Differential Splicing of Pre‐Messenger RNA Produces Multiple Forms of Mature Caprine α_s1‐Casein

Abstract

The identity of multiple forms of caprine a,,-casein in variants A, B, and C has been determined by structural characterisation using mass spectrometry, automated Edman degradation and peptide mapping. Mature goat a,,-casein exists as a mixture of at least four molecular species which differ in peptide chain length. The main component corresponds to the 199-residues form already described. The other three, in lesser amounts, were shorter forms of a,,-casein and differed for the deleted peptides 141 -148, as shown previously for ovine a,,-casein, peptide 110-1 17, or Gln78. Analysis of a,,-casein mRNA from milk somatic cells demonstrated that these forms originated from skipping events at the level of exon 13 (codifying for peptide 210-117) and 16 (codifying for peptide 141-148) and from the presence of a cryptic splice site within exon 11 (whose first CAG triplet encodes Gln78) during primary transcript processing. The finding of these splicing abnormalities in the three common variants A, B, and C suggests that this is a general feature of a,,-casein in goat.A further source of heterogeneity of caprine a,,-casein was identified in the discrete phosphorylation of seryl residues. Eight serine residues (at positions 44, 46, 64 to 68 and 75) are fully phosphorylated (except in variant A because of the replacement Glu77-+Gln which prevents phosphorylation of Ser7.5). Conversely, SerllS and Ser41 are phosphorylated only to about SO% and 20%, respectively. Ser12, although located in a consensus triplet, is never phosphorylated, similarly to the ovine a,,-casein variants.These results confirm that there are stabilised mechanisms of simultaneous synthesis of a,,-casein at different length and of post-translational modification in both caprine and ovine species.Keywords: a,,-casein ; phosphorylation ; differential splicing ; non-allelic deletion ; polymorphism Previous studies performed on the goat milk protein fraction have shown an extensive qualitative polymorphism of a,,-casein, associated with quantitative variation (Boulanger et al., 1984; Grosclaude et a]., 1987). The most recent genoinic data showed the existence of at least 14 autosomal alleles at the goat a5,-casein locus, distributed in seven different classes of protein variants associated with four levels of expression, ranging between 0 (a,,-Cad) and 3.6 g/l (a,,-CasA, B and C)/allele (Martin, 1993;Martin and Leroux, 1994). a,,-casein A, B, C and E variants differ from each other by amino acid substitutions, while a,,-casein D, F and G variants, associated with a low level of protein synthesis, are internally deleted (Brignon et al., 1989(Brignon et al., , 1990. The establishment of the overall organisation of the goat a,,-casein gene (19 exons scattered along 17 kb), the characterisation of alleles F Martin and Leroux, 1994) and E (Jansa Perez et al., 1994) at the genomic level as well as a detailed analysis of their transcription products demonstrated that the deletion occurring within variant F arises from the outsplicing of three exons (9, 10, and 11)...

The impact of genetic polymorphisms on the protein composition of ruminant milks

“…This "codon skipping", which is likely due to an erroneous 3'cryptic splice site usage when exons 10 and 11 are spliced (Fig. 7), has been found, at the protein level, in the four major ruminant species: caprine [25], ovine [24] bovine and water buffalo [26]. Interestingly, such a cryptic splice site slippage does not only occur in ruminants, since a similar event, affecting another glutaminyl residue (Q 37 in the mature protein) encoded by the first codon of exon 6', has been reported in the human a s1 -casein [41,61].…”

“…However, the extent of this heterogeneity depends on the species. While a s1 -casein consists of a mixture of two forms (199 and 198 amino acid residues) in cattle and water buffalo, due to the alternative deletion of Q 78 , there are, in sheep and goats, at least seven molecular forms which differ in their peptide chain length, regardless of the genetic polymorphism [25]. The main com-449 Figure 8.…”

Qualitative and quantitative analysis of wheat gluten proteins by liquid chromatography and electrospray mass spectrometry

“…Quantitative analysis of components was performed by integration of the multiple charged ions relative to each species. 26 …”

Real-time RT-PCR and cDNA macroarray to study the impact of the genetic polymorphism at the αs1-casein locus on the expression of genes in the goat mammary gland during lactation

“…The splicing of a s1 -CasF primary transcripts seems to be deeply disturbed since at least 9 different mRNA have been detected [16]. Nevertheless, alternative splicing also exists for a s1 -CasA allele but is much less complex and involves only two sequences of 24 nucleotides corresponding to exons 13 and 16 which are outspliced during the course of the maturation of the primary transcripts [17]. At the end of the 1980's, defective alleles were preponderant in French flocks.…”