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

Esteban M Cordero,Nobuko Yoshida,Cristian Cortez,José Franco Da Silveira

doi:10.1038/s41598-019-43743-0

Esteban M Cordero, Nobuko Yoshida + Show 2 more

Open Access

https://doi.org/10.1038/s41598-019-43743-0

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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.

Highlights

GP82 belongs to the trans-sialidase superfamily, the largest T. cruzi multigene family, encoding important virulence factors out of 1,430 members[11]
Some of these cDNA sequences contain a portion of the spliced-leader (SL) sequence at the 5′-end indicating that these molecules possess a full 5′-UTR
In the absence of other signals, the initiation of protein translation at an up-stream start codon implies that the signal peptide (SP) became internal, acting as transmembrane domain

Summary

Introduction

GP82 belongs to the trans-sialidase superfamily, the largest T. cruzi multigene family, encoding important virulence factors out of 1,430 members[11]. SPs are tripartite targeting signals arranged in the following order: a positively charged n-region, a central hydrophobic h-region and a terminal c-region composed of small polar amino acids. In the case of CRP-10, for which the mature N-terminus is known[21], it was suggested that the primary translation product initiates at the third starting codon, which is embedded in a Kozak context and encodes a canonical SP. The same criterion was applied to define the primary translation product of TSA-1, its mature N-terminus was not known[24], which would start at the SP (second ATG codon) Both TSA-1 and CRP-10 open reading frames contain additional in-frame starting codons located upstream from those adjacent to the SP encoding stretches of 37 and 38 amino acids, respectively[21,24].

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