Biochimica et Biophysica Acta, 351 (1974) 325-332 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

BBA 36726

r-CASEIN FROM BOVINE COLOSTRUM*

JEAN GUI~RIN a, CHARLES ALA1S a, JACQUELINE JOLLIES b and PIERRE JOLLIES b "Laboratory of Biochemistry, University of Nancy, F-54037 Nancy and bLaboratory of Biochemistry, University of Paris VI, F-75272 Paris 06 (France)

(Received November 14th, 1973) (Revised manuscript received February 21st, 1974)

SUMMARY

Whole casein from colostrum contains more r-casein than normal casein. Structural studies have shown that the caseinoglycopeptide (C-terminal moiety) of r- casein seems to be already present at parturition. Colostrum r-casein contains about two times more carbohydrate than normal casein and the presence of N-acetylglucos- amine together with N-acetylgalactosamine suggests structural differences of its pros- thetic group. The behaviour of the sugars after parturition is discussed.

INTRODUCTION

The main components of bovine whole casein, as1-,/3- and r-caseins accounting for 40, 30 and 15 ~ , respectively, are now well known. Their primary structures were recently established [1-7]. Only r-casein contains carbohydrates (N-acetylgalactos- amine, galactose and N-acetylneuraminic acid) and some structural features of the sugar part as well as the linkage between the peptide and sugar moieties have also been determined [5, 8-10].

Few data are available for colostrum casein and the changes in its composition. Most investigations have been concerned with sialic acid and several authors noted that colostrum casein contained more sialic acid than normal casein and that its amount decreased during the colostral period [11-13]. It was also more sensitive to the action of chymosin (or rennin, EC 3.4.4.3) during this period [12]. These results suggested some differences in the composition of casein at the beginning of the lacta- tion period, more particularly a higher content of the r-component.

Casein micelles are not formed in the first colostrum. They could be visualized by electron microscopy only two days after calving [14], though colostrum seems to be rich in r-casein which plays a major role in the structure of miceiles.

We have studied the changes in the r-component in whole casein from bovine colostrum from calving by gel electrophoresis and by analysis of the r-casein and of the "caseinoglycopeptide". This latter peptide represents the C-terminal portion of r-

* 31st communication on caseins.

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casein, released after the specific cleavage of a Phe-Met bond by chymosin [15, 16]; it contains all the carbohydrate residues of the molecule and the genetic substitution of two amino acids; its preparation is much less tedious than that of K-casein.

MATERIALS AND METHODS

Colostrum from individual cows was skimmed by centrifugation and diluted with water (1:2, v/v) at 35 °C; whole casein was precipitated by 4 M HCI until the pH was 4.6. This dilution increased precipitation, as has been found with human milk [17]. The precipitate was washed three times with distilled water, dissolved at pH 7.0 by 1 M NaOH and precipitated again by 1 M HC1. After three washings, the precipitate was dissolved and the solution was lyophilized.

The caseinoglycopeptide was prepared following Alais and Joll~s [18] by the action of chymosin (Chr. Hansen Lab., Copenhagen) (enzyme/substrate ratio, 1:37 000) on a 2 ~o solution of whole casein at 25 °C for 30 rain. The reaction was stopped by the addition of trichloroacetic acid at a final concentration of 12%; the supernatant contained the caseinoglycopeptide.

Colostrum K-casein was obtained from purified whole colostrum casein by gel filtration on Sephadex G-150, according to Yaguchi et al. [19].

The non-protein nitrogen released from whole casein by chymosin was measured following Alais et al. [20]. The trichloroacetic acid filtrate before the action of chymosin was used as a reference.

The determination of phosphorus was performed following Bamann et al. [2 l]; neutral sugars were determined according to Schultze et al. [22] after acid hydrolysis (1 M HCI, 3 h, 100 °C), amino sugars according to Rondle and Morgan [23] after acid hydrolysis (2 M HCI, 3 h, 100 °C) and sialic acid following Warren's procedure [24] after acid hydrolysis (0.1 M H2SO4, I h, 85 °C). The N-acetyl groups were deter- mined by the method of Reissig, as modified by Ghuysen and Salton [25].

Quantitative amino acid analyses were performed after total hydrolysis of the samples (18,48 h, 6 M HCI, 110 °C, sealed tubes under vacuum) using a Technicon autoanalyzer.

For the identification of the amino sugars, column chromatography was run on Dowex 50 W-X8 (200-400 mesh), following the method of Gardell [26] ; the sugars were determined according to Rondle and Morgan [23] or after degradation by nin- hydrin according to Stoffyn and Jeanloz [27].

Urea-starch gel electrophoresis was run following Wake and Baldwin [28] at pH 8.6 in the presence of 2-mercaptoethanol, according to Neelin [29].

Digestion by carboxypeptidase A (EC 3.4.2.1) (Worthington) was carried out in 0.1 M NH4HCO3 at 37 °C for 1 h.

Automated Edman degradation [30] was carried out in a Socosi sequencer, model PS 100, by the quadrol method. The thiazolinones were converted into PTH- amino acids and these were characterized with an amino acid autoanalyzer after regeneration of the free amino acid, or by thin-layer chromatography. The following solvents were employed: (A) chloroform-methanol (90:10, v/v) followed by chloro- form alone; (B) heptane-propionic acid-dichlorethane (58:17:25, by vol.).

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RESULTS

Electrophoresis The characteristic bands of K-casein [17] were absent from the starch gel

electrophoretic pattern of whole casein from bovine colostrum, at least at the begin- ning of the post-partum period (Fig. 1). A blurred zone occupied the place of the typi- cal bands, in spite of the presence of 2-mercaptoethanol. In addition, a smaller num- ber of the bands of c~s- and fl-caseins were visible at the beginning of the lactation period. Under similar conditions, purified colostrum K-casein always gave a diffuse zone though normal K-casein gave well separated bands.

- - a

- b

- - t

- - 8

- [

-@

- - h

Fig. 1. Electrophoretic pattern of colostrum casein: whole casein (a) 15 min, (b) 6 h (c) 18 h, (d) 30 h, (e) 42 h and (f) 60 h after claving; K-casein of colostrum (g, h) and of normal (i) milk.

Action of chymosin on colostrum whole casein As far as the behaviour of whole casein with chymosin (Fig. 2a) is concerned,

the post-partum period can be divided into two phases; a first one, from 0 to 30 h after calving, where the percentage of non-protein nitrogen released by chymosin was rather high (about 2 %), a second one, after 30 h, where the amount of non-protein nitrogen became normal (about 1%).

328

z

~ o.1

c 2

._c ©

g 2 :

1

02 (b )

0 2'4 L / / ' 120 Time after calving (h)

Fig. 2. Evolution of non-protein nitrogen (in percentage total nitrogen) released by chymosin from colostrum whole casein (mean values for three cows). (a) Non-protein nitrogen released by chymosin. (b) Non-protein nitrogen soluble in 12 % trichloroacetic acid before the action of chymosin.

The solubility of colostrum whole casein in 12 % trichloroacetic acid was nearly constant and comparable to that of normal whole casein (Fig. 2b).

The mean amount of K-casein in whole casein from prime colostrum was esti- mated to about 24 %, based on the amount of non-protein nitrogen released by chym- osin from normal K-casein being 8.3 % [31], and from colostrum whole casein being about 2 % instead of about 1% for normal whole casein (see Fig. 2a).

Compositions of K-casein and caseinoglycopeptide from colostrum The amino acid analyses of 12 samples of colostrum caseinoglycopeptide pre-

pared independently from the milk of two cows did not show any significant variations during 65 h after parturition (Table I). Aromatic amino acids, as well as cysteine and arginine remained absent in the colostrum caseinoglycopeptide as was previously observed for the normal caseinoglycopeptide [32].

The phosphorus content ofcolostrum caseinoglycopeptide hardly varied during the first 24 h after parturition (Fig. 3d).

The neutral sugar content rose to a maximum 6 h after parturition and then decreased to a normal level 66 h after calving (Fig. 3a). The amino sugar content was high at parturition and also decreased to a normal level 66 h after parturition (Fig. 3b). The sialic acid content decreased during the first 24 h (Fig. 3c). Glucosamine together with galactosamine were characterized after acid hydrolysis and Table II summarizes the changes observed.

The carbohydrate content of colostrum K-casein was higher when compared to the normal protein: however, the sugar ratios and the phosphorus content remained nearly unchanged (Table III). The amino sugars were N-acetylated.

In a series of colostrum whole casein samples from three different cows, taken from parturition until 120 h after calving, we found a positive correlation (significant

TABLE I

AMINO ACID COMPOSITION OF COW COLOSTRUM CASE1NOGLYCOPEPTIDE Residues/mole of molecular weight 6750.

Amino acid Colostrum* Normal milk [4, 5]

Hydrolysis

18 h 48 h Nearest integer

Aspartic acid 3.6 3.4 4** 4 Threonine 9.0 6.5 11 ** 11 Serine 4.9 4.0 6** 6 Glutamic acid 8.4 7.2 10** 10 Praline 6.9 7.5 8 8 Glycine 1.2 1.1 1 1 Alanine 4.3 5.8 6 6 Valine 4.7 5.5*** 6 6 Methionine 0.7 0.65 1 1 Isoleucine 5.4 6.5*** 7 7 Leucine 1.3 1.2 1 1 Lysine 2.9 2.8 3 3

* Mean values of 12 samples of caseinoglycopeptide from two different cows. ** Values extrapolated to zero time hydrolysis.

*** Values after 72-h hydrolysis.

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0 6 b- ~0

5

~ 4 E ~ a

o

\

(c)

gl

0 0 24 418 G5 Time after calving (h~

Fig. 3. Composition of the non-peptidic moiety of colostrum caseinoglycopeptide (in residues for a peptide moiety of mol. wt 6750). a, neutral sugars; b, amino sugars; c, sialic acid; d, phosphorus.

TABLE II

AMINO SUGAR COMPOSITION OF COLOSTRUM CASEINOGLYCOPEPTIDE ( ~ ) DETERMINED AFTER ACID HYDROLYSIS

Determinations Galactosamine Glucosamine Galactosamine: achieved glucosamine ratio

15 mjn after calving 7.22 1.87 1:0.26 18 h after calving 5.61 1.83 1:0.33 Full lactation* 4.65 0 --

* Mean value of 9 samples.

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TABLE Ill

COMPOSITION OF COW COLOSTRUM to-CASEIN: COMPARISON WITH CASEIN*

Colostrum**

Phosphorus 1.18 Neutral sugars 3.12 Amino sugars* 1.77 Sialic acid** 0.87

1 .4 6 1 .52 0.69 0.57

In residues per mole of apoprotein of tool. wt 18 500. ¢¢

** Mean values of two analyses of two different caseins (15 rain after calving). *** Mean values of 18 analyses of nine different caseins.

t Expressed as N-acetylglucosamine. ,, Expressed as N-acetylneuraminic acid.

NORMAL K-

to 1 ~o) between the amoun t of non-protein nitrogen split from casein by chymosin and the sialic acid content. This correlation has already been reported by Marier et al. [33] for normal casein.

Determination of the C-terminal amino acids and the N-terminal sequence of the caseinoglycopeptide from colostrum

The amino acids released by carboxypeptidase A, valine, alanine, threonine and serine ( l .0 ; 0.82; 0.62; 0.26 residues/mole, respectively), were the same as those released from normal caseinoglycopeptide, showing the similarity of the C-terminal sequences in both cases.

Table IV indicates in detail the establishment of the N-terminal sequence (42

TABLE IV

N-TERMINAL SEQUENCE OF K-CASEINOGLYCOPEPTIDE FROM BOVINE COLOSTRUM DETERMINED WITH A SEQUENCER a, PTH-amino acid determined by thin-layer chromatography in Solvent A; b, PTH-Alanine and PTH-lysine not separated in Solvent A are determined in Solvent B; c, amino acid determined with an autoanalyzer after regeneration, percent of recovery.

Colostrum 1 Met-Ala lie Pro- Pro- Lys- Lys- Ash- Gln-Asp- Lys- (a) i I 4 " -t i ! L

(b) + - L (c) 45 28 25 20 16 15 18 8 12 lO 8

C o l o s t r u m 12 T h r - G l u - l l e - P r o - T h r - I ] e - A s n - T h r - l l e - A l a - S e r - (a) -I ÷ : ~ + ~ I t : (b) (c) 12 9 6 8 4 6 7

Colostrum 23 G l y - G l u - P r o - T h r - S e r - T h r Pro-Thr l l e - G l u - A l a - (a) i ! 4 L I ~ 4 4 I (b) i (c) 6 6.5 I 0.6

42 C o l o s t r u m 34 Va l G l u - S e r - T h r V a l - A l a - T h r - L e u G i l l . . . . . . . . . .

(a) 4 I I + 4 I I (b) d (c) 0.2

331

residues) of the caseinoglycopeptide from colostrum determined with a sequencer; its structure was the same as that previously established for the normal peptide [5, 10]. This study was done, in particular, to exclude the presence of an N-glycosidic linkage involving an asparagine residue (see Discussion).

DISCUSSION

Starch gel electrophoresis as well as carbohydrate analyses revealed some dif- ferences between colostrum and normal K-caseins. Even in the presence of 2-mercapto- ethanol, the typical bands of K-casein did not appear clearly in colostrum casein electropberograms. Abnormal or incomplete electrophoretic patterns with human colostrum casein have already been observed by us [17]. However, it is possible to conclude that there is a greater amount of K-casein in colostrum, as the release of non- protein nitrogen by chymosin from colostrum whole casein was increased.

With regard to the amino acid composition of the colostrum caseinoglycopep- tide and to the amino acids released by carboxypeptidase A, the C-terminal portion of colostrum K-casein seemed to be already present at parturition.

The results concerning the sugar composition of K-casein (Table III) and of the caseinoglycopeptide (Fig. 3) from prime colostrum were not in agreement with each other; the molar ratios were about two times greater in the caseinoglycopeptide. In fact, K-casein was a mixture of different glucosidic variants. The only peptides released by chymosin and soluble in trichloroacetic acid were those which contained a high amount of sugars [34]. This could explain the differences between these two results.

The carbohydrate moiety of K-casein during the colostral period was charac- terized by the presence of N-acetylglucosamine together with N-acetylgalactosamine, though the former is absent from normal casein [18]. An N-glycosidic linkage between N-acetylglucosamine and asparagine seemed unlikely because the "code sequence" Asn-X-Thr/Ser necessary for such a linkage [35] was absent in this part of K-casein. Indeed after total hydrolysis, the caseinoglycopeptide contained four Asp residues; sequence studies established that three of them were asparagine residues. Two asparagine residues were located in the long sequence as indicated in Table IV; the third one was found to be in the sequence -Asn-Thr-Val which was established by classical methods. Thus glucosamine probably constitutes a part of the glucosidic portion different from that of normal K-casein [10]. However, the possibility that a glycoprotein especially rich in N-acetylglucosamine is bound to the colostrum K-casein as an impurity could not be entirely excluded. In any case, the carbohydrate moiety of colostrum K-casein seems to have a more complex structure than that of normal K- casein. This observation could explain the abnormal behaviour of colostrum x-casein in gel electrophoresis.

ACKNOWLEDGEMENTS

This communication constitutes a part of a thesis which will be submitted by one of the authors (J.G.) in partial fulfillment of the requirements for the degree of "Docteur-Ing~nieur" of the University of Nancy I.

This research was supported in part by the C.N.R.S. (ER 102) and the I.N.S.E.R.M. (group U 116).

332

The excellent technical assis tance o f M r Ly Q u a n Le and Miss M o n i q u e Gros s i

is gra tefu l ly acknowledged .

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