The primary structure of a low-Mr multiphosphorylated variant of beta-casein in equine milk.

The research article discusses the identification and detailed analysis of a specific variant of beta-casein found in equine milk, a variant characterized by a low molecular weight and high levels of phosphorylation.

Identification and Isolation of Casein Variant

• The researchers isolated a form of casein, a protein typically found in milk, that was both highly phosphorylated and of a low molecular weight. This was done using Reverse Phase High Performance Liquid Chromatography (RP-HPLC) on milk obtained from a Haflinger mare, a breed of horse.
• The variant made up 4.0% of the total casein content in the milk sample. Its molecular mass was determined using Liquid Chromatography – Electrospray Ionization – Mass Spectrometry (LC-ESI-MS), both before and after it was treated with alkaline phosphatase, an enzyme that removes phosphate groups from proteins.

Determination of Protein Structure

• The resultant molecular mass of the dephosphorylated form of the protein, referred to as the apo-form, was found to be approximately 10,591 Da, a measurement used to express atomic mass. This was found to match the determined primary structure of the protein, revealing that it is indeed a variant of full-length equine beta-casein.
• The protein variant was found to contain only 94 amino acid residues, significantly less than the 226 residues that make up the full-length protein. This shorter length is due to an extensive deletion (of residues 50-181) that occurs as a result of an unusual splice site usage within exon 7 during the processing of primary transcripts, a process that prepares newly transcribed RNA for translation into protein.

Analysis of Phosphorylation Patterns

• The researchers further analyzed the obtained variant of equine beta-casein by LC-ESI-MS and 2-Dimensional Electrophoresis (2-DE) to examine its phosphorylation patterns.
• The beta-casein variant was found to exist in seven different phosphorylation forms, each carrying between one to seven phosphate groups and having isoelectric points (pIs) ranging between 4.67 to 4.01. ‘Isoelectric point’ is the pH at which a particular molecule or surface carries no net electrical charge.
• The most common isoforms were those that included five or six phosphate groups.