Dataset Information

Experimental and bioinformatic approach to identifying antigenic epitopes in human ?- and ?-enolases.

ABSTRACT: Human ?- and ?-enolases are highly homologous enzymes, difficult to differentiate immunologically. In this work, we describe production, purification and properties of anti-?- and anti-?-enolase polyclonal antibodies. To raise antibodies, rabbits were injected with enolase isoenzymes that were purified from human kidney (?-enolase) and skeletal muscle (?-enolase). Selective anti-?- and anti-?-enolase antibodies were obtained by affinity chromatography on either ?- or ?-enolase-Sepharose columns. On Western blots, antibodies directed against human ?-enolase, did not react with human ?-isoenzyme, but recognized pig and rat ?-enolase. To determine what makes these antibodies selective bioinformatic tools were used to predict conformational epitopes for both enolase isoenzymes. Three predicted epitopes were mapped to the same regions in both ?- and ?-enolase. Peptides corresponding to predicted epitopes were synthesized and tested against purified antibodies. One of the pin-attached peptides representing ?-enolase epitope (the C-terminal portion of the epitope 3 - S262PDDPSRYISPDQ273) reacted with anti-?-enolase, while the other also derived from the ?-enolase sequence (epitope 2 - N193VIKEKYGKDATN205) was recognized by anti-?-enolase antibodies. Interestingly, neither anti-?- nor anti-?-antibody reacted with a peptide corresponding to the epitope 2 in ?-enolase (G194VIKAKYGKDATN206). Further analysis showed that substitution of E197 with A in ?-enolase epitope 2 peptide lead to 70% loss of immunological activity, while replacement of A198 with E in peptide representing ?-enolase epitope 2, caused 67% increase in immunological activity. Our results suggest that E197 is essential for preserving immunologically active conformation in epitope 2 peptidic homolog, while it is not crucial for this epitope's antigenic activity in native ?-enolase.

SUBMITTER: Pietkiewicz J

PROVIDER: S-EPMC6005794 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Experimental and bioinformatic approach to identifying antigenic epitopes in human α- and β-enolases.

Pietkiewicz Jadwiga J Danielewicz Regina R Bednarz-Misa Iwona S IS Ceremuga Ireneusz I Wiśniewski Jerzy J Mierzchala-Pasierb Magdalena M Bronowicka-Szydełko Agnieszka A Ziomek Edmund E Gamian Andrzej A

Biochemistry and biophysics reports 20180617

Human α- and β-enolases are highly homologous enzymes, difficult to differentiate immunologically. In this work, we describe production, purification and properties of anti-α- and anti-β-enolase polyclonal antibodies. To raise antibodies, rabbits were injected with enolase isoenzymes that were purified from human kidney (α-enolase) and skeletal muscle (β-enolase). Selective anti-α- and anti-β-enolase antibodies were obtained by affinity chromatography on either α- or β-enolase-Sepharose columns. ...[more]

PMID: 29922723

Similar Datasets

Project description:UnlabelledHuman bocaviruses (HBoV1 to -4) are emerging pathogens associated with pneumonia and/or diarrhea in young children. Currently, there is no treatment or vaccination, so there is a need to study these pathogens to understand their disease mechanisms on a molecular and structural level for the development of control strategies. Here, we report the structures of six HBoV monoclonal antibody (MAb) fragment complexes, HBoV1-15C6, HBoV2-15C6, HBoV4-15C6, HBoV1-4C2, HBoV1-9G12, and HBoV1-12C1, determined by cryo-electron microscopy and three-dimensional image reconstruction to 18.0- to 8.5-Å resolution. Of these, the 15C6 MAb cross-reacted with HBoV1, HBoV2, and HBoV4, while the 4C2, 12C1, and 9G12 MAbs recognized only HBoV1. Pseudoatomic modeling mapped the 15C6 footprint to the capsid surface DE and HI loops, at the 5-fold axis and the depression surrounding it, respectively, which are conserved motifs in Parvoviridae The footprints for 4C2, 12C1, and 9G12 span the surface loops that assemble portions of the 2-/5-fold wall (a raised surface feature between the 2-fold and 5-fold axes of symmetry) and the shoulder of the 3-fold protrusions. The MAb footprints, cross reactive and strain specific, coincide with regions with high and low sequence/structural identities, respectively, on the capsid surfaces of the HBoVs and identify potential regions for the development of peptide vaccines for these viruses.ImportanceHuman bocaviruses (HBoVs) may cause severe respiratory and gastrointestinal infections in young children. The nonenveloped parvovirus capsid carries determinants of host and tissue tropism, pathogenicity, genome packaging, assembly, and antigenicity important for virus infection. This information is currently unavailable for the HBoVs and other bocaparvoviruses. This study identifies three strain-specific antigenic epitopes on the HBoV1 capsid and a cross-reactive epitope on the HBoV1, HBoV2, and HBoV4 capsids using structures of capsid-antibody complexes determined using cryo-electron microscopy and image reconstruction. This is the first study to report the highly conserved parvovirus DE loop at the 5-fold axis as a determinant of antigenicity. Additionally, knowledge of the strain-specific and conserved antigenic epitopes of the bocaviruses can be instrumental in characterization of the virus life cycle, development of peptide vaccines, and generation of gene delivery vectors for cystic fibrosis given the strict tropism of HBoV1 for human airway epithelial cells.

Dataset Information

Experimental and bioinformatic approach to identifying antigenic epitopes in human ?- and ?-enolases.

Publications

Experimental and bioinformatic approach to identifying antigenic epitopes in human α- and β-enolases.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets