Pasteurella multocida toxin is a major virulence factor of Pasteurella multocida, which causes pasteurellosis in men and animals and atrophic rhinitis in rabbits and pigs. The Ϸ145 kDa protein toxin stimulates various signal transduction pathways by activating heterotrimeric G proteins of the G␣ q, G␣i, and G␣12/13 families by using an as yet unknown mechanism. Here, we show that Pasteurella multocida toxin deamidates glutamine-205 of G␣ i2 to glutamic acid. Therefore, the toxin inhibits the intrinsic GTPase activity of G␣ i and causes persistent activation of the G protein. A similar modification is also evident for G␣ q, but not for the closely related G␣ 11, which is not a substrate of Pasteurella multocida toxin. Our data identify the ␣-subunits of heterotrimeric G proteins as the direct molecular target of Pasteurella multocida toxin and indicate that the toxin does not act like a protease, which was suggested from its thiol protease-like catalytic triad, but instead causes constitutive activation of G proteins by deamidase activity.bacterial protein toxin ͉ Gi protein ͉ posttranslational modification ͉ transglutaminase

Bacterial protein toxins are powerful tools for elucidating signaling mechanisms in eukaryotic cells. A number of bacterial protein toxins, e.g. cholera toxin, pertussis toxin (PTx), or Pasteurella multocida toxin (PMT), target heterotrimeric G proteins and have been used to stimulate or block specific signaling pathways or to demonstrate the contribution of their target proteins in cellular effects. PMT is a major virulence factor of P. multocida causing pasteurellosis in man and animals and is responsible for atrophic rhinitis in pigs. PMT modulates various signaling pathways, including phospholipase C␤ and RhoA, by acting on the heterotrimeric G proteins G␣ q and G␣ 12/13 , respectively. Here we report that PMT is a powerful activator of G i protein. We show that PMT decreases basal isoproterenol and forskolin-stimulated cAMP accumulation in intact Swiss 3T3 cells, inhibits adenylyl cyclase activity in cell membrane preparations, and enhances the inhibition of cAMP accumulation caused by lysophosphatidic acid via endothelial differentiation gene receptors. PMT-mediated inhibition of cAMP production is independent of toxin activation of G␣ q and/or G␣ 12/13 . Although the effects of PMT are not inhibited by PTx, PMT blocks PTx-catalyzed ADP-ribosylation of G i . PMT also inhibits steadystate GTPase activity and GTP binding of G i in Swiss 3T3 cell membranes stimulated by lysophosphatidic acid. The data indicate that PMT is a novel activator of G i , modulating its GTPase activity and converting it into a PTx-insensitive state.

Pasteurella multocida is the causative agent of a number of epizootic and zoonotic diseases. Its major virulence factor associated with atrophic rhinitis in animals and dermonecrosis in bite wounds is P. multocida toxin (PMT). PMT stimulates signal transduction pathways downstream of heterotrimeric G proteins, leading to effects such as mitogenicity, blockade of apoptosis, or inhibition of osteoblast differentiation. On the basis of Gα(i2), it was demonstrated that the toxin deamidates an essential glutamine residue of the Gα(i2) subunit, leading to constitutive activation of the G protein. Here, we studied the specificity of PMT for its G-protein targets by mass spectrometric analyses and by utilizing a monoclonal antibody, which recognizes specifically G proteins deamidated by PMT. The studies revealed deamidation of 3 of 4 families of heterotrimeric G proteins (Gα(q/11), Gα(i1,2,3), and Gα(12/13) of mouse or human origin) by PMT but not by a catalytic inactive toxin mutant. With the use of G-protein fragments and chimeras of responsive or unresponsive G proteins, the structural basis for the discrimination of heterotrimeric G proteins was studied. Our results elucidate substrate specificity of PMT on the molecular level and provide evidence for the underlying structural reasons of substrate discrimination.

To clarify the sorting mechanism of the lysosomal/granular proteoglycan serglycin, we treated human promonocytic U937 cells with p-nitrophenyl-beta-D-xyloside (PNP-xyl) and cycloheximide. In the absence of protein synthesis, the carbohydrate moiety of serglycin was synthesized as PNP-xyl-chondroitin sulfate (CS), and most of it was delivered to lysosomes and degraded. Further, an augmented lysosomal targeting of serglycin in the presence of tunicamycin suggested that a sorting/lectin receptor with multiple specificity was involved with an increased capacity for serglycin in the absence of N-glycosylation. Correspondingly, the cation-independent mannose 6-phosphate receptor (CI-MPR) and sortilin were observed to bind to immobilized CS. These receptors were eluted in the presence of 200-400 mM and 100-250 mM NaCl, respectively. After treating the cells with a cross-linking reagent, a portion of the sulfated proteoglycan was coimmunoprecipitated with the CI-MPR but not with sortilin. In the presence of phorbol ester, lysosomal targeting of serglycin and to a lesser extent, of cathepsin D was inhibited. We conclude that the CI-MPR participates in lysosomal and granular targeting of serglycin and basic proteins such as lysozyme associated with the proteoglycan in hematopoietic cells.