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Repressive processing of antihypertensive peptides, Val-Pro-Pro and Ile-Pro-Pro, in Lactobacillus helveticus fermented milk by added peptides
Journal of Bioscience and Bioengineering VOL. 114 No. 2, 133e137, 2012 www.elsevier.com/locate/jbiosc
Repressive processing of antihypertensive peptides, Val-Pro-Pro and Ile-Pro-Pro, in Lactobacillus helveticus fermented milk by added peptides Taketo Wakai,1 Naoya Yamaguchi,2 Misaki Hatanaka,1 Yasunori Nakamura,1 and Naoyuki Yamamoto1, * Microbiology & Fermentation Laboratory, Calpis, 11-10 5-Chome, Fuchinobe, Chuo-ku, Sagamihara-shi, Kanagawa 252-0206, Japan1 and Hokkaido Research Organization, Tokachi Agricultural Experiment Station, 9-2 Shinsei minami, Memuro, Kasai-gun, Hokkaido 082-0081, Japan2 Received 30 January 2012; accepted 23 March 2012 Available online 15 May 2012
Lactobacillus helveticus can release the antihypertensive peptides, Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP), from casein in fermented milk by a specific proteolytic system. To better understand the regulation of gene expression of the proteolytic enzymes thought to link to the processing of both antihypertensive peptides in L. helveticus, microarray analysis for whole gene expression in the presence and absence of added peptides in the fermented milk was studied. The productivity of both VPP and IPP in L. helveticus CM4 fermented milk was repressed by adding 2% quantity of Peptone as peptide mixture to the milk. Among the selected 13 amino acids, Gly, Ile, Leu, Phe, Met, Ser and Val were effective in the repression of the productivity of VPP and IPP in the fermented milk. The activity of the cell wallassociated proteinase, which may play a key role in the processing of the two antihypertensive peptides, was significantly repressed by the addition of the 2% quantity of Peptone into the fermented milk. By DNA microarray analysis it was found that prtH2 corresponding to the cell wall-associated proteinase gene, most of the endopeptidase genes such as pepE, pepO1, pepO2 and pepO3, most of the oligopeptide transporter genes, such as dppA2, dppB, dppC, dppD and dppF, most likely involved in the processing of VPP and IPP were down-regulated. These results suggest that amino acids released from milk peptides in the fermented milk might down-regulate the gene expressions of some of the proteolytic enzymes and may cause repression of the release of VPP and IPP in L. helveticus fermented milk. Ó 2012, The Society for Biotechnology, Japan. All rights reserved. [Key words: Val-Pro-Pro and Ile-Pro-Pro; Proteolytic system; Transcriptional regulation; Amino acids; Microarray analysis]
Lactic acid bacteria possess a proteolytic system which involves the hydrolysis of milk proteins and allows the bacteria to use peptides and amino acids as nutrients (1,2). Among lactic acid bacteria, Lactobacillus helveticus can grow rapidly in milk because of its high proteolytic activity and resistance to acid stress (3e5). Therefore, L. helveticus can release a large amount of peptides, including bioactive peptides in fermented milk by means of proteolysis of milk proteins (3,6). Among bioactive peptides, the antihypertensive peptides, Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP), that have inhibitory activities against angiotensin I-converting enzyme (kininase II; EC 3.4.15.1) (ACE), which controls blood pressure in vivo, have been extensively studied (7,8). Furthermore, consecutive uptakes of these peptides for more than a week resulted in significant reduction of blood pressure in many clinical trials (9e15). Meta-analysis for these clinical trials showed a significant reduction of blood pressure by treatment with VPP and IPP (16). Moreover, a current double-blind placebo controlled study revealed an improvement of arterial stiffness by oral administration of VPP and IPP for more than 8 weeks (17). For the release of a large amount of VPP and IPP, the L. helveticus CM4 strain with high cell wall-associated proteinase activity was isolated and used in the preparation of the fermented milk product
(EU Patent, 1016709A1, 1991). However, the proteinase activation process was repressed by certain amino acids and peptides released in the fermentation process (18). The regulation of the proteolytic system in response to amino acids, the so called codY system, has been well characterized in lactococci, bacilli and streptococci (19e21). CodY protein senses the intracellular pool of branched-chain amino acids and increases affinity to the codY-box sequence when branched chain amino acids occur. The gene transcription of most proteolytic systems, including cell wall-associated proteinase, some peptidases and transporters that have the codY-box sequences adjacent to the promoter regions of the structural genes, are controlled by CodY protein (19e21). In contrast, there is little information about the regulation of the proteolytic system in L. helveticus, which is most likely involved in the processing of VPP and IPP by peptides and amino acids. So, the objective of the present study is to analyze the regulation of the proteolytic gene expression in L. helveticus during milk fermentation in the presence of peptides by using a microarray for the whole genome of the CM4 strain. Consequently, the key enzymes involved in the processing of VPP and IPP are discussed throughout the transcriptome analysis. MATERIALS AND METHODS
* Corresponding author. Tel.: þ81 42 769 7831; fax: þ81 42 769 7810. E-mail address: [email protected] (N. Yamamoto).
Materials Val-Pro-Pro and Ile-Pro-Pro were purchased from Kurabou (Osaka, Japan). Isotopes for (13C5)Val-(13C5)Pro-Pro (m/z 324.2) and Ile-(13C5)Pro-Pro (m/z
1389-1723/$ e see front matter Ó 2012, The Society for Biotechnology, Japan. All rights reserved. doi:10.1016/j.jbiosc.2012.03.015
WAKAI ET AL.
J. BIOSCI. BIOENG.,
20 10 0 0
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peptides (-)
B
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+peptides 4 2 0 0
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Time (h) FIG. 1. Changes in amounts of Val-Pro-Pro and Ile-Pro-Pro in Lactobacillus helveticus CM4 fermented milk with or without 2% (W/V) of peptides (Peptone; BD). Then, at 0, 2, 3, 4, 6, 8, 14 and 22 h of fermentation after the addition of the peptides, the amounts of VPP (open and closed squares) and IPP (opened and closed squares and circles) in CM4 fermented milk with (closed squares and circles) or without (open squares and circles) peptides were analyzed by LC-MSMS method.
and were repressed to 9% for VPP and 33% for IPP if peptides were added in the milk medium, respectively (Fig. 1A and B). The cell growth with peptides monitored by the optical density was slightly faster than that without peptides. These results strongly suggest a repressive effect of peptides on the proteolytic system involving the processing of both antihypertensive peptides. Repressive effects of amino acids on the release of VPP and IPP To understand the detailed repressive effect of each studied amino acid on the release of VPP and IPP in L. helveticus fermented milk, the amount of VPP and IPP in the L. helveticus fermented milk was measured with or without 13 different amino acids. Most of the tested amino acids showed repression of the production of VPP and IPP. Gly, Ile and Leu showed a higher repressive effect and Phe, Met, Ser and Val were slightly effective as repressers, (Fig. 2). It has been
VPP IPP
Gly
Ile
Leu
Phe
Ser
Met
Val
Tyr
Asn
0%
Pro
100%
Thr
Repressive release of the antihypertensive peptides by added peptides In our previous study, we have found a large amount of the antihypertensive peptides, Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP), in L. helveticus fermented milk (3), but it was suggested that other released peptides in the fermented milk had a repressive effect on cell-wall associated proteinase activity and on the production of both antihypertensive peptides (18). So, the influence of the peptides (and amino acids) on the release of VPP and IPP during the L. helveticus CM4 fermentation was analyzed. As shown in Fig. 1A and B, the release of VPP and IPP in the CM4 fermented milk increased during the fermentation. However, release of VPP and IPP in the fermented milk was significantly repressed by the addition of 2% of peptides (Peptone; BD, NJ, USA) in the fermentation as shown in Fig. 1. Maximum values of VPP and IPP in the fermented milk were observed at 13 h after the fermentation,
+peptides
30
Ala
RESULTS
peptides (+)
Gln
Microarray analysis For the transcriptome analysis of L. helveticus CM4 fermented in milk medium with or without peptides (Peptone; BD, NJ, USA), cells were harvested and added into same volume of RNA Protect Bacteria Reagent (Qiagen K. K., Tokyo, Japan). The cells were collected by a centrifugation at 20,000 rpm for 10 min. The cell pellets were stored at 30 C. Total RNA was purified and was used for analysis of its quality. Untreated cells were used as controls. Total RNA was extracted using an RNeasy Mini Kit (Qiagen, Valencia, CA, USA) after lysis by lysozyme and phenol treatments. The following procedures for microarray analysis were performed according to the protocol of Roche NimbleGen, Inc. (Madison, WI, USA). The whole genome sequence of CM4 strain was completed (unpublished data), therefore we prepared a microarray for L. helveticus CM4 genes and used in this study. cDNA was synthesized from total RNA for use in the hybridization. The hybridized arrays were scanned and normalized using NimbleScan software. Results for particular genes are presented as an n-fold change of expression. We selected genes if they were up-regulated more than 1.88-fold and down-regulated less than 0.32-fold in a comparison of cells cultured with or without peptides.
peptides (-)
40
Control
Cell wall-associated proteinase activity For the measurement of cell wallassociated proteinase activity during the fermentation in milk medium, CM4 cells were harvested at 1, 3, 3.5, 4, 5, 6 and 7 h after fermentation at 37 C maintaining medium pH at 6.0. Then, sodium citrate was added up to 2% (W/V) to clarify the fermented milk. After a centrifugation of the clarified fermented milk at 20,000 g for 10 min, cells were washed with 50 mM b-glycerophosphoric acid at room temperature. The washed cells were suspended in a phosphate saline buffer, pH 7.4, and adjusted to a cell density at 590 nm as 1.0 to measure the cell wall-associated proteinase activity per cell density (OD590nm). The cell wall-associated proteinase activity was measured according to the method described previously (23) using a fluorescein isothiocyanate casein (FITC-casein) as a substrate. The reaction mixture, containing 100 ml of 0.2% FITC-casein (Sigma Aldrich Japan, Tokyo) and 100 ml of the enzyme solution, was incubated at 42 C for 60 min. Soluble peptides were collected in the supernatant after adding 200 ml of 5% TCA, and precipitating unhydrolyzed protein at 15,000 g for 10 min. Fifty ml of the supernatant was neutralized by the addition of 150 ml of 500 mM TriseHCl buffer, pH 8.5. Then, fluorescence was measured by using the Microplate Reader (POWERSCAN HT; Dainippon Pharmaceutical) with an excitation wavelength of 495 nm and an emission wavelength of 525 nm. One unit of proteolytic activity was defined as the amount of enzyme yielding 1% of total initial casein fluorescence as TCA soluble fluorescence after 60 min of hydrolysis.
VPP conc. (µg/mL)
Measurement of VPP and IPP in the fermented milk CM4 strain (EU Patent, 1016709A1, 1991) was pre-cultured in 9% (W/W) low-heated skim milk at 37 C for 24 h. The pre-cultured fermented milk was added up to 5% to a fresh milk medium (9% low-heated skim milk). It was fermented at 37 C for 25 h, with a pH maintained at 6.0 by adding of 50% NaOH, and then Peptone was added to the fermented milk up to 2% at 3 h after the fermentation. VPP and IPP in the fermented milk were measured at 3, 4, 5, 6, 7, 8, 9, 11, 13, 16, 19 and 25 h after the fermentation according to the previous method with some modification (22). Measurements were carried out by using a high performance liquid chromatography-atmospheric pressure ionization multiple reaction monitoring mass spectrometer (HPLC-API-MRM-MS) in positive ionization mode. The internal standards used were isotope (13C5)Val-(13C5)Pro-Pro for VPP and isotope Ile-(13C5)Pro-Pro for IPP, respectively. To understand the influence of various amino acids on the production of VPP and IPP in the fermented milk, a mixture with or without 2% (W/V) of Gln, Ala, Thr, Pro, Tyr, Asn, Val, Ser, Met, Phe, Leu, Ile, Gly was added and fermented at 37 C for 24 h. Then, the production of VPP and IPP in the fermented milk with amino acid was compared with that prepared without amino acid.
A
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332.2) were obtained from SCRUM Inc. (Tokyo, Japan). RNA Protect Bacteria Reagent was purchased from Qiagen K. K. (Tokyo, Japan). Peptone was purchased from BD (NJ, USA). Peptone prepared by enzymatic hydrolysis of animal proteins was used as peptide mixture without Val-Pro-Pro and Ile-Pro-Pro sequence.
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FIG. 2. Repressive effect of various amino acids on the productivity of VPP and IPP in Lactobacillus helveticus CM4 fermented milk. Value represents the ratio comparing with and without the addition of each amino acid.
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reported that branched chain amino acids (BCAA) in a different strain, Lactococcus lactis, has a repressive effect on the proteolytic system regulated by the codY system. CodY is a well studied and important transcriptional regulator system. Taking this into account, it was suggested that there would be a repressive effect of the amino acids including BCAA on the proteolytic system and these would affect the release of VPP and IPP in L. helveticus CM4.
of the excision of N-terminal amino acid of X-Pro sequence. The expression of pepX gene was not changed greatly by the addition of peptides at 0.5 and 2 h (Table 1). For the aminopeptidase, only the pepCE gene and pepN2 gene were effectively down-regulated at 0.5 and 2 h after the addition of peptides. Among the dipeptidase and tripeptidase genes, pepT2 and pepV genes thought to be not involved in the processing of VPP and IPP (Table 1) were also down-regulated. Moreover, all transporter genes, such as dppA1, dppA2, dppB, dppC, dppD and dppF were down-regulated. However, down-regulations of gene expressions for tripeptidase gene (pepT), prolinase gene (pepPN) and proline iminopeptidase (pepI) were not large. From the transcriptome analysis for CM4 genes with or without peptides in the fermented milk, key proteolytic enzyme genes most likely needed for the processing of VPP and IPP, proteinase, endopeptidases, and peptide transporter, were mainly down-regulated (Table 1).
Repressive effect of added peptides on the cell wallassociated proteinase For the understanding of the key enzymes in the proteolytic system, we measured the cell wallassociated proteinase activity on the CM4 cells. This was done on cultured and collected cells from fermented milk after clarification by the addition of 2% of sodium citrate at 1, 3, 3.5, 4, 5, 6 and 7 h during fermentation (Fig. 3). As shown in Fig. 3, the proteinase activity was greatly increased from 1 to 3.5 h during the fermentation without the addition of peptides, however when 2% of the peptides were added at 3 h during fermentation, the proteinase activity was clearly decreased after 3 h (Fig. 3). The release of the VPP and IPP in the CM4 fermented milk (Fig. 1) was correlated with the proteinase activity on CM4 cells (Fig. 3). These results suggest the importance of the cell wall-associated proteinase for the processing of VPP and IPP in the fermented milk, and also repression of the proteinase by peptides in the fermentation.
Proteinase Activity (U/OD)
Gene expression of proteolytic enzymes For a fuller understanding of the repressive effects of peptides on the proteolytic system especially involved in the processing of the antihypertensive peptides, VPP and IPP, we completed a transcriptome analysis for the whole gene sequence corresponding to L. helveticus CM4 strain, and applied this to the CM4 cells collected at 0.5 and 2 h after the addition of peptides into the fermented milk. As shown in Table 1, most of the genes of the proteolytic system were repressed by the addition of the peptides in the fermentation in milk. Among these proteolytic genes, prtY gene corresponding to the cell wallassociated proteinase gene (24) was not repressed. However, it is not clear whether a maturation protein gene is needed for activation of the precursor protein (PrtY). On the other hand, signals for prtH2, reported as a cell wall-associated proteinase gene in CNRZ32 (26), were greatly repressed by the addition of peptides. For the endopeptidases which are considered to be essential for the release of VPP and IPP from the proteinasereleased long peptides, most of the genes were down-regulated by the addition of peptides especially at 0.5 h (Table 1). In more detail, expressions of genes of pepE, pepO1, pepO2 and pepO3 were repressed to 0.33-, 0.75-, 0.07- and 0.77-fold at 0.5 h respectively, and pepO2 gene was still greatly repressed at 2 h by the addition of peptides (Table 1). The study of X-prolyldipeptidyl aminopeptidase (XPDAP) also suggests the importance 400
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200
100
0
0
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3 4 Time (h)
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FIG. 3. Change in the cell wall-associated proteinase activity on Lactobacillus helveticus CM4 cells cultured in skim milk medium with or without the addition of 2% of peptides at 3 h during fermentation as indicated by down arrows. Cells harvested at 0.5 and 2 h after the addition of the peptides (shown by up arrows) were used for microarray analysis.
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Changes of other gene expressions For the understanding of the changes in other gene expressions, except proteolytic system by peptides, repressed genes in CM4 fermented milk by the addition of peptides were summarized in Table S1. Many amino acid transporter genes such as hisM, potE, glnQ, hisJ, rhaT and med were down-regulated by peptides in the milk medium (Table S1). In contrast, some of transcriptional regulator genes, such as tra5, acrR and glpR and part of transporter genes, such as salY, mgtA and lacY were up-regulated at 0.5 h after the addition of peptides in milk medium (Table S2). These down-regulations of the genes were larger at 0.5 h than those at 2 h after cultivation with peptides (Table S1). Moreover, the negative regulations in many amino acid transporter genes were larger in those of proteolytic enzyme genes as shown in Table 1. DISCUSSION The proteolytic system is the most important system in L. helveticus because it allows for its rapid growth in milk and allows for effective utilization of amino acids and peptides originated from milk protein among lactic acid bacteria (3). So, the down-regulation of the proteolytic system would be a significant event in L. helveticus for its metabolism in its work of uptaking and utilizing peptides and amino acids. However, this study is one of very few that has addressed the transcriptome analysis of the proteolytic system in response to peptides in L. helveticus. The transcriptome analysis conducted in the present study, with or without peptides, in the fermented milk supported our consideration of the processing pathway of the unique bioactive peptides, VPP and IPP, in L. helveticus CM4. The cell wall-associated proteinase is the most important enzyme to decompose milk casein and supply long peptides containing the VPP and IPP sequence (23). In the present study, the prtY gene, which was previously shown to play an important role in the release of VPP and IPP, was not down-regulated, so post-transcriptional regulation may be involved in the processing. In contrast it is not clear what role is played in the processing of both antihypertensive peptides by the down-regulation of the prtH2 gene reported as a cell wall-associated proteinase in CNRZ32 (25). Among all the down-regulated endopeptidase genes, pepO1 (26) and pepO2 (27) were considered to be most important enzymes for the C-terminal processing of both peptides. For the N-terminal processing to release VPP and IPP, pepX gene (28) essential for the processing of X-Pro sequence was not affected, but pepCE and pepN2 (29) genes were effectively downregulated by peptides. Most of the oligopeptide transporters, dppB, dppC, dppD and dppF, which most likely play important roles in the processing of VPP and IPP, were also considered to have an effect on the production of VPP and IPP. These results suggest that repressive effect of peptides on the release of VPP and IPP in the CM4 fermented milk might be caused by down-regulations of above important
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TABLE 1. Changes in the expression of genes involved in the proteolytic system of the CM4 strain during fermentation in milk, with or without 2% peptides. Function Proteinase
Aminopeptidase
X-prolyl dipeptidyl peptidase Endopeptidase
Tripeptidase Dipeptidase
Prolidase Prolinase Pro iminopeptidase Peptide transporter (ABC-type)
Gene
Product
0.5 h
2h
MW (KDa)
prtY prtH2 prtM2 pepC2 pepCE pepN pepN2 aminopeptidase I pepX pepE pepE2 pepF pepO1 pepO2 pepO3 pepT pepT2 pepD2 pepD3 pepD4 pepV pepDA pepQ pepQ2 pepPN pepI ddpA1 ddpA2 dppB dppC dppD dppF
Proteinase Proteinase Proteinase maturation protein Aminopeptidase C2 Aminopeptidase CE Aminopeptidase N Aminopeptidase N2 Aminopeptidase I zinc metalloprotease X-prolyl-dipeptidyl aminopeptidase (XPDAP) Endopeptidase E Endopeptidase E2 Oligoendopeptidase F Neutral endopeptidase O1 Neutral endopeptidase O2 Neutral endopeptidase O3 Di- and tripeptidases Di- and tripeptidases Dipeptidase D2 Dipeptidase D3 Dipeptidase D4 Dipeptidase V Dipeptidase A Xaa-Pro aminopeptidase Xaa-Pro aminopeptidase Hydrolases Proline iminopeptidase Dipeptide transport system Dipeptide transport system Dipeptide/oligopeptide/nickel transport systems Dipeptide/oligopeptide/nickel transport systems Dipeptide/oligopeptide/nickel transport system Dipeptide/oligopeptide/nickel transport system
1.04 0.15 1.23 1.18 0.32 1.05 0.31 0.91 1.11 0.33 0.91 0.93 0.75 0.07 0.72 1.03 0.11 1.55 1.51 0.98 0.73 1.28 0.44 1.38 1.30 1.27 0.38 0.07 0.61 0.56 0.66 0.57
0.98 0.20 1.33 0.93 0.72 0.74 0.75 1.15 1.32 1.08 1.26 1.07 1.18 0.36 0.99 1.36 0.70 1.47 1.33 1.12 0.83 0.45 1.07 1.09 1.88 1.15 0.60 0.27 0.98 1.05 1.11 1.02
47.0 181.0 32.8 51.4 53.0 95.8 57.2 40.1 90.5 50.0 50.3 68.1 73.6 73.8 73.1 47.1 48.4 54.9 54.0 53.5 51.5 53.4 41.2 41.4 35.0 33.9 60.7 60.5 33.3 32.2 35.9 34.2
proteolytic genes. Taking into account the above results, the predicted pathway for the processing of both peptides was illustrated in Fig. 4. A cell wall-associated proteinase negative L. helveticus variant strain cannot grow in the milk medium, but shows good growth when amino acids or peptides are added to the milk medium (18). So, the activation of the cell wall-associated proteinase is essential for the rapid growth of the cells in the milk medium. In the previous study with L. helveticus CNRZ32, significant up-regulation of many gene products, such as the proteolytic system, amino acid metabolism, carbohydrate metabolism, etc. by cultivation in milk medium compared to that in peptide rich MRS medium (25) was reported. Then, the peptides released in the medium must be incorporated into the cells mainly by oligopeptide transporter and then
-casein
Maturation protein XPDAP
LPQN IPP LTQTPVV VPP FLQPEVMGVSK
Proteinase
Endopeptidase(↓)
Oligopeptide transporter (↓)
LPQN IPP LTQTPVV VPP FLQPEVMGVSK Aminopeptidase (↓)
QN IPP
V VPP
IPP
VPP
FIG. 4. Predicted processing pathway for the release of Val-Pro-Pro and Ile-Pro-Pro from b-casein by proteolytic enzymes in Lactobacillus helveticus CM4. Key enzyme types, suggested to be importance from the microarray analysis and previous studies, are illustrated. Down-regulated genes are indicated by down arrows. Major peptides detected by LC-MS/MS and predicted peptide processing are shown by dotted arrows.
hydrolyzed to amino acids intracellularly (30). In contrast, the amino acids supplied to the cells would activate some of the regulators that act on the proteolytic genes that negatively regulate the gene expressions as listed in Table 1. It was also observed that the addition of peptides into the fermented milk represses the processing of antihypertensive peptides and cell wall-associated proteinase activity to a greater degree than that of amino acids (data not shown). The down-regulation of the proteolytic gene expression in response to peptides observed in the present study is thought to be caused by incorporated intracellular amino acids, however the added peptides showed bigger impact on the repressive effect on the gene expression than that of amino acids. This may be because of the faster incorporation of the peptides than amino acids into the cell and intracellular quick hydrolysis of the incorporated peptides to amino acids. However, there has been no report on how the regulator protein acts on the transcription of proteolytic systems in response to amino acids in L. helveticus. So, identification of the key components involved in the regulatory system and elucidation of their role in the regulatory system would be needed for detailed understanding of this novel regulatory system. In Lactococcus lactis and Bacillus subtilis, a transcriptional regulatory system, codY system, involved in proteolytic enzymes which was controlled in its response to branched chain amino acids has been reported (19,20). However, there is no codY-like homolog and codYbox-like DNA sequence, which were reported in lactococci (19), at the upstream regions of the proteolytic genes in L. helveticus CM4. Repressive releases of the VPP and IPP in the CM4 fermented milk by specific amino acids including BCAA suggests the down-regulation of the proteolytic system by BCAA as reported in lactococci (19). Studies are now in progress to aid in the full understanding of the regulatory system and in particular the identification of the regulator protein whose existence has been suggested as a result of the CM4 studies. We are purifying the key components that may increase the affinity toward corresponding DNA regions of proteolytic genes observed down-regulations in response to BCAA (Table 1).
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Except for the proteolytic system, many amino acid transporter genes such as hisM, potE, glnQ, hisJ, rhaT and med were down-regulated at 0.5 h after adding of peptides in milk medium as listed in Table S1. In contrast, some of the transcriptional regulator genes, such as tra5, acrR and glpR were up-regulated (Table S2). It has been reported that in Lactococcus lactis the regulation of two aminotransferase genes, araT and bcaP, by CodY depend on intracellular amino acids pools (31). Branched-chain amino acid permease gene, bcaP (ctrA), is also regulated by CodY system in Lactococcus lactis (32). So, the repressive regulation observed in proteolytic system of L. helveticus CM4 in the present study may affect the amino acid transport systems as listed in Table S1. However, the influence of up-regulated transcriptional regulators on the negatively changed gene expressions of amino acid transporters, proteolytic system and other genes listed in Table S1 is not clear. Supplementary data to this article can be found online at doi:10. 1016/j.jbiosc.2012.03.015.
14. Seppo, L., Jauhiainen, T., Poussa, T., and Korpela, R.: A fermented milk high in bioactive peptides has a blood pressure-lowering effect in hypertensive subjects, Am. J. Clin. Nutr., 77, 326e330 (2003). 15. Seppo, L., Kerojoki, O., Suomalainen, T., and Korpela, R.: The effect of a Lactobacillus helveticus LBK-16 H fermented milk on hypertension e a pilot study on humans, Milchwissenschaft, 57, 124e127 (2001). 16. Pripp, H.: Effect of peptides derived from food proteins on blood pressure e a meta-analysis of randomised controlled trials, Food Nutr. Res., 52, 1e9 (2008). 17. Nakamura, T., Mizutani, J., Ohki, K., Yamada, K., Yamamoto, N., Takeshi, M., and Takazawa, K.: Casein hydrolysate containing Val-Pro-Pro and Ile-Pro-Pro improves central blood pressure and arterial stiffness in hypertensive subjects: a randomized, double-blind, placebo-controlled trial, Atherosclerosis, 219, 298e303 (2011). 18. Yamamoto, N., Akino, A., Takano, T., and Shishido, K.: Presence of active and inactive molecules of a cell wall-associated proteinase in Lactobacillus helveticus CP790, Appl. Environ. Microbiol., 61, 698e701 (1995). 19. den Hengst, C. D., van Hijum, S. A., Geurts., J. M., Nauta, A., Kok, J., and Oscar, O. P.: The Lactococcus lactis CodY regulon: identification of a conserved cis-regulatory element, J. Biol. Chem., 280, 34332e34342 (2005). 20. Belitsky, B. R. and Sonenshein, A. L.: Genetic and biochemical analysis of CodY-binding sites in Bacillus subtilis, J. Bacteriol., 190, 1224e1236 (2008). 21. Wouter, T., Hendriksen, Bootsma, H. J., Estevaeo, S., Hoogenboezem, T., de Jong, A., de Groot, R., Kuipers, O. P., and Hermans, P. W. M.: CodY of Streptococcus pneumoniae: link between nutritional gene regulation and colonization, J. Bacteriol., 190, 590e601 (2008). 22. Matuura, K., Mizuno, S., Nishimura, S., Gotou, T., and Yamamoto, N.: Quantitative analysis of antihypertensive peptides, Val-Pro-Pro and Ile-ProPro, in casein hydrolyzate with Aspergillus oryzae protease: an LCeMS method, Milchwissenschaft, 60, 24e27 (2005). 23. Yamamoto, N., Akino, A., and Takano, T.: Purification and specificity of a cellwall-associated proteinase from Lactobacillus helveticus CP790, J. Biochem., 114, 740e745 (1993). 24. Yamamoto, N., Shinoda, T., and Takano, T.: Molecular cloning and sequence analysis of a gene encoding an extracellular proteinase from Lactobacillus helveticus CP790, Biosci. Biotechnol. Biochem., 64, 1217e1222 (1999). 25. Smeianov, V. V., Wechter, P., Broadbent, J. R., Hughes, J. E., Rodíguez, B. T., Christensen, T. K., Ardö, Y., and Steele, J. L.: Comparative high-density microarray analysis of gene expression during growth of Lactobacillus helveticus in milk versus rich culture medium, Appl. Environ. Microbiol., 73, 2662e2672 (2007). 26. Chen, Y. S. and Steele, J. L.: Genetic characterization and physiological role of endopeptidase O from Lactobacillus helveticus CNRZ32, Appl. Environ. Microbiol., 64, 3411e3415 (1998). 27. Chen, Y. S., Christensen, J. E., Broadbent, J. R., and Steele, J. L.: Identification and characterization of Lactobacillus helveticus PepO2, an endopeptidase with post-proline specificity, Appl. Environ. Microbiol., 69, 1276e1282 (2003). 28. Vesanto, E., Savijoki, K., Rantanen, T., Steele, J. L., and Palva, A.: An X-prolyl dipeptidyl aminopeptidase (pepX) gene from Lactobacillus helveticus, Microbiology, 141, 3067e3075 (1995). 29. Varmanen, P., Vesanto, E., Steele, J. L., and Palva, A.: Characterization and expression of the pepN gene encoding a general aminopeptidase from Lactobacillus helveticus, FEMS Microbiol. Lett., 124, 315e320 (1994). 30. Detmers, F. J., Kunji, E. R., Lanfermeijer, F. C., Poolman, B., and Konings, W. N.: Kinetics and specificity of peptide uptake by the oligopeptide transport system of Lactococcus lactis, Biochemistry, 37, 16671e16679 (1998). 31. Chambellon, N. and Yvon, M.: CodY-regulated aminotransferases AraT and BcaT play a major role in the growth of Lactococcus lactis in milk by regulating the intracellular pool of amino acids, Appl. Environ. Microbiol., 69, 3061e3068 (2003). 32. den Hengst, C. D., Groeneveld, M., Kuipers, O. P., and Kok, J.: Identification and functional characterization of the Lactococcus lactis CodY-regulated branched-chain amino acid permease BcaP (CtrA), J. Bacteriol., 188, 3280e3289 (2006).
References 1. Kunji, E. R., Mierau, I., Hagting, A., Poolman, B., and Konings, W. N.: The proteolytic systems of lactic acid bacteria, Antonie Van Leeuwenhoek, 70, 187e221 (1996). 2. Kok, J.: Genetics of the proteolytic system of lactic acid bacteria, FEMS Microbiol. Rev., 7, 15e42 (1990). 3. Yamamoto, N., Akino, A., and Takano, T.: Antihypertensive effect of different kinds of fermented milk in spontaneously hypertensive rats, Biosci. Biotechnol. Biochem., 58, 776e778 (1994). 4. Christensen, J. E., Dudley, E. G., Pederson, J. A., and Steele, J. L.: Peptidases and amino acid catabolism in lactic acid bacteria, Antonie Van Leeuwenhoek, 76, 217e246 (1999). 5. Yamamoto, N., Masujima, Y., and Takano, T.: Reduction of membrane-bound ATPase activity in a Lactobacillus helveticus strain with slower growth at low pH, FEMS Microbiol. Lett., 138, 179e184 (1996). 6. Sasaki, M., Bosman, B. W., and Tan, P. S.: Comparison of proteolytic activities in various lactobacilli, J. Dairy Res., 62, 601e610 (1995). 7. Nakamura, Y., Yamamoto, N., Sakai, K., Okubo, A., Yamazaki, S., and Takano, T.: Purification and characterization of angiotensin I-converting enzyme inhibitors from sour milk, J. Dairy Sci., 78, 777e783 (1995). 8. Nakamura, Y., Yamamoto, N., Sakai, K., and Takano, T.: Antihypertensive effect of sour milk and peptides isolated from it that are inhibitors to angiotensin I-converting enzyme, J. Dairy Sci., 78, 1253e1257 (1995). 9. Aihara, K., Kajimoto, O., Hirata, H., Takahashi, R., and Nakamura, Y.: Effect of powdered fermented milk with Lactobacillus helveticus on subjects with highnormal blood pressure or mild hypertension, J. Am. Coll. Nutr., 24, 257e265 (2005). 10. Hata, Y., Yamamoto, M., Ohni, M., Nakajima, K., Nakamura, Y., and Takano, T.: A placebo-controlled study of the effect of sour milk on blood pressure in hypertensive subjects, Am. J. Clin. Nutr., 64, 767e771 (1996). 11. Jauhiainen, T., Rönnback, M., Vapaatalo, H., Wuolle, K., Kautiainen, H., and Korpela, R.: Lactobacillus helveticus fermented milk reduces arterial stiffness in hypertensive subjects, Int. Dairy J., 17, 1209e1211 (2007). 12. Mizuno, S., Matsuura, K., Gotou, T., Nishimura, S., Kajimoto, O., Yabune, M., Kajimoto, Y., and Yamamoto, N.: Antihypertensive effect of casein hydrolysate in a placebo-controlled study in subjects with high-normal blood pressure and mild hypertension, Br. J. Nutr., 94, 84e91 (2005). 13. Sano, J., Ohki, K., Higuchi, T., Aihara, K., Mizuno, S., Kajimoto, O., Nakagawa, S., Kajimoto, Y., and Nakamura, Y.: Effect of casein hydrolysate, prepared with protease derived from Aspergillus oryzae, on subjects with highnormal blood pressure or mild hypertension, J. Med. Food, 8, 423e430 (2005).
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Repressive processing of antihypertensive peptides, Val-Pro-Pro and Ile-Pro-Pro, in Lactobacillus helveticus fermented milk by added peptides Taketo Wakai,1 Naoya Yamaguchi,2 Misaki Hatanaka,1 Yasunori Nakamura,1 and Naoyuki Yamamoto1, * Microbiology & Fermentation Laboratory, Calpis, 11-10 5-Chome, Fuchinobe, Chuo-ku, Sagamihara-shi, Kanagawa 252-0206, Japan1 and Hokkaido Research Organization, Tokachi Agricultural Experiment Station, 9-2 Shinsei minami, Memuro, Kasai-gun, Hokkaido 082-0081, Japan2 Received 30 January 2012; accepted 23 March 2012 Available online 15 May 2012
Lactobacillus helveticus can release the antihypertensive peptides, Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP), from casein in fermented milk by a specific proteolytic system. To better understand the regulation of gene expression of the proteolytic enzymes thought to link to the processing of both antihypertensive peptides in L. helveticus, microarray analysis for whole gene expression in the presence and absence of added peptides in the fermented milk was studied. The productivity of both VPP and IPP in L. helveticus CM4 fermented milk was repressed by adding 2% quantity of Peptone as peptide mixture to the milk. Among the selected 13 amino acids, Gly, Ile, Leu, Phe, Met, Ser and Val were effective in the repression of the productivity of VPP and IPP in the fermented milk. The activity of the cell wallassociated proteinase, which may play a key role in the processing of the two antihypertensive peptides, was significantly repressed by the addition of the 2% quantity of Peptone into the fermented milk. By DNA microarray analysis it was found that prtH2 corresponding to the cell wall-associated proteinase gene, most of the endopeptidase genes such as pepE, pepO1, pepO2 and pepO3, most of the oligopeptide transporter genes, such as dppA2, dppB, dppC, dppD and dppF, most likely involved in the processing of VPP and IPP were down-regulated. These results suggest that amino acids released from milk peptides in the fermented milk might down-regulate the gene expressions of some of the proteolytic enzymes and may cause repression of the release of VPP and IPP in L. helveticus fermented milk. Ó 2012, The Society for Biotechnology, Japan. All rights reserved. [Key words: Val-Pro-Pro and Ile-Pro-Pro; Proteolytic system; Transcriptional regulation; Amino acids; Microarray analysis]
Lactic acid bacteria possess a proteolytic system which involves the hydrolysis of milk proteins and allows the bacteria to use peptides and amino acids as nutrients (1,2). Among lactic acid bacteria, Lactobacillus helveticus can grow rapidly in milk because of its high proteolytic activity and resistance to acid stress (3e5). Therefore, L. helveticus can release a large amount of peptides, including bioactive peptides in fermented milk by means of proteolysis of milk proteins (3,6). Among bioactive peptides, the antihypertensive peptides, Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP), that have inhibitory activities against angiotensin I-converting enzyme (kininase II; EC 3.4.15.1) (ACE), which controls blood pressure in vivo, have been extensively studied (7,8). Furthermore, consecutive uptakes of these peptides for more than a week resulted in significant reduction of blood pressure in many clinical trials (9e15). Meta-analysis for these clinical trials showed a significant reduction of blood pressure by treatment with VPP and IPP (16). Moreover, a current double-blind placebo controlled study revealed an improvement of arterial stiffness by oral administration of VPP and IPP for more than 8 weeks (17). For the release of a large amount of VPP and IPP, the L. helveticus CM4 strain with high cell wall-associated proteinase activity was isolated and used in the preparation of the fermented milk product
(EU Patent, 1016709A1, 1991). However, the proteinase activation process was repressed by certain amino acids and peptides released in the fermentation process (18). The regulation of the proteolytic system in response to amino acids, the so called codY system, has been well characterized in lactococci, bacilli and streptococci (19e21). CodY protein senses the intracellular pool of branched-chain amino acids and increases affinity to the codY-box sequence when branched chain amino acids occur. The gene transcription of most proteolytic systems, including cell wall-associated proteinase, some peptidases and transporters that have the codY-box sequences adjacent to the promoter regions of the structural genes, are controlled by CodY protein (19e21). In contrast, there is little information about the regulation of the proteolytic system in L. helveticus, which is most likely involved in the processing of VPP and IPP by peptides and amino acids. So, the objective of the present study is to analyze the regulation of the proteolytic gene expression in L. helveticus during milk fermentation in the presence of peptides by using a microarray for the whole genome of the CM4 strain. Consequently, the key enzymes involved in the processing of VPP and IPP are discussed throughout the transcriptome analysis. MATERIALS AND METHODS
* Corresponding author. Tel.: þ81 42 769 7831; fax: þ81 42 769 7810. E-mail address: [email protected] (N. Yamamoto).
Materials Val-Pro-Pro and Ile-Pro-Pro were purchased from Kurabou (Osaka, Japan). Isotopes for (13C5)Val-(13C5)Pro-Pro (m/z 324.2) and Ile-(13C5)Pro-Pro (m/z
1389-1723/$ e see front matter Ó 2012, The Society for Biotechnology, Japan. All rights reserved. doi:10.1016/j.jbiosc.2012.03.015
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20 10 0 0
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peptides (-)
B
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Time (h) FIG. 1. Changes in amounts of Val-Pro-Pro and Ile-Pro-Pro in Lactobacillus helveticus CM4 fermented milk with or without 2% (W/V) of peptides (Peptone; BD). Then, at 0, 2, 3, 4, 6, 8, 14 and 22 h of fermentation after the addition of the peptides, the amounts of VPP (open and closed squares) and IPP (opened and closed squares and circles) in CM4 fermented milk with (closed squares and circles) or without (open squares and circles) peptides were analyzed by LC-MSMS method.
and were repressed to 9% for VPP and 33% for IPP if peptides were added in the milk medium, respectively (Fig. 1A and B). The cell growth with peptides monitored by the optical density was slightly faster than that without peptides. These results strongly suggest a repressive effect of peptides on the proteolytic system involving the processing of both antihypertensive peptides. Repressive effects of amino acids on the release of VPP and IPP To understand the detailed repressive effect of each studied amino acid on the release of VPP and IPP in L. helveticus fermented milk, the amount of VPP and IPP in the L. helveticus fermented milk was measured with or without 13 different amino acids. Most of the tested amino acids showed repression of the production of VPP and IPP. Gly, Ile and Leu showed a higher repressive effect and Phe, Met, Ser and Val were slightly effective as repressers, (Fig. 2). It has been
VPP IPP
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Ile
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Repressive release of the antihypertensive peptides by added peptides In our previous study, we have found a large amount of the antihypertensive peptides, Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP), in L. helveticus fermented milk (3), but it was suggested that other released peptides in the fermented milk had a repressive effect on cell-wall associated proteinase activity and on the production of both antihypertensive peptides (18). So, the influence of the peptides (and amino acids) on the release of VPP and IPP during the L. helveticus CM4 fermentation was analyzed. As shown in Fig. 1A and B, the release of VPP and IPP in the CM4 fermented milk increased during the fermentation. However, release of VPP and IPP in the fermented milk was significantly repressed by the addition of 2% of peptides (Peptone; BD, NJ, USA) in the fermentation as shown in Fig. 1. Maximum values of VPP and IPP in the fermented milk were observed at 13 h after the fermentation,
+peptides
30
Ala
RESULTS
peptides (+)
Gln
Microarray analysis For the transcriptome analysis of L. helveticus CM4 fermented in milk medium with or without peptides (Peptone; BD, NJ, USA), cells were harvested and added into same volume of RNA Protect Bacteria Reagent (Qiagen K. K., Tokyo, Japan). The cells were collected by a centrifugation at 20,000 rpm for 10 min. The cell pellets were stored at 30 C. Total RNA was purified and was used for analysis of its quality. Untreated cells were used as controls. Total RNA was extracted using an RNeasy Mini Kit (Qiagen, Valencia, CA, USA) after lysis by lysozyme and phenol treatments. The following procedures for microarray analysis were performed according to the protocol of Roche NimbleGen, Inc. (Madison, WI, USA). The whole genome sequence of CM4 strain was completed (unpublished data), therefore we prepared a microarray for L. helveticus CM4 genes and used in this study. cDNA was synthesized from total RNA for use in the hybridization. The hybridized arrays were scanned and normalized using NimbleScan software. Results for particular genes are presented as an n-fold change of expression. We selected genes if they were up-regulated more than 1.88-fold and down-regulated less than 0.32-fold in a comparison of cells cultured with or without peptides.
peptides (-)
40
Control
Cell wall-associated proteinase activity For the measurement of cell wallassociated proteinase activity during the fermentation in milk medium, CM4 cells were harvested at 1, 3, 3.5, 4, 5, 6 and 7 h after fermentation at 37 C maintaining medium pH at 6.0. Then, sodium citrate was added up to 2% (W/V) to clarify the fermented milk. After a centrifugation of the clarified fermented milk at 20,000 g for 10 min, cells were washed with 50 mM b-glycerophosphoric acid at room temperature. The washed cells were suspended in a phosphate saline buffer, pH 7.4, and adjusted to a cell density at 590 nm as 1.0 to measure the cell wall-associated proteinase activity per cell density (OD590nm). The cell wall-associated proteinase activity was measured according to the method described previously (23) using a fluorescein isothiocyanate casein (FITC-casein) as a substrate. The reaction mixture, containing 100 ml of 0.2% FITC-casein (Sigma Aldrich Japan, Tokyo) and 100 ml of the enzyme solution, was incubated at 42 C for 60 min. Soluble peptides were collected in the supernatant after adding 200 ml of 5% TCA, and precipitating unhydrolyzed protein at 15,000 g for 10 min. Fifty ml of the supernatant was neutralized by the addition of 150 ml of 500 mM TriseHCl buffer, pH 8.5. Then, fluorescence was measured by using the Microplate Reader (POWERSCAN HT; Dainippon Pharmaceutical) with an excitation wavelength of 495 nm and an emission wavelength of 525 nm. One unit of proteolytic activity was defined as the amount of enzyme yielding 1% of total initial casein fluorescence as TCA soluble fluorescence after 60 min of hydrolysis.
VPP conc. (µg/mL)
Measurement of VPP and IPP in the fermented milk CM4 strain (EU Patent, 1016709A1, 1991) was pre-cultured in 9% (W/W) low-heated skim milk at 37 C for 24 h. The pre-cultured fermented milk was added up to 5% to a fresh milk medium (9% low-heated skim milk). It was fermented at 37 C for 25 h, with a pH maintained at 6.0 by adding of 50% NaOH, and then Peptone was added to the fermented milk up to 2% at 3 h after the fermentation. VPP and IPP in the fermented milk were measured at 3, 4, 5, 6, 7, 8, 9, 11, 13, 16, 19 and 25 h after the fermentation according to the previous method with some modification (22). Measurements were carried out by using a high performance liquid chromatography-atmospheric pressure ionization multiple reaction monitoring mass spectrometer (HPLC-API-MRM-MS) in positive ionization mode. The internal standards used were isotope (13C5)Val-(13C5)Pro-Pro for VPP and isotope Ile-(13C5)Pro-Pro for IPP, respectively. To understand the influence of various amino acids on the production of VPP and IPP in the fermented milk, a mixture with or without 2% (W/V) of Gln, Ala, Thr, Pro, Tyr, Asn, Val, Ser, Met, Phe, Leu, Ile, Gly was added and fermented at 37 C for 24 h. Then, the production of VPP and IPP in the fermented milk with amino acid was compared with that prepared without amino acid.
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332.2) were obtained from SCRUM Inc. (Tokyo, Japan). RNA Protect Bacteria Reagent was purchased from Qiagen K. K. (Tokyo, Japan). Peptone was purchased from BD (NJ, USA). Peptone prepared by enzymatic hydrolysis of animal proteins was used as peptide mixture without Val-Pro-Pro and Ile-Pro-Pro sequence.
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FIG. 2. Repressive effect of various amino acids on the productivity of VPP and IPP in Lactobacillus helveticus CM4 fermented milk. Value represents the ratio comparing with and without the addition of each amino acid.
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reported that branched chain amino acids (BCAA) in a different strain, Lactococcus lactis, has a repressive effect on the proteolytic system regulated by the codY system. CodY is a well studied and important transcriptional regulator system. Taking this into account, it was suggested that there would be a repressive effect of the amino acids including BCAA on the proteolytic system and these would affect the release of VPP and IPP in L. helveticus CM4.
of the excision of N-terminal amino acid of X-Pro sequence. The expression of pepX gene was not changed greatly by the addition of peptides at 0.5 and 2 h (Table 1). For the aminopeptidase, only the pepCE gene and pepN2 gene were effectively down-regulated at 0.5 and 2 h after the addition of peptides. Among the dipeptidase and tripeptidase genes, pepT2 and pepV genes thought to be not involved in the processing of VPP and IPP (Table 1) were also down-regulated. Moreover, all transporter genes, such as dppA1, dppA2, dppB, dppC, dppD and dppF were down-regulated. However, down-regulations of gene expressions for tripeptidase gene (pepT), prolinase gene (pepPN) and proline iminopeptidase (pepI) were not large. From the transcriptome analysis for CM4 genes with or without peptides in the fermented milk, key proteolytic enzyme genes most likely needed for the processing of VPP and IPP, proteinase, endopeptidases, and peptide transporter, were mainly down-regulated (Table 1).
Repressive effect of added peptides on the cell wallassociated proteinase For the understanding of the key enzymes in the proteolytic system, we measured the cell wallassociated proteinase activity on the CM4 cells. This was done on cultured and collected cells from fermented milk after clarification by the addition of 2% of sodium citrate at 1, 3, 3.5, 4, 5, 6 and 7 h during fermentation (Fig. 3). As shown in Fig. 3, the proteinase activity was greatly increased from 1 to 3.5 h during the fermentation without the addition of peptides, however when 2% of the peptides were added at 3 h during fermentation, the proteinase activity was clearly decreased after 3 h (Fig. 3). The release of the VPP and IPP in the CM4 fermented milk (Fig. 1) was correlated with the proteinase activity on CM4 cells (Fig. 3). These results suggest the importance of the cell wall-associated proteinase for the processing of VPP and IPP in the fermented milk, and also repression of the proteinase by peptides in the fermentation.
Proteinase Activity (U/OD)
Gene expression of proteolytic enzymes For a fuller understanding of the repressive effects of peptides on the proteolytic system especially involved in the processing of the antihypertensive peptides, VPP and IPP, we completed a transcriptome analysis for the whole gene sequence corresponding to L. helveticus CM4 strain, and applied this to the CM4 cells collected at 0.5 and 2 h after the addition of peptides into the fermented milk. As shown in Table 1, most of the genes of the proteolytic system were repressed by the addition of the peptides in the fermentation in milk. Among these proteolytic genes, prtY gene corresponding to the cell wallassociated proteinase gene (24) was not repressed. However, it is not clear whether a maturation protein gene is needed for activation of the precursor protein (PrtY). On the other hand, signals for prtH2, reported as a cell wall-associated proteinase gene in CNRZ32 (26), were greatly repressed by the addition of peptides. For the endopeptidases which are considered to be essential for the release of VPP and IPP from the proteinasereleased long peptides, most of the genes were down-regulated by the addition of peptides especially at 0.5 h (Table 1). In more detail, expressions of genes of pepE, pepO1, pepO2 and pepO3 were repressed to 0.33-, 0.75-, 0.07- and 0.77-fold at 0.5 h respectively, and pepO2 gene was still greatly repressed at 2 h by the addition of peptides (Table 1). The study of X-prolyldipeptidyl aminopeptidase (XPDAP) also suggests the importance 400
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200
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FIG. 3. Change in the cell wall-associated proteinase activity on Lactobacillus helveticus CM4 cells cultured in skim milk medium with or without the addition of 2% of peptides at 3 h during fermentation as indicated by down arrows. Cells harvested at 0.5 and 2 h after the addition of the peptides (shown by up arrows) were used for microarray analysis.
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Changes of other gene expressions For the understanding of the changes in other gene expressions, except proteolytic system by peptides, repressed genes in CM4 fermented milk by the addition of peptides were summarized in Table S1. Many amino acid transporter genes such as hisM, potE, glnQ, hisJ, rhaT and med were down-regulated by peptides in the milk medium (Table S1). In contrast, some of transcriptional regulator genes, such as tra5, acrR and glpR and part of transporter genes, such as salY, mgtA and lacY were up-regulated at 0.5 h after the addition of peptides in milk medium (Table S2). These down-regulations of the genes were larger at 0.5 h than those at 2 h after cultivation with peptides (Table S1). Moreover, the negative regulations in many amino acid transporter genes were larger in those of proteolytic enzyme genes as shown in Table 1. DISCUSSION The proteolytic system is the most important system in L. helveticus because it allows for its rapid growth in milk and allows for effective utilization of amino acids and peptides originated from milk protein among lactic acid bacteria (3). So, the down-regulation of the proteolytic system would be a significant event in L. helveticus for its metabolism in its work of uptaking and utilizing peptides and amino acids. However, this study is one of very few that has addressed the transcriptome analysis of the proteolytic system in response to peptides in L. helveticus. The transcriptome analysis conducted in the present study, with or without peptides, in the fermented milk supported our consideration of the processing pathway of the unique bioactive peptides, VPP and IPP, in L. helveticus CM4. The cell wall-associated proteinase is the most important enzyme to decompose milk casein and supply long peptides containing the VPP and IPP sequence (23). In the present study, the prtY gene, which was previously shown to play an important role in the release of VPP and IPP, was not down-regulated, so post-transcriptional regulation may be involved in the processing. In contrast it is not clear what role is played in the processing of both antihypertensive peptides by the down-regulation of the prtH2 gene reported as a cell wall-associated proteinase in CNRZ32 (25). Among all the down-regulated endopeptidase genes, pepO1 (26) and pepO2 (27) were considered to be most important enzymes for the C-terminal processing of both peptides. For the N-terminal processing to release VPP and IPP, pepX gene (28) essential for the processing of X-Pro sequence was not affected, but pepCE and pepN2 (29) genes were effectively downregulated by peptides. Most of the oligopeptide transporters, dppB, dppC, dppD and dppF, which most likely play important roles in the processing of VPP and IPP, were also considered to have an effect on the production of VPP and IPP. These results suggest that repressive effect of peptides on the release of VPP and IPP in the CM4 fermented milk might be caused by down-regulations of above important
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TABLE 1. Changes in the expression of genes involved in the proteolytic system of the CM4 strain during fermentation in milk, with or without 2% peptides. Function Proteinase
Aminopeptidase
X-prolyl dipeptidyl peptidase Endopeptidase
Tripeptidase Dipeptidase
Prolidase Prolinase Pro iminopeptidase Peptide transporter (ABC-type)
Gene
Product
0.5 h
2h
MW (KDa)
prtY prtH2 prtM2 pepC2 pepCE pepN pepN2 aminopeptidase I pepX pepE pepE2 pepF pepO1 pepO2 pepO3 pepT pepT2 pepD2 pepD3 pepD4 pepV pepDA pepQ pepQ2 pepPN pepI ddpA1 ddpA2 dppB dppC dppD dppF
Proteinase Proteinase Proteinase maturation protein Aminopeptidase C2 Aminopeptidase CE Aminopeptidase N Aminopeptidase N2 Aminopeptidase I zinc metalloprotease X-prolyl-dipeptidyl aminopeptidase (XPDAP) Endopeptidase E Endopeptidase E2 Oligoendopeptidase F Neutral endopeptidase O1 Neutral endopeptidase O2 Neutral endopeptidase O3 Di- and tripeptidases Di- and tripeptidases Dipeptidase D2 Dipeptidase D3 Dipeptidase D4 Dipeptidase V Dipeptidase A Xaa-Pro aminopeptidase Xaa-Pro aminopeptidase Hydrolases Proline iminopeptidase Dipeptide transport system Dipeptide transport system Dipeptide/oligopeptide/nickel transport systems Dipeptide/oligopeptide/nickel transport systems Dipeptide/oligopeptide/nickel transport system Dipeptide/oligopeptide/nickel transport system
1.04 0.15 1.23 1.18 0.32 1.05 0.31 0.91 1.11 0.33 0.91 0.93 0.75 0.07 0.72 1.03 0.11 1.55 1.51 0.98 0.73 1.28 0.44 1.38 1.30 1.27 0.38 0.07 0.61 0.56 0.66 0.57
0.98 0.20 1.33 0.93 0.72 0.74 0.75 1.15 1.32 1.08 1.26 1.07 1.18 0.36 0.99 1.36 0.70 1.47 1.33 1.12 0.83 0.45 1.07 1.09 1.88 1.15 0.60 0.27 0.98 1.05 1.11 1.02
47.0 181.0 32.8 51.4 53.0 95.8 57.2 40.1 90.5 50.0 50.3 68.1 73.6 73.8 73.1 47.1 48.4 54.9 54.0 53.5 51.5 53.4 41.2 41.4 35.0 33.9 60.7 60.5 33.3 32.2 35.9 34.2
proteolytic genes. Taking into account the above results, the predicted pathway for the processing of both peptides was illustrated in Fig. 4. A cell wall-associated proteinase negative L. helveticus variant strain cannot grow in the milk medium, but shows good growth when amino acids or peptides are added to the milk medium (18). So, the activation of the cell wall-associated proteinase is essential for the rapid growth of the cells in the milk medium. In the previous study with L. helveticus CNRZ32, significant up-regulation of many gene products, such as the proteolytic system, amino acid metabolism, carbohydrate metabolism, etc. by cultivation in milk medium compared to that in peptide rich MRS medium (25) was reported. Then, the peptides released in the medium must be incorporated into the cells mainly by oligopeptide transporter and then
-casein
Maturation protein XPDAP
LPQN IPP LTQTPVV VPP FLQPEVMGVSK
Proteinase
Endopeptidase(↓)
Oligopeptide transporter (↓)
LPQN IPP LTQTPVV VPP FLQPEVMGVSK Aminopeptidase (↓)
QN IPP
V VPP
IPP
VPP
FIG. 4. Predicted processing pathway for the release of Val-Pro-Pro and Ile-Pro-Pro from b-casein by proteolytic enzymes in Lactobacillus helveticus CM4. Key enzyme types, suggested to be importance from the microarray analysis and previous studies, are illustrated. Down-regulated genes are indicated by down arrows. Major peptides detected by LC-MS/MS and predicted peptide processing are shown by dotted arrows.
hydrolyzed to amino acids intracellularly (30). In contrast, the amino acids supplied to the cells would activate some of the regulators that act on the proteolytic genes that negatively regulate the gene expressions as listed in Table 1. It was also observed that the addition of peptides into the fermented milk represses the processing of antihypertensive peptides and cell wall-associated proteinase activity to a greater degree than that of amino acids (data not shown). The down-regulation of the proteolytic gene expression in response to peptides observed in the present study is thought to be caused by incorporated intracellular amino acids, however the added peptides showed bigger impact on the repressive effect on the gene expression than that of amino acids. This may be because of the faster incorporation of the peptides than amino acids into the cell and intracellular quick hydrolysis of the incorporated peptides to amino acids. However, there has been no report on how the regulator protein acts on the transcription of proteolytic systems in response to amino acids in L. helveticus. So, identification of the key components involved in the regulatory system and elucidation of their role in the regulatory system would be needed for detailed understanding of this novel regulatory system. In Lactococcus lactis and Bacillus subtilis, a transcriptional regulatory system, codY system, involved in proteolytic enzymes which was controlled in its response to branched chain amino acids has been reported (19,20). However, there is no codY-like homolog and codYbox-like DNA sequence, which were reported in lactococci (19), at the upstream regions of the proteolytic genes in L. helveticus CM4. Repressive releases of the VPP and IPP in the CM4 fermented milk by specific amino acids including BCAA suggests the down-regulation of the proteolytic system by BCAA as reported in lactococci (19). Studies are now in progress to aid in the full understanding of the regulatory system and in particular the identification of the regulator protein whose existence has been suggested as a result of the CM4 studies. We are purifying the key components that may increase the affinity toward corresponding DNA regions of proteolytic genes observed down-regulations in response to BCAA (Table 1).
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Except for the proteolytic system, many amino acid transporter genes such as hisM, potE, glnQ, hisJ, rhaT and med were down-regulated at 0.5 h after adding of peptides in milk medium as listed in Table S1. In contrast, some of the transcriptional regulator genes, such as tra5, acrR and glpR were up-regulated (Table S2). It has been reported that in Lactococcus lactis the regulation of two aminotransferase genes, araT and bcaP, by CodY depend on intracellular amino acids pools (31). Branched-chain amino acid permease gene, bcaP (ctrA), is also regulated by CodY system in Lactococcus lactis (32). So, the repressive regulation observed in proteolytic system of L. helveticus CM4 in the present study may affect the amino acid transport systems as listed in Table S1. However, the influence of up-regulated transcriptional regulators on the negatively changed gene expressions of amino acid transporters, proteolytic system and other genes listed in Table S1 is not clear. Supplementary data to this article can be found online at doi:10. 1016/j.jbiosc.2012.03.015.
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