Equine alpha S1-casein: characterization of alternative splicing isoforms and determination of phosphorylation levels.
- Journal Article
Summary
The study investigates the complexity of alpha(S1)-casein, a protein found in Haflinger mare’s milk, revealing that it has multiple isoforms due to posttranscriptional modifications and varying degrees of phosphorylation.
Overview
In this research, the scientists isolated alpha(S1)-casein from Haflinger mare’s milk and discovered that it had several isoforms, or variations of the protein, caused by alternative splicing events and different levels of phosphorylation. They used two different types of phosphatase to completely dephosphorylate the protein and then analyzed the various dephosphorylated versions, or apoforms, via electrospray ionization mass spectrometry.
Findings
After analysis, several important discoveries were made:
- The research discovered a full-length protein and seven variants that resulted from posttranscriptional modifications.
- The posttranscriptional modifications included “exon skipping” involving exon 7, exon 14, or both. Exon skipping is a process in which an exon, or a sequence of DNA coding for a part of the final protein, is left out of the final RNA molecule.
- A “cryptic splice site” that coded for a glutamine residue was also used. A cryptic splice site is a location in the sequence that usually isn’t used for splicing but gets activated due to some mutations or regulatory changes.
Phosphorylation Levels
In addition to finding these various isoforms, the research also measured the degree of phosphorylation in the native isoforms. This was achieved by performing electrospray ionization mass spectrometry after the isoforms had been separated by ion-exchange chromatography. Through this analysis, 36 different variants of equine alpha(S1)-casein were identified. These variants had varying levels of phosphorylation, with phosphate groups ranging from 2 to 6 or 8, depending on whether exon 7 was skipped.
This study gives us a deeper understandingof the complexity of alpha(S1)-casein and the different possibilities for its structure and phosphorylation levels. This could contribute to further research on the protein’s functions and potential uses.
Cite This Article
Publication
Researcher Affiliations
- Unité de Recherche Animal et Fonctionnalités des Produits Animaux (UR AFPA) - Equipe Protéolyse et Biofonctionnalités des Protéines et des Peptides (PB2P), Nancy-Université, Vandoeuvre-lès-Nancy, France.
MeSH Terms
- Alternative Splicing
- Amino Acid Sequence
- Animals
- Caseins / chemistry
- Caseins / genetics
- Caseins / metabolism
- Chromatography
- Exons
- Female
- Horses
- Milk / chemistry
- Molecular Sequence Data
- Phosphorylation
- Protein Isoforms / chemistry
- Protein Isoforms / genetics
Citations
This article has been cited 7 times.- Deracinois B, Matéos A, Romelard A, Boulier A, Auger J, Baniel A, Ravallec R, Flahaut C. Partial-, Double-Enzymatic Dephosphorylation and EndoGluC Hydrolysis as an Original Approach to Enhancing Identification of Casein Phosphopeptides (CPPs) by Mass Spectrometry. Foods 2021 Sep 9;10(9).
- Rout PK, Verma M. Post translational modifications of milk proteins in geographically diverse goat breeds. Sci Rep 2021 Mar 10;11(1):5619.
- Ryskaliyeva A, Henry C, Miranda G, Faye B, Konuspayeva G, Martin P. Alternative splicing events expand molecular diversity of camel CSN1S2 increasing its ability to generate potentially bioactive peptides. Sci Rep 2019 Mar 27;9(1):5243.
- Cieslak J, Wodas L, Borowska A, Pawlak P, Czyzak-Runowska G, Wojtowski J, Puppel K, Kuczynska B, Mackowski M. 5'-flanking variants of equine casein genes (CSN1S1, CSN1S2, CSN2, CSN3) and their relationship with gene expression and milk composition. J Appl Genet 2019 Feb;60(1):71-78.
- Wang X, Zhao X, Huang D, Pan X, Qi Y, Yang Y, Zhao H, Cheng G. Proteomic analysis and cross species comparison of casein fractions from the milk of dairy animals. Sci Rep 2017 Feb 27;7:43020.
- Shokrollahi B, Choi JY, Won M, Kim ET, Lee SE, Ham JS. Koumiss (Fermented Mare's Milk) as a Functional Food: Bioactive Proteins, Peptides, and Future Perspectives. Foods 2025 Nov 18;14(22).
- Mayerl CJ, German RZ. Evolution, diversification and function of the maternal-infant dyad in mammalian feeding. Philos Trans R Soc Lond B Biol Sci 2023 Dec 4;378(1891):20220554.
