Posttranslational modifications of caseins

C H A P T E R

5 Posttranslational modifications of caseins Etske Bijla, John W. Hollandb, Mike Bolandc a

Dairy Science and Technology, Food Quality and Design Group, Wageningen University and Research, Wageningen, The Netherlands bDrug Toxicology Unit, Forensic and Analytical Science Service, NSW Health Pathology, North Ryde, NSW, Australia cThe Riddet Institute, Massey University, Palmerston North, New Zealand

Introduction The caseins are phosphoproteins and constitute about 80% of the protein in bovine milk (Swaisgood, 2003; Farrell et al., 2004). They are assembled in a colloidal complex with calcium phosphate and small amounts of other minerals. Although obviously important for the provision of amino acids, calcium, and phosphorus for infant nutrition, the structure of the casein micelle is also critical in determining the physical properties of milk. The stability of the micelle, or its controlled destabilization in the case of cheese and yogurt manufacture, is of primary concern to the dairy industry. A number of reviews of micelle structure have been published (Rollema, 1992; Holt and Horne, 1996; Horne, 1998, 2002; Walstra, 1999; Farrell et al., 2006; Dalgleish and Corredig, 2012; Holt et al., 2013). In simple terms, the micelle is a network of protein molecules held together by a combination of hydrophobic interactions between protein molecules and electrostatic interactions between phosphoserine-rich regions of the α- and β-caseins and micellar calcium phosphate. Whereas the internal structure is still the subject of debate (Horne, 1998; Walstra, 1999; Chapter 6 of this volume), there is general acceptance of the “hairy micelle” concept, in which the hydrophilic C-terminal portion of κ-casein extends from the surface, providing steric and electrostatic repulsion, which prevents micelle aggregation. A critical factor in micelle formation and stability is the presence of posttranslational modifications (PTMs) such as phosphorylation on the αs1-, αs2-, and β-caseins and glycosylation on κ-casein. PTMs on secreted proteins such as the caseins occur in the endoplasmic reticulum and/or Golgi complex after the synthesis of the polypeptide chain. As such, they are not encoded by the genes per se but may be dependent on protein (and hence gene) sequence

Milk Proteins https://doi.org/10.1016/B978-0-12-815251-5.00005-0

173

# 2020 Elsevier Inc. All rights reserved.

174

5. Posttranslational modifications of caseins

motifs that are necessary, but of themselves not sufficient, for modification to take place. A number of other factors determine whether or not a PTM occurs, including expression of the genes encoding the enzymes necessary for the modification, the availability of their substrates, and the accessibility of the modification site on the protein, especially after folding. Therefore, although it may be possible to predict the theoretical sites of modification on proteins, determination of the actual sites and the degree to which they are modified requires considerable experimental characterization. Advances in our understanding of complex systems such as the casein micelle are frequently preceded by advances in technology. In recent years, the development of proteomic technologies has greatly enhanced our ability to analyze milk proteins, particularly with respect to PTMs. Gel-based separation strategies, in particular two-dimensional electrophoresis (2-DE), have provided a high-resolution methodology for displaying the heterogeneity of the major milk proteins. As can be seen in Fig. 5.1, genetic variants, phosphovariants, and glycovariants of the caseins can be resolved on a single 2-D gel. In addition, gel-free separation strategies, such as liquid chromatography (LC), are sensitive and robust methods to separate the caseins. Advances in mass spectrometry (MS) have enhanced our ability to analyze the proteins arrayed on 2-D gels or separated by LC methods. Therefore, not only it is possible to resolve many proteins and their isoforms, but also it is possible to characterize

FIG. 5.1 Two-dimensional gel of bovine milk. Section of a 2-D gel highlighting the major protein forms found in bovine milk. Csn, casein; α-Lac, α-lactalbumin; β-Lg, β-lactoglobulin. Adapted from Holland, J.W., Deeth, H.C., Alewood, P.F., 2004. Proteomic analysis of kappa-casein micro-heterogeneity. Proteomics 4, 743–752.

The caseins

175

them, particularly with respect to the many PTMs that affect the properties of the caseins such as charge, mass, hydrodynamic radius, and hydrophobicity. As discussed in this chapter, the four caseins are present in many diverse forms as a result of differential PTMs. The biological reasons behind the diversity are not clear. However, what is clear is that the PTMs of the caseins are critical for their function in micelle formation and stability. The first part of this chapter summarizes what is known about the PTMs of the αand β-caseins. The second part focuses on κ-casein, which has been the subject of recent proteomic studies, and includes an extended discussion on the functional significance of κ-casein heterogeneity. The discussion in these sections is specific to the caseins of the cow, Bos taurus. However, as we have noted that increasing attention is being paid to PTMs of the caseins in the milks from alternative species, such as human, horse, goat, and even elephant, a short review on this topic is provided in the last section.

The caseins The caseins are phosphoproteins that are generally well characterized in terms of their PTMs and have been the subject of a number of reviews (Mercier, 1981; Ginger and Grigor, 1999; Farrell et al., 2004). In fact, caseins have been used as model phosphoproteins in the development of proteomic techniques to examine global phosphorylation patterns (e.g., Cox et al., 2005; Kapkova et al., 2006; Sweet et al., 2006; Zhou et al., 2006; Wu et al., 2007). The multitude of techniques developed for the analysis of phosphorylation is beyond the scope of this chapter but has been well reviewed (Bodenmiller et al., 2007; Collins et al., 2007). Indeed, the topic of “phosphoproteomics” has become a field of study in its own right. The PTMs of the αs1-, αs2-, and β-caseins are summarized in the sections in the succeeding text. An important note concerns the numbering of the amino acids in the casein sequences described in subsequent sections. The numbering of residues is based on the UniProt database entry for the relevant protein (see Figs. 5.2 and 5.3) and includes the signal peptide, which is normally removed during processing to generate the mature protein. This differs from the numbering in most of the dairy literature, in which the N-terminal amino acid of the mature protein is numbered 1.

αs1-Casein The predominant form of αs1-casein in bovine milk contains eight phosphate groups. The phosphates are attached to hydroxyamino acids occurring in the sequence motif Ser/ThrXxx-Glu/Asp/pSer (Mercier, 1981). However, the vast majority of casein phosphorylation sites, including the eight sites in the major form of αs1-casein, occur in the more restricted Ser-Xxx-Glu/pSer motif. Phosphorylation of threonine or of serine in the Ser-Xxx-Asp motif is relatively uncommon. A minor form of αs1-casein with nine phosphates, originally called αs0-casein, also occurs in bovine milk. It contains one extra phosphate on Ser56, which occurs in a Ser-Xxx-Asp motif (Manson et al., 1977). The reference protein for αs1-casein is αs1-CN B-8P, where B-8P signifies the B genetic variant with eight phosphates (Farrell et al., 2004).

176

5. Posttranslational modifications of caseins P02662|CASA1_BOVIN Alpha-S1-casein - Bos taurus (Bovine)

1

MKLLILTCLV AVALARPKHP IKHQGLPQEV LNENLLRFFV APFPEVFGKE

50

51

KVNELSKDIG SESTEDQAME DIKQMEAESI SSSEEIVPNS VEQKHIQKED 100

101 VPSERYLGYL EQLLRLKKYK VPQLEIVPNS AEERLHSMKE GIHAQQKEPM 150 151 IGVNQELAYF YPELFRQFYQ LDAYPSGAWY YVPLGTQYTD APSFSDIPNP 200 201 IGSENSEKTT MPLW

214

P02663|CASA2_BOVIN Alpha-S2-casein - Bos taurus (Bovine)

1

MKFFIFTCLL AVALAKNTME HVSSSEESII SQETYKQEKN MAINPSKENL

51

CSTFCKEVVR NANEEEYSIG SSSEESAEVA TEEVKITVDD KHYQKALNEI 100

50

101 NQFYQKFPQY LQYLYQGPIV LNPWDQVKRN AVPITPTLNR EQLSTSEENS 150 151 KKTVDMESTE VFTKKTKLTE EEKNRLNFLK KISQRYQKFA LPQYLKTVYQ 200 201 HQKAMKPWIQ PKTKVIPYVR YL

222

P02666|CASB_BOVIN Beta-casein - Bos taurus (Bovine)

1

MKVLILACLV ALALARELEE LNVPGEIVES LSSSEESITR INKKIEKFQS

51

EEQQQTEDEL QDKIHPFAQT QSLVYPFPGP IPNSLPQNIP PLTQTPVVVP 100

50

101 PFLQPEVMGV SKVKEAMAPK HKEMPFPKYP VEPFTESQSL TLTDVENLHL 150 151 PLPLLQSWMH QPHQPLPPTV MFPPQSVLSL SQSKVLPVPQ KAVPYPQRDM 200 201 PIQAFLLYQE PVLGPVRGPF PIIV

224

FIG. 5.2 Amino acid sequences of the bovine α- and β-caseins. Swiss-Prot accession numbers, entry names, protein names, species, and sequences for the α- and β-caseins. The database sequences are for the B variant of αs1-casein, the A variant of αs2-casein, and the A2 variant of β-casein. Potential phosphorylation sites are shown in bold type and those that have been experimentally confirmed are underlined. The signal peptides are shown in italics.

A number of genetic variants of αs1-casein have been described (Farrell et al., 2004). Only the less common forms, D and F, are likely to have altered phosphorylation profiles. Variant D has Ala68 substituted with Thr, which generates a phosphorylation site of the form Thr-Xxx-Glu. Phosphorylation of this residue was detected when the variant was first identified (Grosclaude et al., 1972a). Variant F has Ser81 substituted with Leu, which disrupts the serine cluster, Ser79-Ile-Ser-Ser-Ser-Glu-Glu85, and eliminates the secondary phosphorylation sites at Ser79 and Ser81. The amino acid sequence and the modifications of αs1-casein are shown in Fig. 5.2.

αs2-Casein The αs2-casein component of bovine milk is more varied than the αs1-casein component. It generally presents as a mixture of five phosphoforms with 10–14 phosphates (Vincent et al., 2016),

The caseins

1 51

↓

MMKSFFLVVT ILALTLPFLG AQEQNQEQPI RCEKDERFFS DKIAKYIPIQ

177

50

YVLSRYPSYG LNYYQQKPVA LINNQFLPYP YYAKPAAVRS PAQILQWQVL 100

101 SNTVPAKSCQ AQPTTMARHP HPHLSFMAIP PKKNQDKTEI PTINTIASGE 150 I A 151 PTSTPTTEAV ESTVATLEDS PEVIESPPEI NTVQVTSTAV

190

FIG. 5.3 Amino acid sequence of κ-casein A (Swiss-Prot accession number P02668). The N-terminal signal sequence (1–21) is shown in italics. The arrow indicates the amino terminus of the mature protein. Recognized sites of potential phosphorylation and glycosylation are indicated in bold, underlined text. Amino acid substitutions distinguishing the B variant are shown above the main sequence.

and phosphoforms with 9 or 15 phosphates have been detected in trace amounts (Fang et al., 2016). The reference protein for αs2-casein is αs2-CN A-11P (Farrell et al., 2004). The A variant has 12 serine residues in Ser-Xxx-Glu/pSer motifs and four threonine residues in Thr-XxxGlu motifs (Fig. 5.2). Consequently, up to 16 phosphates are theoretically possible. Presumably, the 12 serine residues are the first to be phosphorylated. However, it is not known whether specific residues remain unphosphorylated in the different forms, but given the consistent appearance of the forms in milk, it is likely to be the case. Unfortunately, it is not possible to draw any conclusions with regard to phosphorylation site occupation until the individual phosphoforms are analyzed. This should be possible with the newly emerging proteomic techniques. Only four genetic variants of αs2-casein have been described (Farrell et al., 2004), and in each case, an altered phosphorylation profile would be expected. In variant B, Ser23 is changed to Phe as a result of a single nucleotide substitution (Ibeagha-Awemu et al., 2007). This causes loss of a phosphorylation site in the first phosphoserine cluster. Variant C has three amino acid changes: Glu48 is changed to Gly, with loss of the phosphorylation site at Ser46; Ala62 is changed to Thr, creating a potential site with the motif Thr62-Xxx-Glu64; Thr145 is changed to Ile, with loss of the potential site at Thr145. Variant D has a deletion of nine amino acids as a result of skipping exon VIII (Bouniol et al., 1993). This results in loss of the first three serines from the second phosphoserine cluster. A second PTM on αs2-casein is the formation of an intramolecular disulfide bond between the two cysteine residues in the protein (Rasmussen et al., 1994). The functional role of disulfide bonding is not clear at this stage, but it may contribute to micelle stability and is discussed further in the section on κ-casein.

β-Casein Bovine β-casein is usually present as a single form with five phosphates, indicating that all five Ser-Xxx-Glu/pSer sites in the sequence are constitutively phosphorylated. The reference protein is β-CN A2-5P (Farrell et al., 2004). Some 12 genetic variants of β-casein have been characterized, but only two variants appear to have altered phosphorylation profiles.

178

5. Posttranslational modifications of caseins

Variant C has a Glu to Lys substitution at residue 52, which removes the phosphorylation site at Ser50. Variant D has a Ser to Lys substitution at residue 33, which removes the primary phosphorylation site at Ser33. Variation in β-casein arises primarily as a result of proteolysis rather than PTMs. The sequence and the phosphorylation sites of β-casein are summarized in Fig. 5.2.

κ-Casein κ-Casein does not contain any phosphoserine clusters and probably plays little part in calcium binding. Its major feature is a variable degree of glycosylation. The keen interest in κ-casein arises largely from its key role as a stabilizer of the micelle structure. In mice, the absence of κ-casein causes a failure of lactation, as the lumina of the mammary gland become clogged with aggregated caseins (Shekar et al., 2006). κ-Casein is usually thought of as having two distinct domains that are separated by the very specific Phe-Met (126–127) bond that is cleaved by chymosin. The N-terminal para-κ-casein is the hydrophobic part that remains in the micelle and contains the disulfide bonds. The C-terminal caseinomacropeptide is the part that extends into the solution and is removed to become part of the whey fraction during cheesemaking. It contains the glycosylation sites, which is why this peptide is often referred to as the glycomacropeptide, although strictly speaking, this term pertains only to forms that are actually glycosylated. The PTMs of κ-casein have been the subject of more recent research and are covered here in much greater detail than those of the other caseins. The full amino acid sequence of bovine κ-casein was first reported in 1973 (Mercier et al., 1973). The mature protein consists of a single chain of 169 amino acids (Fig. 5.3) and has a theoretical molecular weight of 19,037 Da and a theoretical pI of 5.45–5.77 (A variant with one SerP residue) (Farrell et al., 2004). The amino terminal glutamine is cyclized to form a pyrrolidone glutamic acid residue. The bovine κ-casein gene sequence was published in 1988 (Alexander et al., 1988). It consists of five exons spread over about 14 kilobases, with most of the protein-coding region located in exon 4. A cleavable amino terminal signal sequence of 21 amino acids directs secretion of the mature protein. A number of polymorphisms in the κ-casein gene have been identified, resulting in one or more amino acid substitutions in the mature protein. The most common variants are the A and B variants, which differ by two amino acids in the caseinomacropeptide region (Asp169/Thr157 in variant A and Ala169/Ile157 in variant B). The genetic variants of κ-casein are summarized in Table 5.1. A number of polymorphisms in the noncoding region have also been identified (Schild et al., 1994; Keating et al., 2007). Although these do not affect the amino acid sequence, they have the potential to affect expression levels. The reference protein for κ-casein is κ-CN A-1P, UniProt P02668 (Farrell et al., 2004). Phosphorylation κ-Casein appears to be constitutively phosphorylated at Ser170 and only partially phosphorylated at Ser148 (Talbot and Waugh, 1970; Mercier et al., 1973). A minor triphosphorylated form has also been detected (Vreeman et al., 1986; Molle and Leonil, 1995). Other studies have managed to detect only monophosphorylated forms (Rasmussen et al., 1997; Riggs et al., 2001), although, in one of these (Riggs et al., 2001), the phosphorylation site appears to have been identified incorrectly. Phosphorylation has also been examined by

179

The caseins

TABLE 5.1 Genetic variants of bovine κ-casein Varianta

Amino acid changes (relative to the A variant)

A B B2 C

Thr Thr

157

Ser

F1 F2 G1 G2 H I

to Ile, Asp

169

to Ile, Asp

118

176

to Ala to Ala, Ile

157

to His, Thr

(Mercier et al., 1973) 174

to Thr

169

to Ile, Asp

to Ala

to Gly

Asp

(Gorodetskii et al., 1983) (Miranda et al., 1993) (Schlieben et al., 1991)

to Val

(Sulimova et al., 1992)

Arg

31

Arg

118

to Cys

(Prinzenberg et al., 1996)

169

to His

(Prinzenberg et al., 1996)

to Ala

(Sulimova et al., 1996)

156

to Ile

(Prinzenberg et al., 1999)

125

to Ala

Asp Thr

Thr

A(1)

169

169

Ser

J

a

(Grosclaude et al., 1972b) 157

Arg

E

Reference

157

(Prinzenberg et al., 1999) 169

to Ile, Asp 150

Silent (Pro

to Ala, Ser

, CCA to CCG)

176

to Arg

(Mahe et al., 1999) (Prinzenberg et al., 1999)

Other variants have been described but have not been confirmed or have been proven to be identical to those given.

MS of intact κ-casein extracted from 2-D gels. Both mono- and diphosphorylated forms were observed, and phosphorylation at Ser170 was confirmed by tandem MS (Claverol et al., 2003). However, the electrophoretic mobility of some phosphoforms was not consistent with the MS analysis and probably reflected artefactual modification (e.g., deamidation) during purification. Using 2-DE with isoelectric focusing as the first dimension, phosphorylation variants in whole milk can be easily resolved because of the pI shifts caused by the acidic phosphate groups (Holland et al., 2004). The two main phosphorylation sites have been confirmed by tandem MS sequencing of peptic peptides released from protein forms separated by 2-DE. Triphosphorylated forms of both the A variant and the B variant have been observed, and the third phosphorylation site has been identified as Thr166 (Holland et al., 2006). This site is consistent with the general observation of phosphorylation on the Ser/Thr-Xxx-Glu/pSer motif, with only relatively low levels of phosphorylation on threonine residues. A recent study has identified two minor phosphorylation sites at Thr166—as above—and also at Ser187 (Hernandez-Hernandez et al., 2011). Glycosylation Glycosylation is a PTM that is present in all domains of life (Moremen et al., 2012). In milk, two types of protein glycosylation are observed: O-glycosylation (serine- or threonine-linked glycans) and N-glycosylation (asparagine-linked glycans) (Kirmiz et al., 2018). Only O-linked glycans are found in the κ-casein fraction. About 60% of κ-casein has been estimated to be glycosylated, and 40% is nonglycosylated (Vreeman et al., 1986). The presence of carbohydrates on κ-casein was recognized as long ago as 1961 (Alais and Jolles, 1961), and during

180

5. Posttranslational modifications of caseins

the 1970s and 1980s, a large number of studies were directed at elucidating the mono- and oligosaccharide composition, sequence, and sites of attachment to the protein. The major glycan is a tetrasaccharide composed of galactose (Gal), N-acetylgalactosamine (GalNAc), and sialic or neuraminic acid (NeuAc) of the form NeuAcα(2–3)Galβ(1–3)[NeuAcα(2–6)]GalNAc; however, monosaccharide (GalNAc), disaccharide (Galβ(1–3)GalNAc), and trisaccharide (NeuAcα(2–3)Galβ(1–3)GalNAc or Galβ(1–3)[NeuAcα(2–6)]GalNAc) are also found (Fournet et al., 1975, 1979; van Halbeek et al., 1980; Fiat et al., 1988; Saito and Itoh, 1992). A more recent study also detected a disaccharide with NeuAcα(2–6)GalNAc (Nwosu et al., 2010). Therefore, in theory, each glycosite could contain one of these six glycans. The relative amounts of the first five glycans mentioned earlier have been determined by highperformance liquid chromatography (HPLC) as 56.0% tetrasaccharide (948 Da), 36.9% trisaccharide (18.4% linear and 18.5% branched) (656 Da), 6.3% disaccharide (365 Da), and 0.8% monosaccharide (203 Da) (Saito and Itoh, 1992). NeuAcα(2-6)GalNAc was not discovered yet in that study. Considering this distribution, the average molecular weight of a glycosylated side chain is 798 Da. It is not known whether the minor forms arise from incomplete synthesis of the tetrasaccharide in mammary epithelial cells or are products of degradation after synthesis and/or secretion of κ-casein into the lumen of the mammary gland. Establishment of the attachment site(s) of the glycans has been more controversial. On the basis of Edman sequencing of short glycopeptides obtained by enzymatic digestion, Jolles et al. (1973) proposed Thr152 or Thr154 as the glycan attachment site. Kanamori et al. (1980) proposed Thr152, Thr154, and Thr156 (or Thr157) after analyzing a glycopeptide that was derived from κ-casein and that contained three GalNAc residues. Work from the same laboratory on bovine κ-casein from colostrum also indicated glycosylation at Thr152, Thr154, and Thr156 (Doi et al., 1980). Subsequently, using a different peptide fraction prepared from κ-casein of normal milk, glycosylation at Thr154 and Ser162 was reported (Kanamori et al., 1981). Meanwhile, further work from Jolles’ laboratory identified Thr152 as the glycan attachment site on κ-casein from normal milk and Thr152 and Thr163 as the attachment sites on κ-casein from colostrum (Fiat et al., 1981). Zevaco and Ribadeau-Dumas (1984) suggested that glycans could be attached to any of the previously identified sites (Thr152, Thr154, Thr156, Thr157, Ser162, or Thr163), but their published study contained no conclusive evidence for any site. All these studies were limited by the technology available at the time. In normal Edman sequencing, glycosylated serine or threonine residues are not detected, and their presence is inferred from a blank in the sequencing cycle where serine or threonine is expected (for a more detailed discussion, see Pisano et al., 1994). This is further complicated by the fact that serine and threonine are themselves low yield amino acids. Thus, in assigning O-glycosylation sites, Edman sequencing data can easily be misinterpreted. Conclusive identification of glycosylation sites in κ-casein was achieved using solid-phase Edman sequencing, which allows the direct detection of glycosylated serine and threonine residues (Pisano et al., 1994). Variable levels of glycosylation at Thr142, Thr152, Thr154, Thr157 (A variant only), Thr163, and Thr186 were detected, and no evidence of glycosylation at any serine residue was obtained. However, even this study did not give the full picture of κ-casein glycosylation, as it could detect only average glycosylation site occupancy in a crude mixture of glycoforms. Holland et al. (2004) have shown that κ-casein glycoforms can be separated by 2-DE, and the resolution obtained is shown in Fig. 5.4. When the glycosylation site occupancy of individual glycoforms was investigated using tandem MS sequencing of chemically tagged peptides,

The caseins

181

FIG. 5.4 Heterogeneity of κ-casein in cows’ milk. Two-dimensional gel showing multiple forms of κ-casein. The genetic variant, number of phosphate residues, and glycosylation state are indicated. Numbers in parentheses indicate the extra negatively charged residues relative to κ-casein B-1P. Adapted from Holland, J.W., Deeth, H.C., Alewood, P.F., 2004. Proteomic analysis of kappa-casein micro-heterogeneity. Proteomics 4, 743–752.

an interesting pattern was observed. The monoglycoform was glycosylated exclusively at Thr152, the diglycoform was glycosylated at Thr152 and Thr163, and the triglycoform was glycosylated at Thr152, Thr154, and Thr163 (Holland et al., 2005). Further studies using enriched fractions of κ-casein separated on 2-D gels showed up to six glycans on κ-casein B, where only five sites had been previously identified. Tandem MS analysis provided evidence for glycosylation at Thr166 on the tetraglycoform (Holland et al., 2006). The remaining two glycosylation sites were not confirmed but were presumably on Thr142 and Thr186, as proposed earlier (Pisano et al., 1994). This pattern is illustrated in Fig. 5.5. As Thr166 can be phosphorylated or glycosylated, there is potential for competition at this site. However, as both the triphosphate form and the tetraglycoform are very minor forms, it may be of little significance. Using negative ion mode MS, Nwosu et al. (2010) detected glycans on Thr142, Thr152, Thr154, Thr163, and Thr186, but no glycoform was detected on Thr166. Interestingly, out of the five glycosylated sites, only on position Thr142 was the tetraglycoform not detected. The disaccharide NeuAcα(2-6)GalNAc was found only on this position. Positions Thr152 and Thr154 were highly glycosylated, and all known glycan types were detected on these sites, although no distinction could be made between the branched and linear trisaccharide. Overall, the glycosylation of κ-casein in the mammary epithelial cells appears to take place in a highly controlled manner, and this suggests that it is a rather important process with considerable functional significance. Disulfide bonding κ-Casein purified from bovine milk occurs as both monomeric forms and oligomeric forms with up to eight or more monomers linked by disulfide bonds (Swaisgood et al., 1964; Talbot and Waugh, 1970). A more recent study has shown that reduced and carboxymethylated κ-casein can form large fibrillar structures, although these do not occur in milk (Farrell et al., 2003).

182

5. Posttranslational modifications of caseins

MAIPPKKNQDKTEIPTINTIASGEPTSTPTIEAVESTVATLEASPEVIESPPEINTVQVTSTAV

O-Phos NH2

MAIPPKKNQDKTEIPTINTIASGEPTSTPTIEAVESTVATLEASPEVIESPPEINTVQVTSTAV

O-Phos NH2

MAIPPKKNQDKTEIPTINTIASGEPTSTPTIEAVESTVATLEASPEVIESPPEINTVQVTSTAV

O-Phos NH2

MAIPPKKNQDKTEIPTINTIASGEPTSTPTIEAVESTVATLEASPEVIESPPEINTVQVTSTAV

O-Phos NH2

FIG. 5.5 Attachment of tetrasaccharide units to the four major glycoforms of κ-casein. Schematic representation of κ-casein highlighting the glycomacropeptide portion and the attachment sites of phosphate and sugar residues in the major glycoforms. ( ) GalNAc, (●) Gal, and (♦) NeuAc.

▪

The nature of the disulfide-linked complexes has been examined by a number of authors (Groves et al., 1992, 1998; Rasmussen et al., 1992, 1994, 1999; Farrell et al., 2003). There are only two cysteine residues (Cys32 and Cys109) in bovine κ-casein (Mercier et al., 1973), both occurring in the para-κ-casein part of the molecule, and they appear to be randomly linked by disulfide bonds in oligomeric forms (Rasmussen et al., 1992). In monomeric κ-casein, the two cysteines form an intramolecular disulfide bond (Rasmussen et al., 1994). As can be seen in Fig. 5.6, disulfide-bonded monomers, dimers, and trimers can be resolved on 2-D gels of whole milk when reducing agents are omitted (Holland et al., 2008). It is not clear whether these higher-order complexes of κ-casein have any importance in micellar structure, but it would be expected that they would be less likely to dissociate from the micelles.

183

Sources and significance of casein heterogeneity

P MAIPPKKNQDKTEIPTINTIASGEPTSTPTIEAVESTVATLEASPEVIESPPEINTVQVTSTAV

S-S

P P NH 2

FIG. 5.6 Potential modifications on κ-casein B. This schematic summarizes the potential PTMs on κ-casein B. (P) Phosphorylation sites, ( ) GalNAc, (●) Gal, and (♦) NeuAc. Note that trisaccharides and disaccharides lacking one or both NeuAc residues also occur. The monomer has an intramolecular disulfide bond between Cys32 and Cys109.

▪

The cysteine residues are not well conserved across species, with human, porcine, and rodent κ-caseins containing only a single cysteine, precluding the formation of disulfide-linked oligomers larger than dimers (Rasmussen et al., 1999; Bouguyon et al., 2006). However, the ability of κ-casein to form disulfide-linked complexes with itself or with other proteins during heat treatment is relevant to dairy processing (see later). The combined PTMs of bovine κ-casein are summarized in Fig. 5.7.

Sources and significance of casein heterogeneity Although the heterogeneity of the caseins has been recognized for many decades and the structural elements are now fairly well defined, the source of the heterogeneity, particularly in phosphorylation of the αs-caseins and glycosylation of κ-casein, and their functional and biological role(s) are not well understood.

αs-Casein phosphorylation Studies on the sources and the functionality of phosphorylation of the αs-caseins have emerged only in the past decade, within the scope of several milk genomics projects. These studies are discussed here. Sources of heterogeneity One of the sources of variation in phosphorylation of the caseins is gene regulation. A genome-wide association study to identify genomic regions associated with αs1-casein-8P and αs1-casein-9P showed that these two phosphorylation states are not regulated by the same set of genes (Bijl et al., 2014b). αs1-Casein-8P was associated with protein variants of β-lactoglobulin and the β-lactoglobulin concentration, whereas αs1-casein-9P was associated with diacylglycerol acyltransferase 1 (DGAT1) K232 polymorphism. This polymorphism not only influences the milk fat percentage and composition but also is associated with the total αs1-casein and αs2-casein concentration and the protein percentage of milk (Schopen et al., 2011). Bijl et al. (2014b) proposed that substrate specificity of the enzymes on phosphorylation sites might be an important factor in explaining why αs1-casein-8P and αs1-casein-9P are

184

5. Posttranslational modifications of caseins

22---------C----37

22---------C----37

22---------C---------42

90------------------C--------118 Voyager Spec #1=>BC=>RSM500=>MC=>TR=>TR[BP = 5564.3, 1441]

22---------C----37

Voyager Spec #1=>BC=>RSM500=>MC=>TR=>TR[BP = 4013.9, 1583]

4014 1583 22---------C------------45 100 5568 80 60 40 5182 4223 4655 4967 20 0 0 3900 4300 4700 5100 5500 5900

22---------C----37

22---------C----37

22---------C---------42

22---------C------------45 5563 2980

Voyager Spec #1=>BC=>RSM500=>MC=>TR[BP = 5562.2, 2981]

100 80 4015 60 40 20 0 3900 4300

4650 4963 4700

5100

5507 5500

0 5900

22---------C----37 5565 1441 100 90------------------C--------118 80 60 4014 40 5183 4514 20 0 0 3900 4300 4700 5100 5500 5900

22---------C----37

Voyager Spec #1=>BC=>RSM500=>MC=>TR[BP = 5568.6, 844]

5567 843.6 90------------------C--------118 100 4014 5184 80 60 40 4517 20 0 0 3900 4300 4700 5100 5500 5900

FIG. 5.7 Distribution of disulfide-linked isoforms of κ-casein on a nonreducing 2-D gel. Dimers, trimers, and tetramers of κ-casein are labeled to show the participating monomeric forms. The dimers and trimers run as doublets depending on the disulfide linkages. Matrix-assisted laser desorption/ionization time of flight mass spectra show the disulfide-linked peptides obtained from tryptic digests of the homodimer and homotrimer of κ-casein B-1P.

Sources and significance of casein heterogeneity

185

genetically different traits. The unique Ser-Xxx-Asp motif of Ser56 in αs1-casein-9P might be phosphorylated by a different enzyme than that in αs1-casein-8P, because protein kinase enzymes differ in their specificity for phosphorylation sites (Ubersax and Ferrell, 2007). The Golgi-enriched fraction casein kinase (GEF-CK) has been recognized to phosphorylate caseins (Moore et al., 1985). This fraction has a consensus sequence for the Ser-Xxx-Glu/pSer motif and fails to recognize Asp in a Ser-Xxx-Asp motif (Lasa-Benito et al., 1996). In addition, it was shown that the kinase FAM20C, which is one of the candidates for the GEK-CK fraction, specifically phosphorylates the Ser-Xxx-Glu motif (Tagliabracci et al., 2012). It is not known which enzyme could be responsible for the phosphorylation of the Ser-Xxx-Asp motif in αs1-casein-9P. Although αs1-casein and αs2-casein are two different proteins that exhibit different phosphorylation patterns, they seem to be connected by the phosphorylation regulatory system in the mammary gland. The degree of phosphorylation of αs1-casein was found to be correlated to that of αs2-casein (Heck et al., 2008; Fang et al., 2018). Further studies on phenotypic correlations and hierarchical clustering (Fig. 5.8) between αs1-casein with 8 and 9 phosphate groups and αs2-casein with from 9 to 14 phosphate groups suggest that there are at least two regulatory systems for αs-casein phosphorylation (Fang et al., 2016). One system would be responsible for αs-casein with low levels of phosphorylation (αs1-casein-8P, αs2-casein-10P,

FIG. 5.8 Dendrogram of phenotypic correlations among αs-caseins with different levels of phosphorylation. Reprinted from Fang, Z.H., Visker, M.H.P.W., Miranda, G., Delacroix-Buchet, A., Bovenhuis, H., Martin, P., 2016. The relationships among bovine alpha(S)-casein phosphorylation isoforms suggest different phosphorylation pathways. J. Dairy Sci. 99, 8168–8177. https://doi.org/10.3168/jds.2016-11250.

186

5. Posttranslational modifications of caseins

and αs2-casein-11P), and one would be responsible for higher levels of phosphorylation (αs1casein-9P, αs2-casein-12P, αs2-casein-13P, and αs2-casein-14P). Fang et al. (2016) suggested that the αs-caseins with higher levels of phosphorylation may be phosphorylated by another mammary casein kinase on its four Thr-Xxx-Glu motifs. Parity, stage of lactation, genetic variation of cows, and breeds have been indicated in changes in the degree of phosphorylation of the αs-caseins (Buitenhuis et al., 2016; Fang et al., 2017, 2018). Functional significance A few decades ago, it was not possible to determine the variation in the degree of phosphorylation of the αs-caseins; however, with the use of capillary zone electrophoresis or improved separation profiles by liquid chromatography, this is feasible today (Heck et al., 2008; Bonfatti et al., 2009). The use of these techniques has opened up the possibility of studying the functional significance of the phosphorylation of αs-casein. The coagulation properties of milk are important for cheesemaking. Interesting correlations between the phosphorylation of αscasein and the coagulation properties of milk have been found (Frederiksen et al., 2011; Jensen et al., 2012b; Poulsen et al., 2016). Improved milk coagulation was associated with higher fractions of αs1-8P and αs2-11P. Variation in the phosphorylation of αs1-casein also seems to affect the hydrolysis of milk by plasmin (Zhang et al., 2018), which can play a role in the destabilization of UHT milk during storage. UHT milk incubated with plasmin showed a faster decrease in αs1-9P than αs1-8P during the onset of gelation. Bijl et al. (2014c) determined proteolysis during ripening in a model cheese system. Chymosin-induced hydrolysis of αs1-casein-8P and αs1-casein-9P was monitored over time in milk gels. It was found that, at 50% hydrolysis, 15% more αs1-8P than αs1-9P was hydrolyzed. The authors suggested that changes in the physical conformation of the caseins were responsible for the observed differences. When the secondary structure of αs1-8P is compared with that of αs1-9P, the ninth phosphate group is positioned just in front of an alpha-helix region (Creamer et al., 1981; Kumosinski et al., 1991). Therefore, it is likely that the additional phosphate group on this specific position will impact secondary structure formation. Additionally, the ninth phosphate group might affect calcium phosphate nanocluster formation because it can form a strong binding site together with two other phosphorylated residues in close proximity (Holt, 2004; Bijl et al., 2018).

κ-Casein heterogeneity Although the heterogeneity of κ-casein has been recognized for many decades and the structural elements are now fairly well defined, less is known about the source of the heterogeneity, particularly its glycosylation, and its functional role(s). One of the reasons that this subject has not been well studied has been the absence of high-throughput characterization methods. Rapid developments in the field of “glycoproteomics” are leading to new insights on the topic. The available techniques are beyond the scope of this chapter but have been summarized elsewhere (Wuhrer et al., 2007; Le et al., 2017; Mulagapati et al., 2017; Darula and Medzihradszky, 2018).

Sources and significance of casein heterogeneity

187

Early studies on the influence of the glycosylation of κ-casein on its functional and biological properties have been reviewed previously (Dziuba and Minikiewicz, 1996). They highlighted a number of studies addressing factors that could influence the degree of glycosylation and the influence that glycosylation might have on micelle stability. The following sections cover some of those studies again and highlight more recent work related to the sources, functional significance, and biological significance of κ-casein heterogeneity. Sources of heterogeneity A large number of studies have examined the influence of milk protein polymorphism on milk composition and yield, and these have been extensively reviewed (e.g., Ng-Kwai-Hang, 1997; Martin et al., 2002; Heck et al., 2009). However, in many cases, the results have been inconsistent, which is probably a reflection of the multifactorial nature of milk production. It is difficult to isolate the effects of the polymorphism of a single protein from those of the other major milk proteins, especially as there appears to be a substantial degree of coordination of their expression. There are also a number of environmental or cow-related factors such as feed type and lactation stage that are frequently interrelated, as they all vary with the seasonal changes in dairy farming. Studies on specific effects of κ-casein variants have largely focused on the common A and B variants, and there appears to be a consensus that milk from B variant cows contains more fat, protein, casein, and κ-casein than milk from A variant cows (Bovenhuis et al., 1992; Ng-Kwai-Hang, 1997; Bobe et al., 1999). Progress has been made in studies relating to the glycosylation status of κ-casein. Robitaille et al. (1991a) identified a number of factors that appeared to contribute to variation in the neuraminic acid (NeuAc) content of bovine κ-casein. The NeuAc content was higher in cows with the κ-casein AB phenotype than in cows with the AA phenotype; it decreased with increasing parity and varied over the course of lactation, dropping to a minimum at 2–3 months after calving before increasing over the next 9–10 months. They also examined the association between the glycosylation of κ-casein and milk production/composition (Robitaille et al., 1991b). Although there appeared to be a statistically significant association between the NeuAc content of κ-casein and the milk yield, the most striking result of these investigations was the variability of the NeuAc/κ-casein measurements (mean, 64
21 μg/mg; range, 23–166 μg/mg), which suggests that other factors could have had a large impact on glycosylation or that the inherent variability in the assay masked any true associations. Limited 2-D gel analyses suggest that the pattern of glycosylation is far more consistent than these measurements indicate (Holland et al., 2004, 2005). Significant differences in the content of nonglycosylated κ-casein in milk have been reported for cows of different κ-casein genotypes (Lodes et al., 1996). Nonglycosylated κ-casein (as a percentage of total protein) was determined by electrophoretic analysis. The levels were higher in milk from cows with the B variant than in milk from cows with the A variant. The rarer variants, C and E, were generally associated with lower levels. However, as no measurements of glycosylated κ-casein levels were reported, no effect of genetic variant on glycosylation can be inferred from this report. Electrophoretic and chromatographic techniques have been used to profile the caseinomacropeptide from cows of the AA and BB phenotypes. Coolbear et al. (1996) found that the B variant macropeptide was more highly glycosylated than the A variant macropeptide, with an increased content of both hexosamine (i.e., GalNAc) and sialic acid (i.e., NeuAc). After

188

5. Posttranslational modifications of caseins

anion-exchange HPLC on a MonoQ column, the elution profile of the B variant contained more peaks, suggesting that an increased number of oligosaccharide chains were attached. These results were consistent with other studies suggesting more extensive glycosylation of the B variant than the A variant (Vreeman et al., 1986; Molle and Leonil, 1995; Bijl et al., 2014a), despite the fact that the A variant contains an extra (potential) glycosylation site (Pisano et al., 1994). From these and other results, Coolbear et al. (1996) suggested that there were generally consistent patterns of glycosylation for the genetic variants but that the overall extent of glycosylation could vary. It was also discovered that the effect of the κ-casein variant on the relative contents of glycosylated and nonglycosylated κ-casein was consistent across breeds (Poulsen et al., 2016). Variations in κ-casein glycosylation during lactation have already been touched on. Early studies indicated a higher degree of glycosylation of κ-casein in colostrum than in mature milk as well as the presence of two additional sugar moieties, N-acetylglucosamine (GlcNAc) (Guerin et al., 1974; Fournet et al., 1975) and fucose (Fiat et al., 1988). Subsequently, a number of studies addressed the structure of the oligosaccharides attached to colostral κ-casein and how they varied with time after parturition (Saito et al., 1981a, b, 1982; van Halbeek et al., 1981; Fiat et al., 1988). As well as the structures in normal milk that we have already identified, the following structures have been reported: the acidic hexasaccharide, NeuAcα(2–3)Galβ(1–3) [NeuAcα(2–3)Galβ(1–4)GlcNAcβ(1–6)]GalNAc; the acidic pentasaccharide, NeuAcα(2–3)Galβ (1–3)[Galβ(1–4)GlcNAcβ(1–6)]GalNAc; the acidic tetrasaccharide, GlcNAcβ(1–3)Galβ(1–3) [NeuAcα(2–6)]GalNAc; the neutral pentasaccharide Galβ(1–3)[Galβ(1–4){Fucα(1–3)}GlcNAcβ (1–6)]GalNAc; the neutral tetrasaccharide, Galβ(1–3)[Galβ(1–4)GlcNAcβ(1–6)]GalNAc; and the neutral trisaccharide, Galβ(1–3)[GlcNAcβ(1–6)]GalNAc. This extra complexity is already observable 15 min after parturition but decreases to normal over about 66 h (Fiat et al., 1988). These results suggest changes in the expression profiles of the glycosyltransferases that are responsible for assembling the O-linked glycans on κ-casein. The initial step of attachment of GalNAc to a threonine residue, also known as a mucin-type linkage, is catalyzed by UDPGalNAc:polypeptide N-acetylgalactosaminyltransferase (ppGaNTases) (Ten Hagen et al., 2003). In building the tetrasaccharide, the next steps involve transfer of Gal from UDP-Gal and of Neu5Ac from CMP-Neu5Ac donor sugars by galactosyltransferase and sialyltransferases, respectively (Chokhawala and Chen, 2007). The formation of linear and branched glycans follows the same biosynthetic pathways as other mucin-type O-glycans (Corfield and Berry, 2015). Hydrolysis of the glycosidic bonds is catalyzed by glycosidase enzymes. Elevated levels of glycosidase activities have been detected in bovine colostrum (O’Riordan et al., 2014a). Of seven different glucosidases, N-acetyl-β-D-glucosaminidase, α-Lfucosidase, α-galactosidase, and N-acetyl-neuraminidase appeared to be the most biologically relevant. In mature milk, the levels decreased to a minimal but constant level. Also, between animals, only a low level of variation was observed. It is therefore important to consider both the expression profiles of glycosyltransferases and the activity of glycosidases in studying glycosylation during lactation. Another source of variation in glycosylation status that is related to lactation is the length of the cow’s dry period. In cows with a zero-day dry period, the relative amount of glycosylated κ-casein in the protein fraction, as determined by HPLC, increased from 8% to 12% between 6 and 2 weeks prepartum (de Vries et al., 2015). Furthermore, after calving, glycosylation was higher for cows with a zero-day dry period (6.7%) than for cows with a normal dry period

Sources and significance of casein heterogeneity

189

length of 60 days (5.2%). There was a negative correlation between glycosylation and milk yield 2-week postpartum, which was suggested to be related to a reduced productivity of the mammary epithelial cells. In line with these studies, Maciel et al. (2017) found a significant effect of days before calving (DBC), determined at 180, 90, and 60 days before expected calving, on the level of glycosylation relative to total κ-casein. Between 180 and 90 DBC, the level increased from 42.4% to 47.5%, while the calving interval of either 15 or 18 months did not show significant differences in level of glycosylation. Other seasonal factors related to climate, such as heat stress, drought, and nutrition (e.g., pasture vs. fodder), can have an impact on milk production and composition. However, we are not aware of any specific studies on their effect on κ-casein glycosylation. The genetic background of κ-casein glycosylation (expressed as percentage on total protein) was studied by Bonfatti et al. (2014) and Buitenhuis et al. (2016). Simmental breed had a high heritability of 0.46, also Danish Holsteins showed high heritability of 0.64, while Danish Jerseys had a lower heritability of 0.14. Buitenhuis et al. (2016) detected several SNPs that were specific for κ-casein glycosylation. Although no obvious candidate genes were found for genetic regulation of κ-casein glycosylation, two genes were related to the PTM of caseins. The first gene was casein kinase 1, gamma 3 (CSNK1G3) on BTA7, which is a serine-/threonine-specific protein kinase that phosphorylates caseins (Rowles et al., 1991). The second gene was protein kinase c. theta (PRKCQ) on BTA13, which belongs to the family of protein kinase C (PKC) that are also involved in phosphorylation of serine and threonine. If these genes have a specific role in glycosylation remains to be elucidated. Functional significance Casein micelle size

κ-Casein plays a key role in micelle stability by acting as a hairy layer that provides both steric repulsion and electrostatic repulsion between micelles, preventing aggregation. Glycosylation of κ-casein increases both the size of the hydrophilic C-terminal “hairs” and their charge, because of the bulk of the hydrophilic sugar residues with their hydration shells and the negative charge of the neuraminic acid groups, respectively. Theoretically, the higher is the degree of glycosylation of κ-casein, the greater should be its stabilizing ability. As such, it might be expected that the degree of glycosylation of κ-casein would have a marked effect on both the size and the stability of the casein micelles. Several studies have made use of bulk milk to determine correlations between size and milk composition (McGann et al., 1980; Davies and Law, 1983; Donnelly et al., 1984; Dalgleish et al., 1989). Casein micelles from bulk milk were fractionated into different size classes, and it was shown that their size was inversely related to their κ-casein content (relative to total casein). In studies on bulk milk, there was no clear correlation between micelle size and degree of glycosylation. Slattery (1978) found an apparent inverse relationship between the proportion of glycosylated κ-casein and the micelle size, but it did not apply to all of the size fractions isolated. In contrast, Dalgleish (1985, 1986) found that the proportions of glycosylated and nonglycosylated κ-casein did not vary with the micelle size. The studies of Yoshikawa et al. (1982) and O’Connell and Fox (2000) showed an apparent increase in κ-casein glycosylation with increasing micelle size. Some of these discrepancies are probably the result of differences in the method of fractionation and differences in

190

5. Posttranslational modifications of caseins

the analysis methods of glycosylated κ-casein. Also, variation in the bulk milk itself might have caused the contradictory findings. More recently, the milk of individual cows was studied to determine the link between casein micelle size and milk composition (Bijl et al., 2014a). Two sets of nine cows that produced milk with high or low average casein micelle size were selected in this study, and the detailed milk composition, including the degree of glycosylation of κ-casein, was determined. It was found that the average casein micelle size in milk of individual cows correlated significantly with genetic variants A and B of κ-casein and the degree of glycosylation of κ-casein. Small casein micelles were associated with the B variant of κ-casein, as was also shown in bulk milk studies. Interestingly, small casein micelle size was also associated with a high percentage of glycosylated κ-casein in the total protein. It was suggested that glycosylation influences micelle stabilization during or after the formation of the casein micelle in the mammary gland and thereby its size. The underlying mechanism is unknown and needs further study. One way to approach this is to study the effect of purified fractions on casein micelle stability. Takeuchi et al. (1985) used ion-exchange chromatography to prepare nine fractions of κ-casein A-1P that varied in their level of glycosylation. The ability of these subfractions to stabilize αs1-casein was shown to increase with increasing carbohydrate content. Further study on purified casein fractions may help to increase our understanding of casein micelle stability and the role of PTMs. Functionality in cheese

As already stated, micelle stability, or controlled destabilization in the case of cheese and yogurt manufacture, is of key importance in dairy manufacture. A number of authors have investigated the effects of κ-casein heterogeneity on micellar aggregation and the processing properties of milk. In cheese manufacture, the initial step is the chymosin-(rennet)catalyzed cleavage of the Phe126-Met127 bond in κ-casein, resulting in the release of the hydrophilic caseinomacropeptide from the micelle surface, which leads to micellar aggregation or clotting. Doi et al. (1979) examined the susceptibility to chymosin action of κ-casein preparations with different degrees of glycosylation. They found that more highly glycosylated forms were less susceptible to hydrolysis not only by chymosin but also by other proteases. Others have also found an inverse relationship between glycosylation and chymosin susceptibility for purified κ-casein fractions (Addeo et al., 1984; Vreeman et al., 1986) and in model systems (Addeo et al., 1984; Leaver and Horne, 1996). Compared with these model systems, in milk, the relationship is not always consistent. Using bulk milk, Chaplin and Green (1980) claimed that all κ-casein molecules were hydrolyzed with equal efficiency, whereas van Hooydonk et al. (1984) found that the rate of chymosin-catalyzed hydrolysis decreased with increasing glycosylation. The latter was also found in a study of individual milk samples ( Jensen et al., 2015): the highest rate of hydrolysis occurred in nonglycosylated κ-casein, and all glycosylated κ-casein isoforms had lower reaction rates. Within the nonglycosylated κ-casein group, variant A with one or two phosphate groups had the highest reaction rate, followed by variant B 1-2P and variant E 1-2P. Considering the consistent results between the studies of van Hooydonk et al. (1984) and Jensen et al. (2015), it seems convincing that glycan modifications negatively influence the reaction rate of chymosin hydrolysis. The rennet coagulation time (RCT), the rate of curd firming, and the curd firmness have been measured to assess the effect of κ-casein glycosylation on the coagulation properties

Sources and significance of casein heterogeneity

191

of milk (Robitaille et al., 1993). Whereas no effect on the RCT was observed, the rate of curd firming decreased, and the curd firmness increased at higher glycosylation levels. A more recent study on more than 2000 samples of milk from Simmental cows (Bonfatti et al., 2014) showed that the RCT decreased when the total κ-casein (as a proportion of total casein) and the glycosylated κ-casein increased, whereas nonglycosylated κ-casein exhibited a slightly unfavorable effect on the onset of the coagulation process. A decrease of 2 minutes in the RCT was also observed for milks with a high percentage of glycosylation of κ-casein on the total protein compared with milk with the lowest percentage of glycosylation. A favorable effect of κ-casein, glycosylated κ-casein, and degree of glycosylation on curd firmness was also detected. In another recent study, a detailed characterization of κ-casein isoforms was conducted by 2-D gel electrophoresis coupled with MS on milks with extremes of good or poor coagulation properties, selected from 892 samples of milk from Holstein Friesian and Jersey cows ( Jensen et al., 2012a). Six κ-casein isoforms, varying in phosphorylation and glycosylation levels, from each of the genetic variants of κ-casein were separated and identified, along with an unmodified κ-casein form at low abundance. Relative quantification showed that around 95% of the total κ-casein was phosphorylated with one or two phosphates attached, whereas approximately 35% of the identified κ-casein was glycosylated with from one to three tetrasaccharides. When isoforms from individual samples were compared, a very consistent ĸ-casein isoform pattern was found, with only minor differences in relation to breed, ĸ-casein genetic variant, and milk coagulation ability. The effect of PTMs on the coagulation properties of milk was studied by liquid chromatography/electrospray ionizationmass spectrometry using the same sample set of 892 cows (Poulsen et al., 2016). In Holstein milk, a higher relative content of κ-casein to total protein and a higher content of glycosylated κ-casein were associated with improved milk coagulation. In milk from Danish Jersey cows, the same tendency was observed, although it was not significant. Similar to glycosylated κ-casein as discussed by Poulsen et al. (2016), smaller casein micelles were associated with improved milk coagulation (Glantz et al., 2010). Therefore, the negative correlation between casein micelle size and glycosylated κ-casein (Bijl et al., 2014a) might explain the positive association between the degree of glycosylation and the coagulation properties. However, to date, there have been no studies that have determined the casein micelle size of fresh milk in relation to the coagulation properties to confirm this hypothesis. Differences in rennet coagulation properties have also been observed for genetic variants of κ-casein. Shorter RCTs, higher rates of curd firming, and higher curd firmness have been reported for milk from cows with the BB variant than for milk from cows with the AA variant (Walsh et al., 1998; and references therein). These differences between A and B variant milks were maintained after heat treatments of up to 80°C for 2 min, despite an overall deterioration in the coagulation properties at elevated temperatures (Choi and Ng-Kwai-Hang, 2003). Milks containing the rarer κ-casein C variant form rennet gels even more slowly than the A or B variant milks, possibly because of the substitution of histidine for arginine at residue 118, which may affect chymosin binding (Smith et al., 1997). Similar results have been observed for the κ-casein G variant, which has cysteine at residue 118 (Erhardt et al., 1997), and a similar explanation has been proposed (Smith et al., 1997). The influence of genetic variation in the major milk proteins on individual coagulation properties in 1299 Danish Holstein, Danish Jersey, and Swedish Red cows was determined by Poulsen et al. (2013). The authors showed that κ-casein B, αs1-casein C, and β-casein B were associated with good milk coagulation properties, whereas β-casein A2 had a negative effect.

192

5. Posttranslational modifications of caseins

Also in this case, it would be interesting to follow up whether a link between genetic variants and casein micelle size can explain the shorter RCTs of milk with different genetic variants. Acid coagulation

The coagulation of milk can also be induced by acid, as is the case in yogurt manufacture. Because this step does not involve the cleavage of κ-casein, a lesser or different effect might be expected. There are fewer studies on the effect of the glycosylation of κ-casein on acid coagulation. Cases et al. (2003) found that partial deglycosylation with neuraminidase had little effect on micellar surface charge and solvation but caused a decrease in the acid gelation time, a higher rate of gel firming, and a higher final firmness. Heat treatment

Heat treatment of milk can also destabilize the casein micelle structure. The heat-induced coagulation of milk is a very complex process that is affected by many parameters (O’Connell and Fox, 2003). A number of studies have examined the influence of the genetic variants of κ-casein on heat stability parameters, and it is generally accepted that B variant milks are more stable than A variant milks (FitzGerald and Hill, 1997). The reason may be more related to the effects on κ-casein concentration and micelle size, as already mentioned, than to the structural differences between the variants (Smith et al., 2002). Again, there are fewer studies related to the influence of the glycosylation of κ-casein on heat stability. Using a model system composed of casein micelles in simulated milk ultrafiltrate, Minkiewicz et al. (1993) showed that enzymatic removal of neuraminic acid using neuraminidase caused a decrease in heat stability. However, Robitaille and Ayers (1995), using whole milk, could not find a significant effect of neuraminidase treatment on heat stability. When milk is heated above 65°C, β-lactoglobulin denatures, exposing a previously buried sulfhydryl group that can participate in disulfide exchange reactions with other cysteine-containing proteins including κ-casein. This interaction has been recognized for many decades (Sawyer, 1969) and has been the subject of numerous investigations and reviews over the years; a detailed analysis is beyond the scope of this chapter (for an extensive review, see Chapter 9 of this volume). Recent studies have addressed both the mechanism of formation (Guyomarc’h et al., 2003) and the impact on product quality (Vasbinder et al., 2003) of disulfide-linked complexes. Despite the vast amount of literature on this topic, there do not appear to be any studies that have addressed the impact of the variable glycosylation of κ-casein on its ability to form disulfide-linked complexes either with itself or with β-lactoglobulin; however, this is, perhaps, not surprising because the sulfhydryl amino acids are in the para-κ-casein domain of the molecule, and the glycosylation sites are in the caseinomacropeptide. UHT milk

Heat-induced changes in micelle structure are particularly relevant for the production and storage of UHT milk. The extremes of heat treatment (of the order of 140–145°C for 4–10 s) produce a number of changes in the milk, not least of which is the formation of κ-casein-βlactoglobulin complexes. On storage, UHT-treated milks show a variable tendency to form gels, and this phenomenon, known as age gelation, affects product shelf life (for a review, see Datta and Deeth, 2001). From a theoretical perspective, higher initial levels of

Sources and significance of casein heterogeneity

193

glycosylation may act to temper the deleterious effects of heat treatment through effects on micelle size, micellar stability, and the formation of disulfide-linked complexes. The heat treatment itself may affect the glycosylation level at the surface of the micelle either indirectly, through loss of κ-casein in complex formation with β-lactoglobulin, or directly, through degradation of glycosidic residues (van Hooydonk et al., 1987; as quoted in Dziuba and Minikiewicz, 1996). Subsequent changes in the glycosylation level during storage could be mediated by the action of heat-stable glycosidases originating from psychrotrophic bacteria present in the raw milk (Marin et al., 1984). Release of monosaccharides during the storage of UHT milk has been observed (Recio et al., 1998; Belloque et al., 2001). Thus, both the initial glycosylation level of the κ-casein and the residual amount after UHT treatment may affect the storage properties of UHT-treated milk. As the actions of heat-resistant proteases can contribute to the age gelation of UHT milk, the inhibitory effects of glycosylation on the activity of proteases such as plasmin (Doi et al., 1979) may be important for prolonging shelf life. Unraveling specific effects will require the application of modern proteomic technologies for κ-casein analysis (Claverol et al., 2003; Holland et al., 2004, 2005, 2006; O’Donnell et al., 2004). Using these technologies, it will be possible to elaborate the heterogeneous glycoforms of κ-casein in raw milk, after pretreatment(s), after UHT processing, and during storage leading up to gelation. This will allow a definitive assessment of the functional significance of κ-casein glycosylation. Emulsification

The functional performance of the caseinomacropeptide is of importance both for use of the caseinomacropeptide as a food ingredient in its own right and for the properties it can impart to cheese whey products. Emulsifying performance has been found to vary with glycosylation (Kreuss et al., 2009a). Nonglycosylated caseinomacropeptide showed significantly better emulsifying properties than glycosylated caseinomacropeptide. Whereas nonglycosylated caseinomacropeptide showed an emulsifying activity index of 150.7 g/m2, glycosylated caseinomacropeptide achieved a value of only 98.5 g/m2. The stability of the emulsions was 1.4 times higher for nonglycosylated caseinomacropeptide than for glycosylated caseinomacropeptide. Droplet size measurements and creaming studies showed a marked influence of pH on both fractions, with minimal emulsion stabilities at pH 4.1 (glycosylated caseinomacropeptide) and pH 4.9 (nonglycosylated caseinomacropeptide). Investigation of the flocculation behavior and variations in the ionic strength indicated that the glycan side chains induced a combination of electrostatic, steric, and hydrophilic effects, preventing an ordered adsorption of glycosylated caseinomacropeptide molecules at the oil/water interface. In contrast, nonglycosylated caseinomacropeptide built a stable network at the oil/water interface. Foaming

Caseinomacropeptide fractions, both glycosylated and nonglycosylated, were also studied in detail for their foaming properties (Kreuss et al., 2009b). The nonglycosylated caseinomacropeptide-stabilized foams showed significantly higher foam rigidity and stability than the glycosylated caseinomacropeptide-stabilized foams, whereas both fractions yielded a high foaming ability with overruns of around 600%. The glycosylated caseinomacropeptide-stabilized foams, in particular, were considerably influenced by pH

194

5. Posttranslational modifications of caseins

and showed reduced foaming properties above the pI but superior properties at strong acidic pH, below the pI. This influence was less significant for nonglycosylated caseinomacropeptide. An increase in ionic strength did not appear to influence either fraction. Similar to the effects observed in oil-water emulsions, the combination of electrical, steric, and hydrophilic barriers caused by the glycosylation of glycosylated caseinomacropeptide appeared not to allow an ordered adsorption at the air/water interface. Also in this foam system, the nonglycosylated caseinomacropeptide could build a stable network at the air/water surface. Biological significance One aspect of κ-casein heterogeneity that has not been considered earlier is its influence on the biological properties of milk. This area has been reviewed extensively for cows’ milk (Dziuba and Minikiewicz, 1996). Some other reviews provide a broader perspective on the biological properties of all milk glycoproteins and glycolipids (Peterson et al., 2013; O’Riordan et al., 2014b). Several areas, all of which directly or indirectly link to health-related aspects, need to be considered: digestibility and bioavailability, the nutritional value of glycans, and the structure-function relationship of glycosylated groups and the bioactivity of peptides. Firstly, there is the effect of PTM on digestibility and bioavailability. This area has received attention only relatively recently. The effect of glycosylation on the hydrolysis by chymosin has been discussed earlier and has been studied intensively because of its importance for cheesemaking. The digestion of the resultant glycosylated or nonglycosylated caseinomacropeptide by brush border membrane peptidases has been described by Boutrou et al. (2008). Their key finding was that the digestions of nonglycosylated and glycosylated caseinomacropeptide through the action of exopeptidases were similar but that the activity of endopeptidases on glycosylated caseinomacropeptide was limited, certainly because of the attached O-glycosylations. Consequently, many more peptides were identified from the nonglycosylated caseinomacropeptide than from the glycosylated caseinomacropeptide. In addition, the glycosylation core, as well as the number of the attached glycosylated chains, modified the kinetics of digestion, the most heavily glycosylated forms being digested most slowly. Adding to the complexity, it was found by Petrat-Melin et al. (2016) that different genetic variants of κ-casein A, B, and E that varied in degrees of glycosylation were differentially affected by gastrointestinal digestion. Secondly, there is the nutritional contribution of the carbohydrate residues in κ-casein, particularly neuraminic acid (NeuAc). The importance of NeuAc and its roles in numerous biological functions have been reviewed (Schauer, 2000). NeuAc is commonly found as the terminal sugar residue on mammalian glycoproteins. Although mammals can synthesize NeuAc, the high levels in milk and especially colostrum may be related to a high demand for neonatal growth and development. The normal glycans on κ-casein are part of a class known as the Thomsen-/Friedenreich-related antigens (Dall’Olio and Chiricolo, 2001). The terminal NeuAc residues may play a key role in preventing colonization of the gut by pathogenic organisms by providing alternative binding sites that minimize binding to the normal gut epithelium. In this case, not only is the type of glycan important, but also the structure of the glycan will determine its functionality. The final aspect relates to the enormous interest in bioactive peptides derived from milk proteins (Clare and Swaisgood, 2000; Kilara and Panyam, 2003). Numerous in vitro activities

Caseins from other species

195

have been ascribed to κ-casein, its caseinomacropeptide, or peptides derived from them (Dziuba and Minikiewicz, 1996; Brody, 2000). These activities include prebiotic, antimicrobiological, and immunomodulatory effects (O’Riordan et al., 2014b). Some of these activities appear to be associated with particular forms of κ-casein (Malkoski et al., 2001) and can be glycosylation dependent (Li and Mine, 2004; O’Riordan et al., 2018). Whether or not the same activities occur in vivo is not always clear because it requires both generation and absorption of the active component during digestion, and this is not easy to detect. In vivo production of caseinomacropeptide is known to occur after the ingestion of milk (Ledoux et al., 1999; and references therein) and has been detected in the plasma of infants after milk ingestion (Chabance et al., 1995). Any naturally occurring bioactivity of caseinomacropeptide-derived peptides could be strongly influenced by the glycosylation status of κ-casein either directly, by modifying the activity of the peptide, or indirectly, by affecting proteolysis of κ-casein and hence release of the peptide. For future research, it will be interesting to see what we can learn from the biological significance of other mucin-type O-glycans.

Caseins from other species The biodiversity in the composition of milk between species is remarkable. Large differences can be identified by considering only the casein fraction: Fast growing animals such as rabbits can have milk with 90 g/L of caseins, whereas cows’ milk contains 27 g/L, and human milk contains only 5.8 g/L (Holt et al., 2013). Also, the composition of the casein fraction varies. Whereas the bovine milk protein fraction consists of the four caseins discussed in this chapter, the milk of humans and mares contains no or little αs2-casein, and elephant milk contains only β-casein and κ-casein. Differences between species are highly relevant. Firstly, the milks of some of these species provide an alternative source of nutrition. For the milks of all species, an understanding of between-species differences contributes to our knowledge of casein micelle stability and the significance of the heterogeneity of caseins. The differences between species on a genetic level have been reviewed previously (Ginger and Grigor, 1999), but the information on variation in the PTMs is more scattered. We therefore attempt to give an overview of the between-species variation with emphasis on β-casein phosphorylation and κ-casein glycosylation.

Phosphorylation It is apparent that considerable variations in phosphorylation of the caseins occur between species. Whereas the β-casein in bovine milk is mainly present with five phosphate groups, β-casein phosphorylation in human milk and horse milk is more complex. The biological significance of these variations is currently not known but is probably related to the fact that β-casein is the major casein fraction in the milks of these species. Also, the other species, caprine, ovine, and donkey, show variation in the phosphorylation of β-casein and α-casein compared with that in bovine milk. The only exception here is the water buffalo, which shows high consistency with its bovine counterpart.

196

5. Posttranslational modifications of caseins

Human The β-casein of human milk exists as six different forms with from zero to five phosphates (Greenberg et al., 1984) and is therefore more complex than that of bovine milk. In a recent study, the phosphorylation status of β-casein was investigated in human milk samples over time (Molinari et al., 2013). It varied significantly between term and preterm milk. A longitudinal trend among the term population was observed, with the phosphorylation state of β-casein decreasing during lactation in seven of the eight mothers analyzed. In the preterm population, no change in the distribution of phosphorylated isoforms was observed over time in 10 of the 16 mothers, whereas in the other six mothers, the level of phosphorylation increased during lactation. Horse Equine β-casein also shows variation in phosphorylation, with typically from three to seven phosphates on full length β-casein (Girardet et al., 2006) and from one to seven phosphates on a low-molecular-weight form that arises from an internal deletion (Miclo et al., 2007). The phosphorylation sites for equine milk have recently been reported (Mateos et al., 2010). In the mature protein, the isoform 4P was found to be phosphorylated on residues Ser9, Ser23, Ser24, and Ser25. Addition of phosphate groups on Ser18, Thr12, and Ser10 led to the formation of the isoforms 5P–7P, respectively. The results indicate that the in vivo phosphorylation of equine β-casein follows a sequential path and is not random. Ovine and caprine Ovine β-casein has also been reported to be variably phosphorylated, with from zero to seven phosphates (Ferranti et al., 2001), although the position of the seventh phosphorylation site is not clear. Caprine β-casein appears to be more like bovine β-casein, with the same five phosphorylation sites and an additional site on Thr27 (Neveu et al., 2002) in the mature protein. A more recent study compared caprine milks from the indigenous Greek breed and the international breeds Saanen and Alpine (Moatsou et al., 2008). This study identified a wide range of protein polymorphisms and phosphorylations. Phosphorylation of αs1-casein was 7P, 8P, and 9P, with lesser amounts of 6P and, rarely, 10P; phosphorylation of αs2-casein ranged from 6P to 11P; phosphorylation of β-casein was 5P, 6P, and 7P, with small amounts of lesser phosphorylation; κ-casein was phosphorylated with mostly 2P but some lesser amounts. A recent study using nanoscale liquid chromatography coupled with tandem electrospray MS has identified the majority of the phosphorylation sites of the goats’ milk caseins (Olumee-Shabon and Boehmer, 2013). These are summarized in Table 5.2 (Mercier et al., 1977). TABLE 5.2 Phosphorylation positions for the caprine caseins; data from (Olumee-Shabon and Boehmer, 2013) Casein

Number of phosphorylations

Phosphorylated residues

αs1

9

S61, S63, S130 (six others not detected)

αs2

10

S23, S24, S25, S72, S73, S74, S77, S145, S147, S159

β

5

S32, S33, S34, S37, S50

Caseins from other species

197

Other species A detailed proteomic analysis of water buffalo milk (D’Ambrosio et al., 2008) showed high consistency with the bovine counterpart proteins (residue numbers here based on the mature protein sequence). The study identified phosphopeptides from the following: αSl-casein, where phosphorylation occurred at Ser 41, 46, 48, 64, 66, 67, 68, and 75 in the mature protein, and αS2-casein, where phosphorylation occurred at the same sites as those of the bovine counterpart (Ser 129, 131, and 143); β-casein phosphorylation sites were consistent with the bovine counterpart sites (Ser 15, 17, 18, 19, and 35); the main and secondary sites of phosphorylation in buffalo κ-casein were Ser149 and Ser127, as observed for the bovine protein. In donkey milk “caseome,” Chianese et al. (2010) identified 11 phosphorylated components for κ-casein, six phosphorylated components for β- and αs1-casein, and three main phosphorylated components for αs2-casein. Up to five isoforms of β-casein are present in elephant milk (Madende et al., 2018). Of these isoforms, only a nonphosphorylated isoform and a singly phosphorylated (at Ser9) isoform in the mature protein were confirmed using high-resolution MS analysis.

Glycosylation The full amino acid sequences of κ-casein from more than 60 species are currently in the UniProt database (Table 5.3), with another 114 entries covering subspecies and incomplete sequences. Although the amino acid sequences are known for these species, the variation in the glycosylation of κ-casein is known for only a handful of species, and there is conclusive evidence for the positions of the modification sites for even fewer species. Least is known about the variation in carbohydrate moieties and the structures of κ-casein between species, which has been examined in detail only for bovine, human, and caprine milks. An overview of the variation in glycosylation of different species is provided in Table 5.4. Human In human milk, indirect evidence (the absence of Thr residues during Edman sequencing) showed that up to 10 of the residues were glycosylated (Fiat et al., 1980). Additional experiments confirmed that human milk seemed to carry a greater amount of glycosylated groups than bovine milk. N-acetylgalactosamine, galactose, N-acetylneuraminic acid, fucose, and N-acetylglucosamine were detected in human milk (Azuma et al., 1984). The first three are also common in cows’ milk, whereas N-acetylgalactosamine and fucose have been found only in bovine colostrum. As the content of total glycosylated groups was approximately 40% of the total κ-casein weight (Yamauchi et al., 1981), the amount of glycosylated groups in human milk is much higher than in bovine milk, which is estimated at around 4% (Table 5.4). With TABLE 5.3

Species with complete κ-casein entries in the UniProt database (December 2018)

Ailuropoda melanoleuca (giant panda) Aotus nancymaae (Ma’s night monkey) Balaenoptera acutorostrata scammoni (North Pacific minke whale) (Balaenoptera davidsoni) Continued

198

5. Posttranslational modifications of caseins

TABLE 5.3 Species with complete κ-casein entries in the UniProt database (December 2018)—cont’d Bos mutus grunniens (wild yak) (Bos grunniens) Bos taurus (Bovine) Bubalus bubalis (domestic water buffalo) Callithrix jacchus (white-tufted-ear marmoset) Camelus bactrianus (Bactrian camel) Camelus dromedarius (dromedary) (Arabian camel) Camelus ferus (wild Bactrian camel) (Camelus bactrianus ferus) Canis lupus familiaris (dog) (Canis familiaris) Capra hircus (goat) Capricornis crispus (Japanese serow) (Naemorhedus crispus) Capricornis sumatraensis (Sumatran serow) Capricornis swinhoei (Taiwan serow) (Naemorhedus swinhoei) Cavia porcellus (guinea pig) Cercocebus atys (sooty mangabey) (Cercocebus torquatus atys) Cervus nippon (sika deer) Chlorocebus sabaeus (green monkey) (Cercopithecus sabaeus) Colobus angolensis palliatus (Peters’ Angolan colobus) Delphinapterus leucas (beluga whale) Dipodomys ordii (Ord’s kangaroo rat) Enhydra lutris kenyoni Equus asinus africanus Equus caballus (horse) Erinaceus europaeus (Western European hedgehog) Felis catus (cat) (Felis silvestris catus) Fukomys damarensis (Damaraland mole rat) (Cryptomys damarensis) Gorilla gorilla gorilla (Western lowland gorilla) Heterocephalus glaber (naked mole rat) Homo sapiens (human) Lama glama (llama) Leptonychotes weddellii (Weddell seal) Lipotes vexillifer (Yangtze river dolphin) Macaca fascicularis (crab-eating macaque) (Cynomolgus monkey)

Caseins from other species

TABLE 5.3

Species with complete κ-casein entries in the UniProt database (December 2018)—cont’d

Macaca mulatta (Rhesus macaque) Macaca nemestrina (pig-tailed macaque) Mandrillus leucophaeus (drill) (Papio leucophaeus) Mus musculus (Mouse) Myotis brandtii (Brandt’s bat) Naemorhedus goral (Himalayan goral) Neomonachus schauinslandi (Hawaiian monk seal) (Monachus schauinslandi) Neophocaena asiaeorientalis asiaeorientalis (Yangtze finless porpoise) Nomascus leucogenys (Northern white-cheeked gibbon) (Hylobates leucogenys) Odobenus rosmarus divergens (Pacific walrus) Oreamnos americanus (mountain goat) Ornithorhynchus anatinus (duck-billed platypus) Oryctolagus cuniculus (rabbit) Ovis aries (sheep) Pan paniscus (pygmy chimpanzee) (bonobo) Pan troglodytes (chimpanzee) Papio anubis (olive baboon) Physeter catodon (sperm whale) (Physeter macrocephalus) Pongo abelii (Sumatran orangutan) (Pongo pygmaeus abelii) Pteropus alecto (black flying fox) Rattus norvegicus (rat) Rhinopithecus roxellana (golden snub-nosed monkey) (Pygathrix roxellana) Rupicapra rupicapra (chamois) Saiga tatarica (saiga antelope) Sus scrofa (pig) Tachyglossus aculeatus (Australian echidna) Tarsius syrichta (Philippine tarsier) Trichechus manatus latirostris (Florida manatee) Trichosurus vulpecula (brush-tailed possum) Tursiops truncatus (Atlantic bottle-nosed dolphin) (Delphinus truncatus) Ursus maritimus (polar bear) (Thalarctos maritimus)

199

Scientific name of organism

Length (number of amino acids)

Bovine

Bos taurus

190

142, 152, 153, 154, 157, 163, 170, 186

Dromedary (Arabian camel)

Camelus dromedarius

182

125, 129, 169, 172, 173, 154, 161, 178

Caprine (goat)

Capra hircus

192

152, 155, 156, 159, 165, 172, 188

Horse

Equus caballus

185

143, 161, 178

Human

Homo sapiens

182

133, 143, 148, 151, 157, 167, 169, 178

Fucose, N-acetylglucosamine, galactose, N-acetylgalactosamine, N-acetylneuraminic acid

Ovine (sheep)

Ovis aries

192

152, 155, 156, 159, 172, 188

N-acetylgalactosamine, galactose, N-acetylneuraminic acid, N-glycolylneuraminic acid

Amino acid glycosylation site

Carbohydrate moieties

Structure

Amount

N-acetylgalactosamine, galactose, N-acetylneuraminic acid, N-acetylglucosamine (colostrum only), Fucose (colostrum only)

Mono-, di-, tri(two variants), tetrasaccharides

Estimated to be 4% on total κ-casein weight, assuming 50% of the κ-casein is glycosylated with two oligosaccharide side chains each with a molecular weight of 798 Da on average

N-acetylgalactosamine, galactose, N-acetylneuraminic acid, N-glycolylneuraminic acid

Mono-, di-, tri(four variants), tetrasaccharides (four variants)

The contents of N-acetylneuraminic acid and N-glycolylneuraminic acid were, respectively, 1.07 and 1.17 mg/ 100 mg in caprine caseinomacropeptide Higher amount of carbohydrates and sialic acid residues compared with bovine Estimated to be 40% on total κ-casein weight

Mono-, di-, tri(four variants), tetrasaccharides (four variants)

The contents of N-acetylneuraminic acid and N-glycolylneuraminic acid were, respectively, 0.13 and 0.93 mg/ 100 mg in ovine caseinomacropeptide

5. Posttranslational modifications of caseins

Common name of organism

200

TABLE 5.4 Variation in glycosylation of κ-casein between species in length, glycosylation site, carbohydrate moieties, structure, and amount; organisms and glycosylation sites as stated in the UniProt database; amino acid modifications in italics; modification expected based on similarity to bovine casein sequence or sequence analysis

Caseins from other species

201

respect to changes during lactation, a study on glycosylation in human milk revealed changing patterns of glycosylation for many of the whey proteins, but not for κ-casein (Froehlich et al., 2010). Horse The glycosylation of equine κ-casein has been reviewed by Uniacke-Lowe et al. (2010). Glycosylation of horse milk has been indicated by lectin-binding studies (Iametti et al., 2001). However, the exact positions of the glycosylation sites or the carbohydrate moieties are not conclusive. It has been suggested that the κ-casein of equine milk contains more carbohydrates and more sialic acid residues than that of bovine milk (Egito et al., 2001, 2002). Ovine and caprine The positions of the glycans in ovine milk and caprine milk have not been confirmed. Interestingly, the carbohydrate moieties have been identified and quantified. In the milk of both species, N-acetylgalactosamine, galactose, N-acetylneuraminic acid, and N-glycolylneuraminic acid (NeuGc) were detected (Moreno et al., 2001). The contents of N-acetylneuraminic acid and N-glycolylneuraminic acid were, respectively, 0.13 and 0.93 mg/100 mg in ovine caseinomacropeptide and 1.07 and 1.17 mg/100 mg in caprine caseinomacropeptide. Mono- di-, tri-, and tetrasaccharides were detected in ovine milk (Moreno et al., 2000; Mamone et al., 2003; Casal et al., 2013) and in caprine milk ( Javier Moreno et al., 2001; Casal et al., 2013). The structures of the tri- and tetrasaccharides were elucidated by graphitized carbon liquid chromatographyelectrospray ionization ion trap tandem MS (Casal et al., 2013). Compared with bovine milk and human milk, the presence of N-glycolylneuraminic acid on κ-casein is unique for ovine and caprine species. The presence of this additional carbohydrate moiety results in eight different structures of tri- and tetrasaccharides (Fig. 5.9), whereas only three are present in bovine milk. These eight structures are the trisaccharides (NeuAcα(2–3)Galβ(1-3)GalNAc or Galβ(1–3) [NeuAcα(2–6)]GalNAc), (NeuGcα(2–3)Galβ(1–3)GalNAc or Galβ(1–3)[NeuGcα(2–6)]GalNAc) and the tetrasaccharides NeuAcα(2–3)Galβ(1–3)[NeuAcα(2–6)]GalNAc, NeuGcα(2–3)Galβ (1–3)[NeuAcα(2–6)]GalNAc, NeuAcα(2–3)Galβ(1–3)[NeuGcα(2–6)]GalNAc, NeuGcα(2–3)Galβ (1–3)[NeuGcα(2–6)]GalNAc. FIG. 5.9 Tri- and tetrasaccharides present in ovine and

▪

caprine milk. ( ) GalNAc, (●) Gal, (♦) NeuAc, and ( ) NeuGc.

202

5. Posttranslational modifications of caseins

Other species In water buffalo, diglycosylated forms of κ-casein were identified, but the specific residues modified were not reported; however, there is no reason to believe that they would be different from the bovine sites. In dromedary, also known as Arabian camel, five threonine residues have been proposed to be glycosylated (125, 129, 169, 172, 173) (Kappeler et al., 1998); however, to date, no direct evidence confirms these positions. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis, most of the κ-casein in dromedary milk was found to be of low molecular mass and therefore was a low glycosylated form (Kappeler et al., 1998). This was in disagreement with earlier findings of Mehaia (1987), who found a high content of released sialic acid by neuraminidase (7.35 mg/g camel casein) compared with that for bovine κ-casein (3.02 mg/g bovine casein). The glycan structures of dromedary κ-casein have not been determined as yet (Mati et al., 2017).

Conclusions PTMs such as phosphorylation, glycosylation, and perhaps disulfide bond formation play a critical role in casein micelle formation and stability. It seems somewhat surprising that so much variability occurs in these PTMs on the caseins. Whereas significant functional differences in milk properties have consistently been reported for milks with different genetic variants of the caseins and are well established, the effects reported for variable PTMs have been investigated only more recently, enabled by proteomic methods, and supported by a range of highly sensitive MS techniques. A full understanding of these effects and their implications for dairy processing and products is still at an early stage.

Acknowledgments The authors gratefully acknowledge Hein van Valenberg and Lotte Bach Larsen for helpful discussions and critical reading of this manuscript.

References Addeo, F., Martin, P., Ribadeau-Dumas, B., 1984. Susceptibility of buffalo and cow kappa-caseins to chymosin action. Milchwissenschaft 39, 202–205. Alais, C., Jolles, P., 1961. Comparative study of the caseinoglycopeptides formed by the action of rennin on caseins from the cow, sheep and goat. II. Study of the non-peptide portion. Biochim. Biophys. Acta 51, 315–322. Alexander, L.J., Stewart, A.F., Mackinlay, A.G., Kapelinskaya, T.V., Tkach, T.M., Gorodetsky, S.I., 1988. Isolation and characterization of the bovine kappa-casein gene. Eur. J. Biochem. 178, 395–401. Azuma, N., Kaminogawa, S., Yamauchi, K., 1984. Properties of glycomacropeptide and para-κ-casein derived from human κ-casein and comparison of human and bovine κ-caseins as to susceptibility to chymosin and pepsin. Agric. Biol. Chem. 48, 2025–2031. https://doi.org/10.1080/00021369.1984.10866443. Belloque, J., Villamiel, M., Lopez, F.R., Olano, A., 2001. Release of galactose and N-acetylglucosamine during the storage of UHT milk. Food Chem. 72, 407–412. Bijl, E., de Vries, R., van Valenberg, H., Huppertz, T., van Hooijdonk, T., 2014a. Factors influencing casein micelle size in milk of individual cows: genetic variants and glycosylation of κ-casein. Int. Dairy J. 34, 135–141. https://doi. org/10.1016/j.idairyj.2013.08.001.

References

203

Bijl, E., van Valenberg, H.J.F., Huppertz, T., van Hooijdonk, A.C.M., Bovenhuis, H., 2014b. Phosphorylation of alpha (s1)-casein is regulated by different genes. J. Dairy Sci. 97, 7240–7246. https://doi.org/10.3168/jds.2014-8061. Bijl, E., van Valenberg, H., Sikkes, S., Jumelet, S., Sala, G., Olieman, K., van Hooijdonk, T., Huppertz, T., 2014c. Chymosin-induced hydrolysis of caseins: influence of degree of phosphorylation of alpha-s1-casein and genetic variants of beta-casein. Int. Dairy J. 39, 215–221. https://doi.org/10.1016/j.idairyj.2014.07.005. Bijl, E., Huppertz, T., van Valenberg, H., Holt, C., 2018. A quantitative model of the bovine casein micelle: ion equilibria and calcium phosphate sequestration by individual caseins in bovine milk. Eur. Biophys. J. https://doi.org/ 10.1007/s00249-018-1330-2. Bobe, G., Beitz, D.C., Freeman, A.E., Lindberg, G.L., 1999. Effect of milk protein genotypes on milk protein composition and its genetic parameter estimates. J. Dairy Sci. 82, 2797–2804. Bodenmiller, B., Mueller, L.N., Mueller, M., Domon, B., Aebersold, R., 2007. Reproducible isolation of distinct, overlapping segments of the phosphoproteome. Nat. Methods 4, 231–237. Bonfatti, V., Grigoletto, L., Cecchinato, A., Gallo, L., Carnier, P., 2009. Erratum to “Validation of a new reversed-phase high-performance liquid chromatography method for separation and quantification of bovine milk protein genetic variants” [J. Chromatogr. A, 1195 (2008) 101–106]. J. Chromatogr. A 1216, 1528. https://doi.org/10.1016/j. chroma.2009.01.008. Bonfatti, V., Chiarot, G., Carnier, R., 2014. Glycosylation of kappa-casein: genetic and nongenetic variation and effects on rennet coagulation properties of milk. J. Dairy Sci. 97, 1961–1969. https://doi.org/10.3168/jds.2013-7418. Bouguyon, E., Beauvallet, C., Huet, J.-C., Chanat, E., 2006. Disulphide bonds in casein micelle from milk. Biochem. Biophys. Res. Commun. 343, 450–458. Bouniol, C., Printz, C., Mercier, J.-C., 1993. Bovine αs2-casein D is generated by exon VIII skipping. Gene 128, 289–293. Boutrou, R., Jardin, J., Blais, A., Tome, D., Leonil, J., 2008. Glycosylations of kappa-casein-derived caseinomacropeptide reduce its accessibility to endo- but not exointestinal brush border membrane peptidases. J. Agric. Food Chem. 56, 8166–8173. Bovenhuis, H., Van Arendonk, J.A., Korver, S., 1992. Associations between milk protein polymorphisms and milk production traits. J. Dairy Sci. 75, 2549–2559. Brody, E.P., 2000. Biological activities of bovine glycomacropeptide. Br. J. Nutr. 84 (Suppl. 1), S39–S46. Buitenhuis, B., Poulsen, N.A., Gebreyesus, G., Larsen, L.B., 2016. Estimation of genetic parameters and detection of chromosomal regions affecting the major milk proteins and their post translational modifications in Danish Holstein and Danish Jersey cattle. BMC Genet. 17, 114. https://doi.org/10.1186/s12863-016-0421-2. Casal, E., Lebro´n-Aguilar, R., Chuan-Lee, Y., Noboru, T., Quintanilla-Lo´pez, J.E., 2013. Identification of sialylated oligosaccharides derived from ovine and caprine caseinomacropeptide by graphitized carbon liquid chromatography-electrospray ionization ion trap tandem mass spectrometry. Food Anal. Methods 6, 814–825. https://doi.org/10.1007/s12161-012-9489-0. Cases, E., Vidal, V., Cuq, J.L., 2003. Effect of kappa-casein deglycosylation on the acid coagulability of milk. J. Food Sci. 68, 2406–2410. Chabance, B., Jolles, P., Izquierdo, C., Mazoyer, E., Francoual, C., Drouet, L., Fiat, A.M., 1995. Characterization of an antithrombotic peptide from kappa-casein in newborn plasma after milk ingestion. Br. J. Nutr. 73, 583–590. Chaplin, B., Green, M.L., 1980. Determination of the proportion of kappa-casein hydrolyzed by rennet on coagulation of skim-milk. J. Dairy Res. 47, 351–358. Chianese, L., Calabrese, M.G., Ferranti, P., Mauriello, R., Garro, G., De Simone, C., Quarto, M., Addeo, F., Cosenza, G., Ramunno, L., 2010. Proteomic characterization of donkey milk “caseome”. J. Chromatogr. A 1217, 4834–4840. Choi, J.W., Ng-Kwai-Hang, K.F., 2003. Effects of genetic variants of kappa-casein and beta-lactoglobulin and heat treatment on coagulating properties of milk. Asian Australas. J. Anim. Sci. 16, 1212–1217. Chokhawala, H.A., Chen, X., 2007. Enzymatic approaches to O-glycoside introduction: glycosyltransferases. In: Kamerling, J.P. (Ed.), Comprehensive Glycoscience: From Chemistry to Systems Biology. In: 1. Elsevier, Amsterdam, pp. 415–451. Clare, D.A., Swaisgood, H.E., 2000. Bioactive milk peptides: a prospectus. J. Dairy Sci. 83, 1187–1195. Claverol, S., Burlet-Schiltz, O., Gairin, J.E., Monsarrat, B., 2003. Characterization of protein variants and posttranslational modifications: ESI-MSn analyses of intact proteins eluted from polyacrylamide gels. Mol. Cell. Proteomics 2, 483–493. Collins, M.O., Yu, L., Choudhary, J.S., 2007. Analysis of protein phosphorylation on a proteome-scale. Proteomics 7, 2751–2768.

204

5. Posttranslational modifications of caseins

Corfield, A.P., Berry, M., 2015. Glycan variation and evolution in the eukaryotes. Trends Biochem. Sci. 40, 351–359. https://doi.org/10.1016/j.tibs.2015.04.004. Coolbear, K.P., Elgar, D.F., Ayers, J.S., 1996. Profiling of genetic variants of bovine κ-casein macropeptide by electrophoretic and chromatographic techniques. Int. Dairy J. 6, 1055–1068. Cox, D.M., Zhong, F., Du, M., Duchoslav, E., Sakuma, T., McDermott, J.C., 2005. Multiple reaction monitoring as a method for identifying protein posttranslational modifications. J. Biomol. Tech. 16, 83–90. Creamer, L.K., Richardson, T., Parry, D.A.D., 1981. Secondary structure of bovine αS1- and β-casein in solution. Arch. Biochem. Biophys. 211, 689–696. https://doi.org/10.1016/0003-9861(81)90505-1. Dalgleish, D.G., 1985. Glycosylated kappa-caseins and the sizes of bovine casein micelles. Analysis of the different forms of kappa-casein. Biochim. Biophys. Acta 830, 213–215. Dalgleish, D.G., 1986. Analysis by fast protein liquid chromatography of variants of kappa-casein and their relevance to micellar structure and renneting. J. Dairy Res. 53, 43–51. Dalgleish, D.G., Corredig, M., 2012. The structure of the casein micelle of milk and its changes during processing. Annu. Rev. Food Sci. Technol. 3, 449–467. https://doi.org/10.1146/annurev-food-022811-101214. Dalgleish, D.G., Horne, D.S., Law, A.J.R., 1989. Size-related differences in bovine casein micelles. Biochim. Biophys. Acta 991, 383–387. Dall’Olio, F., Chiricolo, M., 2001. Sialyltransferases in cancer. Glycoconj. J. 18, 841–850. D’Ambrosio, C., Arena, S., Salzano, A.M., Renzone, G., Ledda, L., Scaloni, A., 2008. A proteomic characterization of water buffalo milk fractions describing PTM of major species and the identification of minor components involved in nutrient delivery and defense against pathogens. Proteomics 8, 3657–3666. Darula, Z., Medzihradszky, K.F., 2018. Analysis of mammalian O-glycopeptides—we have made a good start, but there is a long way to go. Mol. Cell. Proteomics 17, 2–17. https://doi.org/10.1074/mcp.MR117.000126. Datta, N., Deeth, H.C., 2001. Age gelation of UHT milk—a review. Food Bioprod. Process. 79, 197–210. Davies, D.T., Law, A.J.R., 1983. Variation in protein composition of bovine casein micelles and serum casein in relation to casein micelle size and milk temperature. J. Dairy Res. 50, 67–75. de Vries, R., van Knegsel, A., Johansson, M., Lindmark-Ma˚nsson, H., van Hooijdonk, T., Holtenius, K., Hettinga, K., 2015. Effect of shortening or omitting the dry period of Holstein-Friesian cows on casein composition of milk. J. Dairy Sci. 98, 8678–8687. https://doi.org/10.3168/jds.2015-9544. Doi, H., Kawaguchi, N., Ibuki, F., Kanamori, M., 1979. Susceptibility of kappa-casein components to various proteases. J. Nutr. Sci. Vitaminol. (Tokyo) 25, 33–41. Doi, H., Kobatake, H., Ibuki, F., Kanamori, M., 1980. Attachment sites of carbohydrate portions to peptide chain of kappa-casein from bovine colostrum. Agric. Biol. Chem. 44, 2605–2611. Donnelly, W.J., McNeill, G.P., Buchheim, W., McGann, T.C., 1984. A comprehensive study of the relationship between size and protein composition in natural bovine casein micelles. Biochim. Biophys. Acta 789, 136–143. Dziuba, J., Minikiewicz, P., 1996. Influence of glycosylation on micelle-stabilising ability and biological properties of C-terminal fragments of cow’s κ-casein. Int. Dairy J. 6, 1017–1044. Egito, A.S., Girardet, J.-M., Miclo, L., Moll
e, D., Humbert, G., Gaillard, J.-L., 2001. Susceptibility of equine κ- and β-caseins to hydrolysis by chymosin. Int. Dairy J. 11, 885–893. https://doi.org/10.1016/S0958-6946(01)00123-6. Egito, A.S., Miclo, L., Lo´pez, C., Adam, A., Girardet, J.-M., Gaillard, J.-L., 2002. Separation and characterization of mares’ milk αs1-, β-, κ-caseins, γ-casein-like, and proteose peptone component 5-like peptides. J. Dairy Sci. 85, 697–706. https://doi.org/10.3168/jds.S0022-0302(02)74126-X. Erhardt, G., Prinzenberg, E.-M., Buchberger, J., Krick-Salek, H., Krause, I., Miller, M., 1997. Bovine kappa-casein G: detection, occurrence, molecular genetic characterisation, genotyping and coagulation properties. In: Milk Protein Polymorphism (IDF Special Issue 9702). International Dairy Federation, Brussels, pp. 328–329. Fang, Z.H., Visker, M.H.P.W., Miranda, G., Delacroix-Buchet, A., Bovenhuis, H., Martin, P., 2016. The relationships among bovine alpha(S)-casein phosphorylation isoforms suggest different phosphorylation pathways. J. Dairy Sci. 99, 8168–8177. https://doi.org/10.3168/jds.2016-11250. Fang, Z.H., Bovenhuis, H., Delacroix-Buchet, A., Miranda, G., Boichard, D., Visker, M.H.P.W., Martin, P., 2017. Genetic and nongenetic factors contributing to differences in alpha(s)-casein phosphorylation isoforms and other major milk proteins. J. Dairy Sci. 100, 5564–5577. https://doi.org/10.3168/jds.2016-12338. Fang, Z.H., Bovenhuis, H., van Valenberg, H.J.F., Martin, P., Huppertz, T., Visker, M.H.P.W., 2018. Genetic parameters for alpha(S1)-casein and alpha(S2)-casein phosphorylation isoforms in Dutch Holstein Friesian. J. Dairy Sci. 101, 1281–1291. https://doi.org/10.3168/jds.2017-13623.

References

205

Farrell Jr., H.M., Cooke, P.H., Wickham, E.D., Piotrowski, E.G., Hoagland, P.D., 2003. Environmental influences on bovine kappa-casein: reduction and conversion to fibrillar (amyloid) structures. J. Protein Chem. 22, 259–273. Farrell Jr., H.M., Jimenez-Flores, R., Bleck, G.T., Brown, E.M., Butler, J.E., Creamer, L.K., Hicks, C.L., Hollar, C.M., NgKwai-Hang, K.F., Swaisgood, H.E., 2004. Nomenclature of the proteins of cows’ milk—sixth revision. J. Dairy Sci. 87, 1641–1674. Farrell, H.M., Malin, E.L., Brown, E.M., Qi, P.X., 2006. Casein micelle structure: what can be learned from milk synthesis and structural biology? Curr. Opin. Colloid Interface Sci. 11, 135–147. https://doi.org/10.1016/J. COCIS.2005.11.005. Ferranti, P., Pizzano, R., Garro, G., Caira, S., Chianese, L., Addeo, F., 2001. Mass spectrometry-based procedure for the identification of ovine casein heterogeneity. J. Dairy Res. 68, 35–51. Fiat, A.M., Jolles, J., Aubert, J.P., Loucheux-Lefebvre, M.H., Jolles, P., 1980. Localisation and importance of the sugar part of human casein. Eur. J. Biochem. 111, 333–339. https://doi.org/10.1111/j.1432-1033.1980.tb04946.x. Fiat, A.M., Jolles, J., Loucheux-Lefebvre, M.H., Alais, C., Jolles, P., 1981. Localisation of the prosthetic sugar groups of bovine colostrum kappa-casein. Hoppe Seylers Zeitsch. Physiol. Chem. 362, 1447–1454. Fiat, A.M., Chevan, J., Jolles, P., De Waard, P., Vliegenthart, J.F., Piller, F., Cartron, J.P., 1988. Structural variability of the neutral carbohydrate moiety of cow colostrum kappa-casein as a function of time after parturition. Identification of a tetrasaccharide with blood group I specificity. Eur. J. Biochem. 173, 253–259. FitzGerald, R.J., Hill, J.P., 1997. The relationship between milk protein polymorphism and the manufacture and functionality of dairy products. In: Milk Protein Polymorphism (IDF Special Issue 9702). International Dairy Federation, Brussels, pp. 355–371. Fournet, B., Fiat, A.M., Montreuil, J., Jolles, P., 1975. The sugar part of kappa-caseins from cow milk and colostrum and its microheterogeneity. Biochimie 57, 161–165. Fournet, B., Fiat, A.M., Alais, C., Jolles, P., 1979. Cow kappa-casein: structure of the carbohydrate portion. Biochim. Biophys. Acta 576, 339–346. Frederiksen, P.D., Andersen, K.K., Hammershoj, M., Poulsen, H.D., Sorensen, J., Bakman, M., Qvist, K.B., Larsen, L.B., 2011. Composition and effect of blending of noncoagulating, poorly coagulating, and well-coagulating bovine milk from individual Danish Holstein cows. J. Dairy Sci. 94, 4787–4799. https://doi.org/10.3168/jds.2011-4343. Froehlich, J.W., Dodds, E.D., Barboza, M., McJimpsey, E.L., Seipert, R.R., Francis, J., An, H.J., Freeman, S., German, J.B., Lebrilla, C.B., 2010. Glycoprotein expression in human milk during lactation. J. Agric. Food Chem. 58, 6440–6448. Ginger, M.R., Grigor, M.R., 1999. Comparative aspects of milk caseins. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 124, 133–145. https://doi.org/10.1016/S0305-0491(99)00110-8. Girardet, J.-M., Miclo, L., Florent, S., Moll
e, D., Gaillard, J.-L., 2006. Determination of the phosphorylation level and deamidation susceptibility of equine β-casein. Proteomics 6, 3707–3717. Glantz, M., Devold, T.G., Vegarud, G.E., Mansson, H.L., Stalhammar, H., Paulsson, M., 2010. Importance of casein micelle size and milk composition for milk gelation. J. Dairy Sci. 93, 1444–1451. https://doi.org/10.3168/ jds.2009-2856. Gorodetskii, S.I., Kershulite, D.R., Korobko, V.G., 1983. Primary structure of cDNA of Bos taurus kappa-casein macropeptide. Bioorg. Khim. 9, 1693–1695. 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. Grosclaude, F., Mahe, M.F., Mercier, J.C., Ribadeau-Dumas, B., 1972a. Characterization of genetic variants of alpha-S1 and beta bovine caseins. Eur. J. Biochem. 26, 328–337. Grosclaude, F., Mahe, M.-F., Mercier, J.-C., Ribadeau-Dumas, B., 1972b. Localization of amino-acid substitutions differentiating the A and B variants of kappa-casein in cattle. Ann. Genet. Select. Anim. 4, 515–521. Groves, M.L., Dower, H.J., Farrell Jr., H.M., 1992. Reexamination of the polymeric distributions of kappa-casein isolated from bovine milk. J. Protein Chem. 11, 21–28. Groves, M.L., Wickham, E.D., Farrell Jr., H.M., 1998. Environmental effects on disulfide bonding patterns of bovine kappa-casein. J. Protein Chem. 17, 73–84. Guerin, J., Alais, C., Jolles, J., Jolles, P., 1974. Kappa-casein from bovine colostrum. Biochim. Biophys. Acta 351, 325–332. Guyomarc’h, F., Law, A.J., Dalgleish, D.G., 2003. Formation of soluble and micelle-bound protein aggregates in heated milk. J. Agric. Food Chem. 51, 4652–4660.

206

5. Posttranslational modifications of caseins

Heck, J.M.L., Olieman, C., Schennink, A., van Valenberg, H.J.F., Visker, M.H.P.W., Meuldijk, R.C.R., van Hooijdonk, A.C.M., 2008. Estimation of variation in concentration, phosphorylation and genetic polymorphism of milk proteins using capillary zone electrophoresis. Int. Dairy J. 18, 548–555. Heck, J.M.L., Schennink, A., van Valenberg, H.J.F., Bovenhuis, H., Visker, M.H.P.W., van Arendonk, J.A.M., van Hooijdonk, A.C.M., 2009. Effects of milk protein variants on the protein composition of bovine milk. J. Dairy Sci. 92, 1192–1202. Hernandez-Hernandez, O., Lebron-Aguilar, R., Quintanilla-Lopez, J.E., Luz Sanz, M., Javier Moreno, F., 2011. Detection of two minor phosphorylation sites for bovine ĸ-casein macropeptide by reversed-phase liquid chromatography-tandem mass spectrometry. J. Agric. Food Chem. 59, 10848–10853. Holland, J.W., Deeth, H.C., Alewood, P.F., 2004. Proteomic analysis of kappa-casein micro-heterogeneity. Proteomics 4, 743–752. Holland, J.W., Deeth, H.C., Alewood, P.F., 2005. Analysis of O-glycosylation site occupancy in bovine kappa-casein glycoforms separated by two-dimensional gel electrophoresis. Proteomics 5, 990–1002. Holland, J.W., Deeth, H.C., Alewood, P.F., 2006. Resolution and characterisation of multiple isoforms of bovine kappa-casein by 2-DE following a reversible cysteine-tagging enrichment strategy. Proteomics 6, 3087–3095. Holland, J.W., Deeth, H.C., Alewood, P.F., 2008. Analysis of disulphide linkages in bovine kappa-casein oligomers using two-dimensional electrophoresis. Electrophoresis 29, 2402–2410. Holt, C., 2004. An equilibrium thermodynamic model of the sequestration of calcium phosphate by casein micelles and its application to the calculation of the partition of salts in milk. Eur. Biophys. J. Biophys. Lett. 33, 421–434. https://doi.org/10.1007/s00249-003-0377-9. Holt, C., Horne, D.S., 1996. The hairy casein micelle: evolution of the concept and its implications for dairy technology. Neth. Milk Dairy J. 50, 85–111. Holt, C., Carver, J.A., Ecroyd, H., Thorn, D.C., 2013. Invited review: Caseins and the casein micelle: their biological functions, structures, and behavior in foods. J. Dairy Sci. 96, 6127–6146. https://doi.org/10.3168/jds.2013-6831. Horne, D.S., 1998. Casein interactions: casting light on the black boxes, the structure in dairy products. Int. Dairy J. 8, 171–177. Horne, D.S., 2002. Casein structure, self-assembly and gelation. Curr. Opin. Colloid Interface Sci. 7, 456–461. Iametti, S., Tedeschi, G., Oungre, E., Bonomi, F., 2001. Primary structure of κ-casein isolated from mares’ milk. J. Dairy Res. 68, 53–61. https://doi.org/10.1017/S0022029900004544. Ibeagha-Awemu, E.M., Prinzenberg, E.M., Jann, O.C., Luhken, G., Ibeagha, A.E., Zhao, X., Erhardt, G., 2007. Molecular characterization of bovine CSN1S2*B and extensive distribution of zebu-specific milk protein alleles in European cattle. J. Dairy Sci. 90, 3522–3529. Javier Moreno, F., Recio, I., Olano, A., L€ opez-Fandin˜o, R., 2001. Heterogeneity of caprine κ-casein macropeptide. J. Dairy Res. 68, 197–208. https://doi.org/10.1017/S002202990100471X. Jensen, H.B., Holland, J.W., Poulsen, N.A., Larsen, L.B., 2012a. Milk protein genetic variants and isoforms identified in bovine milk representing extremes in coagulation properties. J. Dairy Sci. 95, 2891–2903. Jensen, H.B., Poulsen, N.A., Andersen, K.K., Hammershoj, M., Poulsen, H.D., Larsen, L.B., 2012b. Distinct composition of bovine milk from Jersey and Holstein-Friesian cows with good, poor, or noncoagulation properties as reflected in protein genetic variants and isoforms. J. Dairy Sci. 95, 6905–6917. https://doi.org/10.3168/ jds.2012-5675. Jensen, H.B., Pedersen, K.S., Johansen, L.B., Poulsen, N.A., Bakman, M., Chatterton, D.E.W., Larsen, L.B., 2015. Genetic variation and posttranslational modification of bovine kappa-casein: effects on caseinomacropeptide release during renneting. J. Dairy Sci. 98, 747–758. https://doi.org/10.3168/jds.2014-8678. Jolles, J., Fiat, A.M., Alais, C., Jolles, P., 1973. Comparative study of cow and sheep kappa-caseinoglycopeptides: determination of the N-terminal sequences with a sequencer and location of the sugars. FEBS Lett. 30, 173–176. Kanamori, M., Kawaguchi, N., Ibuki, F., Doi, H., 1980. Attachment sites of carbohydrate moieties to peptide chain of bovine kappa-casein from normal milk. Agric. Biol. Chem. 44, 1855–1861. Kanamori, M., Doi, H., Ideno, S., Ibuki, F., 1981. Presence of O-glycosidic linkage through serine residue in kappacasein component from bovine mature milk. J. Nutr. Sci. Vitaminol. (Tokyo) 27, 231–241. Kapkova, P., Lattova, E., Perreault, H., 2006. Nonretentive solid-phase extraction of phosphorylated peptides from complex peptide mixtures for detection by matrix-assisted laser desorption/ionization mass spectrometry. Anal. Chem. 78, 7027–7033.

References

207

Kappeler, S., Farah, Z., Puhan, Z., 1998. Sequence analysis of Camelus dromedarius milk caseins. J. Dairy Res. 65, 209–222. https://doi.org/10.1017/S0022029997002847. Keating, A.F., Davoren, P., Smith, T.J., Ross, R.P., Cairns, M.T., 2007. Bovine κ-casein gene promoter haplotypes with potential implications for milk protein expression. J. Dairy Sci. 90, 4092–4099. Kilara, A., Panyam, D., 2003. Peptides from milk proteins and their properties. Crit. Rev. Food Sci. Nutr. 43, 607–633. Kirmiz, N., Robinson, R.C., Shah, I.M., Barile, D., Mills, D.A., 2018. Milk glycans and their interaction with the infant-gut microbiota. Annu. Rev. Food Sci. Technol. 9, 429–450. https://doi.org/10.1146/annurev-food030216-030207. Kreuss, M., Strixner, T., Kulozik, U., 2009a. The effect of glycosylation on the interfacial properties of bovine caseinomacropeptide. Food Hydrocoll. 23, 1818–1826. Kreuss, M., Krause, I., Kulozik, U., 2009b. Influence of glycosylation on foaming properties of bovine caseinomacropeptide. Int. Dairy J. 19, 715–720. Kumosinski, T.F., Brown, E.M., Farrell, H.M., 1991. 3-dimensional molecular modeling of bovine caseinsalpha-s1-casein. J. Dairy Sci. 74, 2889–2895. 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 determinants. FEBS Lett. 382, 149–152. https://doi.org/ 10.1016/0014-5793(96)00136-6. Le, T.T., Deeth, H.C., Larsen, L.B., 2017. Proteomics of major bovine milk proteins: novel insights. Int. Dairy J. 67, 2–15. https://doi.org/10.1016/j.idairyj.2016.11.016. Leaver, J., Horne, D.S., 1996. Chymosin-catalysed hydrolysis of glycosylated and nonglycosylated bovine kappacasein adsorbed on latex particles. J. Colloid Interface Sci. 181, 220–224. Ledoux, N., Mah
e, S., Dubarry, M., Bourras, M., Benamouzig, R., Tom
e, D., 1999. Intraluminal immunoreactive caseinomacropeptide after milk protein ingestion in humans. Nahrung 43, 196–200. Li, E.W.Y., Mine, Y., 2004. Immunoenhancing effects of bovine glycomacropeptide and its derivatives on the proliferative response and phagocytic activities of human macrophage-like cells, U937. J. Agric. Food Chem. 52, 2704–2708. Lodes, A., Krause, I., Buchberger, J., Aumann, J., Klostermeyer, H., 1996. The influence of genetic variants of milk proteins on the composition and technological properties of milk. 1. Casein micelle size and the content of non-glycosylated κ-casein. Milchwissenschaft 51, 368–373. Maciel, G.D.M., Mogensen, L., Lehmann, J.O., Kidmose, U., Kristensen, T., Larsen, L.B., Poulsen, N.A., 2017. Impaired milk quality and cheese making properties is not a concern for managing cows for 15 or 18 months calving intervals. Int. Dairy J. 70, 2–11. https://doi.org/10.1016/j.idairyj.2016.12.014. Madende, M., Kemp, G., Stoychev, S., Osthoff, G., 2018. Characterisation of African elephant beta casein and its relevance to the chemistry of caseins and casein micelles. Int. Dairy J. 85, 112–120. https://doi.org/10.1016/j. idairyj.2018.05.013. Mahe, M.F., Miranda, G., Queval, R., Bado, A., Zafindrajaona, P.S., Grosclaude, F., 1999. Genetic polymorphism of milk proteins in African Bos taurus and Bos indicus populations. Characterisation of variants alphaS1-casein H and kappa-casein. Genet. Sel. Evol. 31, 239–253. Malkoski, M., Dashper, S.G., O’Brien-Simpson, N.M., Talbo, G.H., Macris, M., Cross, K.J., Reynolds, E.C., 2001. Kappacin, a novel antibacterial peptide from bovine milk. Antimicrob. Agents Chemother. 45, 2309–2315. Mamone, G., Caira, S., Garro, G., Nicolai, A., Ferranti, P., Picariello, G., Malorni, A., Chianese, L., Addeo, F., 2003. Casein phosphoproteome: identification of phosphoproteins by combined mass spectrometry and twodimensional gel electrophoresis. Electrophoresis 24, 2824–2837. https://doi.org/10.1002/elps.200305545. Manson, W., Carolan, T., Annan, W.D., 1977. Bovine αS0 casein; a phosphorylated homologue of αS1 casein. Eur. J. Biochem. 78, 411–417. Marin, A., Mawhinney, T.P., Marshall, R.T., 1984. Glycosidic activities of Pseudomonas fluorescens on fat-extracted skim milk, buttermilk, and milk fat globule membranes. J. Dairy Sci. 67, 52–59. Martin, P., Szymanowska, M., Zwierzchowski, L., Leroux, C., 2002. The impact of genetic polymorphisms on the protein composition of ruminant milks. Reprod. Nutr. Dev. 42, 433–459. Mateos, A., Girardet, J.-M., Molle, D., Corbier, C., Gaillard, J.-L., Miclo, L., 2010. Identification of phosphorylation sites of equine beta-casein isoforms. Rapid Commun. Mass Spectrom. 24, 1533–1542.

208

5. Posttranslational modifications of caseins

Mati, A., Senoussi-Ghezali, C., Si Ahmed Zennia, S., Almi-Sebbane, D., El-Hatmi, H., Girardet, J.-M., 2017. Dromedary camel milk proteins, a source of peptides having biological activities—a review. Int. Dairy J. 73, 25–37. https://doi. org/10.1016/j.idairyj.2016.12.001. McGann, T.C., Donnelly, W.J., Kearney, R.D., Buchheim, W., 1980. Composition and size distribution of bovine casein micelles. Biochim. Biophys. Acta 630, 261–270. Mehaia, M.A., 1987. Studies of camel casein micelles: treatment with soluble and immobilized neuraminidase. Carbohydr. Polym. 7, 361–369. https://doi.org/10.1016/0144-8617(87)90003-8. Mercier, J.C., 1981. Phosphorylation of caseins, present evidence for an amino acid triplet code post translationally recognized by specific kinases. Biochimie 63, 1–17. Mercier, J.C., Brignon, G., Ribadeau-Dumas, B., 1973. Primary structure of bovine kappa B casein. Complete sequence. Eur. J. Biochem. 35, 222–235. Mercier, J.-C., Addeo, F., P
elissier, J.-P., 1977. Structure primaire du cas
einomacropeptide de la caseine K caprine. Biochimie 58, 1303–1310. https://doi.org/10.1016/S0300-9084(77)80013-8. Miclo, L., Girardet, J.M., Egito, A.S., Molle, D., Martin, P., Gaillard, J.L., 2007. The primary structure of a low-M(r) multiphosphorylated variant of beta-casein in equine milk. Proteomics 7, 1327–1335. Minkiewicz, P., Dziuba, J., Muzinska, B., 1993. The contribution of N-acetylneuraminic acid in the stabilization of micellar casein. Polish J. Food Nutr. Sci. 2, 39–48. Miranda, G., Anglade, P., Mahe, M.F., Erhardt, G., 1993. Biochemical characterization of the bovine genetic kappacasein C and E variants. Anim. Genet. 24, 27–31. Moatsou, G., Moschopoulou, E., Molle, D., Gagnaire, V., Kandarakis, I., Leonil, J., 2008. Comparative study of the protein fraction of goat milk from the Indigenous Greek breed and from international breeds. Food Chem. 106, 509–520. Molinari, C.E., Casadio, Y.S., Hartmann, B.T., Arthur, P.G., Hartmann, P.E., 2013. Longitudinal analysis of protein glycosylation and beta-casein phosphorylation in term and preterm human milk during the first 2 months of lactation. Br. J. Nutr. 110, 105–115. Molle, D., Leonil, J., 1995. Heterogeneity of the bovine κ-casein caseinomacropeptide, resolved by liquid chromatography on-line with electrospray ionization mass spectrometry. J. Chromatogr. A 708, 223–230. Moore, A., Boulton, A.P., Heid, H.W., Jarasch, E.-D., Craig, R.K., 1985. Purification and tissue-specific expression of casein kinase from the lactating guinea-pig mammary gland. Eur. J. Biochem. 152, 729–737. https://doi.org/ 10.1111/j.1432-1033.1985.tb09254.x. Moremen, K.W., Tiemeyer, M., Nairn, A.V., 2012. Vertebrate protein glycosylation: diversity, synthesis and function. Nat. Rev. Mol. Cell Biol. 13, 448–462. https://doi.org/10.1038/nrm3383. Moreno, F.J., Recio, I., Olano, A., Lopez-Fandino, R., 2000. Chromatographic characterization of ovine κ-casein macropeptide. J. Dairy Res. 67, 349–359. https://doi.org/10.1017/S0022029900004222. Moreno, F.J., Olano, A., Villamiel, M., 2001. N-acetyl- and N-glycolyl-neuraminic acid content in kappa-casein macropeptides. Milchwissenschaft 56, 669–671. Mulagapati, S., Koppolu, V., Raju, T.S., 2017. Decoding of O-linked glycosylation by mass spectrometry. Biochemistry 56, 1218–1226. https://doi.org/10.1021/acs.biochem.6b01244. Neveu, C., Molle, D., Moreno, J., Martin, P., Leonil, J., 2002. Heterogeneity of caprine beta-casein elucidated by RP-HPLC/MS: genetic variants and phosphorylations. J. Protein Chem. 21, 557–567. Ng-Kwai-Hang, K.F., 1997. A review of the relationship between milk protein polymorphism and milk composition/ milk production. In: Milk Protein Polymorphism. (IDF Special Issue 9702). International Dairy Federation, Brussels, pp. 22–37. Nwosu, C.C., Strum, J.S., An, H.J., Lebrilla, C.B., 2010. Enhanced detection and identification of glycopeptides in negative ion mode mass spectrometry. Anal. Chem. 82, 9654–9662. https://doi.org/10.1021/ac101856r. O’Connell, J.E., Fox, P.F., 2000. The two-stage coagulation of milk proteins in the minimum of the heat coagulation time-pH profile of milk: effect of casein micelle size. J. Dairy Sci. 83, 378–386. O’Connell, J.E., Fox, P.F., 2003. Heat-induced coagulation of milk. In: Fox, P.F., McSweeney, P.L.H. (Eds.), Advanced Dairy Chemistry, Volume 1, Proteins, third ed. Kluwer Academic/Plenum Publishers, New York, NY, pp. 879–945. O’Donnell, R., Holland, J.W., Deeth, H.C., Alewood, P., 2004. Milk proteomics. Int. Dairy J. 14, 1013–1023. O’Riordan, N., Kane, M., Joshi, L., Hickey, R.M., 2014a. Glycosidase activities in bovine milk over lactation. Int. Dairy J. 35, 116–121.

References

209

O’Riordan, N., Kane, M., Joshi, L., Hickey, R.M., 2014b. Structural and functional characteristics of bovine milk protein glycosylation. Glycobiology 24, 220–236. https://doi.org/10.1093/glycob/cwt162. O’Riordan, N., O’Callaghan, J., Butto`, L.F., Kilcoyne, M., Joshi, L., Hickey, R.M., 2018. Bovine glycomacropeptide promotes the growth of Bifidobacterium longum ssp. infantis and modulates its gene expression. J. Dairy Sci. 101, 6730–6741. https://doi.org/10.3168/jds.2018-14499. Olumee-Shabon, Z., Boehmer, J.L., 2013. Detection of casein phosphopeptides in goat milk. J. Proteome Res. 12, 3034–3041. Peterson, R., Cheah, W.Y., Grinyer, J., Packer, N., 2013. Glycoconjugates in human milk: protecting infants from disease. Glycobiology 23, 1425–1438. https://doi.org/10.1093/glycob/cwt072. Petrat-Melin, B., Kristiansen, G.H., Le, T.T., Poulsen, N.A., Larsen, L.B., Young, J.F., 2016. In vitro gastrointestinal digestion of purified bovine kappa-casein variants A, B, and E: effects on antioxidant and angiotensin 1-converting enzyme inhibitory capacity. Int. Dairy J. 57, 44–51. https://doi.org/10.1016/j.idairyj.2016.02.036. Pisano, A., Packer, N.H., Redmond, J.W., Williams, K.L., Gooley, A.A., 1994. Characterization of O-linked glycosylation motifs in the glycopeptide domain of bovine kappa-casein. Glycobiology 4, 837–844. Poulsen, N.A., Bertelsen, H.P., Jensen, H.B., Gustavsson, F., Glantz, M., Mansson, H.L., Andren, A., Paulsson, M., Bendixen, C., Buitenhuis, A.J., Larsen, L.B., 2013. The occurrence of noncoagulating milk and the association of bovine milk coagulation properties with genetic variants of the caseins in 3 Scandinavian dairy breeds. J. Dairy Sci. 96, 4830–4842. https://doi.org/10.3168/jds.2012-6422. Poulsen, N.A., Jensen, H.B., Larsen, L.B., 2016. Factors influencing degree of glycosylation and phosphorylation of caseins in individual cow milk samples. J. Dairy Sci. 99, 3325–3333. https://doi.org/10.3168/jds.2015-10226. Prinzenberg, E.M., Hiendleder, S., Ikonen, T., Erhardt, G., 1996. Molecular genetic characterization of new bovine kappa-casein alleles CSN3F and CSN3G and genotyping by PCR-RFLP. Anim. Genet. 27, 347–349. Prinzenberg, E.M., Krause, I., Erhardt, G., 1999. SSCP analysis at the bovine CSN3 locus discriminates six alleles corresponding to known protein variants (A, B, C, E, F, G) and three new DNA polymorphisms (H, I, A1). Anim. Biotechnol. 10, 49–62. Rasmussen, L.K., Hojrup, P., Petersen, T.E., 1992. The multimeric structure and disulfide-bonding pattern of bovine kappa-casein. Eur. J. Biochem. 207, 215–222. Rasmussen, L.K., Hojrup, P., Petersen, T.E., 1994. Disulphide arrangement in bovine caseins: localization of intrachain disulphide bridges in monomers of kappa- and alpha s2-casein from bovine milk. J. Dairy Res. 61, 485–493. Rasmussen, L.K., Sorensen, 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 caseinomacropeptides: a solid-state phosphorus-31 nuclear magnetic resonance and sequence and mass spectrometric study. J. Dairy Sci. 80, 607–614. Rasmussen, L.K., Johnsen, L.B., Tsiora, A., Sorensen, E.S., Thomsen, J.K., Nielsen, N.C., Jakobsen, H.J., Petersen, T.E., 1999. Disulphide-linked caseins and casein micelles. Int. Dairy J. 9, 215–218. Recio, M.I., Villamiel, M., Martı´nez-Castro, I., Olano, A., 1998. Changes in free monosaccharides during storage of some UHT milks: a preliminary study. Zeitsch. Lebensmitteluntersuch. Forsch. A 207, 180–181. Riggs, L., Sioma, C., Regnier, F.E., 2001. Automated signature peptide approach for proteomics. J. Chromatogr. A 924, 359–368. Robitaille, G., Ayers, C., 1995. Effects of κ-casein glycosylation on heat stability of milk. Food Res. Int. 28, 17–21. Robitaille, G., Ng-Kwai-Hang, K.F., Monardes, H.G., 1991a. Variation of the N-acetylneuraminic acid content of bovine kappa-casein. J. Dairy Res. 58, 107–114. Robitaille, G., Ng-Kwai-Hang, K.F., Monardes, H.G., 1991b. Association of kappa-casein glycosylation with milk production and composition in Holsteins. J. Dairy Sci. 74, 3314–3317. Robitaille, G., Ng-Kwai-Hang, K.F., Monardes, H.G., 1993. Effect of κ-casein glycosylation on cheese yielding capacity and coagulating properties of milk. Food Res. Int. 26, 365–369. Rollema, H.S., 1992. Casein association and micelle formation. In: Fox, P.F. (Ed.), Advanced Dairy Chemistry. Elsevier Applied Science, London, pp. 111–140. Rowles, J., Slaughter, C., Moomaw, C., Hsu, J., Cobb, M.H., 1991. Purification of casein kinase I and isolation of cDNAs encoding multiple casein kinase I-like enzymes. Proc. Natl. Acad. Sci. U. S. A. 88, 9548–9552. https://doi.org/ 10.1073/pnas.88.21.9548. Saito, T., Itoh, T., 1992. Variations and distributions of O-glycosidically linked sugar chains in bovine kappa-casein. J. Dairy Sci. 75, 1768–1774.

210

5. Posttranslational modifications of caseins

Saito, T., Itoh, T., Adachi, S., 1981a. The chemical structure of a tetrasaccharide containing N-acetylglucosamine obtained from bovine colostrum kappa-casein. Biochim. Biophys. Acta 673, 487–494. Saito, T., Itoh, T., Adachi, S., Suzuki, T., Usui, T., 1981b. The chemical structure of neutral and acidic sugar chains obtained from bovine colostrum kappa-casein. Biochim. Biophys. Acta 678, 257–267. Saito, T., Itoh, T., Adachi, S., Suzuki, T., Usui, T., 1982. A new hexasaccharide chain isolated from bovine colostrum kappa-casein taken at the time of parturition. Biochim. Biophys. Acta 719, 309–317. Sawyer, W.H., 1969. Complex between β-lactoglobulin and κ-casein. A review. J. Dairy Sci. 52, 1347–1355. Schauer, R., 2000. Achievements and challenges of sialic acid research. Glycoconj. J. 17, 485–499. Schild, T.A., Wagner, V., Geldermann, H., 1994. Variants within the 50 -flanking regions of bovine milk-proteinencoding genes. I. Kappa-casein-encoding gene. Theor. Appl. Genet. 89, 116–120. Schlieben, S., Erhardt, G., Senft, B., 1991. Genotyping of bovine kappa-casein (kappa-CNA, kappa-CNB, kappa-CNC, kappa-CNE) following DNA sequence amplification and direct sequencing of kappa-CNE PCR product. Anim. Genet. 22, 333–342. Schopen, G.C.B., Visker, M., Koks, P.D., Mullaart, E., van Arendonk, J.A.M., Bovenhuis, H., 2011. Whole-genome association study for milk protein composition in dairy cattle. J. Dairy Sci. 94, 3148–3158. https://doi.org/10.3168/ jds.2010-4030. Shekar, P.C., Goel, S., Rani, S.D.S., Sarathi, D.P., Alex, J.L., Singh, S., Kumar, S., 2006. κ-Casein-deficient mice fail to lactate. Proc. Natl. Acad. Sci. U. S. A. 103, 8000–8005. Slattery, C.W., 1978. Variation in the glycosylation pattern of bovine kappa-casein with micelle size and its relationship to a micelle model. Biochemistry 17, 1100–1104. Smith, M.H., Hill, J.P., Creamer, L.K., Plowman, J.E., 1997. Towards understanding the variant effect on the rate of cleavage by chymosin on κ-caseins A and C. In: Milk Protein Polymorphism (IDF Special Issue 9702). International Dairy Federation, Brussels, pp. 185–188. Smith, M.H., Edwards, P.J., Palmano, K.P., Creamer, L.K., 2002. Structural features of bovine caseinomacropeptide A and B by 1H nuclear magnetic resonance spectroscopy. J. Dairy Res. 69, 85–94. Sulimova, G.E., Sokolova, S.S., Semikozova, O.P., Nguet, L.M., Berberov, E.M., 1992. Analysis of DNA polymorphism of cluster genes in cattle: casein genes and major histocompatibility complex (MHC) genes. Tsitolog. Genet. 26, 18–26. Sulimova, G.E., Badagueva, I.N., Udina, I.G., 1996. Polymorphism of the kappa-casein gene in populations of the subfamily Bovinae. Genetika 32, 1576–1582. Swaisgood, H.E., 2003. Chemistry of the caseins. In: Fox, P.F., McSweeney, P.L.H. (Eds.), Advanced Dairy Chemistry, Volume 1, Proteins, third ed. Kluwer Academic/Plenum Publishers, New York, NY, pp. 139–202. Swaisgood, H.E., Brunner, J.R., Lillevik, H.A., 1964. Physical parameters of κ-casein from cow’s milk. Biochemistry 20, 1616–1623. Sweet, S.M.M., Creese, A.J., Cooper, H.J., 2006. Strategy for the identification of sites of phosphorylation in proteins: neutral loss triggered electron capture dissociation. Anal. Chem. 78, 7563–7569. Tagliabracci, V.S., Engel, J.L., Wen, J.Z., Wiley, S.E., Worby, C.A., Kinch, L.N., Xiao, J.Y., Grishin, N.V., Dixon, J.E., 2012. Secreted kinase phosphorylates extracellular proteins that regulate biomineralization. Science 336, 1150–1153. https://doi.org/10.1126/science.1217817. Takeuchi, M., Tsuda, E., Yoshikawa, M., Sasaki, R., Chiba, H., 1985. Fractionation and characterization of 9 subcomponents of bovine kappa-casein A. Agric. Biol. Chem. 49, 2269–2276. Talbot, B., Waugh, D.F., 1970. Micelle-forming characteristics of monomeric and covalent polymeric kappa caseins. Biochemistry 9, 2807–2813. Ten Hagen, K.G., Fritz, T.A., Tabak, L.A., 2003. All in the family: the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases. Glycobiology 13, 1R–16R. Ubersax, J.A., Ferrell Jr., J.E., 2007. Mechanisms of specificity in protein phosphorylation. Nat. Rev. Mol. Cell Biol. 8, 530–541. Uniacke-Lowe, T., Huppertz, T., Fox, P.F., 2010. Equine milk proteins: chemistry, structure and nutritional significance. Int. Dairy J. 20, 609–629. https://doi.org/10.1016/j.idairyj.2010.02.007. van Halbeek, H., Dorland, L., Vliegenthart, J.F., Fiat, A.M., Jolles, P., 1980. A 360-MHz 1H NMR study of three oligosaccharides isolated from cow kappa-casein. Biochim. Biophys. Acta 623, 295–300. van Halbeek, H., Dorland, L., Vliegenthart, J.F., Fiat, A.M., Jolles, P., 1981. Structural characterization of a novel acidic oligosaccharide unit derived from cow colostrum kappa-casein. A 500 MHz 1H NMR study. FEBS Lett. 133, 45–50.

References

211

van Hooydonk, A.C.M., Olieman, C., Hagedoorn, H.G., 1984. Kinetics of the chymosin-catalysed proteolysis of kappa-casein in milk. Neth. Milk Dairy J. 38, 207–222. van Hooydonk, A.C.M., De Koster, P.G., Boerrigter, I.J., 1987. The renneting properties of heated milk. Neth. Milk Dairy J. 41, 3–18. Vasbinder, A.J., Alting, A.C., Visschers, R.W., De Kruif, C.G., 2003. Texture of acid milk gels: formation of disulfide cross-links during acidification. Int. Dairy J. 13, 29–38. Vincent, D., Elkins, A., Condina, M.R., Ezernieks, V., Rochfort, S., 2016. Quantitation and identification of intact major milk proteins for high-throughput LC-ESI-Q-TOF MS analyses. PLoS One 1. https://doi.org/10.1371/journal. pone.0163471. Vreeman, H.J., Visser, S., Slangen, C.J., Van Riel, J.A., 1986. Characterization of bovine kappa-casein fractions and the kinetics of chymosin-induced macropeptide release from carbohydrate-free and carbohydrate-containing fractions determined by high-performance gel-permeation chromatography. Biochem. J. 240, 87–97. Walsh, C.D., Guinee, T.P., Reville, W.D., Harrington, D., Murphy, J.J., O’Kennedy, B.T., FitzGerald, R.J., 1998. Influence of κ-casein genetic variant on rennet gel microstructure, Cheddar cheesemaking properties and casein micelle size. Int. Dairy J. 8, 707–714. Walstra, P., 1999. Casein sub-micelles: do they exist? Int. Dairy J. 9, 189–192. Wu, H.Y., Tseng, V.S., Liao, P.C., 2007. Mining phosphopeptide signals in liquid chromatography-mass spectrometry data for protein phosphorylation analysis. J. Proteome Res. 6, 1812–1821. Wuhrer, M., Catalina, M.I., Deelder, A.M., Hokke, C.H., 2007. Glycoproteomics based on tandem mass spectrometry of glycopeptides. J. Chromatogr. B 849, 115–128. https://doi.org/10.1016/j.jchromb.2006.09.041. Yamauchi, K., Azuma, N., Kobayashi, H., Kaminogawa, S., 1981. Isolation and properties of human kappa-casein. J. Biochem. 90, 1005–1012. Yoshikawa, M., Takeuchi, M., Sasaki, R., Chiba, H., 1982. Chemical characteristics of bovine casein micelles fractionated by size on CPG-10/300 chromatography. Agric. Biol. Chem. 46, 1043–1048. Zevaco, C., Ribadeau-Dumas, B., 1984. A study on the carbohydrate binding sites of bovine kappa-casein using high performance liquid chromatography. Milchwissenschaft 39, 206–210. Zhang, C., Bijl, E., Hettinga, K., 2018. Destabilization of UHT milk by protease AprX from Pseudomonas fluorescens and plasmin. Food Chem. 263, 127–134. https://doi.org/10.1016/j.foodchem.2018.04.128. Zhou, H., Xu, S., Ye, M., Feng, S., Pan, C., Jiang, X., Li, X., Han, G., Fu, Y., Zou, H., 2006. Zirconium phosphonatemodified porous silicon for highly specific capture of phosphopeptides and MALDI-TOF MS analysis. J. Proteome Res. 5, 2431–2437.