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Separation of a milk acid phosphatase from a purified lactoferrin fraction and identification as a member of the mammalian purple acid phosphatase family
International Dairy Journal 23 (2012) 86e90
Contents lists available at SciVerse ScienceDirect
International Dairy Journal journal homepage: www.elsevier.com/locate/idairyj
Short communication
Separation of a milk acid phosphatase from a purified lactoferrin fraction and identification as a member of the mammalian purple acid phosphatase family Shun Yamaguchi, Takayuki Miura*, Asami Baba, Ryozo Akuzawa Department of Food Science and Technology, Nippon Veterinary and Life Science University, 2-27-5, Musashino, Tokyo 180-8602, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 March 2011 Received in revised form 13 October 2011 Accepted 24 October 2011
Active acid phosphatase (AcP), 37 kDa, was completely separated from the purified lactoferrin fraction of bovine milk (LF-rich fraction). The N-terminal amino acid sequence of the 37 kDa molecule had 84% homology with bovine uteroferrin (Uf)-like protein. The 37 kDa molecule has an optimum pH range of 4.5e5.0 and an optimum temperature of 60 C. The AcP activity of the iron removal 37 kDa molecule (iron-depleted LF-rich fraction) was approximately half that of the iron-containing sample (LF-rich fraction). The activity increased in a time-dependent manner on tryptic digestion. These profiles correspond to the mammalian purple acid phosphatase (PAP) family (Uf, Type-5 and tartrate-resistant acid phosphatase: EC3.1.3.2). It seems reasonable to propose that the active molecule in the LF-rich fraction is an undocumented bovine PAP-family protein. Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved.
1. Introduction Since bovine lactoferrin (LF) is cationic at the pH of milk or cheese whey, as are lactoperoxidase (LPO) and some minor proteins, it can be easily isolated from milk using a cationic exchange resin (Yoshida & Ye, 1991). The LF fraction thus separated is widely used as purified LF for commercial purposes. However, the characterization of LF using proteomic approaches has revealed an unexpected complexity of minor milk proteins and peptides. Some researchers have reported that LF-binding proteins (LF-BP) are co-purified with LF from bovine milk; these include ribonuclease-4, angiogenin, neutrophil gelatinase-associated lipocalin and fibroblast growth factor-binding protein (Fujihara, Tanigawa, Kawakami, & Ohtsuki, 2004; Tanigawa, Fujihara, Sakamoto, Yanahira, & Ohtsuki, 2001). Thus, the purified LF from milk would be better called the ‘LF-rich fraction’. The physiological activities of these minor proteins associated with LF may be involved in multiple functions of LF in vivo. Recently, Miura, Ono, Izumi, and Akuzawa (2010) reported that acid phosphatase (AcP) activity is a new enzymatic activity in the LF-rich fraction. Although the main active AcP molecules in the LF-rich fraction were tentatively identified as LF, this conclusion is controversial because purified LF can bind other minor phosphatases. Therefore, the purpose of the present study was to
* Corresponding author. Tel.: þ81 422 51 6121; fax: þ81 422 51 9984. E-mail address: [email protected] (T. Miura).
clarify whether the lactoferrin or another molecule has AcP activity, using a high-performance size-exclusion chromatography (HPSEC) technique. 2. Materials and methods 2.1. Preparation of LF-rich fraction from raw milk The AcP active fraction was prepared from bovine raw skimmed milk (obtained from Holstein cows at the faculty dairy farm of Nippon Veterinary and Life Science University), using amberlite FPC-3500 (Organo Co., Tokyo, Japan) cation-exchange resin and cellulofine CM 500 (cellulofine CM 500, Kanto Co., Tokyo, Japan) carboxymethyl column (3 10 cm), as described by Miura et al. (2010). These AcP active fractions are considered to be the LF-rich fraction. 2.2. Purification of fraction with AcP activity from LF-rich fraction using high performance size exclusion chromatography 2.2.1. First-step chromatography A high performance size exclusion chromatography (HPSEC) procedure was used with slight modifications as described by Kawakami et al. (2006). The LF-rich fraction (100 mL) was injected onto a TSKgel 2000SWXL gel filtration column (8 250 mm, Tosoh, Tokyo, Japan) using a TOSOH HPLC system (8020 model, Tosoh, Tokyo, Japan). The column was eluted at a flow rate of 1 mL min1 with a fraction collected 30 s (500 mL per fraction), using 10 mM
0958-6946/$ e see front matter Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2011.10.003
S. Yamaguchi et al. / International Dairy Journal 23 (2012) 86e90
87
sodium acetate buffer with 300 mM NaCl (pH 5.2) as elution buffer, and the eluent was monitored at 280 nm.
(DDBJ), National Center for Biotechnology Information (NCBI) and SWISS-PROT on website.
2.2.2. Re-chromatography Subsequently, the highest AcP active fraction obtained from first-step chromatography (no. 17 fraction) was collected in 3 mL (six times via first-step chromatography) and dialyzed against 10 mM acetate buffer (1 L) using a dialysis tube (Viskase Companies, Inc., Darien, IL, USA) for 2 h at 4 C. The dialyzed sample was frozen at 30 C and then freeze-dried for 24 h under vacuum at 10 C. To concentrate the sample, the lyophilized powder was rehydrated to 100 mL volume (30-fold concentration from original volume) using 300 mM NaCl containing 10 mM acetate buffer (pH 5.2) containing 8 M urea as a strong denaturant. The above the sample (100 mL) was applied onto a TSKgel 2000SWXL gel filtration column (re-chromatography), and equilibrated with the elution buffer containing 8 M urea and the eluate was monitored at 280 nm. Resulting eluted fractions (500 mL per fraction) were each dialyzed against 10 mM acetate buffer (1 L) using a dialysis tube for 24 h at 4 C before assay for AcP activity. The total protein concentration was determined according to the method of Lowry, Rosebrough, Farr, and Randall (1951); bovine serum albumin was used as a protein standard.
2.6. Removal of iron from LF-rich fraction An iron-depleted LF-rich fraction was obtained according to the method of Mazurier and Spik (1980). The purified LF-rich fraction was dissolved (10 mg mL1) in 100 mM sodium citrate acid buffer, pH 2.0, containing 40 mM EDTA and allowed to equilibrate at 4 C overnight (24 h). The solution was then dialyzed against various pH levels of buffer for 24 h at 4 C. 2.7. Study of optimum pH and temperature Effects of pH and temperature on AcP activity of LF-rich fraction or iron-depleted sample were investigated as described by Miura et al. (2010). The pH of buffers was adjusted at 50 C by the gradual addition of 0.1 M HCl or NaOH with rapid stirring. For pH studies, the buffers employed were 0.1 M acetic acid for pH values 3e5 and 0.1 M Tris-HCl for pH 6e9; assays were performed at 50 C.
2.3. Assay for phosphatase activity Enzyme activity was determined by a modification of the method used by Akuzawa and Fox (1998), with slight modifications as described by Miura et al. (2010). Enzyme sample (100 mL) was added to 100 mL of 10 mM p-nitrophenylphosphate (p-NPP; Sigma Chemicals, St. Louis, MO, USA) in 10 mM sodium acetate (pH 5.2) and incubated for 30 min at 50 C. The reaction stopped by 200 mL of 1 M NaOH and absorbance was measured at 405 nm. One phosphatase unit was converted from absorbance values to 1 mmol of p-NP 100 mL1 30 min1 using standard curves. 2.4. Sodium dodecyl sulphate polyacrylamide gel electrophoresis and silver staining The molecular mass of the active molecules in the individual fraction tubes was estimated by Coomassie brilliant blue and silver staining after sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE analysis, according to the method of Laemmli (1970), was performed as described by Miura et al. (2010). The AcP active fractions after HPSEC were mixed with an equal volume of SDS-PAGE sample buffer, and boiled for 5 min. Aliquots of mixtures (20 mL) were separated on a 7e20% (w/v) acrylamide gradient separation gel. SDS-PAGE gels were stained for protein using a modified Merril silver stain (Merril, Goldman, Sedman, & Ebert, 1981). Molecular mass was estimated by comparison with standards (Precision of Plus ProteinÔ KaleidoscopeÔ Prestained Standards, Bio-Rad Labolatories, Inc., Hercules, CA 94547, USA). 2.5. N-terminal amino acid sequence determination Active AcP fractions were analyzed by SDS-PAGE and then transferred electrophoretically onto a polyvinylidene difluoride (PVDF) membrane according to the method of Matsudaira (1987). The N-terminal amino acid sequence of the AcP molecule was determined with an automatic protein sequencer model PSQQ-21 (Shimadzu Co., Kyoto, Japan) as described by Miura et al. (2010). Amino acid homology searches were performed using basic local alignment search tool (BLAST) through the DNA Database of Japan
Fig. 1. The first step chromatography on a TSKgel 2000SWXL column of AcP protein isolated from LF-rich fraction (panel A: B, absorbance at 280 nm; C, phosphatase activity; the double headed arrow indicates AcP active fractions 16e18) and SDS-PAGE profiles of fractions 14e20 (panel B: M denotes pre-stained molecular mass standards; double headed arrow indicates AcP active fractions 16e18).
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commercial LF (Morinaga Milk Industry Co., Ltd., Tokyo, Japan) (25 mg) were digested with 50 or 500 mg of trypsin or pepsin (Sigma, St. Louis, MO, USA) in 100 mL of the incubation solution (10 mM sodium phosphate buffer at pH 7.5 or deionized water adjusted by HCl to pH 2.5). Proteolytic digestion was performed at 37 C for 6 h, and terminated by re-adjusting pH to 5.2 using 200 mL of 0.1 M acetic acid buffer (pH 5.2). The AcP activity was expressed as absorbance at 405 nm. 3. Results and discussion 3.1. Purification and identification of active AcP molecules from purified LF-rich fraction
Fig. 2. Re-chromatography on a TSKgel 2000SWXL column of the AcP active fraction number 17 from first step chromatography (panel A: B, absorbance at 280 nm; C, phosphatase activity; the arrow indicates the AcP active fraction, No. 16) and SDS-PAGE profiles of fractions 13e19 (panel B: M denotes pre-stained molecular mass standards; double headed arrow indicates the AcP active fraction 16; single headed arrow indicates the 37 kDa protein).
For temperature studies, 0.1 M acetic acid adjusted to pH 5.2 at 30e80 C was used. For both pH and temperature studies incubations were for 30 min. The specific activity data were expressed as a percentage of the maximum value. 2.8. Proteolytic digestion of LF-rich fraction A proteolytic digestion procedure was used as described by Shimazaki et al. (1993) with slight modifications. LF-rich fraction or
In our previous report, one peak of AcP active fraction (LF-rich fraction) was separated from skim milk using a carboxy methyl cation-exchange column, and it was tentatively suggested that the principal AcP active molecule was the LF (Miura et al., 2010). However, there remain some questions because the active AcP fraction was not completely purified. To clarify whether the LF or another molecule has AcP activity in the LF-rich fraction, the authors investigated purification of the LF-rich fraction using HPSEC. As shown in Fig. 1A, one peak of AcP active fractions consisting of three fractions (no. 16, 17 and 18) was obtained. The activity of the first fraction (Fig. 1A, no. 16) showed very low activity (0.21 U 100 mL1) compared to the other two fractions. This fraction showed many bands, thought to be mainly 80 kDa lactoferrin by silver staining (Fig. 1B, no. 16). The middle fraction (no. 17) showed the highest activity (5.8 U 100 mL1) although bands were not seen (Fig. 1B, no. 17). The activity of the last fraction (no. 18) was about one tenth (0.6 U 100 mL1) of that of the no. 17 fraction and contained two distinct bands (Fig. 1B, no. 18). These results suggested that an unknown molecule has the main AcP activity rather than LF. To completely separate the main true AcP molecule from the most highly active AcP fraction (no. 17), that denatured sample was re-chromatographed. One AcP-active fraction was obtained (Fig. 2, no. 16), and analyzed by SDS-PAGE, which detected only a band at 37 kDa by silver staining (Fig. 2B, no. 16). The purification procedure for the 37 kDa molecule is summarized in Table 1. The specific activity was 15,132 units mg1, and the activity increased 90,704fold compared with that of raw milk. However, the last TSK gel 2000 SWXL procedure showed that the total activity markedly decreased by 81% compared with that of the previous procedure (cellulofine CM 500). Since priority was given to complete purification, it was thought that the overall loss of AcP activity was due to denaturation by urea. 3.2. Determination of N-terminal amino acid sequence When the band at 37 kDa band analyzed by a protein sequencer, the N-terminal amino acid sequence obtained was AAAAATTPAPMLRFFAVDI (Table 2). That amino acid sequence was 84% (16/19 residues) homologous to bovine uteroferrin (Uf)-like protein
Table 1 Purification of 37 kDa protein with AcP activity. Fraction
Volume (mL)
Total protein (mg)
Total activity (units)
Specific activity (units mg1)
Purification (fold)
Yield (%)
Raw milk Skim milk Amberlite FPC-3500 Cellulofine CM 500 TSK gel 2000 SWXL
2000 1500 400 60 1
74,985 60,701 177 46.5 0.01
12,510 6593 7232 5143 180.3
0.167 0.109 41 110 15,132
1 0.65 245 662 90,704
100 53 58 41 1.4
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Table 2 Comparison of amino acid residues of the 37 kDa protein and PAP-family proteins.a
Name
Organism
The 37 kDa protein
Bovine
Uteroferrin-like protein TRAP/Type 5-acid phosphatase TRAP/Type 5-acid phosphatase TRAP/uteroferrin
Bovine Rat Mouse Pig
Amino acid residues A | A L L R
A | A L L T
A | A A T N
A | A H H T
A | A C G R
T | T T T T
T | T A A A
P | P P P P
A | A A T T
P | P S P P
M | M T T I
L | L L L L
R | R R R R
Accession number F | F F F F
F A | V A V A V A V A
V | V V V V
D I G G G G
D D D D
W W W W
XP_002688895 : NCBI P29288 : swiss-prot Q05117 : swiss-prot P09889 : swiss-prot
a Vertical lines indicate the residues identical for the 37 kDa protein and uteroferrin-like protein sequence. Homology with uteroferrin-like protein and other phosphatases (TRAP) is shown by box-like enclosure.
(DDBJ Accession number XP_002688895). The amino acid residue at positions 15, 18 and 19 did not be correspond to the above Uf-like protein sequence data despite being analyzed three or more times. Although the amino acid sequence of Uf-like protein was predicted from the bovine genome, this sequence showed a high homology with the purple acid phosphatases (PAP) family (EC3.1.3.2) such as various mammalian type 5-acid phosphatase (ACP5) and tartrate resistant acid phosphatase (TRAP) using the gene data in UniGene at the National Center for Biotechnology Information (NCBI) (Table 2). All of these proteins exhibit similar molecular masses in the 30e40 kDa range, basic pI and high activity toward responsestabilized substrate, such as p-nitrophenyl phosphate. Uf in bovine milk or the bovine uterus has never been reported. Therefore, it was examined whether this molecule corresponds to the enzymatic character of mammalian PAP/TRAP. 3.3. Comparison of characteristics of 37-kDa protein and PAP Mammalian PAP/TRAPs (EC3.1.3.2) contains dinuclear iron at the active site and such iron ions are important in regulating its activity. Therefore, this study analyzed the effect of iron on AcP activity of 37 kDa protein. Commercial purified bovine LF (Morinaga Milk Industry Co., Ltd) was used as the LF-rich fraction in the present experiment because it was confirmed to contain Uf-like protein (data not shown). The activity profile of the iron-containing sample had an optimum pH of 5.0, and that of the iron-depleted sample was lower (4.5) than that of iron-containing sample (Fig. 3A). The temperature for
maximum activity of each sample under the conditions tested was 60 C (Fig. 3B). The maximum activity of the iron-depleted sample was approximately half that of the iron-containing sample. Such profiles of an optimum pH range and the effect of iron on activity were consistent with other reports relative to PAP/TRAP (Andersson, Ek-Rylander, & Hammarström, 1984), although the optimum temperature could not be compared because little attention has been given to this point in other reports. It has been established that limited proteolysis of native bovine PAP/TRAP (spleen), recombinant human and rat PAP/TRAP with the serine protease trypsin and the cysteine proteases papain and cathepsin B converts the enzymes to a predominantly two-subunit structure and increases their activities several-fold (Funhoff, Klaassen, Samyn, Van Beeumen, & Averill, 2001; Ljusberg, EkRylander, & Andersson, 1999). Therefore, the effects of limited proteolysis using trypsin and pepsin on the activity of the molecule were examined. When the commercial LF was subject to tryptic digestion (1:2, w/w), it increased in a time-dependent manner about six-fold within 6 h (Fig. 4A). Consequently, when digested with 20-fold trypsin against LF (1:20, w/w), the activity rapidly reached a two-fold increase in 1 h, and the activity then decreased after this time (Fig. 4B), suggesting activation of the enzyme upon prolonged tryptic digestion. Digestion with the aspartic enzyme pepsin resulted in a gentle decrease in AcP activity at all concentrations (data not shown). These results are in agreement with the general profile of mammalian PAP/TRAP, although the cleaved fragments were not detected because the AcP molecule was present in a small quantity in this LF-rich fraction (or skim milk).
Fig. 3. Comparison of AcP activity between the LF-rich fraction (C) and the iron-depleted LF-rich fraction (B) in term of effect of (panel A) pH and (panel B) temperature. The results shown are means with standard errors (n ¼ 3).
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S. Yamaguchi et al. / International Dairy Journal 23 (2012) 86e90
Fig. 4. Activity profile of LF-rich fraction after time-dependent tryptic digestion. LF-rich fraction was digested with either 50 (panel A) or 500 (panel B) mg of trypsin. The results shown are means with standard errors (n ¼ 3).
The LF-rich fraction also contained various basic proteins, such as LF, basic fibroblast growth factors (bFGFs) and ribonuclease (RNase) (Kawakami et al., 2006), and these proteins were abundant compared with the 37 kDa molecule. The small amount of 37 kDa molecule became possible to separate by denaturant-containing rechromatography of HPSEC in this study. Considering the presence of AcP in skim milk as PAP/TRAP might help to explain the existence of some isoforms reported in skim milk (Akuzawa & Fox, 2004). Although the physical function of PAP/TRAP is unknown, it has been suggested that PAP/TRAP function as phosphotyrosyl phosphatases in biological systems (Halleen, Kaija, Stepan, Vihko, & Väänänen, 1998; Nuthmann, Dirks, & Drexler, 1993), and they have been implicated as mediators in bone resorption by osteoclasts (Moonga, Moss, Patchell, & Zaidi, 1990; Schindelmeiser, Münstermann, & Witzel, 1987). 4. Conclusion The AcP molecule in the LF-rich fraction from skim milk was completely purified as a 37 kDa molecule, which corresponded to the sequence of Uf-like protein and belongs to the PAP/TRAP family. It seems reasonable to suppose that the active molecule in the LFrich fraction is PAP/TRAP. Further work is in progress to elucidate the origin of milk PAP/TRAP, its relation to biological events, and its significance. Acknowledgment This project was funded by the Morinaga Foundation for Health and Nutrition. References Akuzawa, R., & Fox, P. F. (1998). Purification and characterization of an acid phosphatase from cell membrane fraction of Lactococcus lactis ssp lactis 303. Food Research International, 31, 157e165. Akuzawa, R., & Fox, P. F. (2004). Acid phosphatase in cheese. Animal Science Journal, 75, 385e391. Andersson, G., Ek-Rylander, B., & Hammarström, L. (1984). Purification and characterization of a vanadate-sensitive nucleotide tri- and diphosphatase with acid pH optimum from rat bone. Archives of Biochemistry and Biophysics, 228, 431e438.
Fujihara, M., Tanigawa, K., Kawakami, H., & Ohtsuki, K. (2004). Biochemical characterization of lactoferrin (LF)-binding proteins involved in the multiple physiological functions of LF in vitro. Milk Science, 53, 320e322. Funhoff, E. G., Klaassen, C. H. W., Samyn, B., Van Beeumen, J., & Averill, B. A. (2001). The highly exposed loop region in mammalian purple acid phosphatase controls the catalytic activity. Chembiochem, 2, 355e363. Halleen, J. M., Kaija, H., Stepan, J. J., Vihko, P., & Väänänen, H. K. (1998). Studies on the protein tyrosine phosphatase activity of tartrate-resistant acid phosphatase. Archives of Biochemistry and Biophysics, 352, 97e102. Kawakami, A., Hirayama, K., Kawakami, F., Kawakami, H., Fujihara, M., & Ohtsuki, K. (2006). Purification and biochemical characterization of a fibroblast growth factor-binding protein (FGF-BP) from the lactoferrin fraction of bovine milk. Biochimica et Biophysica Acta, 1760, 421e431. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680e685. Ljusberg, J., Ek-Rylander, B., & Andersson, G. (1999). Tartrate-resistant purple acid phosphatase is synthesized as a latent proenzyme and activated by cysteine proteinases. Biochemical Journal, 343, 63e69. Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement with the folin phenol reagent. Journal of Biological Chemistry, 193, 265e275. Matsudaira, P. (1987). Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. Journal of Biological Chemistry, 262, 10035e10038. Mazurier, J., & Spik, G. (1980). Comparative study of the iron-binding properties of human transferring. I. Complete sequential iron saturation and desaturation of the lactotransferrin. Biochimica et Biophysica Acta, 629, 399e408. Merril, C. R., Goldman, D., Sedman, S. A., & Ebert, M. H. (1981). Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science, 211, 1437e1438. Miura, T., Ono, K., Izumi, T., & Akuzawa, R. (2010). Presence of lactoferrin in a bovine skim milk fraction with acid phosphatase activity. International Dairy Journal, 20, 67e71. Moonga, B. S., Moss, D. W., Patchell, A., & Zaidi, M. (1990). Intracellular regulation of enzyme secretion from rat osteoclasts and evidence for a functional role in bone resorption. Journal of Physiology, 429, 29e45. Nuthmann, V., Dirks, W., & Drexler, H. G. (1993). Transient expression of tartrateresistant acid phosphatase (TRAP) gene in hamster cells: a pilot study. Leukemia, 7, 1960e1964. Schindelmeiser, J., Münstermann, D., & Witzel, H. (1987). Histochemical investigations on the localization of the purple acid phosphatase in the bovine spleen. Histochemistry, 87, 13e19. Shimazaki, K., Tanaka, T., Kon, H., Oota, K., Kawaguchi, A., Maki, Y., et al. (1993). Separation and characterization of the C-terminal half molecule of bovine lactoferrin. Journal of Dairy Science, 76, 946e955. Tanigawa, K., Fujihara, M., Sakamoto, R., Yanahira, S., & Ohtsuki, K. (2001). Characterization of bovine Angiogenin-1 and lactogenin-like protein as a Glycyrrhizin-binding protein and their in vitro phosphorylation by C-kinase. Biological and Pharmaceutical Bulletin, 24, 443e447. Yoshida, S., & Ye, X.-Y. (1991). Isolation of lactoperoxidase and lactoferrins from bovine milk acid whey by carboxymethyl cation exchange chromatography. Journal of Dairy Science, 74, 1439e1444.
Contents lists available at SciVerse ScienceDirect
International Dairy Journal journal homepage: www.elsevier.com/locate/idairyj
Short communication
Separation of a milk acid phosphatase from a purified lactoferrin fraction and identification as a member of the mammalian purple acid phosphatase family Shun Yamaguchi, Takayuki Miura*, Asami Baba, Ryozo Akuzawa Department of Food Science and Technology, Nippon Veterinary and Life Science University, 2-27-5, Musashino, Tokyo 180-8602, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 March 2011 Received in revised form 13 October 2011 Accepted 24 October 2011
Active acid phosphatase (AcP), 37 kDa, was completely separated from the purified lactoferrin fraction of bovine milk (LF-rich fraction). The N-terminal amino acid sequence of the 37 kDa molecule had 84% homology with bovine uteroferrin (Uf)-like protein. The 37 kDa molecule has an optimum pH range of 4.5e5.0 and an optimum temperature of 60 C. The AcP activity of the iron removal 37 kDa molecule (iron-depleted LF-rich fraction) was approximately half that of the iron-containing sample (LF-rich fraction). The activity increased in a time-dependent manner on tryptic digestion. These profiles correspond to the mammalian purple acid phosphatase (PAP) family (Uf, Type-5 and tartrate-resistant acid phosphatase: EC3.1.3.2). It seems reasonable to propose that the active molecule in the LF-rich fraction is an undocumented bovine PAP-family protein. Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved.
1. Introduction Since bovine lactoferrin (LF) is cationic at the pH of milk or cheese whey, as are lactoperoxidase (LPO) and some minor proteins, it can be easily isolated from milk using a cationic exchange resin (Yoshida & Ye, 1991). The LF fraction thus separated is widely used as purified LF for commercial purposes. However, the characterization of LF using proteomic approaches has revealed an unexpected complexity of minor milk proteins and peptides. Some researchers have reported that LF-binding proteins (LF-BP) are co-purified with LF from bovine milk; these include ribonuclease-4, angiogenin, neutrophil gelatinase-associated lipocalin and fibroblast growth factor-binding protein (Fujihara, Tanigawa, Kawakami, & Ohtsuki, 2004; Tanigawa, Fujihara, Sakamoto, Yanahira, & Ohtsuki, 2001). Thus, the purified LF from milk would be better called the ‘LF-rich fraction’. The physiological activities of these minor proteins associated with LF may be involved in multiple functions of LF in vivo. Recently, Miura, Ono, Izumi, and Akuzawa (2010) reported that acid phosphatase (AcP) activity is a new enzymatic activity in the LF-rich fraction. Although the main active AcP molecules in the LF-rich fraction were tentatively identified as LF, this conclusion is controversial because purified LF can bind other minor phosphatases. Therefore, the purpose of the present study was to
* Corresponding author. Tel.: þ81 422 51 6121; fax: þ81 422 51 9984. E-mail address: [email protected] (T. Miura).
clarify whether the lactoferrin or another molecule has AcP activity, using a high-performance size-exclusion chromatography (HPSEC) technique. 2. Materials and methods 2.1. Preparation of LF-rich fraction from raw milk The AcP active fraction was prepared from bovine raw skimmed milk (obtained from Holstein cows at the faculty dairy farm of Nippon Veterinary and Life Science University), using amberlite FPC-3500 (Organo Co., Tokyo, Japan) cation-exchange resin and cellulofine CM 500 (cellulofine CM 500, Kanto Co., Tokyo, Japan) carboxymethyl column (3 10 cm), as described by Miura et al. (2010). These AcP active fractions are considered to be the LF-rich fraction. 2.2. Purification of fraction with AcP activity from LF-rich fraction using high performance size exclusion chromatography 2.2.1. First-step chromatography A high performance size exclusion chromatography (HPSEC) procedure was used with slight modifications as described by Kawakami et al. (2006). The LF-rich fraction (100 mL) was injected onto a TSKgel 2000SWXL gel filtration column (8 250 mm, Tosoh, Tokyo, Japan) using a TOSOH HPLC system (8020 model, Tosoh, Tokyo, Japan). The column was eluted at a flow rate of 1 mL min1 with a fraction collected 30 s (500 mL per fraction), using 10 mM
0958-6946/$ e see front matter Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2011.10.003
S. Yamaguchi et al. / International Dairy Journal 23 (2012) 86e90
87
sodium acetate buffer with 300 mM NaCl (pH 5.2) as elution buffer, and the eluent was monitored at 280 nm.
(DDBJ), National Center for Biotechnology Information (NCBI) and SWISS-PROT on website.
2.2.2. Re-chromatography Subsequently, the highest AcP active fraction obtained from first-step chromatography (no. 17 fraction) was collected in 3 mL (six times via first-step chromatography) and dialyzed against 10 mM acetate buffer (1 L) using a dialysis tube (Viskase Companies, Inc., Darien, IL, USA) for 2 h at 4 C. The dialyzed sample was frozen at 30 C and then freeze-dried for 24 h under vacuum at 10 C. To concentrate the sample, the lyophilized powder was rehydrated to 100 mL volume (30-fold concentration from original volume) using 300 mM NaCl containing 10 mM acetate buffer (pH 5.2) containing 8 M urea as a strong denaturant. The above the sample (100 mL) was applied onto a TSKgel 2000SWXL gel filtration column (re-chromatography), and equilibrated with the elution buffer containing 8 M urea and the eluate was monitored at 280 nm. Resulting eluted fractions (500 mL per fraction) were each dialyzed against 10 mM acetate buffer (1 L) using a dialysis tube for 24 h at 4 C before assay for AcP activity. The total protein concentration was determined according to the method of Lowry, Rosebrough, Farr, and Randall (1951); bovine serum albumin was used as a protein standard.
2.6. Removal of iron from LF-rich fraction An iron-depleted LF-rich fraction was obtained according to the method of Mazurier and Spik (1980). The purified LF-rich fraction was dissolved (10 mg mL1) in 100 mM sodium citrate acid buffer, pH 2.0, containing 40 mM EDTA and allowed to equilibrate at 4 C overnight (24 h). The solution was then dialyzed against various pH levels of buffer for 24 h at 4 C. 2.7. Study of optimum pH and temperature Effects of pH and temperature on AcP activity of LF-rich fraction or iron-depleted sample were investigated as described by Miura et al. (2010). The pH of buffers was adjusted at 50 C by the gradual addition of 0.1 M HCl or NaOH with rapid stirring. For pH studies, the buffers employed were 0.1 M acetic acid for pH values 3e5 and 0.1 M Tris-HCl for pH 6e9; assays were performed at 50 C.
2.3. Assay for phosphatase activity Enzyme activity was determined by a modification of the method used by Akuzawa and Fox (1998), with slight modifications as described by Miura et al. (2010). Enzyme sample (100 mL) was added to 100 mL of 10 mM p-nitrophenylphosphate (p-NPP; Sigma Chemicals, St. Louis, MO, USA) in 10 mM sodium acetate (pH 5.2) and incubated for 30 min at 50 C. The reaction stopped by 200 mL of 1 M NaOH and absorbance was measured at 405 nm. One phosphatase unit was converted from absorbance values to 1 mmol of p-NP 100 mL1 30 min1 using standard curves. 2.4. Sodium dodecyl sulphate polyacrylamide gel electrophoresis and silver staining The molecular mass of the active molecules in the individual fraction tubes was estimated by Coomassie brilliant blue and silver staining after sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE analysis, according to the method of Laemmli (1970), was performed as described by Miura et al. (2010). The AcP active fractions after HPSEC were mixed with an equal volume of SDS-PAGE sample buffer, and boiled for 5 min. Aliquots of mixtures (20 mL) were separated on a 7e20% (w/v) acrylamide gradient separation gel. SDS-PAGE gels were stained for protein using a modified Merril silver stain (Merril, Goldman, Sedman, & Ebert, 1981). Molecular mass was estimated by comparison with standards (Precision of Plus ProteinÔ KaleidoscopeÔ Prestained Standards, Bio-Rad Labolatories, Inc., Hercules, CA 94547, USA). 2.5. N-terminal amino acid sequence determination Active AcP fractions were analyzed by SDS-PAGE and then transferred electrophoretically onto a polyvinylidene difluoride (PVDF) membrane according to the method of Matsudaira (1987). The N-terminal amino acid sequence of the AcP molecule was determined with an automatic protein sequencer model PSQQ-21 (Shimadzu Co., Kyoto, Japan) as described by Miura et al. (2010). Amino acid homology searches were performed using basic local alignment search tool (BLAST) through the DNA Database of Japan
Fig. 1. The first step chromatography on a TSKgel 2000SWXL column of AcP protein isolated from LF-rich fraction (panel A: B, absorbance at 280 nm; C, phosphatase activity; the double headed arrow indicates AcP active fractions 16e18) and SDS-PAGE profiles of fractions 14e20 (panel B: M denotes pre-stained molecular mass standards; double headed arrow indicates AcP active fractions 16e18).
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commercial LF (Morinaga Milk Industry Co., Ltd., Tokyo, Japan) (25 mg) were digested with 50 or 500 mg of trypsin or pepsin (Sigma, St. Louis, MO, USA) in 100 mL of the incubation solution (10 mM sodium phosphate buffer at pH 7.5 or deionized water adjusted by HCl to pH 2.5). Proteolytic digestion was performed at 37 C for 6 h, and terminated by re-adjusting pH to 5.2 using 200 mL of 0.1 M acetic acid buffer (pH 5.2). The AcP activity was expressed as absorbance at 405 nm. 3. Results and discussion 3.1. Purification and identification of active AcP molecules from purified LF-rich fraction
Fig. 2. Re-chromatography on a TSKgel 2000SWXL column of the AcP active fraction number 17 from first step chromatography (panel A: B, absorbance at 280 nm; C, phosphatase activity; the arrow indicates the AcP active fraction, No. 16) and SDS-PAGE profiles of fractions 13e19 (panel B: M denotes pre-stained molecular mass standards; double headed arrow indicates the AcP active fraction 16; single headed arrow indicates the 37 kDa protein).
For temperature studies, 0.1 M acetic acid adjusted to pH 5.2 at 30e80 C was used. For both pH and temperature studies incubations were for 30 min. The specific activity data were expressed as a percentage of the maximum value. 2.8. Proteolytic digestion of LF-rich fraction A proteolytic digestion procedure was used as described by Shimazaki et al. (1993) with slight modifications. LF-rich fraction or
In our previous report, one peak of AcP active fraction (LF-rich fraction) was separated from skim milk using a carboxy methyl cation-exchange column, and it was tentatively suggested that the principal AcP active molecule was the LF (Miura et al., 2010). However, there remain some questions because the active AcP fraction was not completely purified. To clarify whether the LF or another molecule has AcP activity in the LF-rich fraction, the authors investigated purification of the LF-rich fraction using HPSEC. As shown in Fig. 1A, one peak of AcP active fractions consisting of three fractions (no. 16, 17 and 18) was obtained. The activity of the first fraction (Fig. 1A, no. 16) showed very low activity (0.21 U 100 mL1) compared to the other two fractions. This fraction showed many bands, thought to be mainly 80 kDa lactoferrin by silver staining (Fig. 1B, no. 16). The middle fraction (no. 17) showed the highest activity (5.8 U 100 mL1) although bands were not seen (Fig. 1B, no. 17). The activity of the last fraction (no. 18) was about one tenth (0.6 U 100 mL1) of that of the no. 17 fraction and contained two distinct bands (Fig. 1B, no. 18). These results suggested that an unknown molecule has the main AcP activity rather than LF. To completely separate the main true AcP molecule from the most highly active AcP fraction (no. 17), that denatured sample was re-chromatographed. One AcP-active fraction was obtained (Fig. 2, no. 16), and analyzed by SDS-PAGE, which detected only a band at 37 kDa by silver staining (Fig. 2B, no. 16). The purification procedure for the 37 kDa molecule is summarized in Table 1. The specific activity was 15,132 units mg1, and the activity increased 90,704fold compared with that of raw milk. However, the last TSK gel 2000 SWXL procedure showed that the total activity markedly decreased by 81% compared with that of the previous procedure (cellulofine CM 500). Since priority was given to complete purification, it was thought that the overall loss of AcP activity was due to denaturation by urea. 3.2. Determination of N-terminal amino acid sequence When the band at 37 kDa band analyzed by a protein sequencer, the N-terminal amino acid sequence obtained was AAAAATTPAPMLRFFAVDI (Table 2). That amino acid sequence was 84% (16/19 residues) homologous to bovine uteroferrin (Uf)-like protein
Table 1 Purification of 37 kDa protein with AcP activity. Fraction
Volume (mL)
Total protein (mg)
Total activity (units)
Specific activity (units mg1)
Purification (fold)
Yield (%)
Raw milk Skim milk Amberlite FPC-3500 Cellulofine CM 500 TSK gel 2000 SWXL
2000 1500 400 60 1
74,985 60,701 177 46.5 0.01
12,510 6593 7232 5143 180.3
0.167 0.109 41 110 15,132
1 0.65 245 662 90,704
100 53 58 41 1.4
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Table 2 Comparison of amino acid residues of the 37 kDa protein and PAP-family proteins.a
Name
Organism
The 37 kDa protein
Bovine
Uteroferrin-like protein TRAP/Type 5-acid phosphatase TRAP/Type 5-acid phosphatase TRAP/uteroferrin
Bovine Rat Mouse Pig
Amino acid residues A | A L L R
A | A L L T
A | A A T N
A | A H H T
A | A C G R
T | T T T T
T | T A A A
P | P P P P
A | A A T T
P | P S P P
M | M T T I
L | L L L L
R | R R R R
Accession number F | F F F F
F A | V A V A V A V A
V | V V V V
D I G G G G
D D D D
W W W W
XP_002688895 : NCBI P29288 : swiss-prot Q05117 : swiss-prot P09889 : swiss-prot
a Vertical lines indicate the residues identical for the 37 kDa protein and uteroferrin-like protein sequence. Homology with uteroferrin-like protein and other phosphatases (TRAP) is shown by box-like enclosure.
(DDBJ Accession number XP_002688895). The amino acid residue at positions 15, 18 and 19 did not be correspond to the above Uf-like protein sequence data despite being analyzed three or more times. Although the amino acid sequence of Uf-like protein was predicted from the bovine genome, this sequence showed a high homology with the purple acid phosphatases (PAP) family (EC3.1.3.2) such as various mammalian type 5-acid phosphatase (ACP5) and tartrate resistant acid phosphatase (TRAP) using the gene data in UniGene at the National Center for Biotechnology Information (NCBI) (Table 2). All of these proteins exhibit similar molecular masses in the 30e40 kDa range, basic pI and high activity toward responsestabilized substrate, such as p-nitrophenyl phosphate. Uf in bovine milk or the bovine uterus has never been reported. Therefore, it was examined whether this molecule corresponds to the enzymatic character of mammalian PAP/TRAP. 3.3. Comparison of characteristics of 37-kDa protein and PAP Mammalian PAP/TRAPs (EC3.1.3.2) contains dinuclear iron at the active site and such iron ions are important in regulating its activity. Therefore, this study analyzed the effect of iron on AcP activity of 37 kDa protein. Commercial purified bovine LF (Morinaga Milk Industry Co., Ltd) was used as the LF-rich fraction in the present experiment because it was confirmed to contain Uf-like protein (data not shown). The activity profile of the iron-containing sample had an optimum pH of 5.0, and that of the iron-depleted sample was lower (4.5) than that of iron-containing sample (Fig. 3A). The temperature for
maximum activity of each sample under the conditions tested was 60 C (Fig. 3B). The maximum activity of the iron-depleted sample was approximately half that of the iron-containing sample. Such profiles of an optimum pH range and the effect of iron on activity were consistent with other reports relative to PAP/TRAP (Andersson, Ek-Rylander, & Hammarström, 1984), although the optimum temperature could not be compared because little attention has been given to this point in other reports. It has been established that limited proteolysis of native bovine PAP/TRAP (spleen), recombinant human and rat PAP/TRAP with the serine protease trypsin and the cysteine proteases papain and cathepsin B converts the enzymes to a predominantly two-subunit structure and increases their activities several-fold (Funhoff, Klaassen, Samyn, Van Beeumen, & Averill, 2001; Ljusberg, EkRylander, & Andersson, 1999). Therefore, the effects of limited proteolysis using trypsin and pepsin on the activity of the molecule were examined. When the commercial LF was subject to tryptic digestion (1:2, w/w), it increased in a time-dependent manner about six-fold within 6 h (Fig. 4A). Consequently, when digested with 20-fold trypsin against LF (1:20, w/w), the activity rapidly reached a two-fold increase in 1 h, and the activity then decreased after this time (Fig. 4B), suggesting activation of the enzyme upon prolonged tryptic digestion. Digestion with the aspartic enzyme pepsin resulted in a gentle decrease in AcP activity at all concentrations (data not shown). These results are in agreement with the general profile of mammalian PAP/TRAP, although the cleaved fragments were not detected because the AcP molecule was present in a small quantity in this LF-rich fraction (or skim milk).
Fig. 3. Comparison of AcP activity between the LF-rich fraction (C) and the iron-depleted LF-rich fraction (B) in term of effect of (panel A) pH and (panel B) temperature. The results shown are means with standard errors (n ¼ 3).
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Fig. 4. Activity profile of LF-rich fraction after time-dependent tryptic digestion. LF-rich fraction was digested with either 50 (panel A) or 500 (panel B) mg of trypsin. The results shown are means with standard errors (n ¼ 3).
The LF-rich fraction also contained various basic proteins, such as LF, basic fibroblast growth factors (bFGFs) and ribonuclease (RNase) (Kawakami et al., 2006), and these proteins were abundant compared with the 37 kDa molecule. The small amount of 37 kDa molecule became possible to separate by denaturant-containing rechromatography of HPSEC in this study. Considering the presence of AcP in skim milk as PAP/TRAP might help to explain the existence of some isoforms reported in skim milk (Akuzawa & Fox, 2004). Although the physical function of PAP/TRAP is unknown, it has been suggested that PAP/TRAP function as phosphotyrosyl phosphatases in biological systems (Halleen, Kaija, Stepan, Vihko, & Väänänen, 1998; Nuthmann, Dirks, & Drexler, 1993), and they have been implicated as mediators in bone resorption by osteoclasts (Moonga, Moss, Patchell, & Zaidi, 1990; Schindelmeiser, Münstermann, & Witzel, 1987). 4. Conclusion The AcP molecule in the LF-rich fraction from skim milk was completely purified as a 37 kDa molecule, which corresponded to the sequence of Uf-like protein and belongs to the PAP/TRAP family. It seems reasonable to suppose that the active molecule in the LFrich fraction is PAP/TRAP. Further work is in progress to elucidate the origin of milk PAP/TRAP, its relation to biological events, and its significance. Acknowledgment This project was funded by the Morinaga Foundation for Health and Nutrition. References Akuzawa, R., & Fox, P. F. (1998). Purification and characterization of an acid phosphatase from cell membrane fraction of Lactococcus lactis ssp lactis 303. Food Research International, 31, 157e165. Akuzawa, R., & Fox, P. F. (2004). Acid phosphatase in cheese. Animal Science Journal, 75, 385e391. Andersson, G., Ek-Rylander, B., & Hammarström, L. (1984). Purification and characterization of a vanadate-sensitive nucleotide tri- and diphosphatase with acid pH optimum from rat bone. Archives of Biochemistry and Biophysics, 228, 431e438.
Fujihara, M., Tanigawa, K., Kawakami, H., & Ohtsuki, K. (2004). Biochemical characterization of lactoferrin (LF)-binding proteins involved in the multiple physiological functions of LF in vitro. Milk Science, 53, 320e322. Funhoff, E. G., Klaassen, C. H. W., Samyn, B., Van Beeumen, J., & Averill, B. A. (2001). The highly exposed loop region in mammalian purple acid phosphatase controls the catalytic activity. Chembiochem, 2, 355e363. Halleen, J. M., Kaija, H., Stepan, J. J., Vihko, P., & Väänänen, H. K. (1998). Studies on the protein tyrosine phosphatase activity of tartrate-resistant acid phosphatase. Archives of Biochemistry and Biophysics, 352, 97e102. Kawakami, A., Hirayama, K., Kawakami, F., Kawakami, H., Fujihara, M., & Ohtsuki, K. (2006). Purification and biochemical characterization of a fibroblast growth factor-binding protein (FGF-BP) from the lactoferrin fraction of bovine milk. Biochimica et Biophysica Acta, 1760, 421e431. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680e685. Ljusberg, J., Ek-Rylander, B., & Andersson, G. (1999). Tartrate-resistant purple acid phosphatase is synthesized as a latent proenzyme and activated by cysteine proteinases. Biochemical Journal, 343, 63e69. Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement with the folin phenol reagent. Journal of Biological Chemistry, 193, 265e275. Matsudaira, P. (1987). Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. Journal of Biological Chemistry, 262, 10035e10038. Mazurier, J., & Spik, G. (1980). Comparative study of the iron-binding properties of human transferring. I. Complete sequential iron saturation and desaturation of the lactotransferrin. Biochimica et Biophysica Acta, 629, 399e408. Merril, C. R., Goldman, D., Sedman, S. A., & Ebert, M. H. (1981). Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science, 211, 1437e1438. Miura, T., Ono, K., Izumi, T., & Akuzawa, R. (2010). Presence of lactoferrin in a bovine skim milk fraction with acid phosphatase activity. International Dairy Journal, 20, 67e71. Moonga, B. S., Moss, D. W., Patchell, A., & Zaidi, M. (1990). Intracellular regulation of enzyme secretion from rat osteoclasts and evidence for a functional role in bone resorption. Journal of Physiology, 429, 29e45. Nuthmann, V., Dirks, W., & Drexler, H. G. (1993). Transient expression of tartrateresistant acid phosphatase (TRAP) gene in hamster cells: a pilot study. Leukemia, 7, 1960e1964. Schindelmeiser, J., Münstermann, D., & Witzel, H. (1987). Histochemical investigations on the localization of the purple acid phosphatase in the bovine spleen. Histochemistry, 87, 13e19. Shimazaki, K., Tanaka, T., Kon, H., Oota, K., Kawaguchi, A., Maki, Y., et al. (1993). Separation and characterization of the C-terminal half molecule of bovine lactoferrin. Journal of Dairy Science, 76, 946e955. Tanigawa, K., Fujihara, M., Sakamoto, R., Yanahira, S., & Ohtsuki, K. (2001). Characterization of bovine Angiogenin-1 and lactogenin-like protein as a Glycyrrhizin-binding protein and their in vitro phosphorylation by C-kinase. Biological and Pharmaceutical Bulletin, 24, 443e447. Yoshida, S., & Ye, X.-Y. (1991). Isolation of lactoperoxidase and lactoferrins from bovine milk acid whey by carboxymethyl cation exchange chromatography. Journal of Dairy Science, 74, 1439e1444.