1 Roslin Institute Edinburgh, Roslin, Midlothian EH25 9PS and 3 Hannah Research Institute, Ayr KA6 5HL, UK
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Abstract |
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Keywords: ß-casein/ß-lactoglobulin/milk/phosphorylation/trans-genic mice
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Introduction |
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Milk proteins are traditionally divided into two classes, the caseins and the serum or whey proteins. The caseins comprise a family of relatively unstructured or rheomorphic phosphoproteins (Holt and Sawyer, 1993) which, under physiological conditions, are predominantly located in calcium phosphate-containing colloidal aggregates, the casein micelles (Holt, 1992
; Rollema, 1992
). In contrast, the bovine whey proteins such as ß-lactoglobulin (BLG),
-lactalbumin and lactoferrin, which, together, typically constitute about 20% of the total protein in the milk, are non-phosphorylated, globular molecules. The lack of phosphorylation of BLG requires explanation since it is thought to follow the same intracellular secretory pathways as the caseins (Mepham et al. 1992
) It has two threonyl residues and one seryl residue that could, in principle, be phosphorylated by the mammary gland casein kinases. Part of the explanation for the lack of secondary modification may be the relatively low activity of the kinases in phosphorylating threonyl residues, even in casein (Mepham et al., 1992
). It was of interest, therefore, to investigate whether a sequence of ß-casein, which is invariably almost fully phosphorylated in the native protein (i.e. the so-called phosphate centre of ß-casein), would also undergo phosphorylation on introduction into a surface site of BLG.
We have shown previously that the major sheep whey protein, BLG, is expressed abundantly in the milk of transgenic mice (Simons et al., 1987; McClenaghan et al., 1995
). Here, we describe the first example of the genetically engineered phosphorylation of a normally unphosphorylated milk protein and report its successful expression and secretion in transgenic mouse lines.
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Materials and methods |
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The ovine BLG cloned gene (pSS1tgXS) comprises 4 kb DNA 5' to the CAP site, 4.9 kb BLG transcription unit and 1.8 kb 3' flanking sequences (Simons et al., 1987). The BLG transcription unit has a unique ClaI site at the extreme 3' end of exon 3 which lies in an external ß-turn in the native folded protein (Brownlow et al., 1997
). This restriction site was used to insert a DNA sequence derived from bovine ß-casein which codes for a phosphorylation centre (Figure 1a
) (Bonsing et al., 1988
). The point of insertion of the amino acid sequence GluSerLeuSerSerSerGluGluSer forming the phosphorylation centre (PC) in the modified BLG protein is shown in Figure 1b
. In order to enhance the incorporation of the PC DNA sequence, the oligomers were designed at the 5' and 3' termini to disrupt the ClaI site upon successful ligation into pSS1tgXS; allowance was also made for bias of codon usage. The plasmid was incubated overnight at room temperature with the annealed PC oligomer in the presence of ClaI and T4 DNA ligase and the products transformed into Escherichia coli DL 43.
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PC/BLG transgenic mice were generated by pronuclear injection of fertilized eggs obtained from superovulated C57BL/6xCBA (F1) mature female mice which had been mated with F1 stud males. Founder transgenic animals were identified by PCR analysis of tail digests (Whitelaw et al., 1991) and transgenic lines were maintained by breeding to F1 mice. Transgenic mice from established BLG lines 7 and 45 (Simons et al., 1987
) were used as a source of mouse milk containing unmodified ovine BLG.
Sample collection and preparation
Milk samples were collected from mice at 11 days of lactation (Simons et al., 1987) and either used fresh or stored at 20°C. Whey fractions were prepared from defatted, fresh milk by centrifugation at 11 600 g in an Eppendorf centrifuge for 15 min.
Polyacrylamide gel electrophoresis, phosphatase treatment and Western blotting
Native PAGE was carried out in Trisglycine buffer at pH 8.6 by the method of Davies and Law (1977) using the Phast system (Amersham International, Little Chalfont, UK). To test for the presence of phosphorylation, protein samples were pre-incubated with either 0.4 or 0.04 units of alkaline phosphatase (Type VII-N from bovine intestinal mucosa, Sigma-Aldrich, Poole, Dorset, UK) for either 30 min or 1 h before loading on the gel.
Analysis of milk and whey samples by SDSPAGE (Laemmli, 1970) was performed using 15% discontinuous gels of 1.5 mm thickness, followed by Coomassie Brilliant Blue G-250 staining. Samples were prepared as described previously (Hitchin et al., 1996
). BLG was purified from fresh, pooled sheep milk (Aschaffenburg and Drewry, 1957
), checked for purity by gel and Western blot analysis and the protein content determined by the micro-Kjeldahl technique (McClenaghan et al., 1995
). The molecular masses of the native ovine BLG and the whey PC/BLG were estimated by plotting the migration distance as a function of the logarithm of the molecular mass of the standards.
Proteins were transferred from gels to nitrocellulose filters by semi-dry Western blotting (Khyse-Anderson, 1984). Filters were blocked with horse serum, reacted serially with rabbit anti-ovine BLG and anti-rabbit IgG horseradish peroxidase and developed with diaminobenzidine and hydrogen peroxide (Dobie, 1996
). For detection of phosphate groups, filters were incubated with mouse anti-phosphoserine monoclonal antibody (Sigma-Aldrich) followed by anti-mouse IgG horseradish peroxidase; these filters were developed using the ECL chemiluminescence method supplied by Amersham Life Science (Amersham International). Rainbow molecular mass markers were also obtained from Amersham Life Science.
Protein fractionation by FPLC
Whey proteins from native and transgenic mice were separated by cation-exchange FPLC on a Mono S column (Pharmacia, Uppsala, Sweden) as detailed elsewhere (Stevenson and Leaver, 1994).
Mass spectrometric analysis
Fractions corresponding to ovine BLG and PC/BLG were collected from cation-exchange FPLC separation of centrifuged whey from transgenic milks. Proteins were desalted by reversed-phase HPLC (Hitchin et al., 1996) and freeze-dried. After redissolving in acetonitrile and H2O (1:1, v/v) containing 0.1% trifluoroacetic acid, the proteins were mixed and their molecular masses determined by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry using an
-cyano-4-hydroxycinnamic acid matrix (Hitchin et al., 1996
).
Infrared spectroscopy
Infrared spectra were recorded at room temperature on KBr discs containing ~0.5% of freeze-dried protein or peptide in the mid-infrared region (4000400 cm1) at a spectral resolution of 2 cm1 and with triangular apodization using a Mattson Galaxy 7000 spectrophotometer. Integration of peak areas and all other manipulations and analyses of the recorded spectra were performed using the FIRST software package supplied with the instrument.
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Results |
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Analysis of milks by SDSPAGE (Figure 2a) showed that the novel protein present in PC/BLG transgenic mouse whey migrated more slowly and hence had a significantly higher molecular mass (~22 900 Da) than native BLG (~18 600 Da). This difference could not be accounted for entirely by the small increase in molecular mass (936 Da) caused by the insertion of nine additional amino acids. Phosphorylation of the protein would explain the aberrant migration of modified BLG observed by SDSPAGE since phosphorylation has been shown to decrease migration in SDSPAGE systems (Green and Pastewka, 1976
). Faint bands were also seen in this region in non-transgenic and BLG 45 milks. These bands were not seen in the whey fractions of these milks, nor did they react with either anti-BLG or anti-phosphoserine antibodies. This indicates that they do not represent trace amounts of phosphorylated BLG in these milks, but are probably minor casein components. The purified PC/BLG isolated from the milk whey migrated as a single band, faster than the ovine protein, on a native gel and this mobility was reduced by pre-incubation with alkaline phosphatase to form, eventually, another single band (Figure 2b
). Throughout this reaction, a maximum of two bands were detected, suggesting only one phosphorylated state is present. Western blotting, using specific, polyclonal antiserum, confirmed that the novel protein was a form of BLG (Figure 3a
) and its reaction with anti-phosphoserine monoclonal antibody provided additional qualitative evidence of phosphorylation (Figure 3b
).
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Discussion |
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A number of experiments with transgenic mice have shown that heterologous and exogenous proteins can also be subjected to complex secondary processing. For example, the human plasma protein -1-antitrypsin requires glycosylation and factor IX,
-carboxylation and ß-hydroxylation, but both are secreted as functional proteins in transgenic mouse milk (Archibald et al., 1990
; Yull et al., 1995
). We have also shown that bovine ß-casein is expressed in its fully phosphorylated form in transgenic mice and incorporated into the endogenous milk micelles (Hitchin et al., 1996
).
By the introduction of a mammary gland casein kinase recognition sequence in the form of a potential casein phosphate centre into the ovine BLG gene, we have demonstrated that this normally unphosphorylated milk protein is phosphorylated in the transgenic mouse mammary gland. Why phosphorylation of the native BLG does not occur and why the chimeric protein is phosphorylated to only a small degree requires explanation.
According to the N + 2 rule for the phosphorylation of caseins by the mammary gland casein kinases (Mercier, 1981), the recognition site for phosphorylation at position N is Ser/ThrXxxGlu/Asp/SerP, where Xxx is any amino acid, although phosphorylation may be reduced if Xxx is bulky and, in general, the extent of phosphorylation is reduced at threonyl sites and at seryl sites if Asp is in the N + 2 position. In a typical casein phosphate centre, such as that used in the present work, the sites form a compact cluster such as SerPLeuSerPSerPSerPGluGlu, but many variations on this sequence motif are found in other caseins.
The physico-chemical methods indicate an average degree of phosphorylation of the whey PC/BLG of between 1.4 and 1.7, suggesting that a mixture of 1-P and 2-P, with possibly smaller proportions of more highly phosphorylated forms, is present. However, the single band seen in the native gel (Figure 2b) appears to demonstrate that only a single degree of phosphorylation is present. Likewise, only two bands were seen at any stage of the dephosphorylation with the alkaline phosphatase, consistent with one phosphorylated residue per molecule. Clearly, in this regard, more work needs to be done.
On the basis of the N + 2 rule, there are three potential sites of phosphorylation in native bovine BLG. These are at Thr49 where the sequence is ThrProGlu, Ser110 where the sequence is Ser-Ala-Glu and Thr125 where the sequence is ThrProGlu. None is known to be phosphorylated in the native bovine protein, as indicated by mass spectrometry (Léonil et al., 1997). A possible reason is that the hydroxyamino acid in the N position or the acidic residue in the N + 2 position are sterically hindered in accepting the phosphate moiety from the kinase. To form a judgement on this, the accessibilities of the relevant residues to water were computed for bovine BLG in the latticexcrystal structure (Brownlow et al., 1997
). The crystal structure of the ovine BLG has been partially determined (Rocha et al., 1996
) and appears, as expected, to be closely similar to the bovine homologue. In the triclinic lattice, the two monomers in the BLG dimer are non-equivalent and in the conventional numbering scheme, residue numbers above 200 belong to the second monomer such that a residue N has a molecular dyad-related pair residue at position 200 + N. Solvent accessibilities for the six sites are given in Table I
. Thr49 lies in a ß-turn conformation between ß-strands B and C, Ser110 lies in a loop between ß-strands G and H, which also includes a single turn helix, and Thr125 also lies in a loop connecting ß-strand H to the 3-turn
-helix. All these sites are reasonably accessible to solvent, as would normally be expected for such hydrophilic residues and are therefore potential phosphorylation sites (Figure 1
). Clearly, some factor other than the N + 2 rule of Mercier (1981) and ready accessibility to the kinase can influence whether a potential site is phosphorylated in practice.
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The PC/BLG does not appear to be incorporated into casein micelles to any significant extent, as demonstrated by the pelleting experiment (Figure 2a). Phosphoseryl groups play a key role in the formation of complexes between
s1-,
s2- and ß-caseins and micellar calcium phosphate, but unlike the highly phosphorylated
s1-,
s2- and ß-caseins, which in bovine milk contain 89, 1113 and 5 phosphate groups, respectively, the low level of whey PC/BLG phosphorylation may be insufficient for incorporation into micelles. Such a conclusion is supported by the evidence of phosphoproteins with a low degree of phosphorylation in the whey of milks of a number of species. Thus, the abundant rat WAP contains the sequence SerSerSerGluAsp which closely resembles a casein phosphate centre and is, indeed, singly phosphorylated (Hennighausen et al., 1982
). In contrast, the corresponding mouse protein sequence is AlaSerProIleGly, which does not undergo phosphorylation. In human milk, the ß-casein occurs in forms whose extent of phosphorylation varies between 0 and 5 mol P/mol and whereas the more highly phosphorylated forms (3-P to 5-P) are found in pelleted casein (Greenberg et al., 1984
) the unphosphorylated form is predominantly in the whey (Monti and Jollés, 1982
). Likewise, in experiments on the formation of artificial casein micelles formed from whole human casein and colloidal calcium phosphate, the micelles were found to contain only 5-P and 4-P forms of the ß-casein together with a trace of 3-P (Aoki et al, 1992
). The conclusion was that more than three phosphorylated residues are required for an interaction with the colloidal calcium phosphate of casein micelles. The low level of PC/BLG in the pelleted casein micelle fraction of the transgenic mouse milk can therefore be ascribed to a low proportion of 3-P and 4-P phosphorylated forms. Although phosphatase inhibitors were occasionally added to freshly collected milk samples without any apparent influence on the degree of phosphorylation of the PC/BLG, more extensive kinase-catalysed phosphorylation of the protein followed by partial dephosphorylation by phosphatase activity, within the mammary gland, cannot be ruled out.
The next phase of this work will involve the analysis of the structure and physical properties of this novel phosphorylated protein.
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Acknowledgments |
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Notes |
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4 To whom correspondence should be addressed.E-mail: leaverj{at}hri.sari.ac.uk
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References |
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Received July 6, 1998; revised November 23, 1998; accepted November 27, 1998.