Stability improvement of the fatty acid binding protein Sm14 from S. mansoni by Cys replacement: structural and functional characterization of a vaccine candidate.
The Schistosoma mansoni fatty acid binding protein (FABP), Sm14, is a vaccine candidate against, S. mansoni and F. hepatica. Previously, we demonstrated the importance of a correct fold to achieve protection in immunized animals after cercariae challenge [[10]. C.R.R. Ramos, R.C.R. Figueredo, T.A. Pertinhez, M.M. Vilar, A.L.T.O. Nascimento, M. Tendler, I. Raw, A. Spisni, P.L. Ho, Gene structure and M20T polymorphism of the Schistosoma mansoni Sm14 fatty acid-binding protein: structural, functional and immunoprotection analysis. J. Biol. Chem. 278 (2003) 12745-12751.]. Here we show that the reduction of vaccine efficacy over time is due to protein dimerization and subsequent aggregation. We produced the mutants Sm14-M20(C62S) and Sm14-M20(C62V) that, as expected, did not dimerize in SDS-PAGE. Molecular dynamics calculations and unfolding experiments highlighted a higher structural stability of these mutants with respect to the wild-type. In addition, we found that the mutated proteins, after thermal denaturation, refolded to their active native molecular architecture as proved by the recovery of the fatty acid binding ability. Sm14-M20(C62V) turned out to be the more stable form over time, providing the basis to determine the first 3D solution structure of a Sm14 protein in its apo-form. Overall, Sm14-M20(C62V) possesses an improved structural stability over time, an essential feature to preserve its immunization capability and, in experimentally immunized animals, it exhibits a protection effect against S. mansoni cercariae infections comparable to the one obtained with the wild-type protein. These facts indicate this protein as a good lead molecule for large-scale production and for developing an effective Sm14 based anti-helminthes vaccine.
Ramos
Celso R R
CR
Biotechnology Center, Butantan Institute, Av. Vital Brasil 1500, 05503-900, São Paulo, SP, Brazil.
Spisni
Alberto
A
Oyama
Sérgio
S
Sforça
Mauricio L
ML
Ramos
Henrique R
HR
Vilar
Mônica M
MM
Alves
Adriana C
AC
Figueredo
Rita C R
RC
Tendler
Míriam
M
Zanchin
Nilson I T
NI
Pertinhez
Thelma A
TA
Ho
Paulo Lee
PL
D000818:Animals; D003198:Computer Simulation; D050612:Fatty Acid Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D005260:Female; D015801:Helminth Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D051379:Mice; D008958:Models, Molecular; D009154:Mutation; D017510:Protein Folding; D055503:Protein Multimerization; D055550:Protein Stability; D012550:Schistosoma mansoni / Q000737:chemistry* / Q000235:genetics / Q000254:growth & development / Q000276:immunology; D012555:Schistosomiasis mansoni / Q000469:parasitology / Q000517:prevention & control; D014612:Vaccines / Q000008:administration & dosage / Q000737:chemistry
D050612:Fatty Acid Transport Proteins; D015801:Helminth Proteins; D014612:Vaccines; C068709:SM14 protein, Schistosoma mansoni