Isolated Hypervariable Regions Derived from Streptococcal M Proteins Specifically Bind Human C4b-Binding Protein: Implications for Antigenic Variation

Abstract

Antigenic variation in microbial surface proteins represents an apparent paradox, because the variable region must retain an important function, while exhibiting extensive immunological variability. We studied this problem for a group of streptococcal M proteins in which the ∼50-residue hypervariable regions (HVRs) show essentially no residue identity but nevertheless bind the same ligand, the human complement regulator C4b-binding protein (C4BP). Synthetic peptides derived from different HVRs were found to retain the ability to bind C4BP, implying that the HVR corresponds to a distinct ligand-binding domain that can be studied in isolated form. This finding allowed direct characterization of the ligand-binding properties of isolated HVRs and permitted comparisons between different HVRs in the absence of conserved parts of the M proteins. Affinity chromatography of human serum on immobilized peptides showed that they bound C4BP with high specificity and inhibition experiments indicated that different peptides bound to the same site in C4BP. Different C4BP-binding peptides did not exhibit any immunological cross-reactivity, but structural analysis suggested that they have similar folds. These data show that the HVR of streptococcal M protein can exhibit extreme variability in sequence and immunological properties while retaining a highly specific ligand-binding function.

Human C4b-binding Protein, Structural Basis for Interaction with Streptococcal M Protein, a Major Bacterial Virulence Factor

“…1D) to measure the binding affinity between C4BP12 and a dimerised peptide corresponding to the hypervariable region of M4 (residues 1-45 of M4 protein with a C-terminal Cys added. A dimer (M4-N) was created because previous studies had shown dimerization of M4 strongly enhanced C4BP binding (32). The results are consistent with a 1:1 stoichiometry, and K d ϭ 0.5 M. Taken together, the data summarized in Fig.…”

“…1 prove that the N-terminal two CCP modules of C4BP ␣-chain along with the four residues that link them are necessary and sufficient for M4 recognition. The data confirm that M4-N constitutes a useful model of the C4BP-binding region of the bacterial protein (32). The Structure of C4BP12 Has Been Determined-To further characterize the M4-binding site of C4BP, the structure of C4BP12 was solved using NMR.…”

Host–pathogen interactions in Streptococcus pyogenes infections, with special reference to puerperal fever and a comment on vaccine development

“…A clue to this problem was obtained through the finding that many HVRs specifically bind the same ligand, in spite of the extreme sequence variability between different HVRs. The bound ligand is human C4b-binding protein (C4BP) [8,9], a ~570 kDa plasma protein that inhibits activation of the classical pathway of complement activation [10,11]. More than 50% of all S. pyogenes strains bind C4BP [12], and all available evidence indicates that C4BP binds to the HVR of the M protein expressed by these strains [8,9].…”

“…The bound ligand is human C4b-binding protein (C4BP) [8,9], a ~570 kDa plasma protein that inhibits activation of the classical pathway of complement activation [10,11]. More than 50% of all S. pyogenes strains bind C4BP [12], and all available evidence indicates that C4BP binds to the HVR of the M protein expressed by these strains [8,9]. It is important to note, however, that many M proteins do not bind C4BP, and the exact molecular function of the HVR in these M proteins remains unknown.…”

“…Thus, the key property of a protective antibody may be its ability to inhibit binding of ligand (s) readily be studied in soluble form and different ligand-binding regions can be studied as synthetic peptides [9,18]. The evolution of extreme sequence divergence in the Nterminal region of M proteins is of considerable interest, because the variability appears to be at least as great as that observed in HIV-1 and influenza virus [9].…”