Abstract
BackgroundWhey acidic protein (WAP) is a major protein identified in the milk of several mammalian species with cysteine-rich domains known as four-disulfide cores (4-DSC). The organization of the eutherian WAP genes is highly conserved through evolution. It has been proposed that WAP could play an important role in regulating the proliferation of mammary epithelial cells. A bacteriostatic activity was also reported. Conversely to the other mammalian species expressing WAP in their milk, camel WAP contains 4 additional amino acid residues at the beginning of the second 4-DSC domain, introducing a phosphorylation site. The aim of this study was to elucidate the origin of this specificity, which possibly impacts its physiological functions.ResultsUsing LC-ESI-MS, we identified in Camelus bactrianus from Kazakhstan a phosphorylated whey protein, exhibiting a molecular mass (12,596 Da), 32 Da higher than the original WAP (12,564 Da) and co-eluting with WAP. cDNA sequencing revealed a transition G/A, which modifies an amino acid residue of the mature protein (V12 M), accounting for the mass difference observed between WAP genetic variants. We also report the existence of two splicing variants of camel WAP precursors to mRNA, arising from an alternative usage of the canonical splice site recognized as such in the other mammalian species. However, the major camel WAP isoform results from the usage of an unlikely intron cryptic splice site, extending camel exon 3 upstream by 12-nucleotides encoding the 4 additional amino acid residues (VSSP) in which a potentially phosphorylable Serine residue occurs. Combining protein and cDNA sequences with genome data available (NCBI database), we report another feature of the camel WAP gene which displays a very rare GC-AG type intron. This result was confirmed by sequencing a genomic DNA fragment encompassing exon 3 to exon 4, suggesting for the GC donor site a compensatory effect in terms of consensus at the acceptor exon position.ConclusionsCombining proteomic and molecular biology approaches we report: the characterization of a new genetic variant of camel WAP, the usage of an unlikely intron cryptic splice site, and the occurrence of an extremely rare GC-AG type of intron.
Highlights
Whey acidic protein (WAP) is a major protein identified in the milk of several mammalian species with cysteine-rich domains known as four-disulfide cores (4-DSC)
Nucleotide sequence of camel WAP cDNA Using the rabbit and rodents WAP encoding gene sequences as references, we searched for the expected fourth exon of the WAP gene in the camel genome sequence (NCBI, LOC105095719)
Identification and characterization of a new WAP genetic variant in Bactrian camel milk LC-ESI-Liquid chromatography coupled to tandem mass spectrometry (MS) analysis of a Bactrian camel milk from the Shymkent region (Fig. 3), revealed the presence, in peak III (Table 2), of two molecular masses differing by 80 Da 12,596 Da and 12,676 Da, besides the cognate WAP
Summary
Whey acidic protein (WAP) is a major protein identified in the milk of several mammalian species with cysteine-rich domains known as four-disulfide cores (4-DSC). The casein fraction comprises 4 caseins (αs1-, αs2-, β- and κ-casein) encoded by four autosomal genes (CSN1S1, CSN1S2, CSN2 and CSN3, respectively) mapped on chromosome 6 in cattle and goat [5, 6]. This fraction is rather complex with many splicing variants and post-translational modifications [3]. WAP is a major whey protein identified in the milk of several species from eutherians as well as marsupial and monotremes [7]. The WAP has been identified in rabbits [10], porcine [11], wallaby [12], brushtail possum [13]
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