title: Lack of immunological cross-reactivity between parasite-derived and recombinant forms of ES-62, a secreted protein of Acanthocheilonema viteae.
abstract: The longevity of filarial nematodes is dependent on secreted immunomodulatory products. Previous investigation of one such product, ES-62, has suggested a critical role for post-translationally attached phosphorylcholine (PC) moieties. In order to further investigate this, ES-62 lacking PC was produced, using the Pichia pastoris recombinant gene expression system. Unlike parasite-derived ES-62, which is tetrameric the recombinant material was found to consist of a mixture of apparently stable tetramers, dimers and monomers. Nevertheless, the recombinant protein was considered to be an adequate PC-free ES-62 as it was recognized by existing antisera against the parasite-derived protein. However, subsequent to this, recognition of parasite-derived ES-62 by antibodies produced against the recombinant protein was found to be absent. In an attempt to explain this, recombinant ES-62 was subjected to structural analysis and was found to (i) contain 3 changes in amino acid composition; (ii) demonstrate significant alterations in glycosylation; (iii) show major differences in protein secondary structure. The effects of these alterations in relation to the observed change in immunogenicity were investigated and are discussed. The data presented clearly show that recognition by existing antibodies is insufficient proof that recombinant proteins can be used to mimic parasite-derived material in studies on nematode immunology and vaccination.
authors.affiliation: Department of Immunology, University of Strathclyde, Glasgow G4 0NR, UK.
authors.forename: C A
authors.initials: CA
authors.lastname: Egan
authors.forename: K M
authors.initials: KM
authors.lastname: Houston
authors.forename: M J C
authors.initials: MJ
authors.lastname: Alcocer
authors.forename: A
authors.initials: A
authors.lastname: Solovyova
authors.forename: R
authors.initials: R
authors.lastname: Tate
authors.forename: G
authors.initials: G
authors.lastname: Lochnit
authors.forename: I B
authors.initials: IB
authors.lastname: McInnes
authors.forename: M M
authors.initials: MM
authors.lastname: Harnett
authors.forename: R
authors.initials: R
authors.lastname: Geyer
authors.forename: O
authors.initials: O
authors.lastname: Byron
authors.forename: W
authors.initials: W
authors.lastname: Harnett
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism / Q000379:methods; D003429:Cross Reactions; D004153:Dipetalonema / Q000235:genetics / Q000276:immunology* / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006031:Glycosylation; D015801:Helminth Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D007074:Immunoglobulin G / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D016297:Mutagenesis, Site-Directed / Q000379:methods; D010767:Phosphorylcholine / Q000737:chemistry / Q000378:metabolism; D010843:Pichia / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D017433:Protein Structure, Secondary / Q000502:physiology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology*; D013997:Time Factors; D014461:Ultracentrifugation / Q000379:methods
chemical_list: C435884:ES-62 protein, Acanthocheilonema viteae; D015801:Helminth Proteins; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D010767:Phosphorylcholine