Getting into the sulfoglycoproteome of Trypanosoma cruzi

M.N. RODRIGUEZ; M. LANDONI; L. L. SOPRANO; V. G.DUSCHAK; A. S. COUTO

virtual

Congreso; Reunion Conjunta SAIB-SAMIGE; 2021

SAIB

Chagas disease or American Trypanosomiasis caused by the protozoan Trypanosoma cruzi is a neglected disease constituting a serious problem in Central and South America. During T. cruzi developmental stages, glycoproteins play important roles in the host-parasite interaction, such as cellular recognition, host cell invasion and adhesion, and immune evasion. Previously, we have described for the first time the presence of sulfated glycoproteins in Trypanosomatids. Within the analysis of T. cruzi proteins, we have structurally characterized by mass spectrometry analysis, two sulfated glycoproteins present in epimastigote forms: cruzipain (Cz) and serincarboxypeptidase (SCP). Sulfation is a significant modification of glycoproteins that plays a key role in biological processes. In this sense, we have characterized the structure of the sulfated NAc-Glc6SO3-epitope (sulfotope) present in Cz and provided strong evidence that sulfotopes are involved in the generation of heart muscle tissue damage in BALB/c mice, in absence of infection, and might play a role in the immunopathogenesis of chronic Chagas disease. Remarkably, IgG2-specific antibody levels to sulfated epitopes of Cz, inversely correlates with the degree of cardiac dysfunction. The presence of sulfated glycoproteins indicates that the parasite contains sulfotransferase activity suggesting there may be other sulfated glycoproteins still not characterized with relevant biological roles yet unknown. The goal of this project is to unravel the sulfoglycoproteome of epimastigote and trypomastigote forms of T. cruzi to establish potential differences between the developmental forms as well as to determine its biological implications.Epimastigote forms were lysed and after centrifugation, the lysate was subjected to a DEAE-Sepharose column chromatography and eluted by steps with buffer Tris containing 0 %, 0.2 % and 0.6 % NaCl. Fractions named F0, F0.2, F0.6 respectively analyzed by SDS-PAGE. Different bands were detected when developed with blue Coomassie for proteins and with PAS for glycoproteins. Samples of F0.2 were further digested in parallel with sulfatase and phosphatase and analyzed by SDS-PAGE. Interestingly apart from Cz, at least two proteins in F0.2 were clearly modified by the sulfatase treatment but not with phosphatase. Those two proteins were further cut from the gel, trypsinized and subjected to MS analysis for identification. Treated and untreated F0.2 were further compared by bidimentional electrophoresis. When the same fractionation and analysis was performed on trypomastigote forms, fraction F0.2 showed by SDS-PAGE a major band of aprox 66 kDa that was partially modified by the sulfatase treatment. The identification and characterization of novel sulfotopes that might be able to play essential roles would represent an important advance in our understanding on the immunopathogenesis and infection associated with Chagas disease.