Cargando…

Characterization of an epimastigote-stage-specific hemoglobin receptor of Trypanosoma congolense

BACKGROUND: Since Trypanosoma spp. lack a complete heme synthesis pathway, the parasites are totally dependent on their host for heme throughout all of the stages of their life-cycle. We herein report the identification and characterization of a T. congolense epimastigote form (EMF)-specific hemoglo...

Descripción completa

Detalles Bibliográficos
Autores principales: Yamasaki, Shino, Suganuma, Keisuke, Yamagishi, Junya, Asada, Masahito, Yokoyama, Naoaki, Kawazu, Shin-ichiro, Inoue, Noboru
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4877808/
https://www.ncbi.nlm.nih.gov/pubmed/27216446
http://dx.doi.org/10.1186/s13071-016-1563-9
_version_ 1782433451452923904
author Yamasaki, Shino
Suganuma, Keisuke
Yamagishi, Junya
Asada, Masahito
Yokoyama, Naoaki
Kawazu, Shin-ichiro
Inoue, Noboru
author_facet Yamasaki, Shino
Suganuma, Keisuke
Yamagishi, Junya
Asada, Masahito
Yokoyama, Naoaki
Kawazu, Shin-ichiro
Inoue, Noboru
author_sort Yamasaki, Shino
collection PubMed
description BACKGROUND: Since Trypanosoma spp. lack a complete heme synthesis pathway, the parasites are totally dependent on their host for heme throughout all of the stages of their life-cycle. We herein report the identification and characterization of a T. congolense epimastigote form (EMF)-specific hemoglobin (Hb) receptor. The gene was initially reported to encode a T. congolense haptoglobin (Hp)-Hb complex receptor (TcHpHbR) based on its similarity to a gene encoding a T. brucei Hp-Hb complex receptor (TbHpHbR). METHODS: Trypanosoma congolense IL3000 was used in this study. A TcHpHbR gene was PCR amplified from the parasite genome. The recombinant protein was used as an immunogen to raise antibodies for immunofluorescence assay and immunoblotting. Hemoglobin uptake by the parasite was examined by using Alexa 488 labelled Hb and visualized by confocal laser scanning microscopy. The qualitative and quantitative interaction between TcHpHbR and its ligand were measured using a surface plasmon resonance assay. RESULTS: We found that, unlike TbHpHbR, TcHpHbR was exclusively expressed in the EMF stage at RNA and protein levels. The recombinant TcHpHbR (rTcHpHbR) was co-precipitated with free-Hb in a GST-pull down assay. Surface plasmon resonance revealed that rTcHpHbR binds free-Hb with high affinity (dissociation constant (K(d)) = 2.1×10(-8) M) but free-Hp with low affinity (K(d) = 2.2×10(-7) M). Furthermore, Alexa 488-labelled-Hb was only taken up by the EMF and co-localized with tomato lectin, which is a marker of endocytic compartments (flagellar pocket and lysosome). CONCLUSION: We conclude that the T. congolense EMF takes up free-Hb via TcHpHbR, a receptor which is specific to this developmental stage. We therefore propose renaming TcHpHbR as T. congolense EMF-specific Hb receptor (TcEpHbR). ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13071-016-1563-9) contains supplementary material, which is available to authorized users.
format Online
Article
Text
id pubmed-4877808
institution National Center for Biotechnology Information
language English
publishDate 2016
publisher BioMed Central
record_format MEDLINE/PubMed
spelling pubmed-48778082016-05-25 Characterization of an epimastigote-stage-specific hemoglobin receptor of Trypanosoma congolense Yamasaki, Shino Suganuma, Keisuke Yamagishi, Junya Asada, Masahito Yokoyama, Naoaki Kawazu, Shin-ichiro Inoue, Noboru Parasit Vectors Research BACKGROUND: Since Trypanosoma spp. lack a complete heme synthesis pathway, the parasites are totally dependent on their host for heme throughout all of the stages of their life-cycle. We herein report the identification and characterization of a T. congolense epimastigote form (EMF)-specific hemoglobin (Hb) receptor. The gene was initially reported to encode a T. congolense haptoglobin (Hp)-Hb complex receptor (TcHpHbR) based on its similarity to a gene encoding a T. brucei Hp-Hb complex receptor (TbHpHbR). METHODS: Trypanosoma congolense IL3000 was used in this study. A TcHpHbR gene was PCR amplified from the parasite genome. The recombinant protein was used as an immunogen to raise antibodies for immunofluorescence assay and immunoblotting. Hemoglobin uptake by the parasite was examined by using Alexa 488 labelled Hb and visualized by confocal laser scanning microscopy. The qualitative and quantitative interaction between TcHpHbR and its ligand were measured using a surface plasmon resonance assay. RESULTS: We found that, unlike TbHpHbR, TcHpHbR was exclusively expressed in the EMF stage at RNA and protein levels. The recombinant TcHpHbR (rTcHpHbR) was co-precipitated with free-Hb in a GST-pull down assay. Surface plasmon resonance revealed that rTcHpHbR binds free-Hb with high affinity (dissociation constant (K(d)) = 2.1×10(-8) M) but free-Hp with low affinity (K(d) = 2.2×10(-7) M). Furthermore, Alexa 488-labelled-Hb was only taken up by the EMF and co-localized with tomato lectin, which is a marker of endocytic compartments (flagellar pocket and lysosome). CONCLUSION: We conclude that the T. congolense EMF takes up free-Hb via TcHpHbR, a receptor which is specific to this developmental stage. We therefore propose renaming TcHpHbR as T. congolense EMF-specific Hb receptor (TcEpHbR). ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13071-016-1563-9) contains supplementary material, which is available to authorized users. BioMed Central 2016-05-23 /pmc/articles/PMC4877808/ /pubmed/27216446 http://dx.doi.org/10.1186/s13071-016-1563-9 Text en © Yamasaki et al. 2016 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research
Yamasaki, Shino
Suganuma, Keisuke
Yamagishi, Junya
Asada, Masahito
Yokoyama, Naoaki
Kawazu, Shin-ichiro
Inoue, Noboru
Characterization of an epimastigote-stage-specific hemoglobin receptor of Trypanosoma congolense
title Characterization of an epimastigote-stage-specific hemoglobin receptor of Trypanosoma congolense
title_full Characterization of an epimastigote-stage-specific hemoglobin receptor of Trypanosoma congolense
title_fullStr Characterization of an epimastigote-stage-specific hemoglobin receptor of Trypanosoma congolense
title_full_unstemmed Characterization of an epimastigote-stage-specific hemoglobin receptor of Trypanosoma congolense
title_short Characterization of an epimastigote-stage-specific hemoglobin receptor of Trypanosoma congolense
title_sort characterization of an epimastigote-stage-specific hemoglobin receptor of trypanosoma congolense
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4877808/
https://www.ncbi.nlm.nih.gov/pubmed/27216446
http://dx.doi.org/10.1186/s13071-016-1563-9
work_keys_str_mv AT yamasakishino characterizationofanepimastigotestagespecifichemoglobinreceptoroftrypanosomacongolense
AT suganumakeisuke characterizationofanepimastigotestagespecifichemoglobinreceptoroftrypanosomacongolense
AT yamagishijunya characterizationofanepimastigotestagespecifichemoglobinreceptoroftrypanosomacongolense
AT asadamasahito characterizationofanepimastigotestagespecifichemoglobinreceptoroftrypanosomacongolense
AT yokoyamanaoaki characterizationofanepimastigotestagespecifichemoglobinreceptoroftrypanosomacongolense
AT kawazushinichiro characterizationofanepimastigotestagespecifichemoglobinreceptoroftrypanosomacongolense
AT inouenoboru characterizationofanepimastigotestagespecifichemoglobinreceptoroftrypanosomacongolense