title: Characterization of an epimastigote-stage-specific hemoglobin receptor of Trypanosoma congolense.
abstract: 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).
  
  
  
  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.
  
  
  
  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).
  
  
  
  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).
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada, Obihiro, Hokkaido, 080-8555, Japan.
authors.forename: Shino
authors.initials: S
authors.lastname: Yamasaki
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada, Obihiro, Hokkaido, 080-8555, Japan.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Suganuma
authors.affiliation: Research Center for Zoonosis Control, Hokkaido University, Sapporo, Hokkaido, 001-0020, Japan.
authors.forename: Junya
authors.initials: J
authors.lastname: Yamagishi
authors.affiliation: Department of Protozoology, Institute of Tropical Medicine (NEKKEN), Nagasaki University, Sakamoto, Nagasaki, 852-8523, Japan.
authors.forename: Masahito
authors.initials: M
authors.lastname: Asada
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada, Obihiro, Hokkaido, 080-8555, Japan.
authors.forename: Naoaki
authors.initials: N
authors.lastname: Yokoyama
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada, Obihiro, Hokkaido, 080-8555, Japan.
authors.forename: Shin-Ichiro
authors.initials: S
authors.lastname: Kawazu
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada, Obihiro, Hokkaido, 080-8555, Japan. ircpmi@obihiro.ac.jp.
authors.forename: Noboru
authors.initials: N
authors.lastname: Inoue
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D005260:Female; D006242:Haptoglobins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006454:Hemoglobins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008018:Life Cycle Stages; D051379:Mice; D008813:Mice, Inbred ICR; D015800:Protozoan Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins; D016415:Sequence Alignment; D014348:Trypanosoma congolense / Q000235:genetics / Q000378:metabolism*
chemical_list: D006242:Haptoglobins; D006454:Hemoglobins; D015800:Protozoan Proteins; D011994:Recombinant Proteins; C035492:haptoglobin-hemoglobin complex
keywords: Epimastigote; Hemoglobin receptor; Trypanosoma congolense