Jacqueline Hanocq-Quertier

A novel heterodimeric transferrin receptor encoded by a pair of VSG expression site-associated genes in T. brucei

In T. brucei, a transferrin-binding protein has been found to share sequence homology with pESAG-7 and -6, the products of two related genes present in the VSG gene polycistronic transcription unit. When expressed in Xenopus oocytes, they appear as N-glycosylated proteins secreted in the medium (pESAG-7) and GPI anchored to the membrane (pESAG-6). These proteins are able to homo- or heterodimerize, probably through association in the same orientation. Only heterodimers can bind Tf, possibly two molecules per dimer. A comparison of Tf binding to pESAG-7/6-expressing oocytes and trypanosomes suggests that pESAG-7/6 is the Tf receptor of the parasite. In trypanosomes, the majority of pESAG-7/6 is released from the membrane and associates, together with Tf, with a glycosylated matrix present in the lumen of the flagellar pocket. Both pESAG-7/6 and Tf are internalized via coated pits and vesicles. These observations suggest a novel mode of Tf binding and uptake in trypanosomes.

Characterization of the ligand‐binding site of the transferrin receptor in Trypanosoma brucei demonstrates a structural relationship with the N‐terminal domain of the variant surface glycoprotein

The Trypanosoma brucei transferrin (Tf) receptor is a heterodimer encoded by ESAG7 and ESAG6, two genes contained in the different polycistronic transcription units of the variant surface glycoprotein (VSG) gene. The sequence of ESAG7/6 differs slightly between different units, so that receptors with different affinities for Tf are expressed alternatively following transcriptional switching of VSG expression sites during antigenic variation of the parasite. Based on the sequence homology between pESAG7/6 and the N‐terminal domain of VSGs, it can be predicted that the four blocks containing the major sequence differences between pESAG7 and pESAG6 form surface‐exposed loops and generate the ligand‐binding site. The exchange of a few amino acids in this region between pESAG6s encoded by different VSG units greatly increased the affinity for bovine Tf. Similar changes in other regions were ineffective, while mutations predicted to alter the VSG‐like structure abolished the binding. Chimeric proteins containing the N‐terminal dimerization domain of VSG and the C‐terminal half of either pESAG7 or pESAG6, which contains the ligand‐binding domain, can form heterodimers that bind Tf. Taken together, these data provided evidence that the T.brucei Tf receptor is structurally related to the N‐terminal domain of the VSG and that the ligand‐binding site corresponds to the exposed surface loops of the protein.

Abrupt RNA changes precede the first cell division during the differentiation of Trypanosoma brucei bloodstream forms into procyclic forms in vitro

We have monitored the timing of DNA and RNA synthesis during the synchronous differentiation of Trypanosoma brucei bloodstream forms into procyclic forms in vitro. Both are triggered after a lag period of 4 h and reach a first peak after 9 h. The division of the kinetoplast precedes that of the nucleus by about 4 h. The first cell divisions are observed after 10 h, and the cell number is doubled after 20 h. The total RNA content per cell increases sharply between 4 and 10 h, then progressively decreases as cell division progresses. The increase in RNA content cannot be due solely to accumulation of rRNA since it is also observed for mRNAs such as actin. The VSG mRNA has almost disappeared within 2 h, while the procyclin mRNA accumulates soon after the triggering of differentiation, with a strong peak between 4 and 6 h. At this moment, the amount of procyclin mRNA per cell is at least 20-fold higher than in established procyclic culture forms. The loss of the VSG and the appearance of procyclic-specific proteins essentially occur before the first cell division. These observations contrast with the progressive transition observed when monomorphic slender forms are induced to transform under the same conditions.

Transient adenylate cyclase activation accompanies differentiation of Trypanosoma brucei from bloodstream to procyclic forms.

Pleomorphic bloodstream forms of Trypanosoma brucei differentiate synchronously into procyclic forms when cultivated at 27 degrees C in the presence of citrate/cis-aconitate. The activity of adenylate cyclase was monitored during this process. Two phases of transient stimulation were observed. The first phase occurred 6-10 h after the triggering of differentiation, a period which immediately follows the release of the bulk of the VSG and immediately precedes both the first cell division and the loss of the bloodstream-specific ESAG 4 transmembrane adenylate cyclase. The second phase occurred between 20 and 40 h, when the cells that emerged from the first division began to proliferate. These observations suggest that cAMP may be involved in differentiation/proliferation of the parasite.

MILD ACID STRESS AS A DIFFERENTIATION TRIGGER IN TRYPANOSOMA BRUCEI

In vitro differentiation of Trypanosoma brucei from the bloodstream to the procyclic form is efficiently induced by the combination of cold shock from 37 to 27 degrees C and the addition of citrate/cis-aconitate (CCA) to the incubation medium. Here it is reported that exposure of pleomorphic bloodstream trypanosomes to mild acidic conditions (pH 5.5 for 2 h at 37 degrees C) not only accelerated the process of morphological transformation from long slender and intermediate to short stumpy bloodstream forms but also allowed their subsequent differentiation into procyclic forms even in the absence of CCA. This process appeared to involve the glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC), since null GPI-PLC mutants (PLC-) appeared to be largely refractory to acid stress-induced differentiation. However, an effective response was restored upon reintegration of the GPI-PLC gene in the genome (PLC+).

Families of adenylate cyclase genes in Trypanosoma brucei

Four genes for adenylate cyclase have been characterized in Trypanosoma brucei. One of them, esag 4 (for expression site associated gene 4) is present in different VSG (variant surface glycoprotein) gene expression sites and, thus, is only expressed in the bloodstream form of the parasite. The others, termed gresag 4.1, 4.2 and 4.3 (for genes related to esag 4) are expressed in both bloodstream and procyclic forms. In addition, we cloned a esag 4-related gene from T. congolense. Here we characterize the genomic organization of gresag 4.1 and 4.3. While gresag 4.3 is unique, gresag 4.1 exists as a multigenic family of at least nine members located on a 3-Mb chromosome. Six of them are clustered in a region of 300 kb, three copies being tandemly linked. The determination of the nucleotide sequence of a conserved 1.6 kb PstI fragment demonstrated the presence of two separate subgroups in this family. This gene arrangement is present in different isolates of T.b. brucei/rhodesiense/gambiense. Several gresag 4.1 copies are transcribed in both bloodstream and procyclic forms.

Adenylate cyclase and cyclic amp-phosphodiesterase activities during the early phase of maturation Xenopus laevis oocytes

Maturation (meiosis) is the transformation, upon hormonal stimulation, of a diploid ovarian oocyte into a haploid and fertilizable egg (review [ 11). In amphibians, in vitro maturation of oocytes dissected out of the ovary can generally be induced by a short incubation of the oocytes with progesterone. In Xenopus laevis, the process of maturation extends over 6-10 h; it involves several different steps the most important of which, from a biological viewpoint, is the appearance of the maturation promoting factor (MPF) after 4-6 h hormonal treatment [2]. An essential step in the initiation of maturation is the dephosphorylation of certain classes of proteins. This phenomenon is believed to result from the following sequence of events: an intracellular release of membrane-bound Ca2* would be instantaneously triggered by progesterone; Ca’+and calmodulin would activate a cyclic AMP (CAMP)-phosphodiesterase. A drop of CAMP content would follow; as a result, a cAMPdependent protein kinase activity would decrease with a subsequent dephosphorylation of proteins. Some of these steps are well substantiated by experimental data, whereas others are still hypothetical: it has been established that progesterone treatment induces the release of Ca2+from the membrane within 30-90 s [3]; a calmodulin-like protein has been isolated from Xenopus oocytes [4]; moreover, Ca2+-calmodulin injected into oocytes has been reported to induce their maturation [5], but it has not yet been proved that Ca’+and calmodulin activate in vivo an oocytic CAMP-phosphodiesterase. However, in [6] protein dephosphorylation is clearly indicated as an essential event for the initiation of maturation. Injec-

Heat-shock response in Xenopus oocytes during meiotic maturation and activation

After a 60 min heat-shock at 36 degrees C, Xenopus oocytes are still able to accomplish a complete meiotic maturation in response to a progesterone treatment. The 36 degrees C heat-shock applied to maturing oocytes strongly enhances the synthesis of a single heat-shock protein of approx. 70 000 molecular weight (hsp70); after activation with the Ca2+-ionophore A 23187, matured oocytes still display the ability to synthesize hsp70 and to survive a heat-shock. A cycloheximide treatment combined with a heat-shock induces, during the recovery period, the synthesis of two heat-shock proteins, of approx. 70 000 and 83 000 molecular weight.

Cytological Effects of Heat‐Shocks on Xenopus Oocytes and Eggs

Heat‐shocks (80 min at 34°c) induce the appearance of aster‐like fibrous structures (cytasters) in maturing Xenopus oocytes. Cytaster formation is suppressed by treatments with colchicine or nocodazole of heat‐shocked maturing oocytes. Heat‐shocks destroy the meiotic spindle, but have no effect on cytasters induced by D2O treatment.