Tw. Pearson et al., The major cell surface glycoprotein procyclin is a receptor for induction of a novel form of cell death in African trypanosomes in vitro, MOL BIOCH P, 111(2), 2000, pp. 333-349
Bloodstream forms (BSF) and procyclic culture forms (PCF) of African trypan
osomes were incubated with a variety of lectins in vitro. Cessation of cell
division and profound morphological changes were seen in procyclic forms b
ut not in BSF after incubation with concanavalin A (Con A): wheat germ aggl
utinin and Ricinus communis agglutinin. These lectins caused the trypanosom
es to cease division, become round and increase dramatically in size, the l
atter being partially attributable to the formation of what appeared to be
a large 'vacuole-like structure' or an expanded flagellar pocket. Con A was
used in all further experiments. Spectrophotometric quantitation of extrac
ted DNA and flow cytometry using the DNA intercalating dye propidium iodide
showed that the DNA content of Con A-treated trypanosomes increased dramat
ically when compared to untreated parasites. Examination of these cells by
fluorescence microscopy showed that many of the Con A-treated cells were mu
ltinucleate whereas the kinetoplasts were mostly present as single copies,
indicating a disequilibrium between nuclear and kinetoplast replication. Im
munofluorescence experiments using monoclonal antibodies (mAb) specific for
paraflagellar rod proteins and for kinetoplastid membrane protein-11 (KMP-
11), showed that the Con A-treated parasites had begun to duplicate the fla
gellum but that this had only proceeded along part of the length of the cel
ls, suggesting that the cell division process was initiated but that cytoki
nesis was subsequently inhibited. Tunicamycin-treated wild-type trypanosome
s and mutant trypanosomes expressing both high levels of non-glycosylated p
rocyclins and procyclin isoforms with truncated N-linked sugars were resist
ant to the effects of Con A, suggesting that N-linked carbohydrates on the
procyclin surface coat were the ligands for Con A binding. This was support
ed by data obtained using mutant parasites created by deletion of all three
EP procyclin isoforms, two of which contain N-glycosylation sites, by homo
logous recombination. The knockout mutants showed reduced binding of fluore
scein-labelled Con A as determined by flow cytometry and were resistant to
the effects of Con A. Taken together the results show that Con A induces mu
ltinucleation, a disequilibrium between nuclear and kinetoplast replication
. and a unique form of cell death in procyclic African trypanosomes and tha
t the ligands for Con A binding are carbohydrates on the EP forms of procyc
lin. The possible significance of these findings for the life cycle of the
trypanosomes in the tsetse fly vector is discussed. (C) 2000 Elsevier Scien
ce B.V. All rights reserved.