title: Substrate specificity of Pasteurella multocida toxin for α subunits of heterotrimeric G proteins.
abstract: 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.
authors.affiliation: Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany. joachim.orth@pharmakol.uni-freiburg.de
authors.forename: Joachim H C
authors.initials: JH
authors.lastname: Orth
authors.forename: Ines
authors.initials: I
authors.lastname: Fester
authors.forename: Peter
authors.initials: P
authors.lastname: Siegert
authors.forename: Markus
authors.initials: M
authors.lastname: Weise
authors.forename: Ulrike
authors.initials: U
authors.lastname: Lanner
authors.forename: Shigeki
authors.initials: S
authors.lastname: Kamitani
authors.forename: Taro
authors.initials: T
authors.lastname: Tachibana
authors.forename: Brenda A
authors.initials: BA
authors.lastname: Wilson
authors.forename: Andreas
authors.initials: A
authors.lastname: Schlosser
authors.forename: Yasuhiko
authors.initials: Y
authors.lastname: Horiguchi
authors.forename: Klaus
authors.initials: K
authors.lastname: Aktories
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity*; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity*; D001483:Base Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000235:genetics; D044385:GTP-Binding Protein alpha Subunits / Q000737:chemistry* / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005973:Glutamine / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016979:Pasteurella multocida / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity*; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction / Q000187:drug effects; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D018076:DNA, Complementary; D044385:GTP-Binding Protein alpha Subunits; C062258:Pasteurella multocida toxin; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D005973:Glutamine; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D043802:GTP-Binding Protein alpha Subunits, Gq-G11