DENATURATION AT INTERFACES AFFECTS IMMUNOREACTIVITY OF β-LACTOGLOBULIN AND ITS CELLULAR UPTAKE BY MONOCYTES

Bovine -lactoglobulin (β-LG) is the major protein in bovine whey, but it is not found in human milk. It belongs to the lipocalin protein family, and is one of the major milk allergens. At physiological pH BLG is a dimer consisting of two identical amino acid chains (162 amino acids) each having a molecular mass about 18 kDa. β-LG is an exceptionally acid-stable protein. At pH 2 it dissociates reversibly into monomers, but its structure remains native. Furthermore, native β-LG is almost totally resistant to pepsin degradation at low pH. Resistance to acid hydrolysis as well as to proteases allows some of the β-LG to remain intact after digestion. This increases the probability that intact β-LG as well as its digested fragments will be absorbed as antigens. The spontaneous adsorption of protein molecules on interfaces is an ubiquitous phenomenon in natural and man-made systems. The structural rearrangement caused by the direct contact with the sorbent phase may affect protein biological activity, including bioavailability, and ability to bind micro- and macromolecular ligands. Moreover, protein immunoreactivity has been assessed to change if protein molecules interact with an hydrophobic phase. The aim of this work is to understand whether and how the β-LG conformational changes derived from the interaction with a hydrophobic interfaces - that consist in our case in an oil-in-water nanoemulsion - can modulate its immunoreactivity and its absorption behavior by human cells involved in the immuno response Competitive ELISA measurements, carried out using specific monoclonal antibodies, prove that β-LG increase dramatically its immunoreactivity after interaction with the hydrophobic surface, giving the evidences that the protein, after the conformational rearrangement coming from the absorption, should be recognised in different way compared with the native one. The interfacial denaturation changes also the protein uptake behaviour by human monocytes. We carried out experiments using monocytes from a MonoMac6 cell line and a FITC-labeled β-LG. Uptake kinetics were recorded using flow cytometry and cell confocal microscopy pictures were taken. Results show that the native free protein is internalized by the cells, and a heat denaturation process seems to do not interferer on the internalizeted β-LG final amount and kinetics. Moreover the presence of 10 and 50 molar excess of unlabeled free protein seems to not influence the protein uptake. Surface denatured β-LG is internalizated by monocytes more efficiently and rapidity. The uptake of this protein form is influenced by the presence unlabeled free β-LG, as visible by a general slowing-down of the internalization rate. Furthermore the internalization of the FITC-labeled β-LG is not influenced by the presence of unlabeled protein staked on the oil droplets. All these results point the importance of the physical form of a potential allergen on its immunoreactivity response and uptake behaviour, as the fact that the natural form of this allergen in whole milk and dairy products is often in complex with a lipid matrix. Future studies will be aimed to study the behaviour of other cells from the immunosystem after the contact with β-LG adsorbed on this hydrophobic surface.