The phosphorylation pattern of human a s1 -casein is markedly different from the ruminant species Esben S. Sørensen, Lise Møller, Maria Vinther, Torben E. Petersen and Lone K. Rasmussen* Protein Chemistry Laboratory, Department of Molecular Biology, University of Aarhus, Denmark Caseins are highly phosphorylated milk proteins assembled in large colloidal structures termed micelles. In the milk of ruminants, a s1 -casein has been shown to be extensively phosphorylated. In this report we have determined the phosphorylation pattern of human a s1 -casein by a combi- nation of matrix-assisted laser desorption mass spectrometry and amino acid sequence analysis. Three phosphorylation variants were identified. A nonphosphorylated form, a variant phosphorylated at Ser18 and a variant phosphory- lated at Ser18 and Ser26. Both phosphorylation sites are located in the amino acid recognition sequence of the mammary gland casein kinase. Notably, no phosphoryla- tions were observed in the conserved region covering resi- dues Ser70–Glu78, which is extensively phosphorylated in the ruminant a s1 -caseins. Keywords: a s1 -casein; human milk; mammary gland casein kinase; phosphorylation. Caseins are the predominant milk proteins of most mammalian species [1]. In ruminants, about 75% of the milk protein content is constituted of caseins. The corresponding figure for human milk is only about 40% [2]. In the milk of ruminants, caseins interact with calcium phosphate forming large stable colloidal particles termed micelles. These micellar complexes make it possible to maintain a supersaturated calcium phosphate concentra- tion in milk, providing the newborn with sufficient calcium phosphate for the mineralization of the rapidly growing calcified tissues. In this context, the phosphory- lation of the individual caseins plays a significant role in the interaction with calcium phosphate and thereby the organization of the micelles. The ruminant caseins, which are the most intensely studied, comprise a s1 -, a s2 -, b-and j-casein. Their phosphorylation pattern has been the basis of many studies and the general feature is that they are highly phosphorylated proteins, phosphorylated by the mammary gland casein kinase [3–7]. The primary requirement for phosphorylation by this kinase is a glutamate, a phospho- serine or an aspartate two residues to the C-terminal side of the phosphoacceptor site (S-x-E/Sp/D) [8,9]. Compared with ruminants, human milk contains a very low concentration of calcium phosphate and the function of casein in delivering calcium to the neonate is therefore muted in this species. In human milk, the predominant caseins are j-andb-casein, which differ from its ruminant counterpart by a lower degree of phosphorylation [10]. For many years it was generally accepted that a s1 -casein was absent or present in only very small amounts in human milk [2]. In the mid-1990s, two groups isolated and sequenced a minor 27-kDa casein component that was identified as being the human counterpart of a s1 -casein [11,12]. In addition, it was shown that this a s1 -casein component forms disulfide-bonded heteromultimers with j-casein in human milk [12]. The molecular cloning and sequencing of mRNA transcripts revealed the presence of three forms of a s1 -casein in human milk [13,14]. In the present study, we report the phosphorylation pattern of human a s1 -casein. Materials and methods Materials Trypsin (EC 3.4.21.4) was obtained from Worthington Biochemical Corporation (Freehold, NJ, USA). Vydac C 4 and C 18 reverse-phase resins were from The Separations Group (Hesperia, CA, USA) and the RP C 2 /C 18 column was from Amersham Biosciences AB (Uppsala, Sweden). Reagents used for sequencing were from Applied Biosys- tems (Foster City, CA, USA). All other reagents were of analytical reagent grade. Purification of human a s1 -casein Human a s1 -casein was purified from human milk as described [12]. During this procedure, the protein was reduced and alkylated to dissociate the disulfide-linked complex consisting of a s1 -andj-casein. To remove small residual amounts of j-casein, the protein was subjected to reverse-phase chromatography on a Vydac C 4 reverse-phase column. The purity of the resulting a s1 -casein was verified by SDS/PAGE and N-terminal amino acid sequence analysis. Correspondence to E. S. Sørensen, Protein Chemistry Laboratory, Department of Molecular Biology, University of Aarhus, Science Park, Gustav Wieds Vej 10, DK-8000 Aarhus, Denmark. Fax: + 45 8 6136597, Tel.: + 45 8 9425092, E-mail: ess@imsb.au.dk Enzyme: Trypsin (EC 3.4.21.4) *Present address: Symphogen A/S, DK-2800 Denmark. (Received 5 May 2003, revised 14 July 2003, accepted 16 July 2003) Eur. J. Biochem. 270, 3651–3655 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03755.x Generation, separation and characterization of peptides Approximately 300 lg of reduced and alkylated human a s1 -casein was digested with trypsin using a ratio of enzyme to substrate of 1 : 100 (w/w) in 0.1 M ammonium bicar- bonate, pH 8.1, at 37 °C for 6 h. Separation of the peptides was carried out by reverse-phase HPLC on a Vydac C 18 column and detected in the effluent by measuring the absorbency at 226 nm (as described in the legend to Fig. 1). Fraction 35 (Fig. 1) was rechromatographed by reverse- phase HPLC on a SMART-system equipped with a 2.1 · 100 mm C 2 /C 18 RPC column using a gradient of acetonitrile in 0.05% heptafluorobutyric acid at 25 °C. Peptides were characterized by mass spectrometric- and amino acid sequence analysis. Mass spectrometric analyses of the peptides were performed using a MALDI-TOF mass spectrometer (Voyager DE PRO, Applied Biosystems Inc.). Theoretical peptide masses were calculated using the GPMAW program (Lighthouse Data, Odense, Denmark). Amino acid sequence analysis was performed on an automated amino acid sequencer (ABI 477A/120A; Applied Biosystems Inc.). To locate phosphoserines in the sequence, phosphopeptides were treated with ethanethiol to convert phosphoserine into S-ethylcysteine [15] which can be identified by amino acid sequence analysis as PTH- S-ethylcysteine after its release in the corresponding cycle. PTH-S-ethylcysteine eluted just before the diphenylthiourea peak in the system used [16]. Results and discussion Human a s1 -casein was purified in a reduced and carboxy- methylated state as described [12]. The protein was digested with trypsin and the resulting peptides were separated by reverse-phase chromatography (Fig. 1). Fractions were collected and the peptides were characterized by mass spectrometric- and sequence analysis. The combined results are shown in Table 1. The amino acid sequence of human a s1 -casein is shown in Fig. 2. Peptides identified by mass spectrometric analysis and/or sequence analysis are under- lined. As seen in the Fig. 2, peptides covering the entire Fig. 1. Reversed-phase separation of a trypsin digest of human a s1 -casein. Human a s1 -casein was digested with trypsin as described in Materials and methods. Peptides were eluted with a gradient of 80% acetonitrile in 0.1% trifluoroacetic acid (dotted line) on a Vydac C 18 (10 lm) column (4 · 250 mm). The col- umn was operated at 40 EC and the flow rate was 0.85 mLÆmin )1 . Peptides were detected in the effluent by recording the absorbance at 226 nm (solid line), collected manually and characterized as described in the text. Table 1. Characterization of peptides from the tryptic digest of human a s1 -casein. Peak numbers designations correspond to those of Fig. 1. The amino acid sequence was identified by sequence analysis and/or MALDI-TOF MS. Calculated MH + , calculated protonated mono- isotopic masses; observed MH + , molecular monoisotopic protonated mass determined by MALDI-TOF MS. Peak number Sequence Calculated MH + Observed MH + 11 45–50 733.37 733.29 16 8–11 564.28 564.25 17 43–50 990.51 990.55 19 45–53 1105.55 1105.5 23 1–7 879.59 879.6 25 84–90 972.36 972.29 25 54–67 1605.71 1605.77 26 39–42 515.33 515.27 26 28–36 1143.46 1143.38 28 4–7 498.34 498.24 35–1 a 12–27+2P 1942.82 1942.78 35–2 a 68–83 1718.75 1718.75 36 4–11 1043.6 1043.62 37 12–27+1P 1862.86 1862.78 38 12–27 1782.89 1782.86 41 12–36+2P 3067.34 3067.26 43 12–36+1P 2987.34 2987.26 44 91–109 2267.16 2267.15 49 164–171 904.44 904.38 53 142–163 2580.21 2580.44 54 111–132 2591.24 2591.18 54 133–141 1170.57 1170.5 62 133–163 3731.75 3731.12 65 111–163 6303.97 6303.61 a Peaks from rechromatography of fraction 35 from Fig. 1. 3652 E. S. Sørensen et al. (Eur. J. Biochem. 270) Ó FEBS 2003 sequence of human a s1 -casein have been identified and characterized in this study. Glycosylation Three asparagine residues in human a s1 -casein (Asn14, Asn54, Asn154) are located in the putative glycosylation sequence Asn-X-Ser/Thr. In the case of Asn14 and Asn154, a neighbouring proline residue in position X corrupts the glycosylation sequence and renders it unfit for glycosylation. Regarding Asn54, this study did not show any evidence for glycosylation of this residue in human a s1 -casein. Mass spectrometric analysis of peak 25 (Fig. 1) containing the peptide Asn54–Lys67 showed a mass of 1605.77 Da which corresponds to the calculated protonated monoisotopic mass (1605.71 Da) of the unmodified peptide sequence, thereby showing that Asn54 was not glycosylated in human a s1 -casein. Likewise in this study, we observed no O-glycosylations in human a s1 -casein. Phosphorylation Human a s1 -casein contains 16 serines and four threonines, where nine of the serines and one threonine are located in the recognition sequence of the mammary gland casein kinase [8,9]. The recognition sequence (Ser/Thr-X-Glu/ Ser(P)/Asp), comprises an acidic residue, glutamic acid, aspartic acid or a phosphorylated residue, as the second amino acid to the C-terminal side of the serine or threonine to be targeted. Especially interesting, human a s1 -casein contains a serine rich region, SSISSSSEE(70–78), where five of the serines are located in the recognition sequence of the mammary gland casein kinase. This region is highly conserved among all species with known a s1 -casein sequences (for alignment see [13]), and in all analyzed species a high degree of phosphorylation has been observed in this region [3–5]. Our laboratory has much experience of employing MALDI-TOF mass spectrometric analysis for identification and localization of phosphorylation sites in proteins [16–18]. MALDI-TOF mass spectrometric analysis of a phospho- peptide results in a spectrum with an easily identifiable fragmentation pattern which is characteristic for phospho- rylated serines. These spectra contain a series of peaks separated by approximately 98 Da, which represents the fragmentation of a phosphoserine to dehydroalanine. In this work, we have analyzed all fractions from the reverse-phase HPLC separation of the tryptic digest of human a s1 -casein (Fig. 1) by MALDI-TOF mass spectro- metric and N-terminal sequence analysis. We identified four fractions with the characteristic fragmentation pattern of peaks at 35, 37, 41 and 43. A representative MALDI-TOF spectrum of peak 41 showing the characteristic fragmenta- tion of a phosphopeptide is shown in Fig. 3. Peak 35 was found to contain two peptides which potentially could be phosphorylated, thus the fraction was rechromatographed by reversed-phase HPLC on a SMART HPLC system to separate the two components, 35–1 and 35–2. Peak 35–2 gave a mass spectrum with only one mass at 1718.75 Da which is identical with the calculated protonated mass for the tryptic peptide covering residues 68–83, thereby showing that this peptide is not phosphorylated in human a s1 -casein. This observation was confirmed by Edman sequencing of the peptide, which showed normal yields of PTH-serine in all relevant cycles. Mass analysis of peak 35–1 showed a mass of 1942.78 Da, as well as two populations of ions at approximately )98 Da and )196 Da. This triplet of ions, each separated by approximately 98 Da, indicates that peak 35–1 contains a phosphopeptide with two phosphoserines. Furthermore, the observed mass at 1942.78 Da correlates with the calculated protonated mass (1942.82 Da) for the tryptic peptide covering residues 12–27 and containing two phosphorylations (159.93 Da). Mass analysis of peak 37 showed a mass of 1862.78 Da, and a single fragmentation ion at )98 Da was observed, indicating the presence of a single phosphorylation in the peptide. Furthermore, the Fig. 2. Localization of phosphorylations in human a s1 -casein. The amino acid sequence was deduced from the cDNA sequence [11]. Solid lines indicate isolated and characterized peptides (Table 1). P denotes identified phosphorylation. Peptides are numbered according to the reversed-phase elution profile in Fig. 1. Fig. 3. MALDI-TOF MS of peak 41 from Fig. 1. The protonated mass at m/z 3067.26 corresponds to the peptide 12–36 including to phosphorylations. The characteristic fragmentation pattern confirms the presence of two phosphorylations in the peptide. Ó FEBS 2003 Phosphorylation of human a s1 -casein (Eur. J. Biochem. 270) 3653 mass 1862.78 Da correlates with the calculated protonated mass of residues 12–27 containing a single phosphorylation (1862.86 Da). Finally, mass analysis of peak 38 showed a mass which correlates with the mass of the peptide covering residues 12–27 without any modifications. In conclusion, we have observed the peptide 12–27 in three different forms, with zero, and one and two phosphate groups attached. Peaks 41 and 43 represent peptide 12–36 with two and one phosphorylated groups, respectively. These peptides, result- ing from incomplete cleavage at Arg27, do not contain any additional serines or threonines compared with peaks 35 and 37, and thus they were not characterized further. The peptide, LQNPSESSEPIPLESR(12–27) (peak 35–1), contains three serines located in the recognition sequence of the mammary gland casein kinase (Ser16, Ser18 and Ser26). To determine which of the serines are in fact phosphorylated, we subjected the two peptides to an ethanethiol treatment followed by Edman sequencing as outlined in Materials and methods. The ethanethiol treat- ment converts the labile phosphoserine residues into S-ethylcysteine, which is more stable and able to withstand the relatively harsh Edman chemistry during automated sequencing [15]. Furthermore, PTH-S-ethylcysteine elutes in an open window just before the diphenylthiourea peak in the on-line HPLC system used in these studies. Sequence analysis of peptide 35–1 succeeding the ethanethiol treat- ment revealed PTH-S-ethylcysteine in cycles 7 and 15, corresponding to Ser18 and Ser26 in human a s1 -casein. Likewise, sequence analysis of peptide 37, after the ethanethiol treatment gave PTH-S-ethylcysteine in sequence cycle 7, corresponding to Ser18 in human a s1 -casein. The yields of PTH-serine in cycles corresponding to Ser16 and Ser19 were as expected, indicating that these residues were not phosphorylated. As a control experiment (data not shown) human and bovine b-casein were purified, tryptic digests were generated and these were separated by reversed-phase HPLC using the system and column described in Materials and methods. In MALDI-TOF MS analyses of the human b-casein digest, two peptides with protonated masses of 2407.85 and 2327.80 were identified. These masses correspond to the expected masses of the peptide 1–18 of human b-casein with four phosphorylations (2408.00) and three phosphoryla- tions (2328.00), respectively. Likewise in the digest of bovine b-casein, a peptide with a protonated mass of 3122.27 was observed, corresponding to the peptide 1–25 of bovine b-casein with four phosphorylations (3122.40 Da). These results indicate that the protocol used for identification of phosphorylation sites is capable of handling highly phos- phorylated peptides. Furthermore, the methods used in the present study have previously been used for identification of phosphorylation sites in several milk proteins in our laboratory, most prominently the 28 phosphorylation sites in bovine milk osteopontin [16]. Therefore it is not likely that the lack of identification of a highly phosphorylated peptide in a s1 -casein is due to limitations of the techniques used. Finally, MALDI-TOF MS analysis of native human a s1 -casein showed ions corresponding to a mass of approxi- mately 20 232 Da, which correlates well with the calculated mass of human a s1 -casein including two phosphate groups (20246 Da) (Fig. 4). In conclusion, these studies show that human a s1 -casein exists in three phosphorylation variants. A nonphosphory- lated form, a variant containing a single phosphorylation at Ser18 and a variant phosphorylated at Ser18 and Ser26. It is difficult to determine the quantitative relation between the three phosphorylation variants, but judged by the reversed-phase HPLC trace in Fig. 1, the variant containing a single phosphorylation at Ser18 is the major variant ( 50%), followed by the nonphosphorylated form ( 30%) and the doubly phosphorylated variant ( 20%). The degree of identity between human and other known a s1 -casein sequences is overall low (alignment of sequences is shown in [6,13]). The phosphorylations have been charac- terized in ovine (Ovis aries), caprine (Capra hircus), bovine (Bos taurus), water buffalo (Bubalus bubalis)andcamel (Camelus dromedarius) a s1 -casein. Generally, all of these species have been reported to have a significant higher number of phosphorylations than shown to be the case for human a s1 -casein in this study. Bovine a s1 -casein is phos- phorylated at up to nine positions depending on the genetic variants [3], the ovine a s1 -casein is phosphorylated at up to 11 positions [5], the water buffalo a s1 -casein is phospho- rylated at 6–8 positions [6], the caprine counterpart is phosphorylated at 9–10 positions [4], and camel a s1 -casein is phosphorylated at up to six serines [7]. The most striking difference in the phosphorylation pattern between human a s1 -casein and its ruminant counterparts is the shortage of phosphorylation in the serine-rich region consisting of residues Ser70–Glu78 in the human sequence. This region has been shown to be highly phosphorylated in all the above mentioned species. In this study, we have isolated the tryptic peptide covering residues Met68–Lys83, which contains the serine-rich region, in a nonphosphorylated form, and no traces of a phosphorylated form of this peptide were observed. The lack of phosphorylation at other positions reported to be modified in ovine, caprine and bovine a s1 -casein (serines 41, 46, 48, 75 and 115 in all species, and Ser12 in ovine a s1 -casein, all numbers referring to the ruminant sequences), can simply be explained by sequence substitu- tions at these positions, leaving no hydroxyamino acids to be phosphorylated at these positions in human a s1 -casein. The region containing the phosphorylated residues, Ser18 Fig. 4. MALDI-TOF MS of intact human a s1 -casein. The peak at m/z 20233 corresponds well to the calculated mass of human a s1 -casein including two phosphate groups (20246 Da). The peak at m/z 10121 represents the doubly protonated species (M2H + ). 3654 E. S. Sørensen et al. (Eur. J. Biochem. 270) Ó FEBS 2003 and Ser26, in human a s1 -casein is not especially well conserved among the other analyzed species. The sequence containing Ser18 in the human a s1 -casein is part of exon 3 in the human a s1 -casein gene, which is not present in the ruminant species [13]. Ser26, situated in exon 5 of the human a s1 -casein gene, is not conserved in any other species except the wallaby, in which the phosphorylation pattern has not been determined [19]. During the review of these results, we were puzzled by the lack of phosphorylation in the conserved region Ser70– Glu78, which is so extensively phosphorylated in the ruminant species. To test whether our results were repre- sentative, milk from three different women was analyzed. a s1 -Casein was purified and the reversed-phase traces of tryptic digests of the protein were compared and found to be identical in all cases, thereby showing similar phosphoryla- tion of the protein in different individuals. The phosphory- lation pattern of human a s1 -casein described here, and especially the lack of phosphorylations in the region Ser70– Glu78, is therefore unlikely to be a result of intra-species post-translational polymorphism in the protein. However, it should be emphasized that it is more difficult to show the absence of a modification convincingly than its presence; hence the existence of minor species partially phosphory- lated at the region discussed can not be entirely excluded. The deletion of 11 amino acids at positions 59–69 and of 37 amino acids at positions 59–95 in caprine a s1 -casein leads to the variants D and F. In both cases these deletions, which start at the same position of the polypeptide chain, include the major phosphorylation site of the protein [20]. In ruminant milk, a s1 -casein, as well as the other three caseins a s2 -, b-andj-casein is present in micellar structures responsible for the calcium transport to the neonates. Compared with ruminant milk, the milk of primates holds a much lower concentration of calcium and a function of a s1 - casein in calcium transport in human milk is not likely. Recent studies of caprine a s1 -casein suggest that the protein interacts with the other caseins in the rough endoplasmic reticulum and that the formation of this complex is required for their efficient export to the Golgi apparatus [21]. Whether a similar scenario exists in the human system remains to be elucidated. Acknowledgments Special thanks to H. Breinholt and K E. Højbjerg, Department of Obstetrics and Gynaecology, University Hospital of Aarhus, for providing the individual milk samples. References 1. Jennes, R. & Holt, C. (1987) Casein and lactose in milk of 31 species are negatively correlated. Experentia 43, 1015–1018. 2. Kunz, C. & Lo ¨ nnerdal, B. (1990) Casein and casein subunits in preterm milk, colostrum, and mature human milk. J. Pediatr. Gastroenterol. Nutr. 10, 454–461. 3. Mercier, J.C., Grosclaude, F. & Ribadeau-Dumas, B. (1971) Structure primaire de la case ´ ine a s1 -bovine. Eur. J. Biochem. 23, 41–51. 4. Ferranti, P., Addeo, F., Malorni, A., Chianese, L., Leroux, C. & Martin, P. (1997) Differential splicing of pre-messenger RNA produces multiple forms of mature caprine a s1 -casein. Eur. J. Biochem. 249, 1–7. 5. Ferranti, P., Malorni, A., Nitti, G., Laezza, P., Pizzano, R., Chi- anese, L. & Addeo, F. (1995) Primary structure of ovine a s1 - caseins: localization of phosphorylation sites and characterization of genetic variants A, C and D*. J. Dairy Res. 62, 281–296. 6. Ferranti, P., Scaloni, A., Caira, S., Chianese, L., Malorni, A. & Addeo, F. (1998) The primary structure of water buffalo a s1 -and b-casein: characterization of a novel b-variant. J. Protein Chem. 17, 835–844. 7. Kappeler, S., Farah, Z. & Puhan, Z. (1998) Sequence analysis of Camelus dromedarius milk caseins. J. Dairy Res. 65, 209–222. 8. Mercier, J.C. (1981) Phosphorylation of caseins, present evidence for an amino acid triplet code posttranslationally recognized by specific kinases. Biochimie (Paris) 63, 1–17. 9.Lasa-Benito,M.,Marin,O.,Meggio,F.&Pinna,L.A.(1996) Golgi apparatus mammary gland casein kinase: monitoring by a specific peptide substrate and definition of specificity determi- nants. FEBS Lett. 382, 149–152. 10. Greenberg, R., Groves, M.L. & Dower, H.J. (1984) Human beta- casein: amino acid sequence and identification of phosphorylation sites. J. Biol. Chem. 259, 5132–5138. 11. Cavaletto, M., Cantisani, A., Gluffrida, G., Napolitano, L. & Conti, A. (1994) Human a s1 -casein like protein: purification and N-terminal sequence determination. Biol. Chem. Hoppe-Seyler 375, 149–151. 12. Rasmussen, L.K., Due, H.A. & Petersen, T.E. (1995) Human a s1 - casein: purification and characterization. Comp. Biochem. Physiol. 111B, 75–81. 13. Johnsen, L.B., Rasmussen, L.K., Petersen, T.E. & Berglund, L. (1995) Characterization of three types of human a s1 -casein mRNA transcripts. Biochem. J. 309, 237–242. 14. Martin,P.,Brignon,G.,Furet,J.P.&Leroux,C.(1996)Thegene encoding a s1 -casein is expressed in human mammary epithelial cells during lactation. Lait 76, 523–535. 15. Meyer, H.E., Hoffmann-Posorske, E., Korte, H. & Heilmyer, M.G. Jr (1986) Sequence analysis of phosphoserine-containing peptides: modification for picomolar sensitivity. FEBS Lett. 204, 61–66. 16. Sørensen, E.S., Højrup, P. & Petersen, T.E. (1995) Posttransla- tional modifications of bovine osteopontin: identification of twenty-eight phosphorylation and three O-glycosylation sites. Protein Sci. 4, 2040–2049. 17.Sørensen,E.S.&Petersen,T.E.(1994)Identificationoftwo phosphorylation motifs in bovine osteopontin. Biochem. Biophys. Res. Commun. 198, 200–205. 18. Rasmussen, L.K., Sørensen, E.S., Petersen, T.E., Nielsen, N.C. & Thomsen, J.K. (1997) Characterization of phosphate sites in native ovine, caprine and bovine casein micelles and their case- inomacropeptides: a solid-state 31 P NMR and sequence and mass spectrometric study. J. Dairy Sci. 80, 607–614. 19. Ginger, M.R., Piotte, C.P., Otter, D.E. & Grigor, M.R. (1999) Identification, characterisation and cDNA cloning of two caseins from the common brushtail possum (Trichosurus vulpecula)1. Biochim. Biophys. Acta 1427, 92–104. 20. Brignon, G., Mahe, M.F., Ribadeau-Dumas, B., Mercier. J.C. & Grosclaude, F. (1990) Two of the three genetic variants of goat alpha s1-casein which are synthesized at a reduced level have an internal deletion possibly due to altered RNA splicing. Eur. J. Biochem. 193, 237–241. 21. Chanat, E., Martin, P. & Ollivier-Bousquet, M. (1999) a s1 -casein is required for the efficient transport of b-andj-casein from the endoplasmic reticulum to the Golgi apparatus of the mammary epithelial cells. J. Cell Sci. 112, 3399–3412. Ó FEBS 2003 Phosphorylation of human a s1 -casein (Eur. J. Biochem. 270) 3655 . The phosphorylation pattern of human a s1 -casein is markedly different from the ruminant species Esben S. Sørensen, Lise Møller, Maria Vinther,. transcripts revealed the presence of three forms of a s1 -casein in human milk [13,14]. In the present study, we report the phosphorylation pattern of human a s1 -casein. Materials