INTRODUCTION

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Due to their limited capacity for synthesizing amino acids (3), lactococci have to utilize exogenous nitrogen sources for optimal growth. The amino acid requirements appear to be strain specific, but most Lactococcus lactis strains need at least Leu, Ile, Val, and His for growth (15, 28). In milk, the concentrations of several essential amino acids, especially those of Ile and Leu (less than 1 mg liter¹), are very low (10, 18). In addition, milk peptides are a poor source of Leu and Met (16). Thus, for optimal growth in milk, lactococci depend on utilization of casein (18, 29). A complex proteolytic system is needed for casein hydrolysis. According to proposed models, lactocepin (EC 3.4.21.96; previously named cell envelope proteinase PrtP) (34) is involved in the first step of casein degradation. Only some of the oligopeptides released by lactocepin are taken up by the oligopeptide transport system (Opp) and subsequently cleaved into amino acids by intracellular peptidases (for recent reviews, see references 17 and 22).

Two different types of lactocepins (lactocepin I and lactocepin III) have been identified in lactococci on the basis of their specificity for caseins (44). Lactocepin I cleaves -casein preferentially and -casein to a lesser extent. In contrast, lactocepin III cleaves -, -, and _s1-caseins. There are only 44 differences in the amino acid sequences of lactocepins I and III, and 5 of them are responsible for the differences in specificity between the two lactocepins (7, 40).

Lactocepin purification requires release of the enzyme from the cell (30), which results from autoproteolysis of the protein in a Ca²⁺-free buffer (24, 25). Autoproteolysis takes place in the C-terminal part of the protein, presumably in the B domain of the protein, and results in a 145-kDa enzyme, compared to the 186-kDa cell-anchored lactocepin III (1). The action of purified lactocepins towards caseins has been studied extensively (32, 33, 37, 38). In particular, most, if not all, of the peptides released from -casein by purified lactocepin I have been identified by on-line coupling of liquid chromatography (LC) to mass spectrometry (MS) (19). Nevertheless, autoproteolysis of lactocepin may result in a conformational change in the enzyme. It is worth noting that the B domain of lactocepin III, which presumably contains the autoproteolysis site, has been reported to play an important role in the stability of the enzyme (1). Thus, the question of the specificity of the anchored form of lactocepins has to be addressed.

The use of a genetically engineered strain of L. lactis made it possible to analyze the pool of peptides released from -casein by the anchored form of lactocepin I (21). The composition of the pool of peptides was clearly identical to that obtained with the purified enzyme, indicating that autoproteolysis of lactocepin I did not affect the specificity of the enzyme towards -casein, as suggested elsewhere (8). In contrast, there has been no clear demonstration of such conservation (or alteration) of specificity after autoproteolysis of lactocepin III. A previous study suggested that the peptides released by the anchored form of lactocepin III might differ from those released by the purified form (8). Nevertheless, there has been no report on the composition of the peptide pool released from caseins by the native form of lactocepin III. The aim of the present study was, therefore, to analyze in great detail the peptides released from -casein by the anchored form of lactocepin III. A comparison of the data obtained with previously published data indicated that autoproteolysis of lactocepin III affects its specificity towards -casein.

	MATERIALS AND METHODS

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Bacterial strains, plasmids, and culture conditions. The bacterial strains and plasmids used in the present study are listed in Table 1. L. lactis GF100, an autolysin- and oligopeptide transport-deficient derivative of L. lactis MG1363 (21), was a generous gift from B. Poolman (University of Groningen, Groningen, The Netherlands). Plasmids pNZ521 and pNZ511, which encode a wild-type lactocepin III and a C-terminally truncated lactocepin III that is completely secreted into the growth medium, respectively (4), were generous gifts from W. de Vos (Netherlands Institute for Dairy Research, Ede, The Netherlands). Plasmids were introduced into recipient strains by transformation by the procedure of Dornan and Collins (5). The presence of plasmids in transformed strains was checked by agarose gel electrophoresis. Lactococcal strains were grown at 30°C in M17 broth (41) or in chemically defined medium (34) supplemented with 1% (wt/vol) glucose or 1% (wt/vol) lactose and chloramphenicol (5 µg ml¹) when required. The pH of the culture medium was controlled at 6.5. The strains were stored at 80°C in M17 broth containing 0.5% (wt/vol) glucose, 10% (vol/vol) glycerol, and the appropriate antibiotics (each at a concentration of 5 µg ml¹).

Lactocepin isolation. The procedure used to isolate lactocepin III from L. lactis SK11 relied on the autoproteolytic properties of the enzyme (24). Cells were washed twice in 50 mM Tris-HCl (pH 8) containing 30 mM CaCl₂. Lactocepin was released from the cell envelope by incubation for 30 min at 30°C in 50 mM Tris-HCl (pH 8). Cells were removed by centrifugation (10,000 × g for 10 min at 4°C), and CaCl₂ (final concentration, 2 mM) was added to the lactocepin-containing supernatant. Further purification of lactocepin was achieved by anion-exchange chromatography using a Mono Q HR 5/5 column (Pharmacia, Uppsala, Sweden) and a linear 0 to 0.35 M NaCl gradient for 50 min.

-Casein purification. The method used for purification of -casein was adapted from the method of Guillou et al. (13). Total casein was obtained by acidic precipitation of defatted milk produced by a cow selected because it produced a homozygote -casein (A₂ variant). Caseins (4 g) were resuspended in 60 ml of 5 M Tris-HCl buffer (pH 8) containing urea (4.5 M) and dithiothreitol (0.8 mM) and then loaded onto a Q-Sepharose Fast Flow column (Pharmacia) and eluted with a linear 0 to 0.35 M NaCl gradient at 20°C. Eluted fractions containing -casein were detected by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) analysis (see below). Pure -casein-containing fractions obtained from successive runs were pooled, dialyzed against MilliQ water (Millipore Corp., Bedford, Mass.) and freeze-dried (Sublivac RP12; Serail, Argenteuil, France).

PAGE and immunoblotting. SDS-PAGE was performed as previously described (27) by using acrylamide contents in the running gel of 8% (vol/vol) in the case of lactocepin-containing samples and 15% (vol/vol) in the case of casein-containing samples. After electrophoresis, casein-containing gels were stained with Coomassie brilliant blue, whereas lactocepin-containing gels were either silver stained (46) or used for Western blotting as previously described (26). Antibodies raised against lactocepin were obtained from IMEnz Bioengineering, Haren, The Netherlands.

-Casein hydrolysis. Exponentially growing cells of L. lactis GF1004 (optical density at 650 nm [OD₆₅₀], 0.7) were washed twice in 100 mM MES (morpholineethanesulfonic acid)-KOH (pH 6.5) containing 2 mM CaCl₂. The cell suspension (OD₆₅₀, 24; corresponding to 4.8 mg of protein ml¹) was added to a 5 mM Tris-HCl (pH 9) solution containing 0.4% (wt/vol) -casein and incubated at 30°C. Hydrolysis was stopped by removing the cells by centrifugation (10,000 × g for 10 min at 4°C) and then adding trifluoroacetic acid (TFA) (1%, vol/vol). Hydrolysis of casein by purified lactocepin (either from L. lactis SK11 or from L. lactis GF1005) was also performed in the presence of CaCl₂ (2 mM), and the reaction was stopped by adding 1% TFA.

Cell lysis. The possible cell lysis during casein degradation was estimated from the release into the external medium of the X-prolyl-dipeptidyl-aminopeptidase PepX by using Gly-Pro-p-nitroanilide as the substrate. An estimate of the percentage of lysis was obtained by comparing the PepX activity in the supernatant to that in a cell extract, as previously described (18, 21). Considering the cell density used in the hydrolysis experiments, the sensitivity threshold of this method corresponded to lysis of about 0.01% of the population.

HPLC analysis. The peptides soluble in 1% TFA were separated at 40°C by high-performance liquid chromatography (HPLC) on a reverse-phase C₁₈ column (Nucleosil; 250 by 4.6 mm; Shandon HPLC, Cheshire, United Kingdom). Solvents A and B were 0.11% (vol/vol) TFA and 0.1% (vol/vol) TFA-60% (vol/vol) acetonitrile in MilliQ water, respectively. A linear 0 to 60% solvent B gradient was applied for 40 min. The flow rate was 1 ml min¹. The eluted peptides were detected simultaneously by on-line absorbance at 214 nm and fluorescence after postcolumn derivatization of the eluted peptides with o-phthalaldehyde, as previously described (14). UV detection was monitored prior to peptide derivatization. For detection of fluorescence, the excitation and emission wavelengths were 340 and 425 nm, respectively. The free amino acid content of the peptide solution was determined as previously described (18).

LC-MS analysis. The peptides soluble in 1% TFA were identified by on-line coupling of HPLC to MS, essentially as previously described (19, 21). Peptides were separated at 40°C on a reverse-phase C₁₈ HPLC column (150 by 0.5 mm; Perkin-Elmer Corp., Norwalk, Conn.). Solvents A and B were 0.1% (vol/vol) formic acid-4 mM ammonium acetate and 0.1% (vol/vol) formic acid-4 mM ammonium acetate-90% (vol/vol) acetonitrile in MilliQ water, respectively. A linear 5 to 57% solvent B gradient was applied for 100 min. The flow rate was 5 µl min¹. By flow splitting the eluate, about 5% of the sample was introduced into a single quadrupole MS (API 100; Perkin-Elmer Corp.). The MS was used in the positive-ion mode, and full-scan spectra were recorded at mass-to-charge ratios (m/z) between 200 and 2,000. To generate multiply charged ions without fragmentation, a low nozzle voltage (40 V) was used; to generate fragment ions by collision-induced dissociation from peptides, a higher nozzle voltage (170 V) was used.

Transport experiment. The transport assays used were adapted from previously described assays (11, 23). Cells were grown to an OD₆₅₀ of approximately 0.8 in chemically defined medium containing 17 free amino acids as the nitrogen source (34). Prior to transport assays, cells were deenergized for 30 min at 30°C with 10 mM 2-deoxy-D-glucose. For each transport assay, cells (OD₆₅₀, 1; corresponding to 0.2 mg of protein ml¹) were preincubated for 5 min in the presence of 25 mM glucose. Peptide uptake was monitored by determining the increase in the intracellular concentration of free amino acids after dansyl chloride derivatization by using HPLC analysis. The dansylated amino acids were separated at 40°C on a reverse-phase C₁₈ column (Nucleosil; 150 by 4.6 mm; Shandon HPLC). Solvent A was 10 mM sodium citrate (pH 6.2). Solvent B was acetonitrile-25 mM sodium citrate (60:40) (pH 6.2). A linear 0 to 75% solvent B gradient was applied for 75 min. The flow rate was 1 ml min¹. For detection of fluorescence, the excitation and emission wavelengths were 340 and 530 nm, respectively.

	RESULTS

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Hydrolysis of -casein by cell-anchored and autoproteolyzed forms of lactocepin III. Purified -casein was digested for 3 h by lactocepin III purified from L. lactis SK11 or by the autolysin-defective strain L. lactis GF1004 carrying the lactocepin III genes of L. lactis SK11. On the one hand, lactocepin III was purified by a two-step procedure (release from the cell envelope by autodigestion, followed by ion-exchange chromatography). SDS-PAGE and immunoblotting experiments indicated that active fractions contained the purified proteinase and several autoproteolysis fragments (24). No other proteins could be detected in the lactocepin preparation. In particular, lactocepin-containing fractions were free of peptidase activity. This preparation of lactocepin is called autoproteolyzed lactocepin below. On the other hand, when L. lactis GF1004 was used, no lysis of the cells could be detected during the incubation period, as reported previously for a related strain (L. lactis GF200) (2, 21), and no release of lactocepin into the incubation mixture could be detected. Hydrolysis experiments were repeated at least three times and yielded similar results.

View larger version (26K): [in this window] [in a new window]   FIG. 1.   TFA (1%)-soluble peptides released from -casein by autoproteolyzed lactocepin III (bottom line) and cell-anchored lactocepin III (top line). Autoproteolyzed lactocepin III was obtained by incubating L. lactis SK11 cells in a Ca2+-free buffer and was further purified by ion-exchange chromatography.

Additional -casein hydrolysis experiments. Additional -casein degradation experiments were performed by using L. lactis GF100, the L. lactis GF1004 parental Prt strain (Table 1). As expected, no TFA-soluble peptides could be detected when -casein was incubated in the presence of only L. lactis GF100. Addition of autoproteolyzed lactocepin III to the incubation mixture resulted in liberation of peptides. HPLC analysis of this pool of peptides revealed a chromatographic trace similar to that obtained in the presence of -casein and the autoproteolyzed lactocepin (Fig. 2). In particular, the peaks eluting at 19.0, 23.3, 35.4, and 36.3 min, which were specific for the autoproteolyzed form of the enzyme, were detected, whereas the peaks specific for the cell-anchored form of lactocepin III (16.5, 19.7, 22.3, 33.8, and 38.8 min) were not detected. Similarly, the pool of peptides released from -casein by the autoproteolyzed lactocepin was not modified when it was incubated in the presence of L. lactis GF100, as indicated by HPLC analysis (data not shown).

View larger version (23K): [in this window] [in a new window]   FIG. 2.   TFA (1%)-soluble peptides released from -casein by autoproteolyzed lactocepin III in the presence of proteinase-negative strain L. lactis GF100. Autoproteolyzed lactocepin III was obtained by incubating L. lactis SK11 cells in a Ca2+-free buffer and was further purified by ion-exchange chromatography.

Identification of peptides released from -casein by the anchored form of lactocepin. The main peptides released from -casein by autoproteolyzed lactocepin III have already been identified (38). Consequently, only the peptides released by cell-anchored lactocepin III were identified by on-line coupling of HPLC to MS by using scan nozzle voltages ranging from 70 to 170 V. At a low nozzle voltage, multiply charged ions were generated, so that a mass could be assigned to each eluted compound (even in the case of coeluting materials). Increasing the nozzle voltage resulted in collision-induced dissociation of eluted peptides. Analysis of the dissociation fragments made it possible to identify the initial peptides (19, 21). About 100 different peptides were identified in the pool, indicating that a large number of -casein peptide bonds were cleaved (about 55%). The locations of the peptides from -casein suggested that they did not originate from a particular region of the substrate (Fig. 3). More than 50% of the peptides released from -casein contained eight or fewer amino acids. To ensure that these peptides were not excreted by the cells, L. lactis GF1004 was incubated for 3 h in the absence of -casein, and the external medium was subjected to LC-MS analysis. Although several peaks were detected by UV after HPLC separation, only a few masses were recorded, suggesting that most of the compounds could not be ionized. Nevertheless, none of the recorded masses could be assigned to -casein fragments. These results, therefore, indicated that the peptides effectively resulted from degradation of -casein by the cell-anchored lactocepin, whereas the cells incubated in the absence of -casein did not release significant amounts of peptides.

View larger version (33K): [in this window] [in a new window]   FIG. 3.   Localization of the peptides released by the action of cell-anchored lactocepin III on -casein (A2 variant). The dotted arrows indicate peptides identified as peptides that were also released by autoproteolyzed lactocepin III (data from reference 38).

Specificity of the cell-anchored lactocepin III towards -casein. Lactocepin has a significant binding area consisting of eight amino acids (31). Consequently, the environment of each cleavage site has been studied. The presence of a Phe or His residue at position P₄ (i.e., the fourth residue on the C-terminal side of the cleavage site), the presence of a Tyr residue at position P₁, or the presence of an Asn at position P'₂ (i.e., the second residue on the N-terminal side of the cleavage site) prevented hydrolysis of the peptide bond by the cell-anchored lactocepin. In contrast, the presence of an Asp or Gly residue at position P₆, the presence of a Thr residue at position P₃, the presence of a Met residue at position P₁, or the presence of a Phe, Ser, or Asn residue at position P'₁ resulted in cleavage of the peptide bond. According to these preferences, only two cleavages (Pro₆₇-Asn₆₈ and Gln₁₆₇-Ser₁₆₈) were expected (due to the positive action of Asn and Ser residues at position P'₁, respectively) and not observed, whereas only one peptide bond (Glu₅-Leu₆) was cleaved even though it was expected to remain intact (due to the negative action of Asn at position P'₂).

	DISCUSSION

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The use of an autolysin-defective derivative of L. lactis MG1363 carrying the proteinase genes of L. lactis SK11 made it possible to study the action of the cell-anchored lactocepin III towards -casein. More than 100 different oligopeptides released from -casein were identified, whereas no di- or tripeptides could be detected. Such a high number of peptides suggests broad specificity of the enzyme, which was confirmed by the analysis of the proximity of the cleaved bonds. Only particular rules could be identified (positive influence of specific residues at positions P₆, P₃, P₁, and P'₁ during cleavage), which made it possible to explain only 38% of the observed cleavage sites.

On-line coupling of LC to MS resulted in identification of a considerably larger number of peptides than previously reported (38). This is in fact not surprising, since previously identification was performed by N-terminal sequencing of peptides. Such an approach requires pure peptides, and that is the reason why only 20 peptides (the most abundant) were identified. Nevertheless, these peptides were expected to be some of the 105 peptides which we identified. Very surprisingly, we detected only 3 of these 20 peptides, namely, RELEEL, DKIHPF, and SLTLTDVE. Previous studies were performed by using autoproteolyzed lactocepin III, whereas in the present study we used cell-anchored lactocepin III. This comparison clearly indicates that the peptides released by cell-anchored lactocepin III are different from those released by the autoproteolyzed form of the enzyme. That is the reason why the HPLC traces differed when these two forms of lactocepin III were used.

The question is, why are the peptides different? One explanation could be that the 17 abundant peptides which were detected only when autoproteolyzed lactocepin III was used are transported by L. lactis GF1004. Since the Opp oligopeptide transport system has been inactivated in this strain, this suggests that these peptides are translocated by an as-yet-unknown peptide transport mechanism. Such possibility is very unlikely, since (i) transport of oligopeptides, including -casein-derived peptides, by L. lactis, has never been observed in the absence of a functional Opp system (9, 22, 23) and (ii) none of these peptides has been detected in the cells and the intracellular concentrations of amino acids did not match the amino acid composition of these peptides (i.e., a large excess of Glu, Gln, and Leu).

An alternative explanation could be the presence of another surface-associated protease, since at least one other protease is located on the cell surface of L. lactis (35). Such a protease would not act on casein, since the lactocepin-negative strain did not release any peptide from -casein. Thus, this protease should act synergistically with lactocepin, by further degrading the peptides released by the lactocepin. This hypothesis is not consistent with (i) the fact that the pool of peptides released by the autoproteolyzed lactocepin is not modified when it is subjected to the action of a lactocepin-negative strain and (ii) the fact that the secreted lactocepin produced an HPLC trace identical to that obtained with the cell-anchored enzyme. Altogether, these arguments rule out the hypothesis that another cell proteinase explains the differences observed between the cell-anchored lactocepin and the autoproteolyzed lactocepin.

The only remaining explanation for the differences is that autoproteolysis of lactocepin III affects its specificity towards -casein. As a matter of fact, autoproteolyzed lactocepin III has been reported to release Gln₁₉₄-Val₂₀₉ as one of the major peptide products (38). The Tyr₁₉₃-Gln₁₉₄ peptide bond was not cleaved by the cell-anchored lactocepin III. Seven other peptide bonds were found to be cleaved by the autoproteolyzed form of lactocepin III, but they were not hydrolyzed by the lactocepin anchored to the cell; these bonds were the Leu₁₆-phosphoSer₁₇, Leu₅₈-Val₅₉, Tyr₆₀-Pro₆₁, Ile₇₄-Pro₇₅, Trp₁₄₃-Met₁₄₄, His₁₄₈-Gln₁₄₉, and Ser₁₆₈-Lys₁₆₉ bonds (36). These observations clearly indicate that there is a difference in specificity between the two forms of the enzyme (i.e., cell-anchored lactocepin III and autoproteolyzed lactocepin III).

The model proposed for autoproteolysis of lactocepin involves a change in the conformation of the enzyme in the absence of calcium. This change results in exposure of an as-yet-unidentified autoproteolysis site, which is masked in the presence of calcium (6). According to Bruinenberg et al. (1), the autoproteolysis site is most probably located in the B domain of the protein, about 500 amino acids from the C-terminal anchor. Interestingly, the secreted form of lactocepin III, which displayed specificity towards -casein identical to that of the cell-anchored form, lacks the last 311 C-terminal residues (45). Therefore, the data strongly suggest that (i) the specificity of the cell-anchored form of lactocepin III does not depend on an interaction of the enzyme with the cell wall and (ii) the amino acids involved in the change in specificity are located in this deleted region. It is worth noting that the C-terminal region has been reported to play an important role in the stability of the enzyme, as it contains a calcium-binding site (1, 39). On the other hand, a comparison of the data of Juillard et al. (19) and Kunji et al. (21) clearly indicates that both cell-anchored and autoproteolyzed forms of lactocepin I release similar peptides from -casein. The last 311 C-terminal residues of lactocepin III, which are apparently involved in the change in specificity of the enzyme, exhibit some amino acid variations compared to the C-terminal part of lactocepin I, namely, the presence of a duplicate 60-residue region in lactocepin III and seven amino acid substitutions (4, 20). Lactocepin engineering should make it possible to identify which of these differences between lactocepins I and III are involved in the change in specificity of lactocepin III during autoproteolysis.