The primary structure of a low‐M_r multiphosphorylated variant of β‐casein in equine milk

Abstract

Highly phosphorylated casein with a low molecular mass was isolated from Haflinger mare's milk by RP-HPLC. It accounts for 4.0% of the casein content. Its mass was determined by LC-ESI-MS before and after treatment by alkaline phosphatase. The molecular mass found for the apo-form (10,591 +/- 2 Da) is in agreement with its primary structure, which was established by ESI-MS/MS from tryptic peptides. It appeared that this short protein (94 amino acid residues) is an internally truncated form of the full-length equine beta-casein (226 residues). This low-Mr variant of equine beta-casein displays a large deletion (residues 50-181), due to a cryptic splice site usage occurring within exon 7 during the course of primary transcripts processing. The phosphorylation pattern of this equine beta-casein variant was investigated by LC-ESI-MS and 2-DE. Seven phosphorylation forms were identified with one to seven phosphate groups with pIs ranging between 4.67 and 4.01. The major isoforms carry five and six phosphate groups.

Evolution of major milk proteins in Mus musculus and Mus spretus mouse species: a genoproteomic analysis

“…This large deletion is due to the usage of a cryptic splice site occurring in exon 21, 57 nucleotides downstream from an AG defining the proper end of intron 20, in frame and following a rather strong polypyrimidine tract (n = 20) although interrupted by a triplet of contiguous G. The same deletion was also reported in the α s1 -casein from the mouse FVB/N strain (GenBank:AAH40246). Even though such an event seems to be relatively rare, a casual improper splicing using an exon cryptic splice site and leading to the loss of 132 aa residues was also reported in the equine CSN2 mRNA [24]. On the other hand, the loss of a CAG, induced by an error-prone junction sequence, is much more frequent.…”

Equine milk proteins: Chemistry, structure and nutritional significance

“…Equine Lf also contains the AsneGly sequence and may be susceptible to spontaneous deamidation (Girardet et al, 2006). Unique to equine milk and apparently absent from the milk of other species, including ruminants, is a low molecular mass multiphosphorylated b-casein variant which accounts for 4% of the total casein (Miclo et al, 2007). This short protein (94 amino acid residues) is the result of a large deletion (residues 50e181) from fulllength equine b-casein.…”

“…This short protein (94 amino acid residues) is the result of a large deletion (residues 50e181) from fulllength equine b-casein. No spontaneous deamidation of this low molecular mass form of b-casein has been found (Miclo et al, 2007).…”

Donkeys’ milk protein fraction investigated by electrophoretic methods and mass spectrometric analysis

“…Equidae milk protein fraction was also investigated and characterized by different mass spectrometric approaches (Cunsolo et al, 2006;Girardet et al, 2006;Iametti et al, 2001;Miclo et al, 2007;Neveu et al, 2002).…”