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Delineation of the third antigenic site of lysozyme by application of a novel 'surface-simulation' synthetic approach directly linking the conformationally adjacent residues forming the site.

ABSTRACT: We have previously shown that an antigenic site in native lysozyme resides around the disulphide bridge 30-115 and incorporates Lys-33 and Lys-116 and one or both of Tyr-20 and Tyr-23. These residues fall in an imaginary line circumscribing part of the surface of the molecule and passing through the spatially adjacent residues Tyr-20, Arg-21, Tyr-23, Lys-116, Asn-113, Arg-114, Phe-34 and Lys-33. The identity of the site was confirmed by demonstrating that the synthetic peptide Tyr-Arg-Tyr-Gly-Lys-Asn-Arg-Gly-Phe-Lys (which does not exist in lysozyme but simulates a surface region of it), and an analogue in which glycine replaced Tyr-23, possessed remarkable immuno-chemical reactivity that accounted entirely for the expected reactivity of the site in native lysozyme. Tyr-23 is not part of the site, and its contribution was satisfied by a glycine spacer. The novel approach presents a powerful technique for the delineation of antigenic (and other binding) sites in native proteins and confirms that these need not always comprise residues in direct peptide linkage.

SUBMITTER: Lee CL

PROVIDER: S-EPMC1164041 | biostudies-other | 1976 Oct

REPOSITORIES: biostudies-other

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Project description:1. Previous reports from this laboratory have shown that both Lys-33 and Lys-116 are parts of an antigenic site in native lysozyme. Similar studies of tyrosine derivatives indicated that one or both of Tyr-20 and Tyr-23 are located in or very close to an antigenic site in lysozyme. The site, which was located around the disulphide bridge 30-115, was recently shown unequivocally to include the residues Tyr-20, Arg-21, Lys-116, Asn-113, Arg-114, Phe-34 and Lys-33. This was confirmed by the ;surface-simulation' synthetic approach that we have recently developed, in which the foregoing eight surface residues were directly linked via peptide bonds, with intervening spacers where appropriate, into a single peptide. The peptide does not exist in native lysozyme, but simulates a surface region of it. 2. In the present work several surface-simulation peptides were synthesized representing various parts of the region, to determine the minimum structural feature that retains full antigenic reactivity and to investigate if the spatially constructed antigenic site has a preferred direction. 3. The peptide Lys-Asn-Arg-Gly-Phe-Lys exhibited a remarkable inhibitory activity towards the immune reaction of lysozyme and accounted entirely for the maximum expected reactivity of the site in the native protein (i.e. about one-third of the total lysozyme reactivity). An immunoadsorbent of the peptide bound about one-third of the total antibody to lysozyme. 4. The residues Tyr-20 and Arg-21 are not part of the site. The previously reported immunochemical effect observed on nitration of Tyr-20 was due to a deleterious ionic effect exerted by the modified tyrosine residue on the adjacent Lys-96, which is in an entirely different antigenic site of lysozyme. Thus the modification of Tyr-20 impairs the reactivity of an adjacent antigenic site, even though the residue itself is not part of a site. The conformational and immunochemical implications of this finding are discussed. 5. The antigenic site therefore comprises the five spatially adjacent residues Lys-116, Asn-113, Arg-114, Phe-34, Lys-33. The antigenic site exhibited a preferred direction (Lys-116 to Lys-33), since the reverse surface-simulation synthetic sequence was immunochemically inefficient. The site describes a line which circumscribes part [2.1nm in C((alpha))-C((alpha)) distance from Lys-116 to Lys-33] of the surface of the molecule.

Project description:UNLABELLED:Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype are genetically highly variable and have diversified into multiple phylogenetic clades over the past decade. Antigenic drift is a well-studied phenomenon for seasonal human influenza viruses, but much less is known about the antigenic evolution of HPAI H5N1 viruses that circulate in poultry. In this study, we focused on HPAI H5N1 viruses that are enzootic to Indonesia. We selected representative viruses from genetically distinct lineages that are currently circulating and determined their antigenic properties by hemagglutination inhibition assays. At least six antigenic variants have circulated between 2003, when H5N1 clade 2.1 viruses were first detected in Indonesia, and 2011. During this period, multiple antigenic variants cocirculated in the same geographic regions. Mutant viruses were constructed by site-directed mutagenesis to represent each of the circulating antigenic variants, revealing that antigenic differences between clade 2.1 viruses were due to only one or very few amino acid substitutions immediately adjacent to the receptor binding site. Antigenic variants of H5N1 virus evaded recognition by both ferret and chicken antibodies. The molecular basis for antigenic change in clade 2.1 viruses closely resembled that of seasonal human influenza viruses, indicating that the hemagglutinin of influenza viruses from different hosts and subtypes may be similarly restricted to evade antibody recognition. IMPORTANCE:Highly pathogenic avian influenza (HPAI) H5N1 viruses are responsible for severe outbreaks in both commercial and backyard poultry, causing considerable economic losses and regular zoonotic transmissions to humans. Vaccination is used increasingly to reduce the burden of HPAI H5N1 virus in poultry. Influenza viruses can escape from recognition by antibodies induced upon vaccination or infection through genetic changes in the hemagglutinin protein. The evolutionary patterns and molecular basis of antigenic change in HPAI H5N1 viruses are poorly understood, hampering formulation of optimal vaccination strategies. We have shown here that HPAI H5N1 viruses in Indonesia diversified into multiple antigenic variants, that antigenic differences were due to one or a very few substitutions near the receptor binding site, and that the molecular basis for antigenic change was remarkably similar to that for seasonal human influenza viruses. These findings have consequences for future vaccination and surveillance considerations and contribute to the understanding of the antigenic evolution of influenza viruses.

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Delineation of the third antigenic site of lysozyme by application of a novel 'surface-simulation' synthetic approach directly linking the conformationally adjacent residues forming the site.

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