A possible molecular mechanism for parasitic inhibition by lactoferrin

Transferrins (Tfs) belong to a family of iron-binding glycoproteins possessing similar aminoacid sequence though they have different biological functions and locations. Lactoferrin (Lf) is expressed and secreted from glandular epithelial cells and from mature neutrophiles of mammalian and it is an important component of the aspecific host defence or natural immunity, including resistance to parasitic infections. Serum transferrin (sTf) is synthesized by the liver of mammals and secreted into the blood stream; its primary function is iron transport. Ovotransferrin (Otrf), synthesized by avians, displays both iron transport and protective functions. Parasites synthesize papain-like cysteine proteases that are relevant for the virulence and pathogenicity of parasites, being involved in several aspects of the parasite life cycle, it is therefore possible that the antiparasitic activity of Lf could be due to the inhibition of parasitic papain-like cysteine protease that we have recently observed. In this study we have investigated the thermodynamic parameters of hLf, bLf and Otrf inhibition of the parasitic papain-like type I cysteine proteases from Leishmania infantum, Trypanosoma cruzi and Trypanosoma brucei. bLf, hLf and Otrf, both in the apo- and olo-forms, showed time- and concentration-dependent inhibition of the catalytic activity of papain and of type I proteases from L.infantum, T. cruzi and T. brucei. The KI values observed for bLf and hLf inhibition of L. infantum, T. cruzi and T. brucei proteases were in the nanomolar range (KI = 3.1 nM), lower than KI values observed for papain inhibition (KI = 24 nM). Otrf showed lower inhibition of cysteine proteases (KI = 0.6 µM). On the contrary, sTf did not display any inhibition towards parasitic proteases, according to its different role in mammals. The inhibition of parasitic cysteine proteases by hLf, bLf and Otrf appeared to conform to a competitive mechanism. The observed pH optimum for bLf inhibition of parasitic proteases was around neutrality, while it was acidic for hLf and alkaline for Otrf. The further quantitative analysis of pH dependence of the intrinsic ligand-independent inhibition constant KI allowed the evaluation of pKa values that define the acid-base equilibrium of amino acidic residue(s) modulating the enzyme(s)-inhibitor recognition events. SDS-PAGE showed that hLf, bLf and Otrf were easily degraded by either papain or parasitic type I protease during the assay incubation time (few minutes) and it is likely that one or more protease inhibitory peptides were generated from protein hydrolysis. As a matter of fact, a sequence present near the C-terminus of human (hLf) and bovine (bLf) lactoferrin shows homology with the sequence of the active site of cystatins, which are competitive inhibitors of papain-like cysteine proteases. The same sequence is present, though with lower homology, in Otrf and, with even lower homology, in sTf. Therefore, we have charactherized by MALDI-TOF the profile of Lf cleavage by papain and preliminary data suggest the presence of a cystatin-like peptide in two proteolytic fragments of hLf and in one proteolytic fragment of bLf.