Reconstitution of a surface transferrin binding complex in insect form Trypanosoma brucei.
In the bloodstream of the mammalian host, Trypanosoma brucei takes up host transferrin by means of a high-affinity uptake system, presumably a transferrin receptor. Transferrin-binding activity is seen in the flagellar pocket and is absent in insect form trypanosomes. By transfection we have reconstituted a transferrin-binding complex in insect form trypanosomes. Formation of this complex requires the products of two genes that are part of a variant surface glycoprotein expression site, expression site-associated gene (ESAG) 6 (encoding a protein with GPI-anchor) and ESAG 7 (encoding a protein without any obvious membrane attachment). This complex can be precipitated by transferrin-Sepharose and by an antibody directed only against the ESAG 6 protein. Transfection of ESAG 6 or 7 alone did not result in transferrin binding. In the transfected trypanosomes, the products of ESAG 6 alone and the combination of ESAG 6 and 7 did not exclusively localize to the flagellar pocket, but were present all over the surface of the trypanosome. The reconstituted transferrin-binding complex also did not result in the uptake of transferrin. Additional proteins present in bloodstream trypanosomes, but not in sufficient amounts in insect form trypanosomes, may therefore be required for the correct routing of the transferrin-binding complex to the flagellar pocket, and for its rapid internalization after ligand binding.
The Netherlands Cancer Institute, Division of Molecular Biology, Amsterdam.
M J
MJ
Ligtenberg
W
W
Bitter
R
R
Kieft
D
D
Steverding
H
H
Janssen
J
J
Calafat
P
P
Borst
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D001483:Base Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry; D004705:Endocytosis; D005407:Flagella / Q000737:chemistry; D005786:Gene Expression Regulation / Q000235:genetics; D017125:Genes, Protozoan / Q000235:genetics*; D006023:Glycoproteins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011233:Precipitin Tests; D011485:Protein Binding; D015800:Protozoan Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D011995:Recombination, Genetic; D014168:Transferrin / Q000235:genetics / Q000378:metabolism; D014346:Trypanosoma brucei brucei / Q000737:chemistry / Q000378:metabolism*
D006023:Glycoproteins; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D014168:Transferrin; C078011:ESAG 7 protein, Trypanosoma brucei