Signal peptide recognition in Trypanosoma cruzi GP82 adhesin relies on its localization at protein N-terminus

Abstract

Trypanosoma cruzi , the causative agent of Chagas disease, has a dense coat of GPI-anchored virulence factors. T . cruzi GPI-anchored adhesin GP82 is encoded by a repertoire of transcripts containing several in-frame initiation codons located up-stream from that adjacent to the predicted signal peptide (SP). Transfection of T . cruzi epimastigotes with constructs encoding GP82 starting at the SP or from the farthest up-stream methionine confirmed protein expression on the parasite cell surface, comparable to the native GP82. Proteins were fully functional, inducing parasite adhesion to HeLa cells and lysosome mobilization, events required for parasite invasion. Transgenic and native GP82 proteins showed indistinguishable electrophoretic mobility, suggesting similar processing of the SP. Deletion of SP generated a ~72 kDa protein devoid of N -linked oligosaccharides allowing irrefutable identification of GP82 precursor. SP transposition to an internal region of GP82 rendered the signal unrecognizable by the signal peptidase and incapable to direct the nascent protein for ER-membrane association. Altogether our data strongly suggests that GP82 SP fails to function as transmembrane domain and its recognition by the signal peptidase shows strict dependence on the signal localization at protein N-terminus. This report presents the first experimental characterization of the full-length GP82 and its signal peptide.

Extracellular Vesicles in Trypanosomatids: Host Cell Communication

“…EVs of metacyclic-trypomastigotes also contain major surface components of the parasite, including the gp35/50 mucin-like glycoproteins and members of trans-sialidase family of glycoproteins ( Bayer-Santos et al, 2013 ). These surface components participate in the cell adhesion and invasion of the metacyclic-trypomastigotes ( Schenkman et al, 1993 ; Santori et al, 1996 ; Neira et al, 2002 ; Neira et al, 2003 ; Eickhoff et al, 2010 ; Cortez et al, 2012 ; Cortez et al, 2014 ; Cordero et al, 2019 ). Gp82 glycoprotein, a metacyclic specific member of the gp85/trans-sialidase family activates mTOR signaling cascades that allow lysosome migration and fusion with the parasitophorous vacuole producing acidification and parasite escape to the cytosol ( Cortez et al, 2012 ; Cortez et al, 2014 ; Cordero et al, 2019 ).…”

“…These surface components participate in the cell adhesion and invasion of the metacyclic-trypomastigotes ( Schenkman et al, 1993 ; Santori et al, 1996 ; Neira et al, 2002 ; Neira et al, 2003 ; Eickhoff et al, 2010 ; Cortez et al, 2012 ; Cortez et al, 2014 ; Cordero et al, 2019 ). Gp82 glycoprotein, a metacyclic specific member of the gp85/trans-sialidase family activates mTOR signaling cascades that allow lysosome migration and fusion with the parasitophorous vacuole producing acidification and parasite escape to the cytosol ( Cortez et al, 2012 ; Cortez et al, 2014 ; Cordero et al, 2019 ). It also binds to LAMP-2 receptor favoring parasite internalization ( Andrade and Andrews, 2005 ; Albertti et al, 2010 ; Andrade, 2019 ).…”

Ablation of the P21 Gene of Trypanosoma cruzi Provides Evidence of P21 as a Mediator in the Control of Epimastigote and Intracellular Amastigote Replication

“…cruzi epimastigotes (Y strain) in the early exponential phase were washed with phosphate-buffered saline (PBS; pH 7.4) and transfected with 25 µg of plasmid (sgRNA/Cas9/pTREX-n) diluted in P3 Primary Cell 4D-Nucleofector (V4XP-3024) solution according to the manufacturer's instructions. The parasites were electroporated with two pulses (EH100 program) delivered by the 4D Nucleofector system (Lonza) (Cordero et al, 2019). Then, the parasites were maintained in LIT medium supplemented with 20% FBS under drug selection (250 µg/mL G418).…”

The endoplasmic reticulum of trypanosomatids: An unrevealed road for chemotherapy

“…In T. cruzi the translocation of the surface GPI-anchored GP82 trans-sialidase involves a cleavable N-terminal signal peptide followed by N-glycosylation and cleavage/addition of a GPI anchor at C-terminus. By analogy with T. brucei , it is possible that this protein would be transported post-translationally by an SRP-independent pathway and the SP cleaved by signal peptidase complex ( Cordero et al., 2019 ). The silencing of SEC63 , which encodes the SEC63 protein, required for co- and post translational translocation, prevents both polytopic proteins and GPI-anchored proteins from entering the ER, suggesting that in trypanosomes this factor is essential for both translocation routes ( Goldshmidt et al., 2008 ).…”