Agric. Biol. Chem., 44 (11), 2605 `2611, Attachment Sites of Carbohydrate Portions to Peptide Cha. of K-Casein from Bovine Colostrum õ

Transcription

1 Agric. Biol. Chem., 44 (11), 2605 `2611, Attachment Sites of Carbohydrate Portions to Peptide Cha of K-Casein from Bovine Colostrum õ in Hiroshi Doi, Hiroshi KOBATAKE,* Fumio IBUKI and Masao KANAMORI Department of Agricultural Chemistry, Kyoto Prefectural University, Shimogamo, Kyoto 606, Japan *Kyoto General Medico -Chemical Laboratory, Yamashina, Kyoto 607, Japan Received May 21, 1980 Short glycopeptides were prepared from bovine colostral v-casein treated with cyanogen bromide and proteases (pronase P and thermolysin), followed by gel filtrations and ion exchange chromatography. It was confirmed by Edman degradation that glycopeptide I among short glycopeptides obtained was homogeneous. From the effect of alkali treatment, it was assumed that three polysaccharide chains of glycopeptide I were attached to the peptide chain through OH groups of threonines. By chemical procedures and carboxypeptidase P treatment, the amino acid sequence of glycopeptide I was established to be Ser-Gly-Glu-Pro-Thr-Ser-Thr-Pro-Thr-Thr-Glu-Ala-Val. Threonine residues of No. 5, 7 and 9 were bound to the carbohydrate chains through galactosamine. The sugar chain bound to the threonine residue at No. 7 contained glucosamine. Glycopeptide I corresponded to residues of No. 127 `139 in K-casein A from normal milk. Colostrum is a very important food for a baby not only in nutrition but also in immunity. There are many reports on the hormones and immunoglobulins of colos trum.l `3) However, the number of reports on casein components from colostrum is only a few.4,5) K-Casein is a sole glycoprotein in casein fractions, and stabilizes casein micelles in the presence of calcium ion. The function of the carbohydrate portion is not clear, although many works have been carried out.6,7) The primary structure of K-casein was clarified by Mercier et al.8) Fournet et al.91 indicate that two major carbohydrate struc tures existed in normal K-casein. They were composed of N-acetylgalactosamine, galactose and N-acetylneuraminic acid. On the other hand, there are a few reports on K-casein from colostrum.10) It was indicated by Guerin et al.10) that K-casein from bovine colostrum had N-acetylglucosamine, which * presented at the Annual Meeting of Agricultural Chemical Society of Japan, Fukuoka, April, was not contained in normal K-casein. This result suggests that the carbohydrate structure of colostral K-casein is different from that of normal one. K-Casein from colostrum is also heterogeneous due to the carbohydrate moiety as well as that from normal milk.11) From these facts, it is considered that K-casein from colostum has various carbohydrate chains. It has been reported by Fournet et al.11)i that four polysaccharide fractions called Sc0, Se1, Sc2, Sc3 were characterized from bovine colostrum caseinoglycopeptides after gel filtration on Bio-Gel P-4 and preparative paper chromatog raphy. Every sugar fraction isolated after alkaline borohydride treatment contained N acetylgalactosaminitol. This result indicates that N-acetylgalactosamine of the carbohy drate portion of colostrum K-casein attaches O-glycosidically to threonine or serine of pep tide chain. In the case of normal K-casein, Jolles and his coworkers11 `16) have studied on the linkage between carbohydrate portion and peptide chain. They have indicated that threonine

2 2606 H. DOI, H. KOBATAKE, F. IBuKI and M. KANAMORI residues at No. 131 and 133 might be linked to the sugar part. However, the study on the attachment site of polysaccharide of colostrum tc-casein has not been reported. It is the purpose of this paper to make clear the binding site of the carbohydrate moiety to peptide chain. MATERIALS AND METHODS Materials. Colostrum was obtained from an individual Holstein cow between 12 and 36 hr after parturition, v Casein was prepared from bovine colostrum by a modification17) of the method described by Zittle and Custer.18) Carboxypeptidase P purified from Penicillium janthinellum was obtained from Protein Research Foundation (Osaka). Precoated silica gel plate 60 F254 was purchased from Merck (Darmstadt). All other chemicals were of special or reagent grade. Preparation of caseinoglycopeptides from colostrum rc casein. Cow K-caseinoglycopeptides were purified from colostrum k-casein by the method shown in Scheme I. methionyl residue) were added. The reaction mixture was stirred in the tightly stoppered brown flask. After the reaction at 25 Ž for 24 hr, the reaction mixture was diluted approximately 10-fold with distilled water and lyophilized. Lyophilized sample was applied on a Sephadex G-50 (fine) column (4.0 ~ 95 cm) equilibrated with the eluent (formic acid-acetic acid water= 25 : 87 : 888, v/v), and then eluted with the same eluent. Fraction S, a fraction positive to phenol-sulfuric acid, was pooled and lyophilized. ii) Digestion by pronase P and thermolysin. Fraction S was digested by pronase P and thermolysin (pronase P thermolysin-substrate=l : 1 : 100, w/w) in 20 mm Tris HC1 buffer, ph 8.0, at 37 Ž for 24hr. Digestion was stopped by the addition of 1 N HCI to ph 2.2, and the reaction mixture was applied onto a column of Dowex 50W ~ 2 (2.2 ~ 40 cm) equilibrated with the eluent-dl (0.2M sodium citrate-hci buffer, ph 2.2, containing 0.17M ƒà-thiodiglycol and 1 % BRIJ-35). Elution was carried out with each 120 ml of the eluent-dl, the eluent-1 (0.2M sodium citrate-hci buffer, ph 3.25, containing 0.04M ƒà-thiodiglycol, 1.7M ethyl alcohol and 1 % BRIJ 35), the eluent-2 (0.2M sodium citrate-hci buffer, ph 4.25, containing 0.04M ƒà-thiodiglycol and 1%, BRIJ-35) and the eluent-4 (0,35M sodium citrate-hci buffer, ph 5.28, containing 0.046M benzyl alcohol and 1% BRIJ-35) and the eluent-rg (0.2M NaOH containing 1% BRIJ-35). Ninhydrin and phenol-sulfuric acid reactions-positive fractions were lyophilized and applied onto a column of Bio-Gel P-2 (2 ~ 135 cm) for purification and desalination. Chemical analyses. Amino acid analysis was carried out with a Hitachi amino acid analyzer, model KLA-5, after acid hydrolysis in 6N HCI for 20hr at 110 Ž in a sealed tube under vacuum. Amino sugar was identified after acid hydrolysis in 2 N HCl at 100 Ž for 3 hr with an amino acid analyzer using the buffer system described by Yaguchi and Perry.21) Alkali treatment under reducing condition. 5 mg of glycopeptide I was treated with 2 ml of 0.1 N NaOH in the SCHEME I. Preparation of Short Glycopeptides from Casein from Bovine Colostrum. K- presence of I M sodium borohydride at 37 Ž. After 0, 5, 10, 25 and 50 hr, 0.1 ml of the mixture was taken out and neutralized with I N acetic acid. Then amino acid composition was determined after acid hydrolysis de Methionyl peptide bond of K-casein from colostrum was scribed above. cleaved by the treatment with cyanogen bromide in acidic condition, and the fraction S containing carbohydrates obtained by filtration on a Sephadex G-50 (fine) was digested by pronase P and thermolysin. Dowex 50W ~ 2 and Bio-Gel P-2 were used to fractionate glycopeptides. Fractions were assayed by the manual analyses of aliquots with the ninhydrin reagent after alkaline hy drolysis19) and with the phenol-sulfuric acid.20) i) Cleavage with cyanogen bromide. K-Casein from colostrum was treated with cyanogen bromide in 70% formic acid at a concentration of 500 mg per 50 ml g of crystalline cyanogen bromide (500-fold excess to Determination of C-terminal amino acid residues of glycopeptide I after alkaline borohydride treatment. 5 mg of glycopeptide I, from which the carbohydrate moiety was removed by the alkaline borohydride treatment for 30 hr, was reacted with 15ƒÊg of carboxypeptidase Pin 0.6 ml of 50 mm sodium acetate buffer, ph 5.2, containing 0.5 ƒêmol of norleucine as the internal standard at 37 Ž. 0.1 ml of aliquots were taken out after 0, 0.25, 0.5, 1 and 2 hr and 0.1 ml of 3 N NaOH was added to stop the reaction. 0.5 ml of the mixture was applied to an amino acid analyzer after the addition of 0.8 ml of the eluent-dl.

3 Attachment Sites of Carbohydrates of Colostral x-casein 2607 Analysis of N-terminal amino acid sequence of glycopep tide I. Edman degradation procedure of Iwanaga et al.22), was used to determine the amino acid sequence of glycopeptide I. N-terminal amino acids were identified by the thin layer chromatography using silica gel plate 60 F254. Thin layer chromatogram was run in the solvent I (chloroform-methanol=9: 1, v/v) and in the solvent U (chloroform-formic acid= 100: 5, v/v). RESULTS Preparation of glycopeptide fractions from colostrum K-casein In order to prevent the clotting by proteases, K-casein was treated with cyanogen bromide in acidic solution, prior to the digestion by pronase P and thermolysin. Methionyl peptide bond is selectively cleaved by cyanogen bro mide under acidic condition.23) Figure 1 shows the Sephadex G-50 (fine) gel filtration pattern of K-casein treated with cyanogen bromide. Fraction S was eluted ahead of main peak of ninhydrin reaction. Enough amount of cyanogen bromide was added to cleave all methionyl peptide bonds. There may be some interactions between glycopeptides and carbohydrate free peptides Fraction S was pooled and lyophilized. This sample was digested by pronase P and thermolysin in order to cleave FIG. 2. Dowex 50W ~ 2 Ion Exchange Chromatogram of Fraction S Digested by Pronase P and Thermolysin. 230 mg of Fraction S after filtration on a Sephadex G-50 (fine) were digested by each 2.3 mg. of pronase P and thermolysin in 10 ml of 20 mm Tris-HCI buffer, ph 8.0, at 37 Ž for 24 hr. Digestion was stopped by the addition of 1 N HCI to ph 2.2 and lyophilized. The sample dissolved in 3.5 ml of the eluent-dl (0.2M sodium citrate-hci buffer, ph 2.2, containing 0.17 M ƒà-thiodiglycol and 1% BRIJ-35) was applied onto a Dowex 50W ~ 2 column (2.2 ~ 40 cm) equilibrated and eluted with the same buffer at room temperature. A flow rate was 34.2 ml /hr. One tube contained 4 ml. Elution was carried out with the eluent-1, the eluent-2, the eluent-4 and the eluent-rg following the eluent-dl. absorbance at 490 nm; ---, absorbance at 570 nm. the peptide chain. Figure 2 shows the Dowex 50W ~ 2 ion exchange chromatogram of the sample digested by proteases. Three peaks (D1, D2 and D3) were detected by phenol-sulfuric acid reaction. Much more peptides not con taining sugar part were eluted with high ph buffers. Fraction D1, D2 and D3 were pooled and lyophilized. Each fraction was dissolved in a small amount of water and the solution was applied onto a Bio-Gel P-2 column for purification and desalination. The gel filtration profiles were automatically monitored by absorbance at 220 nm with a flow cell attached to a spectrophotometer. Gel filtration patterns are shown in Fig. 3. Six peaks (I, U-1, U-2, V-1 FIG. 1. Sephadex G-50 (fine) Gel Filtration Pattern of Colostrum k-casein Treated with Cyanogen Bromide. 500 mg of K-casein from colostrum were dissolved in 50 ml of 70% formic acid. 2.5 g of crystalline cyanogen bromide were added to react with K-casein at 25 Ž for 24 hr. Lyophilized sample after cyanogen bromide cleavage was applied onto a Sephadex G-50 (fine) column (4.0 ~ 95 cm) equilibrated with the eluent (formic acid-acetic acid-water=25 : 87 : 888, v/v). A flow rate was 36 ml/hr. One tube contained 5.7 m absorbance at 490 nm; ---, absorbance l at 570 nm., V-2 and V-3) were obtained by the gel filtration of fractions D1, D2 and D3. Amino sugar and amino acid compositions Amino sugar and amino acid analyses of each glycopeptide were carried out. Table I shows the contents of amino acid and amino sugar. The content was calculated based on one residue of glycine in a molecule. Amino acid analysis of glycopeptide V-1 was not

4 2608 H. Doi, H. KOBATAKE, F. IBUKI and M. KANAMORI FIG. 3. Bio-Gel P-2 Gel Filtration Profiles of Fraction DI (a), D2 (b) and D3 (c). Each fraction after Dowex 50W ~ 2 chromatography was lyophilized and applied onto a Bio-Gel P-2 column (2 ~ 135 cm) at room temperature. Glycopeptides were eluted with water at a flow rate of 12 ml/hr. One tube contained 4 ml. Detection was by absorbance at 220 nm with a flow cell system attached to a TABLE 1. AMINO ACID AND AMINO SUGAR COMPOSITIONS OF GLYCOPEPTIDES FROM (residue/mol) Ala-Val) of,k-casein A from normal milk. N Glycosidic linkage may not exist in glycopep tide I, because aspartic acid was not contained. Alkali treatment under reducing condition of glycopeptide I Glycopeptide I was treated with 0.1 N NaOH in the presence of 1 M sodium borohy dride at 37 Ž. O-Glycosidic linkage is labile in alkaline solution and the amino acid bound 0 glycosidically to sugar chain is converted to Number of residue was calculated based on one residue of glycine. carried out, as fraction V-1 did not contain amino sugar. The yield of glycopeptide V-3 was very small. Glycopeptide I was used in the following experiments, because this glycopeptide con tained more galactosamine and because its amino acid composition was simple compared with glycopeptide U-2. Glycopeptide I was pure fraction as indicated later from the result of Edman degradation of glycopeptide I. Three residues of galactosamine were con tained in one molecule of glycopeptide I. Amino acid composition of glycopeptide I corresponded to the residues 127 (Ser Gly-Glu-Pro-Thr-Ser-Thr-Pro-Thr-Thr-Glu FIG. 4. Effect of Alkali Treatment under Reducing Condition on Glycopeptide I. 5 mg of glycopeptide I were dissolved in 2 ml of 0.1 N NaOH/l M NaBH, and allowed to incubate at 37 Ž for indicated hours. After 0, 5, 10, 25 and 50 hr, 0.4 m1 portion was taken out and neutralized with 0.48 ml of 1 N acetic acid. Amino acid composition was determined by the procedure described in MATERIALS AND METHODS. Calculation was carried out based on one residue of glycine. œ \ œ, threonine; \, a-aminobutyric acid; \, serine; \, alanine.

5 Attachment Sites of Carbohydrates of Colostral K-Casein 2609 keto acid accompanied by the release of carbohydrate portion. 24) Alkali treatment un der reducing condition coverts serine to ala nine and threonine to ƒ -aminobutyric acid Figure 4 shows the effect of alkali treatment. under reducing condition on glycopeptide I. Native glycopeptide I contained 4 residues of threonine and 2 residues of serine (Table I). The number of serine and alanine was not changed in alkaline solution all through the experimental periods. However, the number of threonine residue decreased from 4 to 1.2, while that of ƒ -aminobutyric acid increased from 0 to 2.2. This result may indicate that carbohydrate portion attached to only thre onine O-glycosidically and that glycopeptide I has three carbohydrate chains in a molecule.24) Edman degradation of glycopeptide I Edman degradation is the method to decide N-terminal amino acid of peptide. Only serine was detected in the first step of Edman degradation. This result shows that glycopep tide I was pure. The N-terminal sequence of glycopeptide I: Ser-Gly-Glu-Pro-X-Ser-X Pro was established by the Edman degra dation. X indicates that the amino acid was not detected. The amino acid bound to the carbohydrate portion is not detected by Edman degradation procedure and the amino acid bound to sugar moiety is contained in aqueous layer. Galactosamine was detected in the aqueous layer of 5th step of Edman degradation of glycopeptide I after acid hydrolysis and galactosamine and gluco samine were in that of 7th step. Therefore, X was considered to be threonine. FIG. 5. Carboxypeptidase P Hydrolysis of Glyco peptides I Treated in Alkaline Solution in the Pres ence of Reducing Agent. 7 mg of glycopeptide I were chromatographed on a Sephadex G-15 column (2 ~ 80 cm) after alkaline borohydride treatment (0.1 N NaOH/ I M NaBH4) at 37 Ž for 30 hr. 5 mg of glycopeptide I, from which the carbohydrate moiety had been removed, were obtained. The sample was reacted with 15 ƒêg of carboxypeptidase P in 0.6 ml of 50 mm sodium acetate buffer, ph 5.2, containing 0.5 ƒêmol of norleucine as the internal standard at 37 Ž. ---, valine; ---, alanine; ---, glutamic acid; - œ, threonine; -, ƒ -aminobutyric acid. œ mg of glycopeptide I treated with 0.1 N NaOH/1 M NaBH4 at 37 Ž for 30 hr. 5.0 mg of glycopeptide I, from which the carbohydrate moiety had been removed, was reacted with carboxypeptidase P at 37 Ž for 2 hr. The release of amino acids from glycopeptide I treated with alkali is shown in Fig. 5. The amino acids were released by carboxypep tidase P in the order of valine, alanine, glutamic acid, threonine and ƒ -aminobutyric acid. This result indicates that the C-terminus of glycopeptide I treated with alkali was Carboxypeptidase P hydrolysis of glycopeptide I after alkali treatment under reducing condition The C-terminal sequence and the third -ABA-Thr-Glu-Ala-Val, where ABA is aminobutyric acid. Therefore, the position ƒ - of ABA of glycopeptide I is the third binding site of the carbohydrate portion of colostral binding site was established by carboxypep tidase P hydrolysis of glycopeptide I after casein. K- alkali treatment under reducing condition. 5.0 mg of peptide was obtained after neutrali zation and Sephadex G-15 gel filtration from 7 DISCUSSION It is important to know the carbohydrate

6 2610 H. Dot, H. KOBATAKE, F. Isutct and M. KANAMORI structure and attachment site for the in vestigation on the function and biosynthesis of glycoproteins. There are many stuides on the carbohydrate structure of glycoproteins.25.26) In general, there are two major types in the linkage of sugar part with peptide chain of N glycosidic linkage through asparagine27) and O-glycosidic linkage through serine and/or threonine.28) In the case of K-casein from normal milk, it is reported by Tran and Baker 29) that the carbohydrate moiety is attached to peptide chain through the OH group of serine or threonine, or both. Further, it has been indicated by Jolles and his coworkers9,11, 14,15) that the binding site of sugar part of normal K casein was threonine residues at No. 131 and/or 133. It is considered from the difference of the carbohydrate composition that K-casein from colostrum has the different carbohydrate structure from that of normal K-casein. The attachment site of sugar part of K-casein from bovine colostrum has not been studied well so far. In this study, a short glycopeptide (glyco peptide I) was isolated from bovine colostral K-casein, and the composition (Table. I), the alkali treatment (Fig. 4), Edman degradation and the carboxypeptidase P hydrolysis (Fig. 5) were examined. Glycopeptide I was composed of 13 residues of amino acids and the carbohydrate portions (Table I). N-Glycosidic linkage in glycopeptide I was ruled out, because this glycopeptide did not contain asparagine. Glycopeptide I was treated with 0.1 N NaOH/l M NaBH4, as 0 glycosidic linkage is labile in alkaline solution. The effect of alkali treatment on glycopeptide I indicated the binding site of the carbohydrate moiety was only threonine. The decrease of 3 residues of threonine in alkaline solution suggested the existence of 3 carbohydrate chains in glycopeptide I. Fournet et al.11) indicated that the carbohydrate chain of colostral K-casein is bound to peptide chain through N-acetylgalactosamine. The number of the carbohydrate chains was in good agreement with the number of galactosamine residues (Table I). 79% of decreased threonine were recovered as ƒ -aminobutyric acid. This low recovery might be caused by incomplete reduction.30) From the results of Edman degradation and the carboxypeptidase P hydrolysis, it was concluded that the amino acid sequence of glycopeptide I was Ser-Gly-Glu-Pro-Thr-Ser Thr-Pro-Thr-Thr-Glu-Ala-Val, and that the attachment sites of the polysaccharide chains were threonine residues at No. 5, No. 7 and No. 9. Threonine residues at No. 7 of glycopeptide I was bound to the carbohydrate portion containing glucosamine. The amino acid sequence of glycopeptide I corresponded to that of the residues of No. 127 `139 of normal K-casein A. Threonine residues at No. 5, No. 7 and No. 9 of glycopeptide I correspond to threonine res idues at No. 131, No. 133 and No. 135 of normal K-casein A, respectively. REFERENCES 1) C. Delouis, Ann. Rech. Vet., 9, 193 (1978). 2) J. E. Butler, Biochim. Biophys. Acta, 251, 435 (1971). 3) R. V. Josephson, E. M. Mikolajcik and V. K. Singh, J. Dairy Sci., 55, 1050 (1972). 4) G. C. Majumdar and N. C. Ganguli, Indian J. Dairy Sci., 23, 179 (1970). 5) M. Hekmati and K. Niroumand, Milchwissenschaft, 33, 24 (1978). 6) C. W. Slattery, Biochemistry, 17, 1100 (1978). 7) S. J. Keller, T. W. Keenan and W. N. Eigel, Biochim. Biophys. Acta, 566, 266 (1979). 8) J. C. Mercier, G. Brignon and B. Ribadeau-Dumas, Eur. J. Biochern., 35, 222 (1973). 9) B. Fournet, A. M. Fiat, C. Alais and P. Jolles, Biochim. Biophys. Acta, 576, 339 (1979). 10) J. Guerin, C. Alais, J. Jolles and P. Jolles, Biochim. Biophys. Acta, 351, 325 (1974). 11) B. Fournet, A. M. Fiat, J. Montreuil and P. Jolles, Biochirnie, 57, 161 (1975). 12) A. M. Fiat, C. Alais and P. Jolles, Chinda, 22, 137 (1968). 13) A. M. Fiat, C. Alais and P. Jolles, Eur. J. Biochem., 27, 408 (1972). 14) J. Jolles, F. Schoentgen, C. Alais, A. M. Fiat and P. Jolles, Hely. Chim. Acta, 55, 2872 (1972). 15) J. Jolles, A. M. Fiat, C. Alais and P. Jolles, FEBS Lett., 30, 173 (1973). 16) P. Jolles, Mol. Cell Biochem., 7, 73 (1975). 17) H. Doi, F. lbuki and M. Kanamori, J. Dairy Sci., 62,

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