Binding of alpha2-macroglobulin to GRAB (Protein G-related alpha2-macroglobulin-binding protein), an important virulence factor of group A streptococci, is mediated by two charged motifs in the DeltaA region

Abstract

GRAB (Protein G-related alpha2M-binding protein) is a surface protein of group A streptococci and exhibits high affinity for alpha2-macroglobulin (alpha2M), a broad-range protease inhibitor. It is the sole alpha2M-binding protein of group A streptococci that has been shown to promote bacterial virulence in a mouse model of skin infection. The binding site for alpha2M was predicted to be in the N-terminal A domain of GRAB. In the present study, the alpha2M-binding domain was first narrowed down to 34 amino acids (amino acids 34-67) using variable truncated N-terminal GRAB fusion proteins. The sequence of the identified domain was used to design overlapping synthetic peptides of different sizes, which were then immobilized on a membrane and assayed for their alpha2M-binding activity. The peptide screening revealed two binding motifs of ten amino acids length, located in the DeltaA (N-terminal part of the A domain) region (amino acids 34-67) with the sequences PRIIPNGGTL (amino acids 41-50) and NAPEKLALRN (amino acids 56-65) respectively. These motifs were used for systematic mutational analysis by generating synthetic peptides containing individual amino acid substitutions at every position of the mapped binding regions. The results indicated a critical role for the arginine residue at position 42 in the first binding domain and at position 64 in the second binding region. Validation of arginine residues as the critical amino acids for alpha2M binding was achieved by site-directed mutagenesis and binding assays. Competitive inhibition assays with GRAB containing amino acid substitutions R42G (Arg42-->Gly), R64G and R42G/R64G indicated differential contribution of the arginine residues at positions 42 and 64 to alpha2M-binding activity and, thus, their involvement in GRAB-induced virulence.

Figure 1. SDS/PAGE analysis of α₂M before and after trypsin treatment

Lane 2 (α₂M) mainly consists of the slow-form α₂M with small amounts of the fast form (double bands at 100 kDa mass). In contrast, the trypsin-pretreated control (α₂M-T) in lane 3 shows the electrophoretically fast-form α₂M. Molecular markers are shown in lane 1.

Figure 2. A model and sequence of GRAB

(A) Schematic representation of GRAB protein (accession no. GI: 4589078). Functional domains are (Ss) signal sequence, (A) α₂M-binding domain with the ΔA region shown hatched, (R) repeat regions, (W) cell-wall attachment site and (M) membrane anchor. (B) Amino acid sequence of GRAB protein. The A region is in bold type and the ΔA region is underlined.

Figure 3. Immunoreactivity and binding of α₂M to recombinant GRAB derivatives

(A) Recombinant GRAB fusion proteins. (B) Coomassie Blue stain of the recombinant GRAB derivatives. (C–E) Immunoblot analysis of GRAB derivatives using (C) anti-GRAB IgG, (D) α₂M and (E) anti-α₂M IgG as the ligands respectively. Lane 1, rGRAB; lane 2, rGST-ΔA; lane 3, rGST-A; lane 4, rGST-AR1; lane 5, GST-AR2; lane 6, GST; and lane 7, α₂M. (F) Competitive inhibition of the binding of ¹²⁵I-α₂M to S. pyogenes isolates KTL3 and A6 by rGST-ΔA (♦) or rGST-A (▪).

Figure 4. Determination of putative α₂M-binding motifs in the A domain of GRAB

(A) The complete amino acid sequence of domain A. (B) Spot-membrane analysis for α₂M-binding activity of the 58 amino acids, divided into 44 overlapping peptides of 15 amino acids each, with an offset of one amino acid each. (C) Serial numbers and sequences of the spots detected positive for binding to α₂M.

Figure 5. Analysis of overlapping peptides of different sizes for α₂M-binding activity

(A, D) Amino acid sequences used for the synthesis of peptides. Binding motifs are underlined. (B) Spot membrane containing eight sets of overlapping peptides with mass ranging from 8 to 15 amino acids, using the initial sequence IIPNGGTL. (E) Spot membrane containing seven sets of synthetic overlapping peptides consisting of 9–15 amino acids each, using NLLGNAPE as the initial sequence. (C, F) Serial numbers and sequences of spots reacting with human α₂M.

Figure 6. Effects of the α₂M-binding motifs PRIIPNGGTL and NAPEKLALRN of GRAB using synthetic peptides containing single amino acid substitutions at every position

(A, C) Spot membranes with peptides analysed in an α₂M ligand binding assay. (B, D) Exchanged amino acids in the motif are underlined. Amino acid substitutions that abolished α₂M binding are indicated in boldface.

Figure 7. Effects of substitutions of the arginine residues in the α₂M-binding motifs PRIIPNGGTL and NAPEKLALRN on α₂M binding to recombinant GRAB proteins

(A) Sequences of the recombinant GRAB proteins with individual amino acid substitutions. (B) Coomassie Blue stain of the rGRAB proteins. (C) Immunoblot analysis with rabbit anti-rGRAB IgG. (D) Blot-overlay assay with human α₂M as the ligand. (E) Immunoblot analysis using rabbit anti-α₂M IgG alone as a control.

Figure 8. Competitive inhibition of the binding of ¹²⁵I-α₂M to the GAS isolate KTL3

GRAB derivatives were added in increasing concentrations to the α₂M-binding reaction. (A) Parent rGRAB, (D) rGRAB⁴² (substitution of Arg⁴²), (B) rGRAB⁶⁴ (substitution of Arg⁶⁴) and (C) rGRAB^42/64 (substitution of Arg⁴² and Arg⁶⁴).