GPI anchor transamidase of Trypanosoma brucei: in vitro assay of the recombinant protein and VSG anchor exchange.
GPI8 from Trypanosoma brucei was cloned and expressed in Escherichia coli. TbGPI8 encodes a 37 kDa protein (35 kDa after removal of the putative signal sequence) with a pI of 5.5. It contains one potential N-glycosylation site near the N-terminus but no C-terminal hydrophobic region. Enzyme activity assays using trypanosomal lysates or recombinant TbGpi8 exhibited cleavage of the synthetic peptide acetyl-S-V-L-N-aminomethyl-coumarine, indicating that TbGpi8 is indeed directly involved in the proteolytic processing of the GPI anchoring signal. Intracellular localization of TbGpi8 within tubular structures, such as the endoplasmic reticulum, was observed by using specific anti-TbGpi8 antibodies. The transamidase mechanism of GPI anchoring was studied in bloodstream forms of Trypanosoma brucei using media containing hydrazine or biotinylated hydrazine. In the presence of the latter nucleophile, part of the newly formed VSG was linked to this instead of the GPI anchor and was not transferred to the cell surface. VSG-hydrazine-biotin was detected by streptavidin in western blots and intracellularly in Golgi-like compartments.
Kang
Xuedong
X
Physiologisch-chemisches Institut, University of Tübingen, Germany.
Szallies
Alexander
A
Rawer
Marc
M
Echner
Hartmut
H
Duszenko
Michael
M
D019881:Aminoacyltransferases / Q000235:genetics / Q000302:isolation & purification*; D000818:Animals; D000940:Antigenic Variation / Q000235:genetics; D001681:Biological Assay; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000302:isolation & purification*; D002451:Cell Compartmentation / Q000502:physiology; D002462:Cell Membrane / Q000378:metabolism*; D003001:Cloning, Molecular; D019084:Fluorescent Antibody Technique, Indirect; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D006834:Hydrazines; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases; D010455:Peptides; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification*; D017386:Sequence Homology, Amino Acid; D014346:Trypanosoma brucei brucei / Q000166:cytology / Q000201:enzymology* / Q000235:genetics; D014353:Trypanosomiasis, African / Q000201:enzymology* / Q000235:genetics; D014643:Variant Surface Glycoproteins, Trypanosoma / Q000378:metabolism*
D015815:Cell Adhesion Molecules; D017261:Glycosylphosphatidylinositols; D006834:Hydrazines; D010455:Peptides; D011993:Recombinant Fusion Proteins; D014643:Variant Surface Glycoproteins, Trypanosoma; C029424:hydrazine; D019881:Aminoacyltransferases; C023175:transamidases; D010447:Peptide Hydrolases