title: Gene identification and comparative molecular modeling of a Trypanosoma rangeli major surface protease.
abstract: Trypanosoma rangeli is a hemoflagellate parasite which is able to infect humans. Distinct from Trypanosoma cruzi, the causative agent of Chagas disease, T. rangeli is non-pathogenic to the vertebrate host. The manner by which the T. rangeli interacts with the host is still unknown, but it certainly depends on the surface molecules. Major surface proteins (MSP) are GPI-anchored, zinc-dependent metalloproteases present in the surface of all trypanosomatids studied so far, which are implicated as virulence factors in pathogenic trypanosomatids, such as Leishmania spp and T. cruzi. The aims of this work were to generate the complete sequence of a T. rangeli MSP (TrMSP) gene and to determine the 3D-structure of the predicted protein by homology modeling. The plasmid bearing a complete copy of a TrMSP gene was completely sequenced and the predicted protein was modeled using Modeller software. Results indicate that TrMSP open reading frame (ORF) codes for a predicted 588 amino acid protein and shows all elements required for its posttranslational processing. Multiple sequence alignment of TrMSP with other trypanosomatids' MSPs showed an extensive conservation of the N-terminal and central regions and a more divergent C-terminal region. Leishmania major MSP (LmMSP), which had its crystal structure previously determined, has an overall 35% identity with TrMSP. This identity allowed the comparative molecular modeling of TrMSP, which demonstrated a high degree of structural conservation between MSPs from other trypanosomatids (TrypMSPs). All modeled MSPs have a conserved folding pattern, apart from structural divergences in the C-domain and discrete differences of charge and topology in the catalytic cleft, and present the same geometry of the canonical HEXXH zinc-binding motif. The determination of surface charges of the molecules revealed that TrMSP is a predominantly positive protein, whereas LmMSP and Trypanosoma cruzi MSP (TcMSP) are negative proteins, suggesting that substrates recognized by TcMSP and LmMSP could not interact with TrMSP. Moreover, the comparison between TrMSP and TcMSP protein sequences has revealed 45 non-neutral amino acid substitutions, which can be further assessed through protein engineering. The characteristics of TrMSP could explain, at least in part, the lack of pathogenicity of T. rangeli to humans and point to the necessity of identifying the biological targets of this enzyme.
authors.affiliation: Departamento de Bioquímica, Farmacologia e Fisiologia, Instituto de Ciências Biológicas e Naturais, Universidade Federal do Triângulo Mineiro, Uberaba, Brazil.
authors.forename: Paulo H M
authors.initials: PH
authors.lastname: Calixto
authors.forename: Mainá
authors.initials: M
authors.lastname: Bitar
authors.forename: Keila A M
authors.initials: KA
authors.lastname: Ferreira
authors.forename: Odonírio
authors.initials: O
authors.lastname: Abrahão
authors.forename: Eliane
authors.initials: E
authors.lastname: Lages-Silva
authors.forename: Glória R
authors.initials: GR
authors.lastname: Franco
authors.forename: Luis E
authors.initials: LE
authors.lastname: Ramírez
authors.forename: André L
authors.initials: AL
authors.lastname: Pedrosa
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D017124:Conserved Sequence; D016054:DNA, Protozoan / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D018320:Leishmania major / Q000737:chemistry / Q000201:enzymology / Q000235:genetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D045726:Metalloproteases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D017510:Protein Folding; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014349:Trypanosoma cruzi / Q000737:chemistry / Q000201:enzymology / Q000235:genetics; D057509:Trypanosoma rangeli / Q000737:chemistry* / Q000201:enzymology / Q000235:genetics
chemical_list: D016054:DNA, Protozoan; D008565:Membrane Proteins; D015800:Protozoan Proteins; D045726:Metalloproteases