizes both the unprocessed and mature form of this enzyme [ 40 ]. Western blot analysis showed that the cruzipain zymogen form (57 kDa) was recognized by the anti-cruzipain antiserum in both WT and in TcγKO epimastigotes, although the latter had increased zymogen levels ( Fig 6A ).…”

“…Lysates containing an equivalent of 1 x 10 6 cells μl -1 were fractionated by SDS-PAGE and transferred onto nitrocellulose membranes (Hybond C, GE Healthcare, PA, USA) according to standard protocols [ 39 ]. Membranes were blocked with 5% non-fat dry milk and 0.05% Tween 20 in PBS (blocking solution) then, they were probed with one of the following primary antibodies (diluted in blocking solution): the monoclonal antibody 211.F7, against the Tcγ hinge domain (1:300); anti-cruzipain (1:1500) antiserum that recognizes both the zymogen and the mature form of cruzipain [ 40 ]; or a mouse anti-TcGAPDH antiserum (1:8,000) [ 41 ], as a loading control. Membranes were then incubated with horseradish peroxidase-conjugated anti-mouse IgG antibodies (Thermo Scientific product #31430) and diluted to 1:7,500 in blocking solution.…”

Treatment of Trypanosoma cruzi with 2-bromopalmitate alters morphology, endocytosis, differentiation and infectivity

“…For co-localization of CZP/transferrin-AlexaFluor-633, the samples incubated with CZP-315.D9 mAb (at 50 μg/ml) [40], were washed three times in PBS and then incubated with a secondary goat anti-mouse-AlexaFluor-488 antibody (1:600 in incubation buffer). For the co-localization with CZP/albumin-AlexaFluor-488, the samples were incubated with CZP-315.D9 mAb and then incubated with a secondary goat anti-mouse-AlexaFluor-594 antibody.…”

Expression of cysteine proteinases and cystatins in parasites and use of cysteine proteinase inhibitors in parasitic diseases. Part III: Protozoa (3): Kinetoplastids

“…Immunolocalization: It was reported that T. cruzi epimastigotes use two endocytic vesicles; cytostome/ cytopharynx complex and the flagellar pocket membrane, to ingest extracellular macromolecules to be stored in reservosomes [122] . Using a monoclonal antibody (mAb) against recombinant CRZ localized a 50 kDa protein in reservosomes [123] . Later, endocytosis was visualized in intracellular amastigotes, and the digested extracellular macromolecules were stored in CRZ-positive lysosome-related structures (LRSs).…”