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.

Figure 1.

Gα_i family members are substrates of PMT. Gα_i proteins were coexpressed with an active PMT fragment and subjected to MS analysis. Left panels: combined extracted ion chromatograms (Q-TOF data) for m/z 455.216 (±10 ppm; solid line) and 455.708 (±10 ppm; dashed line), corresponding to the tryptic peptides MFDVGGQR and MFDVGGER (aa 198–205) of Gα_i1 (A, left panel) and Gα_i3 (B, left panel). The presence of the tryptic peptide MFDVGGER indicates deamidation of Gα_i1 and Gα_i3 at the essential Gln-204 by PMT. Right panels: no relevant deamidation product was detectable when Gα_i1 (A) and Gα_i3 (B) were expressed without PMT.

Figure 2.

Determination of the substrate specificity of PMT using a monoclonal anti-QE antibody that detects toxin-deamidated Gα proteins. A) HEK-293 cells were transfected with pcDNA3-based plasmids expressing the indicated Gα subunits. After 24 h of incubation, the cells were treated with or without PMT (1 nM) for a further 24 h. Cells were lysed and subjected to SDS-PAGE, followed by immunoblotting with monoclonal rat anti-Gα_q Q209E (3G3; anti-QE), polyclonal rabbit anti-Gα_i, polyclonal rabbit anti-Gα_s, and monoclonal mouse anti-tubulin antibody as described in Materials and Methods. Note that control HEK-293 cells (pcDNA) and recombinant Gα_s-expressing cells contain small amounts of endogenous G proteins, which are deamidated by PMT and labeled with anti-QE (double bands in top panel, lanes 2 and 10). The double bands labeled by anti-Gα_s indicate the two isoforms of Gα_s. B) CaCo-2 cells were treated with PMT (1 nM) for 1 h. Toxin-containing medium was discarded, and cells were washed with medium and incubated for indicated times. Cells were lysed and subjected to SDS-PAGE, followed by immunoblotting with monoclonal rat anti-Gα_q Q209E (3G3; anti-QE) and polyclonal rabbit anti-Gα_q/11 antibody.

Figure 3.

PMT substrate specificity with respect to Gα_q and Gα₁₁. A) Combined extracted ion chromatograms (ion trap data) for m/z 431.2 ± 0.2 (solid line) and 431.7 ± 0.2 (dashed line), corresponding to the doubly protonated precursors of the tryptic peptides MVDVGGQR and MFDVGGER (aa 203–210) of Gα_q. Top panel: endogenous Gα_q from untreated WT-MEFs. Bottom panel: endogenous Gα_q from PMT-treated WT-MEFs. B) Combined extracted ion chromatograms (Q-TOF data) for m/z 431.216 ± 20 ppm (solid line) and 431.708 ± 20 ppm (dashed line), corresponding to the doubly protonated precursors of the tryptic peptides MVDVGGQR and MFDVGGER (aa 203–210) of Gα₁₁. Gα₁₁ was precipitated from Gα_q/11-deficient MEFs, which were transduced with retrovirus encoding for Gα₁₁.Top panel: Gα₁₁ from untreated cells. Bottom panel: Gα₁₁ from PMT-treated cells. Deamidation of Gln-209 of Gα_q and Gα₁₁ is complete, and MVDVGGQR is no longer detectable after PMT treatment. C) Immunoblot of endogenous Gα_q after immunoprecipitation with a specific Gα_q antibody. Precipitations were performed from untreated MEF (con) or from PMT-treated MEF (1 nM, 18 h). D) Detection of Gα₁₁ in Gα_q/11-deficient MEFs (−) or in Gα_q/11-deficient MEFs transduced with Gα₁₁-encoding retrovirus (+Gα₁₁). E) Immunoblot of Gα₁₁ after immunoprecipitation with anti-Gα_q/11 antibody from Gα_q/11-deficient MEFs transduced with Gα₁₁-encoding retrovirus.

Figure 4.

PMT substrate specificity with respect to Gα₁₂ and Gα₁₃. A) Combined extracted ion chromatograms for m/z 431.216 ± 10 ppm (solid line) and 431.708 ± 10 ppm (dashed line), corresponding to the doubly protonated precursors of the tryptic peptides MVDVGGQR and MFDVGGER (Gα₁₃: aa 220–227, Gα₁₂: aa 225–232) of Gα₁₃ and Gα₁₂. Top panel: Gα₁₃ from PMT-treated (left) or untreated cells (right; Q-TOF data). Bottom panel: Gα₁₂ from PMT-treated (left) or untreated cells (right; Q-TOF data). The 1-Da shift demonstrates the deamidation of Gln-226 (Gα₁₃) and Gln-231 (Gα₁₂) to Glu. The deamidated form of the tryptic peptide (MVDVGGER) is only detectable when Gα₁₃ or Gα₁₂ is precipitated from PMT-treated cells. In addition, unmodified peptide (MVDVGGQR) is present in precipitates from PMT-treated cells. B) Gα₁₃ cDNA was engineered to harbor an internal flag tag. The construct was ectopically expressed in HEK-293 cells. After treatment with or without PMT (1 nM, 18 h), cells were lysed, and flag-tagged protein was immunoprecipitated, isolated, and subjected to MS analysis. Shown is an immunoblot of Gα₁₃ after immunoprecipitation with anti-flag-tag antibody from PMT-treated or untreated (con) cells. C) Gα₁₂ was ectopically expressed in HEK-293 cells. After treatment with or without PMT (1 nM, 18 h), cells were lysed, and Gα₁₂ protein was immunoprecipitated, using a specific anti-Gα₁₂ antibody, isolated, and subjected to MS analysis. Immunoblot shows Gα₁₂ after immunoprecipitation with anti-Gα₁₂ antibody from PMT-treated or untreated (con) cells. The strong band above Gα₁₂ is part of the antibody used for immunoprecipitation.

Figure 5.

Switch II of G-protein α subunits is not involved in PMT substrate specificity. A) Alignment of amino acid sequences of the switch II region in the α subunits of mouse heterotrimeric GTPases. Nucleotide sequences are obtained from the U.S. National Center for Biotechnology Information (NCBI): Gα_s (P63094), Gα_olf1 (NP_034437), Gα_olf2 (NP_796111), Gα_i1 (NP_034435), Gα_i2 (AAH65159), Gα_i3 (NP_034436), Gα_oA (NP_034438), Gα_oB (P18873.3), Gα_t1 (NP_032166), Gα_t2 (NP_032167), Gα_z (NP_034441), Gα₁₅ (NP_034434), Gα₁₄ (NP_032163), Gα₁₃ (NP_034433), Gα₁₂ (NP_034432), Gα₁₁ (NP_034431), and Gα_q (NP_032165). Numbers below the alignment correspond to the amino acid positions of Gα_q. The active site Gln (at position 209 in Gα_q) is indicated by a gray box, highly conserved flanking residues by boldface type, and flanking residues that result in notable charge differences by are indicated in reverse type on a black box. Asterisks denote identical amino acid residues; colons denote highly conserved residues; periods denote conserved residues. Numbers in the top row indicate positions downstream of the essential Gln. B, C) Combined extracted ion chromatograms (Q-TOF data) for m/z 455.216 ± 10 and 455.708 ± 10 ppm, corresponding to the tryptic peptides MFDVGGQR and MFDVGGER (aa 199–206) of Gα_i2^S207D (B) and Gα_i2^E208Q (C). Gα_i2 mutants were coexpressed with an active PMT fragment and subjected to MS analysis. The presence of the tryptic peptide MFDVGGER indicates deamidation of Gα_i2^S207D and Gα_i2^E208Q at Gln-205 by PMT. No relevant deamidation product was detectable when Gα_i2^S207D and Gα_i2^E208Q were expressed without PMT (right panels).

Figure 6.

PMT-induced activation of Gα_s-containing chimera. A) Scheme of Gα_q-Gα_s chimera. N-terminal part including the HD and switch II of Gα_q (aa 1–216) were fused to the C-terminal effector-binding domain of Gα_s (aa 221–380). B) cAMP accumulation was measured after transfection of Gα_q-Gα_s chimera into HEK-293-cells (squares) or after transfection with an empty pcDNA vector (circles). PMT was added at indicated concentrations for 3 h. Inset: cAMP accumulation of PMT-treated (10 nM, 3 h) cells transfected as indicated. Gα_q-and Gα_s-overexpressing cells show no increase in cAMP levels after PMT incubation. C) Chimera Gα_q-Gα_s was immunoprecipitated from PMT-treated cells (1 nM, 18 h) with a G_s antibody against the C-terminal region. Immunoblot shows Gα_q-Gα_s chimera after immunoprecipitation with anti-Gs antibody. D) Combined extracted ion chromatograms (Q-TOF data) for m/z 431.216 ± 10 and 431.708 ± 10 ppm, corresponding to the doubly protonated precursors of the tryptic peptides MVDVGGQR and MFDVGGER (Gα_q-Gα_s: aa 203–210) of Gα_q-Gα_s. The 1-Da shift demonstrates the deamidation of Gln-209 to Glu. E) Endogenous Gα_s was immunoprecipitated from PMT-treated MEF (1 nM, 18 h) with a G_s antibody against the C-terminal region. Immunoblot shows Gα_s after immunoprecipitation with anti-G_s antibody. F) Combined extracted ion chromatogram (Q-TOF data) for m/z 452.464 ± 20 ppm corresponding to the quadruple protonated precursors of the tryptic peptide VNFHMFDVGGQRDER (Gα_s: aa 203–217) of Gα_s. No deamidation of Gln-213 was detectable.

Figure 7.

PMT effect on Gα_i2 fragments. A) Scheme of Gα_i2 fragments. Gα_i2 consists of the GTPase domain and an HD insert. In the case of Gα_i2^ΔHD, the HD (residues 61–175) was deleted and replaced by a short linker (Ser-Ala-Gly-Ala). The construct Gα_i2^swII comprises only the region around the switch II (residues 184–218). B) GST-fusion proteins of Gα_i2, Gα_i2^ΔHD, and Gα_i2^swII were coexpressed with active or inactive PMT-C and subjected to immunoblot analysis with monoclonal rat anti-Gα_q Q209E (3G3; anti-QE), Gα_pan antibody as described in Materials and Methods. Deamidation of Gα_i2 and Gα_i2^ΔHD was detectable. C) Combined extracted ion chromatograms (Q-TOF data) for m/z 455.216 ± 10 ppm (solid line) and 455.708 ± 10 ppm (dashed line), corresponding to the tryptic peptides MFDVGGQR and MFDVGGER (aa 198–205) of Gα_i2^ΔHD coexpressed with active PMT-C^wt. D) Combined extracted ion chromatograms (Q-TOF data) for m/z 455.216 ± 10 ppm (solid line) and 455.708 ± 10 ppm (dashed line) of Gα_i2^swII coexpressed with active PMT-C^wt. Only the tryptic peptide MFDVGGQR (m/z 455.216), but no MFDVGGER (m/z 455.708), was identified.