Análisis de la propuesta “los viernes al campo”

In the experiments with skim milk and whole milk (Figure 4.3 and Figure 4.5), differences in relative band intensity were observed between the input material and beads A in both, mucin coated and negative control beads. This indicated selective binding of milk proteins from the input material to mucin or Sepharose. The bands representing αS1- and β-caseins were stronger than those for αS2- and κ-casein. However, these differences merely represent the concentration differences in the input material (Table 4.2). A comparison of the ratios of binding caseins (normalised to αS2-casein) with their 1:3:3:1-ratio (αS2-casein:β-casein:αS1-casein:κ-casein) in

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Figure 4.16: Sediment in milks of different fat contents and degrees of processing

SDS-PAGE gel showing 1.5 µl skim milk or commercial skim and whole milk and sediment of 500 µl milk after treatment like in the adhesion assay (30 min at 37°C and 2 or 3 PBS washes). Gel separated at 130 V for 2 hr and stained with CBB G250 overnight.

75 kDa 150 kDa 100 kDa 50 kDa 37 kDa 25 kDa 20 kDa 15kDa 250kDa 10kDa 2kDa

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the casein micelle [493] suggests that αS2- and κ-casein actually showed preferential binding to the beads with exception of the combination of skim milk and EtOH-amine beads.

κ-Casein has only few phosphorylation sites and does not precipitate even at high Ca2+- concentrations [494], indicating low calcium binding. However, a fraction of the κ-casein molecules can be glycosylated [495] and the side chains might introduce functional groups (e.g. hydroxy groups) which can mediate binding. With their phosphoserine (and carboxyl) residues,

αS1-, αS2- and β-caseins are able to bind Ca2+ ions. Mucins can also bind positively charged ions [496], such as calcium [497]. It is possible that these ions act as bridges between milk proteins and mucins and thus are an important factor in muco-ahesion.

Results from Western blot analysis (Figure 4.6 and Figure 4.7) indicate that the binding affinity of caseins is reduced through pepsin hydrolysis. Some of the undigested samples show a difference in band intensity between mucin-coated and negative control beads (more signal on mucin coated beads). After digestion all bands were generally weaker but also the difference in intensity was reduced. This implies a stronger reduction in binding potential of caseins to the mucin beads compared to the negative control beads. However, the effect of gastric digestion on the binding potential of caseins requires further research. A key question here would be whether the partial in vitro gastric digestion decreased the binding potential of casein molecules to

mucin coated Sepharose beads or the size of the adhesive molecule decreased; i.e. were binding sites reduced or were non-binding parts cleaved off? Further, the effect of entropically-driven interactions, e.g. hydrophobic interactions, was not addressed extensively in this study. Although the EtOH wash was included to remove hydrophobically bound molecules, results here are inconclusive. This could be due to the changes in temperature, i.e. adhesion was allowed at 37°C while the washes took place at room temperature. An interesting experimental design could combine hydrophobic interactions and the MFGM-rich fractions.

Further, some proteins showed stronger affinity for one of the two types of beads. These occurred more often in skim milk than in whole milk. For example, the 75 kDa band was

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Table 4.2: Comparison of casein fraction ratios from Figure 4.3 (skim milk; n=1) and Figure 4.5 (whole milk; n=1) and those found in the casein micelle.

25 kDa: αS2-casein. ca. 24 kDa: β-casein. ca. 23 kDa: αS1-casein. 19 kDa: κ-casein. EtOHa: ethanol amine

Band density ratio (normalised to 25 kDa band) Skim milk MUC2 beads Skim milk EtOHa beads Whole milk MUC2 beads Whole milk EtOHa beads Casein micelle [493] 25 kDa 1.0 1.0 1.0 1.0 1 ca. 24 kDa 2.3 2.7 1.9 2.1 3 ca. 23 kDa 2.2 2.9 1.8 2.2 3 19 kDa 1.2 0.9 0.9 0.9 1

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stronger on the MUC2 beads than on the negative control beads after incubation with skim milk; this might show actual binding of the milk protein to MUC2. The molecular weight and position of the band indicate that this protein could be LF (compare Figure 3.1). The binding also showed in the Western blot analysis where a faint band for LF was observed in the mucin- coated beads (Figure 4.7). LF has been shown to bind to mucin glycoproteins (e.g. Soares et al. showed binding to MG2 [498]) but no information specifically to MUC2 binding was found.

Other skim milk proteins with different affinities for mucin and negative control beads were found at 45 kDa and approximately 30 kDa (Figure 4.3). In the case of whole milk, only little difference was observed between the mucin covered and negative control beads. These proteins had molecular weights over 150 kDa which corresponds to multimeric Igs and some MFGM proteins. This agrees with the observation that these proteins showed reduced binding from skim milk which has a considerably reduced content of milk fat. MFGM proteins contain Muc1 (58 kDa protein backbone [499] with glycosylation which was found to separate at approximately 200 kDa on SDS-PAGE gels [500, 501]). Muc1 is a secretory mucin and likely to interact with other mucins because of the similar nature. Xanthine oxidoreductase (146 kDa) was shown in preliminary SDS-PAGE experiments to bind to mucin beads, the experiments were done with sediment in the whey and results also could not be confirmed by Western blot (data not shown). Both of these proteins need further research to confirm their binding potential.

Generally, binding seemed to be dominated by caseins. This could be due to the phosphoproteins sticking to the tube. Figure 4.16 shows that several proteins with molecular weights between 20 kDa and 30 kDa remain in the tubes (tubes contained skim or whole milk but no beads) after up to three washes with PBS. This molecular weight agrees with that of caseins. Further, caseins have been shown to bind to hydrophobic surfaces [502]. However, no specific information about binding to polypropylene, the tube material, could be found. It is also possible that casein micelles cause a thicker binding layer due to micelle clustering [503] and thus show a self-enhancing binding behavior while excluding other proteins from adhering.

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Despite showing indications of specific binding of proteins to MUC2, these results need to be considered with reservation as the bands were weak and sample repetitions were limited. It appeared that proteins bound to the Sepharose beads are what caused high background levels and false positive results. Thus, in the interest of time and the cost of the Sepharose beads, it was decided to continue the investigation of muco-adhesion with mucin producing IEC in culture (Chapter 5). Considering LC-MS/MS results and Western blot, β-LG and caseins were the adherent proteins. Both proteins have been suggested to show oral muco-adhesion before [504]. However, the 75 kDa protein from skim milk, which was analysed but not identified by LC-MS/MS (Table 4.1), showed the greatest accumulation potential at MUC2. Based on its molecular weight this protein could be LF or an Ig-fragment, e.g. secretory component.