Site-directed mutations in the regions of the active site and the interface oligomeric analysis of the Macrophage Migration Inhibitory Factor of Leishmania major (LmMIF2)

Author(s)

Elise Marques Freire Cunha

• Document type: Master's Dissertation
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto (PCARP/BC)
• Defense date: 2011-08-05

Examining board members

Arthur Henrique Cavalcante de Oliveira; Mario Tyago Murakami; Richard John Ward

• Advisor: Arthur Henrique Cavalcante de Oliveira

Abstract

The macrophage migration inhibitory factor (MIF) is considered an important factor in the control of infections caused by pathogens. This protein was first identified in mammals but orthologues have also been found in pathogens and it has been suggested that the protein a modulates the function of the host immune system. The structural and functional characterization from the MIF of Leishmania major may therefore contribute to the understanding of the function of this protein in the host/parasite relationship. This study aimed to use site-directed mutagenesis in the macrophage migration inhibitory factor of L. major (LmMIF2) to investigate the protein structure and function. Site-directed mutagenesis was performed by PCR and the mutants were cloned into the pET21b vector using NdeI/HindIII restriction sites. The recombinant LmMIF2 (rLmMIF2) and P2G, K34E, W66L, W108F and Δ104-113 mutants, all containing a His6-tag, were efficiently expressed in soluble form in E. coli and, subsequently purified from cell lysate by affinity chromatography. A single protein band of 14.5kDa was observed in SDS-PAGE, and gel filtration analysis showed rLmMIF and mutants to be dimeric proteins. Circular dichroism (CD) and intrinsic fluorescence emission of tryptophan (IFTE) were performed to monitor pH-dependent changes in secondary and tertiary structure of these proteins. Far-UV and near-UV-CD analysis showed that the exchange of one amino acid can influence the secondary and tertiary structures of rLmMIF2 and that the mutations did not change the molten globule state formation at low pH. IFTE experiments showed that there is an intrinsic fluorescence quenching that is abolished by the W108 substitution. Compared with the rLmMIF2, the W66L mutant showed a decrease in fluorescence intensity of about 60%, whereas the W108F mutant presented a fluorescence increase of about 25%. Macrophage migration inhibition tests showed that the P2G mutant presented a similar inhibitory activity to the rLmMIF2, while the other mutants showed a decrease in activity of about 50%. These results contribute to elucidate the role of the regions involved in maintaining LmMIF2 structure and its conformational changes, which may be involved in the host immune response modulation. (AU)

• FAPESP's process: 09/04272-0 - Site-directed Mutations in the Active-Site Oligomeric Interface Regions of the macrophage migration inhibitory factor of Leishmania major (LmjMIF2)
• Grantee: Elise Marques Freire Cunha
• Support Opportunities: Scholarships in Brazil - Master