Jean-Claude Mercier

Phosphorylation of caseins, present evidence for an amino acid triplet code posttranslationally recognized by specific kinases

The fifty of so phosphorylated hydroxyamino acid residues hitherto investigated in caseins from different species have been found to occur in tripeptide sequences -Ser/Thr-X-A- where X represents any amino acid residue and A is an acidic residue. This strongly suggests that phosphorylation of caseins involves basically the stepwise enzymatic recognition of primary and secondary anionic amino acid triplets where the determinants are dicarboxylic residues and phosphoseryl residues, respectively. Studies of genetic variants of bovine caseins have provided clear-cut evidence for the actual occurrence of the former recognition sites. The occurrence of the above tripeptide sequences is a necessary but not a sufficient condition for phosphorylation of caseins to occur. Possible factors of constraint such as different intrinsic properties of both phosphate acceptor residues and acidic determinants, the characteristics of the local environment in terms of overall charge and hydrophilicity, secondary structure and steric hindrance, an insufficient available pool of casein kinase(s)... are discussed. All evidence now available supports the concept that phosphorylation of caseins is a posttranslational event and it is suggested that the process may occur during the transfer of completed polypeptide chains from the smooth endoplasmic reticulum to the Golgi apparatus where most of phosphate incorporation is presumably carried out. This organelle is rich in membrane-bound specific cyclic AMP-independent kinase(s) able in vitro to rephosphorylate specifically although not completely phosphatase-treated caseins and caseinophosphopeptides.

Complete amino acid sequence of bovine αS2-casein

In a preceding paper [l] it was shown that the so-called cysZ -, as3 -, as4 and as6 -caseins, which seem to occur in all individual bovine milks, have identical peptide chains and may only differ in their phosphate content (13-10 phosphate groups/mol). Thus, the primary structure of these proteins was studied on a mixture of the four, designated as asZ -casein. Five peptides, CNl to CN5, containing 49,22, 17,4 and 115 residues, respectively, were isolated from a cyanogen bromide hydrolysate of cysZ -casein. They accounted for the 4 methionyl and 207 amino acid residues of the peptide chain. Except for residues 186-187 (His, Gln) of CNl, peptides CNl, CN3 and CN4 were completely sequenced. The alignment of the 5 CNBr peptides, H.CN4-CN2-CNS-CNl-CN3.0H, was determined from sequences studies carried out on these peptides as well as on intact as2 -casein and on some of its 25 tryptic peptides. The sequence of approximately 100 residues (112,24-5 1, 138-207) was given [ 1 ] . The present communication reports the complete primary structure of oLs2 -casein and preliminary data on the location of the phosphate groups.

Comparative study of the amino acid sequences of the caseinomacropeptides from seven species

occur in milk as large and stable calcium phosphate complexes called micelles. When milk is treated with a low amount of a protease such as chymosin (rennin EC 3.4.23.4), the micelles become clotted. This is the result of the hydrolysis of a labile peptide bond in K-casein, which then loses its ability to stabilize casein micelles. The soluble carbo- hydrate-rich C-terminal fragment, caseinomacropeptide (CMP), which is released accounts for about one third of K-casein polypeptide chain. The N-terminal frag- ment, para+-casein, which polymerizes is insoluble. might have been conserved during the course of evo- lution. Such regions may presumably be involved in the stabilization of casein micelles or responsible for the sensitivity of K-casein to chymosin and other proteolytic enzymes. The primary structures of caprine K-casein [9-lo], water buffalo [ 1 l] and porcine [ 121 CMPs have been completely established. That of human CMP [ 121 has been nearly elucidated. Some data concerning the amino acid sequence of bovine K-casein were published in 1970 by Joll& et al.

Complete nucleotide sequence of bovine α-lactalbumin gene: comparison with its rat counterpart

Abstract The nucleotide sequence of the bovine α-lactalbumin gene, whose organization is very similar to that of its rat counterpart, was deduced from the analysis of 2 λ clones isolated from a HindIII genomic bank. The 3090 sequenced nucleotides comprise 738 bp upstream from the transcription unit (∼2 kb) which contains 4 exons of 160, 159, 76 and 330 bp separated by 3 introns of 321, 473 and 504 bp. Comparison with the rat α-lactalbumin gene shows similar percentages of homology between the 4 cognate exons. Since only the first three exons are homologous to the corresponding exons of the lysozyme gene, it is suggested that the 4th exons of α-lactalbumin and lysozyme genes have different origins. The bovine α-lactalbumin mRNA is 725 nucleotides long, excluding the poly(A) tail. The reading frame and the flanking 5′ and 3′ untranslated regions contain 429, 27 and 269 nucleotides, respectively. The derived amino acid sequence differs at 10 positions from that determined directly on mature α-lactalbumin.

Expression analysis of ruminant α-lactalbumin in transgenic mice: Developmental regulation and general location of important cis-regulatory elements

The bovine α‐lactalbumin transgene with 750 bp and 336 bp of the 5′ and 3′ flanking region, respectively, is developmentally regulated as its endogenous counterpart in transgenic mice. Comparative expression analysis of three 5′‐shortened constructs suggests that the region −4771/−220 contains important cis‐acting transcriptional elements. The level of expression of a long caprine α‐lactalbumin transgene encompassing 8.5 kb and 9.5 kb of the 5′ and 3′ flanking region, respectively, was higher but still unrelated to the copy number. Expression of the transgenes and of endogenous milk‐protein genes was tissue‐specific. In contrast with a recent report, only low amounts of the relevant mRNA were detected in some skin samples, which suggests a possible contamination by mammary tissue.

Amino terminal sequences of the precursors of ovine caseins

Abstract The amino terminal sequences of the 4 caseins synthesized by translation of ovine mammary mRNAs in a wheat germ cell-free system have been investigated by automated Edman degradation. The 3 “calcium-sensitive” caseins ( α s1 , α s2 and β) and κ-casein were synthesized as precaseins with amino terminal hydrophobic extensions of 15 and 21 amino acid residues respectively, resembling “signal peptides” of other secretory proteins. The extra pieces of the 4 caseins, which start with a methionyl residue, end with an alanyl residue which may be one of the signals recognized by the mammary membrane-bound enzyme responsible for the specific cleavage of precaseins. The amino terminal extensions of α s1 , α s2 and β-caseins show a high degree of homology suggesting that they have derived from a common ancestor.

Premiers elements de structure primaire des caseines αs2 bovines

The bovine αs2‐, αs3‐, αs4‐ and αs6‐caseins [1] were isolated. The 4 proteins had the same amino‐acid composition and C‐terminal sequence, but different phosphorus contents. From a mixture of these proteins (designated as ‘αs2‐complex’) and from αs3‐casein a single and identical N‐terminal sequence was obtained by Edman degradation. It seems therefore that the 4 proteins have the same peptide chain and only differ in their phosphorus content. For this reason we propose to modify the nomenclature of Annan and Manson [1] and to use in future the single term αs2 to designate the caseins which have been previously called αs2, αs3, αs4 and αs6 by these authors. The study of the primary structure of the peptide chain, which has confirmed these results, was undertaken on the S‐carboxymethylated αs2‐complex. From a cyanogen bromide digest and from a tryptic hydrolyzate of the αs2‐complex, 5 and 25 peptides were obtained respectively, both sets of peptides accounting for the whole peptide chain. Examination of the tryptic peptides containing methionine combined with the N‐ and C‐terminal sequences of the αs2‐complex and some CNBr peptides, gave the order of the CNBr peptides, H.CN4CN2CN5CN1CN3.OH, which contain 4, 22, 115, 49 and 17 residues respectively. A partial sequence accounting for half of the peptide chain of the αs2‐complex is given. This peptide chain is likely composed of 207 amino‐acid residues

Structure primaire du caséinomacropeptide de la caseine K caprine

Summary The amino acid sequence of caprine CMP, the negatively charged C-terminal fragment released by chymosin (rennin EC 3.4.23.4) from goat K-casein at the initial stage of the milk-clotting process, has been investigated. The complete sequence has been determined by analysing chymotryptic and « thermolysinfragments of the CMP. Caprine CMP contains 66 amino acid residues, 2 being phosphorylated, Asp2, Asn5, Thr11, Ser6, SerP2, Glu7, Gln2, Pro6, Ala9, Val5, Met1, Ile6, Lys3, His1, and the carbohydrate-free polypeptide chain has a molecular weight of 6,998 daltons. The occurrence in caprine CMP of an additional phosphate group, linked to serine 168 in the C-terminal region Thr-Ser168-Thr-Glu170-Val.OH of the polypeptide chain, has given support to the phosphorylation code for caseins that we postulated earlier. According to this hypothesis, a specific phosphoryl kinase may recognize an anionic phosphorylation site corresponding to the tripeptide sequence Thr/Ser-X-Glu, X being any amino acid residue. Since the C-terminal sequence of bovine and caprine CMPs differ by the substitution Ala/Glu170 (caprine), phosphorylation of caprine serine 168 could be explained by the occurrence of the new phosphorylation site Ser168-Thr-Glu170.

Identification of the two common alleles of the bovine κ-casein locus by the RFLP technique, using the enzyme Hind III

As could be predicted from a comparison of the cDNA sequences established by STEWART et al. (1984) and GORODETSKIY & KALEDIN (1987) the two common alleles of the bovine K-casein locus, K-Cn’ and K-Cn’, can be identified by the restriction fragment length polymorphism (RFLP) technique using either Hind III or Taq I. The latter endonuclease also detects a polymorphism of the DNA strand carrying the allele K-Cn’. However, for determination of both alleles, the use of Hind III is preferable because, according to the data of the above authors, the RFLP detected by that enzyme is specific for the amino-acid substitution responsible for the difference in charge of the two K-casein variants. When DNA is prepared from blood leucocytes, the occurrence of chimaerism in twins may cause difficulties in interpretation.

Complete nucleotide sequence of ovine β-casein cDNA: inter-species comparison

Abstract The complete nucleotide sequence of ovine β-casein mRNA has been determined by sequencing, according to Sanger-Messing, both a recombinant clone isolated from a mammary cDNA pUC 18 library and a single-stranded cDNA generated by reverse transcription from a synthetic 17-mer primer complementary to the 5′ part of the mRNA coding frame. The 1088 nucleotide long β-casein mRNA, excluding the poly(A) tail, contains a coding frame of 669 nucleotides including the stop codon, flanked by 60 adn 359 nucleotides in the 5′ and 3′ untranslated regions, respectively. It arises from the splicing of 9 exons as deduced from gene sequence data. The deduced amino acid sequence differs at 3 positions from that previously determined by direct sequencing of mature β-casein. Comparison of the ovine, bovine, rat, mouse, and rabbit β-casein mRNA sequences shows a higher homology in the 3′ and 5′ untranslated regions. The most conserved regions in the open reading frame are essentially those encoding the signal peptide and the major phosphorylation site.