Exploring the fine composition of Camelus milk from Kazakhstan with emphasis on protein components

Abstract : The present study aimed to identify, in exploring the protein fraction of camelid milks from several regions of Kazakhstan, original molecules (peptide, proteins) potentially responsible for the properties attributed to camel milk. Nearly 180 milk samples from two camel species (Camelus bactrianus and C. dromedarius, and their hybrids) we collected at different lactation stage, age and calving number, and submitted to different proven analytical techniques and proteomic approaches (SDS-PAGE, LC-MS/MS and LC-ESI-MS). A detailed characterization of 50 protein molecules, relating to genetic variants, isoforms arising from post-translational modifications and alternative splicing events, belonging to 9 protein families (κ-, αs1-, αs2-, β-; and γ-CN, WAP, α-LAC, PGRP, CSA/LPO) was achieved. We reported the occurrence of two unknown isoforms (i1 and i2) of camel αs2-CN arising from alternative splicing events. Using cDNA-sequencing, i1 was characterized as a splicing-in variant of an in-frame 27-nucleotide sequence, of which the presence at the genome level, flanked by canonic motifs defining an exon 13 encoding the nonapeptide ENSKKTVDM, was confirmed. Isoform i2, which appeared to be present at different phosphorylation levels ranging between 8P and 12P, was shown to include an additional decapeptide (VKAYQIIPNL), revealed by LC-MS/MS, encoded by a 3’-extension of exon 16. In addition, we reported, for the first time to our knowledge, the occurrence of a αs2-CN phosphorylation isoform with at least one phosphorylated S/T residue that does not match with the usual canonic sequence (S/T-X-A) recognized by the mammary kinase, suggesting thereby the existence of two kinase systems involved in the phosphorylation of caseins in the mammary gland.As far as camel WAP is concerned, we identified in C. bactrianus a new genetic variant (B), originating from a transition G => A, leading to a codon change (GTG/ATG) in the nucleotide sequence of cDNA, which modifies a single amino acid residue at position 12 of the mature protein (V12M). In addition, we describe the existence of a splicing variant of camel WAP, arising from an alternative usage of the canonical splice site recognized as such in the other mammalian species expressing WAP in their milk. We also report that the WAP isoform predominantly present in camelids milk, first described by Beg et al. (1986) as displaying an additional sequence of 4 amino acid residues (56VSSP59) in the peptide segment connecting the two 4-DSC domains, results from the usage of an unlikely intron cryptic splice site, extending camel exon 3 on its 5’ side by 12-nucleotides. In addition, we confirm that in the camel gene encoding WAP, intron 3 is a GC-AG intron, with a GC donor site showing a compensatory effect in terms of a dramatic increase in consensus at the acceptor exon position.Finally, using an optimized protocol, we isolated camel milk-derived EVs satisfiying the typical requirements for exosomal morphology, size and protein content. We identified a thousand of different proteins representing the first comprehensive proteome of camel milk-derived EVs that appears wider than camel milk proteome, including markers associated with small extracellular vesicles, such as CD63, CD81, HSP70, HSP90, TSG101 and ADAM10. We also identified proteins present in other milk components. This is particularly the case for lactadherin/MFG-E8, Ras-related proteins or CD9 that have been reported to occur in MFG. Our results strongly suggest that milk-derived exosomes have different cellular origin.
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Alma Ryskaliyeva. Exploring the fine composition of Camelus milk from Kazakhstan with emphasis on protein components. Molecular biology. Université Paris-Saclay, 2018. English. ⟨NNT : 2018SACLA016⟩. ⟨tel-02305918⟩

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