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Structure-informed design of an enzymatically inactive vaccine component for group A Streptococcus.

Henningham A, Ericsson DJ, Langer K, Casey LW, Jovcevski B, Chhatwal GS, Aquilina JA, Batzloff MR, Kobe B, Walker MJ - MBio (2013)

Bottom Line: There is no human ortholog of ADI, and we confirm that despite limited structural similarity in the active-site region to human peptidyl ADI 4 (PAD4), ADI does not functionally mimic PAD4 and antiserum raised against GAS ADI does not recognize human PAD4.In this study, we determined the structure of GAS ADI and used this information to improve the vaccine safety of GAS ADI.This example of structural biology informing vaccine design may underpin the formulation of a safe and efficacious GAS vaccine.

View Article: PubMed Central - PubMed

Affiliation: School of Chemistry and Molecular Biosciences, Australian Infectious Diseases Research Centre, University of Queensland, St. Lucia, Qld., Australia.

ABSTRACT

Unlabelled: Streptococcus pyogenes (group A Streptococcus [GAS]) causes ~700 million human infections/year, resulting in >500,000 deaths. There is no commercial GAS vaccine available. The GAS surface protein arginine deiminase (ADI) protects mice against a lethal challenge. ADI is an enzyme that converts arginine to citrulline and ammonia. Administration of a GAS vaccine preparation containing wild-type ADI, a protein with inherent enzymatic activity, may present a safety risk. In an approach intended to maximize the vaccine safety of GAS ADI, X-ray crystallography and structural immunogenic epitope mapping were used to inform vaccine design. This study aimed to knock out ADI enzyme activity without disrupting the three-dimensional structure or the recognition of immunogenic epitopes. We determined the crystal structure of ADI at 2.5 Å resolution and used it to select a number of amino acid residues for mutagenesis to alanine (D166, E220, H275, D277, and C401). Each mutant protein displayed abrogated activity, and three of the mutant proteins (those with the D166A, H275A, and D277A mutations) possessed a secondary structure and oligomerization state equivalent to those of the wild type, produced high-titer antisera, and avoided disruption of B-cell epitopes of ADI. In addition, antisera raised against the D166A and D277A mutant proteins bound to the GAS cell surface. The inactivated D166A and D277A mutant ADIs are ideal for inclusion in a GAS vaccine preparation. There is no human ortholog of ADI, and we confirm that despite limited structural similarity in the active-site region to human peptidyl ADI 4 (PAD4), ADI does not functionally mimic PAD4 and antiserum raised against GAS ADI does not recognize human PAD4.

Importance: We present an example of structural biology informing human vaccine design. We previously showed that the administration of the enzyme arginine deiminase (ADI) to mice protected the mice against infection with multiple GAS serotypes. In this study, we determined the structure of GAS ADI and used this information to improve the vaccine safety of GAS ADI. Catalytically inactive mutant forms of ADI retained structure, recognition by antisera, and immunogenic epitopes, rendering them ideal for inclusion in GAS vaccine preparations. This example of structural biology informing vaccine design may underpin the formulation of a safe and efficacious GAS vaccine.

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Comparison of GAS ADI to human PAD4. (A) Structural alignment of GAS ADI (pink) with human PAD4 (green). The superposition is centered on the α/β-propeller domains, omitting the orthogonal bundle from ADI and the two N-terminal all-β domains from PAD4. ADI active-site residues D166, E220, H275, D277, and C401 align closely with the corresponding residues in PAD4. (B) Unlike human PAD4, GAS ADI does not citrullinate host targets, histone H3, LL-37, or IL-8 in vitro. Host targets were incubated with 5 µM ADI (left lane), 500 nM PAD4 (middle lane), or neither (right lane) for 1 h at 37°C. The reaction was immediately stopped by incubation with SDS-PAGE loading buffer for 15 min at 95°C. Samples containing 1.5 µg of target protein were separated by 15% PAGE and either stained with Coomassie blue (top) or transferred to nitrocellulose membrane for Western blotting with antibodies for the detection of citrulline (bottom). (C) Anti-ADI serum does not recognize recombinant human PAD4 in a Western blot assay. Samples containing 2 µg of ADI (right lane) or PAD4 (left lane) were separated by 15% PAGE and either stained with Coomassie blue (top) or transferred to nitrocellulose membrane for Western blotting (bottom).
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fig4: Comparison of GAS ADI to human PAD4. (A) Structural alignment of GAS ADI (pink) with human PAD4 (green). The superposition is centered on the α/β-propeller domains, omitting the orthogonal bundle from ADI and the two N-terminal all-β domains from PAD4. ADI active-site residues D166, E220, H275, D277, and C401 align closely with the corresponding residues in PAD4. (B) Unlike human PAD4, GAS ADI does not citrullinate host targets, histone H3, LL-37, or IL-8 in vitro. Host targets were incubated with 5 µM ADI (left lane), 500 nM PAD4 (middle lane), or neither (right lane) for 1 h at 37°C. The reaction was immediately stopped by incubation with SDS-PAGE loading buffer for 15 min at 95°C. Samples containing 1.5 µg of target protein were separated by 15% PAGE and either stained with Coomassie blue (top) or transferred to nitrocellulose membrane for Western blotting with antibodies for the detection of citrulline (bottom). (C) Anti-ADI serum does not recognize recombinant human PAD4 in a Western blot assay. Samples containing 2 µg of ADI (right lane) or PAD4 (left lane) were separated by 15% PAGE and either stained with Coomassie blue (top) or transferred to nitrocellulose membrane for Western blotting (bottom).

Mentions: Peptidyl ADI 4 (PAD4) is known to convert arginine residues to citrulline in histones (23) and chemokines (24) within the host. To determine if ADI could be molecularly mimicking the function of human PAD4, we first compared the structure of GAS ADI to that of PAD4 (Protein Data Bank accession no. 2DW5) (25). These superpose with an RMSD of 2.8 Å over 216 residues, with the majority of the deviation coming from the larger α-helical elements present in PAD4. In contrast, the active-site residues of the two structures overlap with an RMSD of <1.0 Å (Fig. 4A). Western blotting indicated that recombinant human PAD4 successfully citrullinated host targets in vitro (Fig. 4B). However, recombinant wild-type ADI did not citrullinate host targets of PAD4 in vitro, including histone H3, LL-37, or interleukin-8 (IL-8) (Fig. 4B). Antiserum raised against wild-type ADI did not recognize human PAD4 in Western blot assays (Fig. 4C). Additionally, incubation of anti-wild-type ADI serum with immobilized PAD4 in an ELISA did not result in a measurable response (data not shown).


Structure-informed design of an enzymatically inactive vaccine component for group A Streptococcus.

Henningham A, Ericsson DJ, Langer K, Casey LW, Jovcevski B, Chhatwal GS, Aquilina JA, Batzloff MR, Kobe B, Walker MJ - MBio (2013)

Comparison of GAS ADI to human PAD4. (A) Structural alignment of GAS ADI (pink) with human PAD4 (green). The superposition is centered on the α/β-propeller domains, omitting the orthogonal bundle from ADI and the two N-terminal all-β domains from PAD4. ADI active-site residues D166, E220, H275, D277, and C401 align closely with the corresponding residues in PAD4. (B) Unlike human PAD4, GAS ADI does not citrullinate host targets, histone H3, LL-37, or IL-8 in vitro. Host targets were incubated with 5 µM ADI (left lane), 500 nM PAD4 (middle lane), or neither (right lane) for 1 h at 37°C. The reaction was immediately stopped by incubation with SDS-PAGE loading buffer for 15 min at 95°C. Samples containing 1.5 µg of target protein were separated by 15% PAGE and either stained with Coomassie blue (top) or transferred to nitrocellulose membrane for Western blotting with antibodies for the detection of citrulline (bottom). (C) Anti-ADI serum does not recognize recombinant human PAD4 in a Western blot assay. Samples containing 2 µg of ADI (right lane) or PAD4 (left lane) were separated by 15% PAGE and either stained with Coomassie blue (top) or transferred to nitrocellulose membrane for Western blotting (bottom).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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fig4: Comparison of GAS ADI to human PAD4. (A) Structural alignment of GAS ADI (pink) with human PAD4 (green). The superposition is centered on the α/β-propeller domains, omitting the orthogonal bundle from ADI and the two N-terminal all-β domains from PAD4. ADI active-site residues D166, E220, H275, D277, and C401 align closely with the corresponding residues in PAD4. (B) Unlike human PAD4, GAS ADI does not citrullinate host targets, histone H3, LL-37, or IL-8 in vitro. Host targets were incubated with 5 µM ADI (left lane), 500 nM PAD4 (middle lane), or neither (right lane) for 1 h at 37°C. The reaction was immediately stopped by incubation with SDS-PAGE loading buffer for 15 min at 95°C. Samples containing 1.5 µg of target protein were separated by 15% PAGE and either stained with Coomassie blue (top) or transferred to nitrocellulose membrane for Western blotting with antibodies for the detection of citrulline (bottom). (C) Anti-ADI serum does not recognize recombinant human PAD4 in a Western blot assay. Samples containing 2 µg of ADI (right lane) or PAD4 (left lane) were separated by 15% PAGE and either stained with Coomassie blue (top) or transferred to nitrocellulose membrane for Western blotting (bottom).
Mentions: Peptidyl ADI 4 (PAD4) is known to convert arginine residues to citrulline in histones (23) and chemokines (24) within the host. To determine if ADI could be molecularly mimicking the function of human PAD4, we first compared the structure of GAS ADI to that of PAD4 (Protein Data Bank accession no. 2DW5) (25). These superpose with an RMSD of 2.8 Å over 216 residues, with the majority of the deviation coming from the larger α-helical elements present in PAD4. In contrast, the active-site residues of the two structures overlap with an RMSD of <1.0 Å (Fig. 4A). Western blotting indicated that recombinant human PAD4 successfully citrullinated host targets in vitro (Fig. 4B). However, recombinant wild-type ADI did not citrullinate host targets of PAD4 in vitro, including histone H3, LL-37, or interleukin-8 (IL-8) (Fig. 4B). Antiserum raised against wild-type ADI did not recognize human PAD4 in Western blot assays (Fig. 4C). Additionally, incubation of anti-wild-type ADI serum with immobilized PAD4 in an ELISA did not result in a measurable response (data not shown).

Bottom Line: There is no human ortholog of ADI, and we confirm that despite limited structural similarity in the active-site region to human peptidyl ADI 4 (PAD4), ADI does not functionally mimic PAD4 and antiserum raised against GAS ADI does not recognize human PAD4.In this study, we determined the structure of GAS ADI and used this information to improve the vaccine safety of GAS ADI.This example of structural biology informing vaccine design may underpin the formulation of a safe and efficacious GAS vaccine.

View Article: PubMed Central - PubMed

Affiliation: School of Chemistry and Molecular Biosciences, Australian Infectious Diseases Research Centre, University of Queensland, St. Lucia, Qld., Australia.

ABSTRACT

Unlabelled: Streptococcus pyogenes (group A Streptococcus [GAS]) causes ~700 million human infections/year, resulting in >500,000 deaths. There is no commercial GAS vaccine available. The GAS surface protein arginine deiminase (ADI) protects mice against a lethal challenge. ADI is an enzyme that converts arginine to citrulline and ammonia. Administration of a GAS vaccine preparation containing wild-type ADI, a protein with inherent enzymatic activity, may present a safety risk. In an approach intended to maximize the vaccine safety of GAS ADI, X-ray crystallography and structural immunogenic epitope mapping were used to inform vaccine design. This study aimed to knock out ADI enzyme activity without disrupting the three-dimensional structure or the recognition of immunogenic epitopes. We determined the crystal structure of ADI at 2.5 Å resolution and used it to select a number of amino acid residues for mutagenesis to alanine (D166, E220, H275, D277, and C401). Each mutant protein displayed abrogated activity, and three of the mutant proteins (those with the D166A, H275A, and D277A mutations) possessed a secondary structure and oligomerization state equivalent to those of the wild type, produced high-titer antisera, and avoided disruption of B-cell epitopes of ADI. In addition, antisera raised against the D166A and D277A mutant proteins bound to the GAS cell surface. The inactivated D166A and D277A mutant ADIs are ideal for inclusion in a GAS vaccine preparation. There is no human ortholog of ADI, and we confirm that despite limited structural similarity in the active-site region to human peptidyl ADI 4 (PAD4), ADI does not functionally mimic PAD4 and antiserum raised against GAS ADI does not recognize human PAD4.

Importance: We present an example of structural biology informing human vaccine design. We previously showed that the administration of the enzyme arginine deiminase (ADI) to mice protected the mice against infection with multiple GAS serotypes. In this study, we determined the structure of GAS ADI and used this information to improve the vaccine safety of GAS ADI. Catalytically inactive mutant forms of ADI retained structure, recognition by antisera, and immunogenic epitopes, rendering them ideal for inclusion in GAS vaccine preparations. This example of structural biology informing vaccine design may underpin the formulation of a safe and efficacious GAS vaccine.

Show MeSH
Related in: MedlinePlus