The ROP2 family ofToxoplasma gondii rhoptry proteins: Proteomic and genomic characterization and molecular modeling

Abstract

Four rhoptry proteins (ROP) of Toxoplasma gondii previously identified with mAb have been affinity purified and analyzed by MS; the data obtained allowed the genomic sequences to be assigned to these proteins. As previously suggested for some of them by antibody crossreactivity, these proteins were shown to belong to a family, the prototype of which being ROP2. We describe here the proteins ROP2, 4, 5, and 7. These four proteins correspond to the most abundant products of a gene family that comprises several members which we have identified in genomic and EST libraries. Eight additional sequences were found and we have cloned four of them. All members of the ROP2 family contain a protein-kinase-like domain, but only some of them possess a bona fide kinase catalytic site. Molecular modeling of the kinase domain demonstrates the conservation of residues critical for the stabilization of the protein-kinase fold, especially within a hydrophobic segment described so far as transmembrane and which appears as an helix buried inside the protein. The concomitant synthesis of these ROPs by T. gondii tachyzoites suggests a specific role for each of these proteins, especially in the early interaction with the host cell upon invasion.

Host cell manipulation by the human pathogen Toxoplasma gondii

“…ROP2 is associated with evacuoles shortly after invasion and ultimately decorates the PVM [40]. Whereas initial experiments indicated that ROP2 is anchored to the PVM by a transmembrane domain [34], this conclusion has been reassessed recently [41,42]. Nonetheless, there is agreement that the N-terminal portion of ROP2 is exposed to the host cytosol.…”

“…The carboxy-terminal portion of ROP4 displays the characteristic kinase domain. However, the lysine (subdomain II) and aspartic acid (subdomain VIb) residues typically necessary for kinase activity are absent [41]. Intriguingly, the mature form of ROP4 is phosphorylated on several serine and threonine residues following the parasite invasion.…”

“…ROP14 presents several predicted transmembrane domains usually found inside proteins involved in transport activity. For this reason, ROP14 is a candidate for the formation of the pore associated with the diffusion of small molecules across the PVM [41]. The dense granule proteins, GRA3, GRA5, GRA7, GRA8 and GRA10, are also localized at the PVM after the invasion of the parasite [56][57][58][59].…”

“…Some of these pseudokinases play a role in the activation or inhibition of other kinases, or in the assembly of kinase in complex [78][79][80][81][82]. While ROP2 subfamily proteins contain a predicted kinase domain, sequence analysis by El Hajj et al [41] shows that ROP2, 4, 5, 7, 8, 11, 2L3 and 2L6 proteins lack important residues usually essential for the catalytic activity. A role of these proteins in the modulation of the host kinase activation remains to be tested.…”

TheToxoplasma gondiirhoptry protein ROP18 is an Irga6‐specific kinase and regulated by the dense granule protein GRA7

“…Although not apparent for an IRG‐specific function of ROP5 in our own experiments (Fleckenstein et al ., 2012), an interaction of ROP5 with ROP18 was reported to control T. gondii virulence by up‐regulation of ROP18 activity (Behnke et al ., 2012). The rop5 locus is represented by a cluster of tandemly repeated genes encoding three different ROP5 isoforms, ROP5A, ROP5B and ROP5C, each containing multiple copies, and all isoforms are predicted to be catalytically inactive (El Hajj et al ., 2006; Reese and Boothroyd, 2011). Individual sequences of these rop5 paralogues are almost identical within strains but differ considerably between T. gondii types I, II and III strains (Behnke et al ., 2011; Reese et al ., 2011; Niedelman et al ., 2012).…”