STRUCTURAL STABILITY OF THE ANTIVIRAL PROTEIN LACTOFERRIN
2
Sanches, D.,2Souza, T. L. F.,1Denani, C. B.,1Santos, R., 1Battistela, K. F.,2Silva, J. L.,2Oliveira, A. C. and
1
Gonçalves, R. B.
1
Departamento de Bioquímica, Instituto Biomédico, Universidade Federal do Estado do Rio de Janeiro,
20211-040, Rio de Janeiro, RJ, Brazil.
2
Programa de Biologia Estrutural, Instituto de Bioquímica Médica, Universidade Federal do Rio de
Janeiro, 21941-590, Rio de Janeiro, RJ, Brazil.
Bovine lactoferrin (bLf) has emerged as the most important whey protein due its several functions
described and its potential applications against several viruses like Influenza, HIV, YFV and others. bLf is
present in a broad range of corporal fluids like blood, saliva and milk and has several antimicrobial and
physiological properties. The three-dimensional structure of bLf was determined and showed that it is
comprised of a single polypeptide chain of 76 kDa divided into two lobes (C and N), linked by an alpha
helix, with two domains in each lobe (C1/C2 and N1/N2). Once the high pressure has been widely used both
for the study of protein and oligomers as in the food industry, we decided to investigate these effects on the
structure of bLf. For this goal we used high hydrostatic pressure, combined with low temperature and
chemical denaturants, to verify the effects of these approaches to promote denaturation of bLf.
Conformational and stability changes were monitored using spectroscopic techniques such as differential
scanning calorimetry (DSC) and fluorescence spectroscopy following intrinsic (tryptophan) fluorescence.
Our results shown that pressure is able to cause around 40% denaturation comparing with the effects of urea.
On the other hand, this process seems to be time dependent and the low temperature was not able to cause
significant changes on tryptophan environment. Sub-denaturing urea concentrations has been a synergic
effect when coupled to high pressure. We believe that this study is important to understand the effects of
pressure on bLf structure and functionality because its crescent applications as a bioactive component in
several products.
Lactoferrina bovina (bLF) tem emergido como a proteína de soro de leite mais importante
devido as suas várias funções descritas. A bLf é uma proteína ligadora de íons ferro e está
presente em uma ampla variedade de fluidos corporais como sangue, saliva, leite e tem
várias propriedades antimicrobianas e fisiológicas. A estrutura tridimensional é composta por
uma única cadeia de polipeptídica de 76 kDa dividido em dois lóbulos (C e N), ligados por
uma hélice alfa, com dois domínios em cada lóbulo (C1/C2 e N1/N2). Estudos prévios com a
lactoferrina humana mostraram que a ligação de ferro leva a mudanças conformacionais na
proteína. Apesar da importância da lactoferrina bovina, existem poucos trabalhos que
demonstrem as diferenças de estabilidade estrutural entre as formas ligada (holo) e não
ligada a ferro (apo). Uma vez que essas mudanças podem afetar a funcionalidade da
lactoferrina, decidiu-se investigar as diferenças na estabilidade das formas apo e holo e, para
tanto, utilizamos a ureia e a alta pressão hidrostática para causar perturbações na estrutura da
bLf e avaliar o efeito protetor deste íon. As mudanças nas estruturas terciária e secundária da
lactoferrina foram monitoradas por técnicas espectroscópicas, como a espectroscopia de
fluorescência e o dicroísmo circular. Nossos resultados mostram claramente que o ferro
estabiliza fortemente a estrutura terciária da bLf bem como preserva a estrutura secundária
da proteína. Além disso, foi possível correlacionar a ligação do ferro na interação da bLf
com células Vero. Observamos que a cinética de internalização da holo-bLf é mais rápida
quando comparada a forma apo. Isto demonstra que a presença de íons ferro favorece a
interação da bLf com a célula. Apesar destes resultados, algumas questões ainda precisam
ser melhor endereçadas principalmente no que diz respeito ao aprofundamento do estudo da
interação da bLf com a célula.
Bovine lactoferrin (bLF) has emerged as the most important whey protein due to its various functions
described. The bLf is an iron binding protein and it is present in a wide variety of body fluids such as blood,
saliva, milk and has several antimicrobial and physiological properties. The three-dimensional structure is
composed of a single chain polypeptide of 76 kDa divided into two lobes (C and N), connected by an alphahelix, with two domains in each lobe (C1/C2 and N1/N2). Previous studies with human lactoferrin showed
that iron-binding leads to conformational changes in the protein.
Despite the importance of bovine lactoferrin, few studies demonstrate the differences in structural stability
between the iron-saturated lactoferrin (holo) and lactoferrin in its iron free state (apo). Since these changes
can affect the functionality of lactoferrin, it was decided to investigate the differences in the stability of apo
and holo forms and, therefore, it was used urea and high hydrostatic pressure to cause disturbances in bLf
structure and evaluate the protective effect this ion. Changes in secondary and tertiary structures of
lactoferrin were monitored by spectroscopic techniques such as fluorescence spectroscopy and circular
dichroism.
Our results clearly show that the iron strongly stabilizes the tertiary structure and preserves the secondary
structure of the protein. Moreover, it was possible to correlate the bLf iron binding interaction with Vero
cells. We observed that the kinetics of internalization of holo-bLf is faster, compared to apo form. It
demonstrates that the presence of iron ions promotes the interaction of the cell with the bLf. Despite these
results, a few issues need to be better addressed primarily with respect to the depth study of the interaction
of the cell with the bLf.
Suported by: FAPERJ, CNPQ and CAPES.
Key word: bovine lactoferrin, iron binding, stability.