Biotecnologia Aplicada Elfos Scientiae ISSN: 0684-4551 Vol. 17, Num. 3, 2000, pp. 198

Biotecnología Aplicada, Volume 17, July-September 2000, p. 198

Primary and Secondary Structure in Antibody Recognition of a Core Protein Epitope from Equine Infectious Anemia Virus

Georgina Tonarelli,¹ Adriana Soutullo,¹ Javier Lottersberger,¹ M Noemi Santi,¹
Jorge L Salvetti,¹ Sergio Garay,¹ Leila M Beltramini²

¹Dpto.Química Orgánica de la Facultad de Bioquímica y Ciencias Biológicas. Universidad Nacional del Litoral. Ciudad Universitaria. Paraje El Pozo. C.C. 242- (3000). Santa Fe. Argentina. E-mail: tonareli@fbcb.unl.edu.ar
²Grupo Biofísica Molecular y Espectroscopía, Instituto de Física de São Carlos-USP, SP-Brasil.

From a selection of papers from Biotecnología Habana`99 Congress.
November 28-December 3, 1999.

Code Number: BA00062

Introduction

The major core protein of EIAV, p26, is one of the primary immunogenic structural proteins during persistent infection and is highly conserved among antigenically variant viral isolates. The detection of antibodies to p26 is the basis of the agar immunodiffusion test (AGID) [1].

The present work was started to determine minimal size epitopes within the C-terminal part of p26. We report our findings about the effect of conformation as an essential requirement for antibody recognition using elongated cycled peptides containing an identified epitope.

Materials and Methods

Peptide synthesis by the Spot Method

Overlapping hexapeptides spanning the C-terminal region of p26, region 277-359 were synthesized on cellulose membranes chemically derivatized with spots of bAla-bAla anchor for the preparation of immobilized peptides. Assembly of the peptides was carried out using Fmoc-chemistry essentially as described [2].

Antibody-binding assay

lt was done with Alkaline Phosphatase (AP)-conjugated as secondary antibody, and the colour reaction was developed by a solution containing BCIP and MTT.

Manual peptide synthesis

A sequence from region 319-346 of EIAV core protein named as p26-1, was manually synthesized on solid phase using Fmoc chemical strategies, purified by semipreparative reverse-phase HPLC (Gilson System) and characterized by FAB-EM.

The thiol groups oxidation was carried out with iodine. The completion of the reaction was monitored by Ellman test.

Antibody binding to the linear and cyclic peptide

lt was identified by an indirect enzyme-linked immunosorbent, assay (ELISA).

Circular Dichroism (CD) measurements

CD spectra were recorded in a Jasco J-720 spectropolarimeter over the wavelength range of 195-250 nm. Measurements were made on peptide samples in the concentration range of 0.3-0.5 mg/mL in 1 mm path length quartz cuvettes. CD measurements were made at 20 ºC, in deionized water.

Results and Discussion

Analysing the series of overlapping hexapeptides recognized by a pool of polyclonal sera of EIAV infected horses, several reactive sequences containing the common epitope MYACRD were identified.

An elongated peptide containing the native sequence from residues 319-346 named as p26-1 (ANEECR NANRHLRPEDTLEEKMYACRDIG) was prepared.

The CD spectrum of p26-1 linear and cyclic showed two minima next to 207 and 223 nm, but presented a different intensity between them. The following secondary structure fractions were calculated from SelCon Program: -p26-1 linear: 9% helix components, 41% b sheet components, 17% turn and 31% unordered; -p26-1 cyclic: 100% helix [3].

A significant conformational change was observed when the peptide was cycled by a bridge between Cys323 and Cys343 that correlated with an improved ability of the cyclic peptide to recognize antibodies in the ELISA test. These results suggest that the conformationally restricted peptide, adequately mimic the native structure of this portion of p26 core protein.

References

Copyright Elfos Scientiae 2000