Structure primaire de la caséine κB bovine
Authors: Mercier Jean‐Claude , Brignon Ghislaine , Ribadeau‐dumas Bruno 
Published: March 2005

In a previous report [1], we have given the complete primary structure of ϰB1‐caseinomacro‐peptide which is the soluble COOH‐terminal fragment split from bovine ϰB1‐casein by rennin. We also reported on the COOH‐terminal sequence of the NH2‐terminal fragment, the so‐called para‐ϰ‐casein.

The present paper deals with the complete amino acid sequence of bovine ϰB‐casein, which has now been achieved by establishing the primary structure of para‐ϰB‐casein of which we discuss the salient features. This work has been reported briefly in a short communication [2]. SCM‐para‐ϰB‐casein and maleyl ϰBCN1, the NH2‐terminal cyanogen bromide fragment of ϰB‐casein [1], were used as starting material. The tryptic and peptic peptides of SCM‐para‐ϰB‐casein and the chymotryptic peptides of ϰBCN1 were isolated on Dowex 50 and Sephadex G‐50 or G‐25, and their sequences were determined either partially or completely by using classical methods and in some cases mass spectrometry. All these peptides and a NBS fragment of SCM‐para‐ϰB‐casein have provided all the overlaps needed for the completion of the amino acid sequence of para‐ϰB‐casein.

Para‐ϰB‐casein is a single polypeptide chain containing 105 amino acid residues: Asp3, Asn4, Thr3, Ser7, PyroGlu1, Glu4, Gln12, Pro12, Gly1, Ala9, Val5, 1/2 Cys2, Met1, Ile6, Leu7, Tyr9, Phe4, Lys6, His3, Trp1, Arg5, and its molecular weight has been calculated to be 12269. The average hydrophobicity, calculated according to Bigelow [3], is 5.48 kJ (1.310 kcal) per residue, and para‐ϰB‐casein can be therefore considered to be a very hydrophobic molecule. Its net positive charge at pH of native milk (about 6.8) is very close to 4.5. The high content (11.5%) and rather uniform distribution of prolyl residues are incompatible with much α‐helical organization of the molecule, as previously shown for ϰ‐casein [4]. Both hydrophobic and charged amino acid residues are distributed non‐uniformly along the chain. Two regions, 1–24 and 80–105, are hydrophilic: the very hydrophilic former, with NH2‐terminal pyroglutamic acid, contains a cysteinyl residue located inside a cluster of eleven ionizable residues including six out of the seven total dicarboxylic amino acids; the 80–105 region, which contains the second cysteinyl residue in position 88, is rather hydrophilic, except at the COOH‐terminal end which is hydrophobic in spite of the presence of a cluster of four basic residues. These two hydrophilic regions are very likely to be on the outisde of the molecule and this may favor the formation of intermolecular S‐S bonds. The very hydro‐phobic central part of the chain, viz., 25–79, where most of the aromatic residues are located, has a para‐ϰ‐casein‐like behaviour in aqueous solution, and it may be responsible for the aggregation ability of para‐ϰ‐casein.

According to the sequence data of both ϰB1‐caseinomacropeptide [1] and para‐ϰ‐casein, it is concluded that bovine ϰB‐casein is made up of a single polypeptide chain containing 169 amino acid residues: Asp4, Asn7, Thr14, Ser12, SerP1, PyroGlu1, Glu12, Gln14, Pro20, Gly2, Ala15, Val11, 1/2 Cys2, Met2, Ile13, Leu8, Tyr9, Phe4, Lys9, His3, Trp1, Arg5, with NH2‐terminal pyroglutamic acid. However, in accordance with the well‐known lability of glutamine residues in NH2‐terminal position [5], such a pyroglutamic acid residue may arise from the subsequent cyclisation of a NH2‐terminal glutamine residue present originally in native ϰ‐casein. The content of proline residues is high (11.8%). The molecular weight of the carbohydrate‐free monomer of ϰB‐casein is 19023. Its net negative charge at pH of native milk is close to 3.5 and its average hydrophobicity is about 5.37 kJ (1.285 kcal) per residue.

We have already reported [6] the location of the two amino acid substitutions differentiating the two known genetic variants : the B variant differs from the A variant by the respective amino acid substitutions Ile 136/Thr and Ala 148/Asp.

Tryptic and chymotryptic peptides isolated by Jollés et al. [7] from ϰA‐casein correspond obviously to the fragments 11–16, 17–25,31–34, 63–67, 1–10 and 86–105 of ϰ‐casein strand, but there is only partial agreement concerning the sequences of amino acid proposed for the last three fragments. More recently, Fiat et al. [8] have studied a short glycopeptide isolated from bovine ϰ‐casein, but neither the amino acid sequence nor the location of the phosphate group agree with our results.