Insertion of a casein kinase recognition sequence induces phosphorylation of ovine ß-lactoglobulin in transgenic mice

M. McClenaghan1, E. Hitchin1,2, E.M. Stevenson3, A.J. Clark1, C. Holt3 and J. Leaver3,4

1 Roslin Institute Edinburgh, Roslin, Midlothian EH25 9PS and 3 Hannah Research Institute, Ayr KA6 5HL, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We have shown that the cellular mechanisms of the mammary gland can be used to produce a phosphorylated form of a normally unphosphorylated milk protein. This was achieved by the insertion of a ß-casein DNA sequence coding for a group of mammary gland casein kinase recognition sites into ovine ß-lactoglobulin. Transgenic mice carrying this modified gene were generated and lactating females were shown to produce a novel ß-lactoglobulin in their milk. The infrared spectrum, reactivity to anti-phosphoserine antibody and reduction of electrophoretic mobility on treatment with alkaline phosphatase showed that the novel protein recovered from the milk whey (serum) was phosphorylated and molecular mass determination by mass spectrometry was consistent with the phosphorylation of one or two residues. A similar level of phosphorylation was measured by quantitative infrared spectroscopy. Centrifugation of the milk to pellet the casein micelles showed that most of the phosphorylated ß-lactoglobulin was in the whey and hence not incorporated into casein micelles.

Keywords: ß-casein/ß-lactoglobulin/milk/phosphorylation/trans-genic mice


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The development of transgenic technology has allowed the direct manipulation of animal genomes with useful outcomes such as the production of bio-pharmaceuticals in ruminant milk (Clark et al., 1989Go; Wright et al., 1991Go). The use of this technology to improve milk quality has been reviewed (Jimenez-Florés and Richardson, 1988Go; Clark, 1992Go; Dalgleish, 1992Go; Martin and Grosclaude, 1993Go; Maga and Murray, 1995Go; Leaver 1996Go). Projects to `humanize' bovine milk are also under way; for example, the expression of human lactoferrin in transgenic animals has been investigated in mice and cattle (Krimpenfort et al., 1991Go; Platenburg et al., 1994Go). While approaches to date have sought to modify the expression levels of endogenous or heterologous proteins (Maga and Murray, 1995Go), the introduction of subtle modifications to milk protein genes that could tailor the functional properties of proteins for use in particular products has not yet been attempted.

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, 1993Go) which, under physiological conditions, are predominantly located in calcium phosphate-containing colloidal aggregates, the casein micelles (Holt, 1992Go; Rollema, 1992Go). In contrast, the bovine whey proteins such as ß-lactoglobulin (BLG), {alpha}-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. 1992Go) 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., 1992Go). 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., 1987Go; McClenaghan et al., 1995Go). 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.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Construct design

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., 1987Go). 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., 1997Go). This restriction site was used to insert a DNA sequence derived from bovine ß-casein which codes for a phosphorylation centre (Figure 1aGo) (Bonsing et al., 1988Go). The point of insertion of the amino acid sequence Glu–Ser–Leu–Ser–Ser–Ser–Glu–Glu–Ser forming the phosphorylation centre (PC) in the modified BLG protein is shown in Figure 1bGo. 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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Fig. 1. (a) Phosphorylation centre/BLG (PC/BLG) construct. The black line represents the BLG gene with exons marked as black boxes and the position of the insert sequence arrowed. The oligomer nucleotide sequences are shown and bold font indicates the inserted 27-mer PC sequence, coding for Glu–Ser–Leu–Ser–Ser–Ser–Glu–Glu–Ser, flanked by BLG sequences (italics); the insert bases which serve to disrupt the BLG ClaI site are underlined. (b) Ribbon 3-D structure of the bovine BLG crystallized protein showing the position in a surface loop of Asp-85 where the casein kinase recognition sequence from ß-casein was inserted.

 
Transgenic mice

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., 1991Go) and transgenic lines were maintained by breeding to F1 mice. Transgenic mice from established BLG lines 7 and 45 (Simons et al., 1987Go) 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., 1987Go) 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 Tris–glycine 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 SDS–PAGE (Laemmli, 1970Go) 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., 1996Go). BLG was purified from fresh, pooled sheep milk (Aschaffenburg and Drewry, 1957Go), checked for purity by gel and Western blot analysis and the protein content determined by the micro-Kjeldahl technique (McClenaghan et al., 1995Go). 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, 1984Go). 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, 1996Go). 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, 1994Go).

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., 1996Go) 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 {alpha}-cyano-4-hydroxycinnamic acid matrix (Hitchin et al., 1996Go).

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 (4000–400 cm–1) at a spectral resolution of 2 cm–1 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.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A mammary gland casein kinase recognition sequence was inserted into the third exon of the ovine BLG gene (PC/BLG, Figure 1aGo), thus incorporating a cluster of four potential phosphorylation sites at the Asp residue at position 85 which lies in a surface ß-turn between ß-strands E and F (Figure 1bGo). This PC/BLG construct was introduced into fertilized mouse eggs by pronuclear injection. Nine G0 transgenic mice were identified and eight transgenic lines were established. Females from six of these lines expressed the transgene protein in milk in amounts ranging from ~1 to 7 mg/ml and the two highest expressing lines, 29 and 66, were selected for further studies.

Analysis of milks by SDS–PAGE (Figure 2aGo) 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 SDS–PAGE since phosphorylation has been shown to decrease migration in SDS–PAGE systems (Green and Pastewka, 1976Go). 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 2bGo). 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 3aGo) and its reaction with anti-phosphoserine monoclonal antibody provided additional qualitative evidence of phosphorylation (Figure 3bGo).




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Fig. 2. (a) SDS–PAGE analysis of transgenic and non-transgenic mouse milk and whey (equivalent loadings). Lane 1, molecular mass markers, phosphorylase b (97 400), albumin (66 200), ovalbumin (45 000), carbonic anhydrase (31 000), trypsin inhibitor (21 500) and lysozyme (14 400); lane 2, non-transgenic mouse milk; lane 3, transgenic BLG 45 mouse milk; lanes 4, 5 and 6, transgenic mouse milks PC/BLG 29.11.8.5.5, 66.13.5.9.4 and 66.13.5.9.6, respectively; lanes 7 and 8, transgenic mouse wheys PC/BLG 66.13.5.9.4 and 66.13.5.9.6, respectively; lanes 9 and 10, non-transgenic mouse wheys; lane 11, 5 µg ovine BLG. The position of the transgene product is arrowed. (b) Native PAGE of recombinant ovine BLG (lanes 1, 10), PC/BLG (lanes 2, 4, 6, 8) and whey PC/BLG preincubated with 0.4 units of alkaline phosphatase for 30 min (lane 3) or 1 h (lane 5) or 0.04 units of alkaline phosphatase for 30 min (lane 7) or 1 h (lane 9).

 



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Fig. 3. (a) Detection of BLG by immunoblotting with a rabbit anti-ovine BLG polyclonal antibody. Amounts equivalent to 20 nl of mouse milk were loaded. Lane 1, Coomassie Brilliant Blue stained molecular mass markers, carbonic anhydrase (31 000), trypsin inhibitor (21 500) and lysozyme, (14 400); lane 2, 1 µg ovine BLG; lane 3, transgenic BLG 45 mouse milk; lanes 4 and 5, transgenic mouse milks PC/BLG 66.13.5.9.4 and 66.13.5.9.6, respectively; lane 6, non-transgenic mouse milk. (b) Detection of phosphorylation by immunoblotting with an anti-phosphoserine monoclonal antibody. Amounts of whey equivalent to 0.320 µl mouse milk were loaded. Lane 1, transgenic BLG 7 mouse whey; lanes 2 and 3, transgenic PC/BLG mouse wheys 66(A) 1.5.2.2 and 66(A) 1.5.2.5, respectively; lane 4, non-transgenic mouse whey; lane 5, 1 µg ovine BLG; lane 6, 1 µg whey PC/BLG; lane 8, 2.5 µg ovine BLG; lane 10, 2.5 µg whey PC/BLG; lane 12, 5 µg whey PC/BLG. Lanes 7, 9, 11 and 13 are unloaded. The positions of molecular mass markers and whey PC/BLG are arrowed.

 
Centrifugation of milks showed that most of the PC/BLG did not sediment with the casein micelles, but remained in the whey (Figure 2aGo). Analysis of the proteins present in the supernatant fraction by FPLC showed a slight decrease in retention time on cation-exchange chromatography for the PC/BLG transgene protein compared with native BLG in mouse whey (Figure 4Go).



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Fig. 4. Cation-exchange FPLC profiles of whey fractions from milks of (A) non-transgenic, (B) PC/BLG transgenic and (C) BLG transgenic mice. Peaks 2 and 3 are whey PC/BLG and ovine BLG, respectively.

 
The phosphate centre inserted into the BLG gene theoretically permits the addition of four phosphate groups to the molecule to give the sequence Glu–SerP–Leu–SerP–SerP–SerP–Glu–Glu–Ser, where SerP is phosphoserine. In order to establish the extent of phosphorylation of the PC/BLG, mass spectrometric analysis was performed on ovine BLG, PC/BLG recovered from the whey fraction of transgenic milks and PC/BLG treated with the alkaline phosphatase. By mixing phosphorylated and either unphosphorylated or dephosphorylated proteins together, a more accurate determination of the mass difference was obtained (Figure 5Go). The absolute molecular mass values for the whey transgene PC/BLG and native BLG were 19 258 (±10) and 18 186 Da, respectively, the mass spectrometer being calibrated on the native ovine BLG. This difference in molecular mass is consistent with 1.7 (±0.1) phosphorylated amino acid residues per protein molecule. The molecular mass difference between the whey PC/BLG and dephosphorylated PC/BLG was 110 ± 20 Da, corresponding to a degree of phosphorylation of 1.4 (±0.2).



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Fig. 5. MALDI-TOF mass spectrometry of mixed ovine BLG and whey PC/BLG purified from transgenic mouse milks.

 
Independent confirmation that the whey PC/BLG molecule was actually phosphorylated and that the sites of phosphorylation were seryl (as expected on the basis of the structure of the recognition site) or threonyl residues, was obtained by infrared spectroscopy. Figure 6Go shows the spectra in the region 900–1150 cm–1 of native BLG (A) and whey PC/BLG (B), both freeze-dried after extensive dialysis against distilled water and therefore close to their isoelectric points. Also shown is the spectrum of the recombinant protein freeze-dried from a solution at pH 7 (C). The presence of a pH-dependent peak at about 980 cm–1 in the whey PC/BLG spectra provides strong evidence that the modified protein is phosphorylated. Many other phosphoproteins and phosphopeptides show this feature, which is assigned to a symmetric or asymmetric PO4 stretching mode in a phosphate ester such as phosphoserine or phosphothreonine (Prescott et al., 1986Go) and it is absent in native BLG (data not shown). The shape of this feature and its variation with pH were very similar to changes seen in the spectrum of the N-terminal 25 amino acid tryptic phosphopeptide of ß-casein, which contains the same phosphate centre, recorded under the same conditions (data not shown). However, the relative intensity of the PO4 stretching mode was reduced, indicating that the phosphate centre in the PC/BLG was incompletely phosphorylated. Support for this conclusion was found in the infrared spectrum at about 1100 cm–1 where additional absorbance indicates a higher proportion of OH moieties in the whey PC/BLG.



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Fig. 6. FTIR spectra of (A) native ovine BLG, (B) whey PC/BLG and (C) whey PC/BLG at pH 7.0. Spectra B and C are offset by 0.1 and 0.2 absorbance units, respectively.

 
A semi-quantitative interpretation of the infrared spectrum was made by integrating the area of the peak at 980 cm–1 using a baseline drawn from 950 to 1000 cm–1. The area was normalized by dividing by the area of the amide I band, which is proportional to the number of peptide bonds. The ratio is therefore proportional to the fraction of phosphorylated residues in the whole molecule. The same ratio was calculated from the infrared spectrum of the ß-casein phosphopeptide which was shown, by MALDI-TOF mass spectrometry, to contain 4.0 phosphoserine residues. Both the peptide and the whey PC/BLG were freeze-dried from a solution at pH 7. The proportion of phosphorylated residues in the whey PC/BLG was thus found to be 1.38 (±0.03) mol P per mole BLG.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
It is well established that the mammary gland has the capacity to synthesize proteins that undergo complex secondary processing. For example, most caseins that have been examined in sufficient detail are phosphorylated, a number (e.g. bovine {kappa}- and {alpha}s2-caseins) are linked by intermolecular disulphide bridges and a few (e.g. bovine {kappa}-casein) are glycosylated. The N-terminal Glu residue in bovine {kappa}-casein is also converted to a pyroglutamate form. Nevertheless, caseins are far from being typical globular proteins; their sequences have diverged rapidly, except for a few relatively short regions of conservation such as the signal peptides and they frequently exhibit sequence insertions/deletions. They have a high rate of point mutational changes, both silent and expressed (Mercier, 1981Go). Even phosphate centres are not invariably conserved, sometimes owing to exon skipping events (Brignon et al., 1990Go). In solution they have a relatively open and flexible conformation for which the term rheomorphic was coined (Holt and Sawyer, 1993Go) and the intermolecular disulphide bridging is rather non-specific (Rasmussen et al., 1992aGo,bGo). In {alpha}s1-casein dimers, for example, both parallel and antiparallel polypeptide chains are found and in {kappa}-casein polymers, interchain linkages Cys11–Cys11, Cys11–Cys88 and Cys88–Cys88 are present. In contrast, the common whey proteins are usually not phosphorylated (rat whey acidic protein, WAP, is an exception) but may be glycosylated (e.g. {alpha}-lactalbumin ) and have a well defined conformation. For example, the WAP proteins are folded to form a 4-disulphide-linked core and in bovine BLG a disulphide bridge is formed between Cys106 and Cys119 but never with the nearby free Cys121. While it may be expected that incorrectly folded whey proteins are recycled or refolded, such can hardly be the case for the caseins. Hence it may be conjectured that some subtle cell sorting operates to process separately the nascent polypeptide chains of whey proteins and caseins.

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 {alpha}-1-antitrypsin requires glycosylation and factor IX, {gamma}-carboxylation and ß-hydroxylation, but both are secreted as functional proteins in transgenic mouse milk (Archibald et al., 1990Go; Yull et al., 1995Go). 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., 1996Go).

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, 1981Go), the recognition site for phosphorylation at position N is Ser/Thr–Xxx–Glu/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 SerP–Leu–SerP–SerP–SerP–Glu–Glu, 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 2bGo) 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 Thr–Pro–Glu, Ser110 where the sequence is Ser-Ala-Glu and Thr125 where the sequence is Thr–Pro–Glu. None is known to be phosphorylated in the native bovine protein, as indicated by mass spectrometry (Léonil et al., 1997Go). 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., 1997Go). The crystal structure of the ovine BLG has been partially determined (Rocha et al., 1996Go) 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 IGo. 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 {alpha}-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 1Go). 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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Table I. Summed and % accessibilities for the total side chains of residues that could be part of a casein mammary gland kinase recognition sequence (Ser/Thr–X–Glu)
 
The accessibilities of the potential sites of phosphorylation in the inserted casein sequence are not known but clearly they are more readily phosphorylated than are the sites in the native protein, at least to the extent of one or two incorporated phosphate moieties. Choosing a surface ß-turn as the site of insertion of the casein sequence was therefore vindicated. The results indicate that the expressed transgene can interact with the phosphokinase(s) in the Golgi apparatus of the mammary secretory cell, but either the process does not go to completion or there is partial reversal by a phosphatase at a later stage.

The PC/BLG does not appear to be incorporated into casein micelles to any significant extent, as demonstrated by the pelleting experiment (Figure 2aGo). Phosphoseryl groups play a key role in the formation of complexes between {alpha}s1-, {alpha}s2- and ß-caseins and micellar calcium phosphate, but unlike the highly phosphorylated {alpha}s1-, {alpha}s2- and ß-caseins, which in bovine milk contain 8–9, 11–13 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 –Ser–Ser–Ser–Glu–Asp– which closely resembles a casein phosphate centre and is, indeed, singly phosphorylated (Hennighausen et al., 1982Go). In contrast, the corresponding mouse protein sequence is –Ala–Ser–Pro–Ile–Gly–, 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., 1984Go) the unphosphorylated form is predominantly in the whey (Monti and Jollés, 1982Go). 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, 1992Go). 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.


    Acknowledgments
 
We are grateful to Roberta Wallace for the generation of transgenic mice and Frances Thompson and Caroline Owen for the maintenance of transgenic mouse lines. We thank Lindsay Sawyer and Sharon Brownlow of Edinburgh University for the calculation of solvent accessibilities of potential phosphorylation sites and for preparing the ribbon diagram in Figure 1Go. We also thank Elaine Little and John Webster for technical assistance and Roddy Field and Elliot Armstrong for photography and graphics. This work was supported by the Biotechnology and Biological Sciences Research Council and by the Scottish Office Agriculture, Environment and Fisheries Department.


    Notes
 
2 Present address: John Innes Centre, Colney, Norwich NR4 7UH, UK Back

4 To whom correspondence should be addressed.E-mail: leaverj{at}hri.sari.ac.uk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received July 6, 1998; revised November 23, 1998; accepted November 27, 1998.





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