Lactoferrin (LF) is an iron-binding glycoprotein belonging to the transferrin family. LF is abundant in milk and is present also in other exocrine fluids (e.g. saliva, tears) and in the secondary granules of neutrophils. LF has a bilobal structure and a high isoelectric point. A cluster of positively-charged amino acids is located at its N-terminus, wherefrom the bioactive peptide lactoferricin (LFcin) is derived. Basic character of LF and its ability to bind iron mediate many beneficial biological properties, which have been attributed to LF – antimicrobial, antiviral and immunomodulatory activities.
Recently, LF has been shown to block activation of plasminogen (Plg), a zymogenic form of serine protease plasmin (Pm) [1]. Plg-Pm system plays an important role in many physiological processes, e.g. fibrinolysis. However, aberrant activation of Plg may aid invasion of tumour cells and certain bacteria [1, 2]. Therefore, inhibition of pathological Plg activation by LF can have a therapeutic potential. It has been shown by surface plasmon resonance (SPR) that holo-LF can bind directly to Plg with a high affinity; however, the interaction does not obey a simple 1:1 mechanism and needs a pre-activation of LF by acids [1]. For an effective exploitation of LF inhibitory activity on Plg activation it will be of major importance to elucidate the molecular structure of LF-Plg complex as well as the pathways of complex formation. These are the aims of our work.
As the apo-LF needs to be exposed to acidic pH before being able to bind Plg, and this treatment can alter its structure and induce oligomerization, we studied in details the conformation of LF in lowering pH by dynamic light scattering. We observed a reversible acidic expansion of LF with no direct implication on its dimerization. Further, we compared reactivity of various forms of LF with Plg by SPR.
The X-ray structures of LF and Plg are known, however, LF was until now crystallized only in conditions employing high concentration of propanol, which can influence the structural features. We have performed large crystallization screening of LF and have found crystallization conditions without alcohol. Finally, because lactoferricin peptide is suspected to mediate the interaction of LF and Plg [1], we have optimized preparation of LFcin by pepsine digestion of LF and purification by a two-step chromatography procedure, and tested the inhibitory activity of purified peptides on Plg activation.