The binding of synthetic retinoids to lipocalin beta-lactoglobulins.

The binding of therapeutically relevant synthetic retinoid derivatives to bovine and reindeer beta-lactoglobulin (betaLG) is demonstrated using fluorescence quenching and ultrafiltration/HPLC methods. Furthermore, synthesis of methyl (E)-3-[4-[(E)-2-(2,6,6-trimethylcyclohex-1-enyl)vinyl]phenyl]-acrylate 4 and (E)-3-[4-[(E)-2-(2,6,6-trimethylcyclohex-1-enyl)vinyl]phenyl]acrylic acid 5 is described. All studied compounds bind to both betaLG homologues with nanomolar K(d) values, and the interaction diminishes the pH-dependent aggregation of retinoids. Thus, betaLG may show benefits in improving the bioavailability of retinoid derivatives.

Binding of phenolic compounds and their derivatives to bovine and reindeer beta-lactoglobulin.

In plant-based food, phenolic compounds usually do not exist in their native form, but as esters, glycosides, or polymers. The native forms, however, require deglycosylation for their intestinal absorption, and aglycone has been considered to be the potential health-protecting/promoting form. The binding of the aglycones of phenolic compounds to bovine and reindeer beta-lactoglobulins (betaLG) using fluorescence quenching was studied. The effects of pH and storage were also studied. Of the compounds investigated, the majority of flavones, flavonols, flavanones, and isoflavones were bound to betaLG. In the pH studies, no significant effects were found. The fact that the phenolic compounds were not released at pH 2 might indicate that they bind to an external part rather than to the central cavity. Studies implicated that betaLG could act as a binder or carrier for phenolic compounds in acidic, basic, or neutral conditions and that the ligand/betaLG complex can remain stable during storage.

Transport properties of bovine and reindeer beta-lactoglobulin in the Caco-2 cell model.

Beta-lactoglobulin (betaLG) is a protein that binds ligands like fatty acids and retinol into the hydrophobic pocket. Our purpose was to study bovine and reindeer betaLG as transporter molecules and compare their transport properties across Caco-2 cell membrane. The reindeer betaLG has more valuable binder characteristics than bovine betaLG because it has only one genetic phenotype and it seems to exhibit better immunological properties. The permeation of betaLG in Caco-2 cells was evaluated by immunoblotting, and the permeation of the model substances retinol, palmitic acid and cholesterol with and without betaLG was determined using [(3)H]-labelled ligands. Both bovine and reindeer betaLG were able to pass across a Caco-2 cell monolayer similarly. Unbound and betaLG-bound [(3)H]retinol and [(3)H]palmitic acid were equally transported across the Caco-2 cell layer, whereas [(3)H]cholesterol could not pass across Caco-2 cells with or without betaLG at any of the studied circumstances. Thus, the bovine and reindeer milk betaLG is not a suitable protein to enhance transport of ligands across the Caco-2 cell membrane, used for predicting intestinal absorption.

Reindeer beta-lactoglobulin crystal structure with pseudo-body-centred noncrystallographic symmetry.

Reindeer beta-lactoglobulin (betaLG) belongs to the lipocalin superfamily. Its DNA and protein sequences have been determined and showed that it had nine residue changes from bovine betaLG. Reindeer betaLG, the structure of which was finally determined at 2.1 A resolution in space group P1, crystallized in a unit cell that is both P2-like and P2(1)-like owing to the presence of an almost perfect (but noncrystallographic) body-centring vector. The non-body-centred data could only be observed using a very bright synchrotron beam and a novel refinement strategy was adopted to enable us to use the weak h + k + l = 2n + 1 reflections.

Microplate screening assay for binding of ligands to bovine or reindeer beta-lactoglobulins.

Several analytical methods have been used to determine whether ligands bind to bovine beta-lactoglobulin (betaLG). The most common methods are based on fluorescence quenching. We have miniaturised this method from a quartz cell to a 96-well plate. The miniaturisation was evaluated using retinol. The binding constants between the two methods demonstrated a good correlation. The 96-well plate method is much faster and allows many references to be used in the same analysis. The miniaturised method was used to study the binding of three different ligands (4-HPR, arotinoid, warfarinyl palmitate) modelled to bind to betaLG. The binding data showed that all of these ligands bound to betaLG. The method was further used to demonstrate that reindeer betaLG could also bind the four ligands in the same way as bovine betaLG. Because one aim is to use bovine and reindeer betaLG as a binder molecule for aliments in e.g. functional food or for drugs, the influence of pH was also studied and demonstrated that short-term acidic conditions had only a slight effect on the binding properties.

Enterocyte and M-cell transport of native and heat-denatured bovine beta-lactoglobulin: significance of heat denaturation.

The three-dimensional structure, digestibility, and immunological properties of bovine beta-lactoglobulin (beta-lg) are modified by heat treatments used in processing of liquid milk products. Because it is not known if such treatments also modify the intestinal transport properties of beta-lg, the transport of native and heat-denatured bovine beta-lg was investigated in experimental cell models using Caco-2 cells and M cells. Transport of beta-lg labeled with a fluorescent marker was followed with fluorometric measurements, electrophoretic analyses, and fluorescence microscopy. The data show that both cell types transported native beta-lg more efficiently than they did heat-denatured beta-lg. In addition, M cells transported native beta-lg more than Caco-2 cells. Transport of native and heat-denatured beta-lg was transcellular. The electrophoretic data also suggest that heat-denatured beta-lg may have degraded more than native beta-lg during the transport.

BCG vaccine modulates intestinal and systemic response to beta-lactoglobulin.

Beta-Lactoglobulin (BLG) is a clinically important antigen in cow's milk and one of the major allergens causing cow's milk allergy. Bacillus Calmette-Guerin (BCG) vaccination has been suggested to modify immune response possibly decreasing the risk of allergy to some antigens in both human and experimental animals. In the present study, we have analyzed whether the early BCG vaccination has any effect on the markers of systemic and gastrointestinal (GI) sensitization to BLG. We immunized two groups of Hooded-Lister rat puppets with intraperitoneal injections of native BLG at 43 and 62 days with pertussis vaccine as adjuvant, one group receiving additionally BCG. The animals were then fed native and denatured milk products twice weekly from 73 to 131 days of age, when they were killed. Control group was not vaccinated and received normal rat forage. Total immunoglobulin E (IgE) levels and BLG-specific IgG(1) and IgG(2a) concentrations were determined in serum samples. Spontaneous interleukin (IL)-4 and interferon (IFN)-gamma production from duodenal specimens were measured, and the inflammatory cells were quantitated in specimens from different sections of the GI tract. Administration of BCG simultaneously with BLG resulted in reduced IgE concentration in serum, while the specific IgG(1) and IgG(2a) antibody responses and the spontaneous secretion of IL-4 and IFN-gamma were not affected. Furthermore, BCG-induced eosinophilic infiltration and increase of intraepithelial lymphocytes (IEL) in the GI mucosa, and a trend toward increased number of lamina propria mononuclear inflammatory cells in the colon (BCG compared with BLG, p = 0.09; BCG compared with controls, p = 0.02). Controls showed increment of IgG(1) response in comparison with the BLG group (p = 0.04) and increase of mucosal eosinophilic infiltration. The BCG modified the response to BLG both at the systemic level as shown by decrease of total IgE and at GI mucosa where increase of eosinophilic infiltration and increased number of IEL were seen. Increment of IgG(1) level and eosinophils in the controls might be related with the lack of modulatory effect of pertussis vaccination. A shift of response toward the lower GI tract after BCG immunization as shown by a trend for increase of mononuclear inflammatory cells in colon lamina propria mimics disease development in some cases of clinical food allergy, and emphasizes the need for evaluation of the changes in the whole GI tract in food allergy models.

Effect of heat denaturation on beta-lactoglobulin-induced gastrointestinal sensitization in rats: denatured betaLG induces a more intensive local immunologic response than native betaLG.

Beta-lactoglobulin (betaLG) is one of the first foreign antigens encountered by a newborn child, and it is the major allergen causing cow's milk allergy. Heat denaturation causes changes to the protein structure, but the significance of heat-induced changes for immunogenicity or allergenicity is not known. To clarify how heat denaturation affects allergenicity and immunogenicity, we immunized Hooded-Lister rat pups with intra-peritoneal injections of native or heat-denatured betaLG at days 43 and 62 after birth. The animals were then fed native and denatured milk products twice weekly from 73 to 101 days of age with a feeding tube, after which they were allowed cheese and milk ad libitum, until they were killed on day 131. Total immunoglobulin (Ig)E and betaLG-specific IgG1 and IgG2a levels were determined from serum samples. Spontaneous interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) production was measured from duodenal specimens, and specimens of gastrointestinal mucosae were studied for the presence of inflammatory cells. The rats immunized with native betaLG had higher levels of total serum IgE than the unimmunized controls or the rats immunized with heat-denatured betaLG, while heat-denatured betaLG induced a significantly more intensive mononuclear inflammatory cell and eosinophil infiltration in the gastroduodenal mucosa. The betaLG-specific IgG antibody and IL-4 and IFN-gamma responses were similar in the two groups of immunized animals. Hence, denaturation modifies the immunogenic and allergenic properties of betaLG. Heat-denatured betaLG induces a more intensive local reaction in the gastrointestinal mucosa, while there is some evidence for enhanced systemic allergic sensitization by native betaLG.