BLBS102-c26 BLBS102-Simpson March 21, 2012 13:51 Trim: 276mm X 219mm Printer Name: Yet to Come
498 Part 4: MilkαS1-CaseinThe amino acid sequence of equineαs1-casein has been deduced
from its cDNA sequence (Lenasi et al. 2003). The protein con-
tains 205 amino acids and has a molecular mass of 26,614.4
Da prior to post-translational modification, that is, it is con-
siderably larger than its bovine or human counterpart (Table
26.3). Two smaller isoforms ofαs1-casein have been identified
in equine milk, which probably result from the skipping of ex-
ons during transcription (Miranda et al. 2004). Equineαs1-casein
contains six potential phosphorylation sites (Lenasi et al. 2003),
five of which are in very close proximity (Ser 75 ,Ser 77 ,Ser 79 ,
Ser 80 ,Ser 81 ) and can thus form a phosphorylation centre, which
is important in the structure of casein micelles. Mat ́eos et al.
(2009a) determined the different phosphorylation levels of the
native isoforms of equineαs1-casein and identified 36 differ-
ent variants with several phosphate groups ranging from two
to six or eight which, like equineβ-casein, present a complex
pattern on one dimensional and two dimensional electrophore-
sis. Bovineαs1-casein contains eight or nine phosphorylation
sites (Swaisgood 2003), which form two phosphorylation cen-
tres (De Kruif and Holt 2003). Bovineαs1-casein contains three
distinct hydrophobic regions, roughly including residues 1–44,
90–113 and 132–199 (Swaisgood 2003). These regions are char-
acterised by positive values for hydropathy. Likewise, equine
αs1-casein has three domains with a high hydropathy value, that
is, around residues 25–30, 95–105 and 150–205 and therefore
it probably has association properties similar to those of bovine
αs1-casein. Furthermore, equineαs1-casein contains two regions
with very low hydropathy, that is, around residues 45–55 and
125–135, which are expected to behave hydrophilically. Human
αs1-casein does not appear to have distinct hydrophobic regions.
Overall, equine and humanαs1-casein have comparable grand
average hydropathy (GRAVY) scores, which are lower than that
of bovineαs1-casein (Table 26.3), indicating an overall higher
hydrophobicity for the latter. GRAVY scores reflect the relative
ratio of hydrophobic and hydrophilic amino acid residues in a
protein, with a positive value reflecting an overall hydrophobic
and a negative value an overall hydrophilic nature of the protein.
Prior to the mid-1990s, it was generally assumed that hu-
man milk contains mainlyβ-andκ-caseins with little or no
αs-casein (Kunz and Lonnerdal 1990). A minor casein compo- ̈
nent has since been identified and is considered to be the human
equivalent ofαs1-casein, although this identification highlights
several inconsistencies in comparison with the equivalent casein
in other species. Uniquely, humanαs1-casein appears to con-
tain at least two cysteine residues and exists as a multimer in
complex withκ-casein (Cavaletto et al. 1994, Rasmussen et al.
1995). Johnsen et al. (1995) identified three cysteine residues
in humanαs1-casein and provided a molecular explanation for
αs1-κ-casein complex formation. Martin et al. (1996) provided
definitive evidence for the presence of a functionalαs1-casein
locus in the human genome which is expressed in the mam-
mary gland during lactation, while Sørensen et al. (2003) de-
termined the phosphorylation pattern of humanαs1-casein. In
bovine milk,αs1- casein is a major structural component of the
casein micelle and plays a functional role in curd formation(Walstra and Jenness 1984). The relatively low level ofαs1-
casein in equine milk (Table 26.2), and similarly in human milk,
may be significant and, coupled with the low protein content,
could be responsible for the soft curd produced in the stom-
ach of the infant or foal (Dr. Ursula Fogarty, National Equine
Centre, Ireland – personal communication). Goat milk lacking
αs1-casein has poor coagulation properties compared to milk
containingαs1-casein (Clark and Sherbon 2000). Bevilac ̧qua
et al. (2001), who assessed the capacity of goat’s milk with a low
or highαs1-casein content to induce milk protein sensitisation
in guinea pigs, found significantly less sensitisation in milk with
lowαs1-casein. This may represent another important attribute
of the lowαs1-casein content of equine milk for use in human
allergology.
Anαs1-like protein of approximately 31–33 kDa has been
identified in asinine milk although Criscione et al. (2009) re-
ported its absence in one Ragusana donkey under investigation.αS2-CaseinThe complete amino acid sequence of equineαs2-casein is un-
known, but Ochirkhuyag et al. (2000) published the sequence of
theN-terminal 15 amino acid residues (Lys-His-Lys-Met-Glu-
His-Phe-Ala-Pro-Xaa-Tyr-Xaa-Gln-Val-Leu, where Xaa is an
unknown amino acid). Only five of these amino acids were con-
firmed by Miranda et al. (2004). Isoelectric focusing showed two
major bands for equineαs2-casein, with isoelectric points in the
pH range 4.3–5.1 (Ochirkhuyag et al. 2000). Bovineαs2-casein
is the most highly phosphorylated casein, usually containing 11
phosphorylated serine residues, with lesser amounts containing
10, 12 or 13 phosphate groups (Swaisgood 2003). There are
no reports on the presence ofαs2-casein in human milk. Using
three different methods for protein identification, Criscione et al.
(2009) could not detectαs2-orκ-casein in asinine milk.β-CaseinThe amino acid sequence of equineβ-casein, derived from
the cDNA, has been reported by Lenasi et al. (2003), and re-
vised by Girardet et al. (2006) with the insertion of eight amino
acids (glutamic acid (Glu 27 )toLys 34 ). The theoretical molecular
mass of this 226 amino acid polypeptide is 25,511.4 Da (Table
26.3). Bovine and humanβ-casein contain 209 and 211 amino
acid residues, respectively (Table 26.3). Two smaller variants
of equineβ-casein, which probably result from casual exon-
skipping during transcription, were reported by Miranda et al.
(2004). The 28C-terminal amino acids contain seven poten-
tial phosphorylation sites (Ser 9 ,Ser 15 ,Ser 18 ,Ser 23 ,Ser 24 ,Ser 25 ,
Ser 28 ) and multiple-phosphorylated isoforms of equineβ-casein
containing three to seven phosphoserine residues have been re-
ported, with the isoelectric point varying from pH 4.74–5.30
(Girardet et al. 2006, Mat ́eos et al. 2009b). Bovineβ-casein,
which contains four or five phosphorylated serine residues,
has an isoelectric point of 5.0–5.5 (Swaisgood 2003). Human
β-casein has up to six levels of phosphorylation, that is, 0, 1, 2, 3,
4 or 5 phosphorylated serine residues (Sood and Slattery 2000).