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An individual sample of donkey CN, before and after chymosin hydrolysis, was analyzed by PAGE-SDS and was shown in Fig. 3.22A after Blue Coomassie staining; with the aim to identify each donkey casein fraction it was immunostained using specific polyclonal antibodies against each donkey casein fraction (αs1-, αs2-, β-,

and κ-CN) (Fig. 3.22B). The electrophoretic profile obtained for donkey casein fraction is shown to be very similar to that reported by Miranda et al. (2004) for mare and goat caseins. In fact, in the casein zone, the heaviest electrophoretic band was that corresponding to αs2-CN, followed by αs1-CN. For the latter, two main

bands were separated, which probably could correspond to its two deleted forms resulting from the skipping of exon 7 and 5; instead β-CN has migrated into the intermediate zone between these two αs1-CN

components. The results of this immuno-electrophoretic technique also showed the fastest mobility of κ-CN towards the anode compared to the other casein fractions, suggesting, therefore, its lower molecular weight between asinine caseins. In addition, κ-CN migrated as a more or less extensive smear because of considerable variation in the level of post-translational glycosylation. This electrophoretic behaviour had previously been highlighted by Miranda et al. (2004) with respect to mare’s milk.

The presence of κ-CN also in whey protein used as reference and corresponding to the casein zone, suggested that this smear would include highly glycosylated κ-CN forms, which could be either the heaviest and less abundant forms.

Moreover, this immuno-electrophoretic technique showed that β- and αs1-CN are the most abundant casein

fractions followed by αs2- and κ-CN, and this feature makes donkey milk more similar to human milk, in

which αs2-CN has not yet been identified. Also peptides derived essentially from β- and αs1-CN hydrolysis

by endogenous enzymes of donkey milk were showed in the immunostained profiles.

The results obtained by this immuno-electrophoretic technique were also confirmed by Fluorescent Glycoprotein Detection (Fig 3.22C). In fact, the κ-CN band was more fluorescent than the other casein bands with a major molecular weight, thus suggesting its high glycosylation degree. It should be mentioned that the migration zone of donkey κ-CN spanned all the other casein bands.

Donkey κ-CN seems to be more glycosylated (about 12 glycosides) than bovine κ-CN which has six glycosylated Thr residues and considering the preferential peptide bond of donkey κ-CN susceptible to chymosin hydrolysis, it could probably be Phe97-Ile98 and not Phe105-Met106 bond (which would reflect differences in the mechanism of clotting of milk in ruminant and non ruminant mammals). In this way, donkey κ-CN, showing more similarities to equine and human κ-CN, compared to bovine κ-CN may belong to the κ-CN group II.

Fig 3.22: Characterization of donkey casein, before and after chymosin hydrolysis, by PAGE-SDS analysis

(A) and immunoblotting with specific polyclonal antibodies against each casein fraction (B) with fluorescent glycoprotein detection (C).

WPD: whey protein donkey; CNC: casein cow; CNC+chim.: casein cow + chymosin; CND+chim.: casein

donkey + chymosin. W P D C N C C N C +c hi m. W P C C N D C N D +c hi m. W P D C N C C N C +c hi m. W P C C N D C N D +c hi m. (A) (B) _(C) C N D +c hi m. C N D +c hi m. C N D +c hi m. C N D +c hi m. C N D C N D C N D C N D α αα αs2 ααααs1 ββββ κκκκ (+) (-)

κ-CN

Conclusions 1

The proteomic approach allowed the identification of the four casein fractions (αs1-, αs2-, β- e κ-CN) in

donkey milk together with their related heterogeneity due to post-translational phenomena such as the different phosphorylation degree of the caseins (αs1-, αs2-, β-CN) and the high glycosylation level of κ-CN,

incorrect splicing of primary transcript in mRNA (non allelic deleted forms of αs1-, αs2-, β-CN) and genetic

polymorphism of αs1- and β-CN (one or more variants in individual samples).

Specifically

• αs1-CN was composed at least of three protein components, characterized by a different length of

their amino acid sequence (210, 202, 197 aa) and also expressed at different intensity (202>197>210), where the minor component with 210 aa was found for the first time. This means, that unlike αs1-CN mare, all exons are expressed in donkey αs1-CN gene. The incorrect splicing of

the primary transcript (210) determining the single and/or contemporary elimination of exons 7 and 5 gave rise to the two deleted components with 202 and 197 aa, respectively. Each of these three components (210, 202, 197) showed a microheterogeneity due both to the different phosphorylation degree (5, 6 e 7P) that the glutamine residue deletion. The screening carried out on the samples by RP-HPLC and MS analysis also revealed a qualitative polymorphism at αs1-CN locus for the finding

of a new αs1-CN variant, in addition to the protein already known in literature (Cunsolo et al.,

2009a), but also a quantitative polymorphism at this locus (paragraph 3.13.3), for the detection of samples characterized by the presence of αs1-CN only in traces, as in goat milk.

• β-CN is always present in donkey milk as in human milk (paragraph 3.13.3). The immuno- electrophoretic and chromatographic analysis of analyzed samples showed three new genetic variants at this locus, each 226 aa long, which we named B, C, e D, while the mass spectrometry allowed to identify the characterizing amino acid substitutions. The structural study has indicated for each new variant the same phosphorylation degree of β-CN A reference (Cunsolo et al., 2009b), indicating that the involved mutations do not affect serine residues in the delegated code sequences. Each new variant, therefore, was heterogeneous for the presence of three phosphorylated components (5, 6, 7P) and for the contemporary presence of a non allelic deleted form lacking the peptide E27SITHINK34, encoded by exon 5, always with the same three phosphorylation degrees. • αs2-CN was identified and characterized in donkey milk by Chianese et al. (2010). Its heterogeneity

is due to its high phosphorylation degree (10, 11, 12 P) and to the presence of deleted forms (Cosenza et al., 2010; Saletti et al., 2012). The electrophoretic and chromatographic analysis also suggested the existence of a possible quali-quantitative polymorphism at αs2-CN locus (paragraph

3.13.3). The absence of αs2-CN in some samples of donkey milk, as in the human milk, together

with a lower expression level of αs1-CN and β-Lg, confirm the compositional similarity of two

milks and represent a scientific basis for donkey milk’s use in nutrition of infants with CMP allergy. • To date, for κ-CN only partial data on its cDNA sequence exist. We have experimental evidences of

κ-CN presence in DM only after specific immunostaining, likely depending either on the low amount of this fraction in the milk or on its lower reactivity to Blue Coomassie staining. As in ruminants, κ-CN is the most heterogeneous casein fraction and probably this phenomenon is generated from its high glycosylation degree. In fact the fluorescent glycoproteins detection showed that κ-CN is not only the casein with the lowest molecular weight but also the most fluorescent, being a highly glycosylated protein. For this reason, it could have more similarity with human and equine κ-CN than other species. Finally, the immunoelectrophoretic profiles also showed the occurrence of a possible genetic polymorphism at this locus and the non-susceptibility of κ-CN to chymosin hydrolysis compared to αs1- and αs2-CN is due to the non para-κ-CN formation. This

result is in agreement with cDNA κ-CN sequence which would lack of the specific site for chymosin action.

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