INTRODUCTION

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Production of a catalytically active cell envelope proteinase (CEP) in Lactococcus lactis subsp. cremoris AM1 and SK11 during growth in a chemically defined medium depends on the presence of calcium ions in the medium; calcium cannot be replaced by other bivalent cations present in the medium to obtain a stable, active CEP (6). A similar dependence on Ca²⁺ ions has been observed with strain Wg2 (19). The CEPs of strains SK11 and Wg2 have been characterized extensively both genetically and biochemically (22, 29). These enzymes represent two of the several types of lactococcal CEPs which have been distinguished on the basis of their specificity towards peptides, namely, a CEP with type III specificity (CEP_III) and a CEP with mixed type I-type III specificity (CEP_I/III) (14). The mature CEPs of strains SK11 and Wg2 are large molecules that are 1,775 or 1,715 amino acid residues long. If the repeat sequence in the C-terminal part is not included, the sequence of the SK11 CEP differs at 44 positions from the sequence of the Wg2 CEP (33). The CEP N-terminal domain consisting of 500 residues exhibits significant sequence similarities with subtilisins and related serine proteinases (the subtilases). One difference between the CEPs and the subtilases is the large C-terminal extension in each CEP whose extreme C terminus anchors the enzyme in the cell envelope. In the subtilases four Ca-binding sites have been recognized; the association of Ca²⁺ ions with these sites is known to influence the activity and thermal stability of the enzymes and to protect the enzymes from autoproteolytic degradation (16, 30, 35). It has been predicted that there are three corresponding Ca²⁺-binding sites in CEP (31). In view of the role of Ca²⁺ in the subtilases, the observed dependence of the production of an active CEP on Ca²⁺ ions during growth may be interpreted either in terms of an active conformation that can be adopted only if Ca²⁺ ions are involved in the folding process or in terms of a requirement for Ca²⁺ ions that are needed to stabilize the active conformation.

Ca²⁺ ions protect cell-bound CEP from detachment from the cells as well. In the absence of Ca²⁺ ions in the suspending buffer, CEP activity can be released (10, 26). It has been suggested that this release occurs following the removal of bound calcium and that, as a result of this removal, local molecular unfolding outside the actual proteinase domain, in the large C-terminal extension, exposes a sequence which is highly susceptible to autoproteolytic attack (23, 24). Consequently, prevention of release of CEP by relatively high concentrations of calcium in the buffer should be due to preservation of the resistant form of the bound enzyme. Complete release of the proteinase from the cells occurs only after repeated resuspension of the cells in fresh Ca-free buffer (10, 24). In fact, this suggests that each subsequent treatment in Ca-free buffer results in the establishment of an equilibrium between free Ca²⁺ and bound Ca²⁺ which causes the release process to slow down and eventually to halt. Only resuspension in fresh Ca-free buffer results in continuation of this process.

In order to better understand the role of Ca²⁺ ions in the protection and functioning of the cell-bound CEP, we used dilute suspensions of cells of strains SK11 and Wg2 which were thoroughly washed with ice-cold Ca-free buffer and investigated the effects of temperature and Ca²⁺ concentration on cell-bound CEP. Significant differences between the two strains with respect to activity, stability, and release of the cell-bound CEPs were observed. As these differences were apparently related to the few substitutions inside and/or outside the proteinase domains of the two CEP molecules, we investigated the possible involvement of different segments of the CEP molecule in the Ca²⁺-related features of the wild-type enzymes by studying recombinant strains which produced hybrid CEPs constructed from the wild types. These experiments established that the C-terminal extension plays an essential role.

(A preliminary account of some of the results has been published in the proceedings of the International Dairy Lactic Acid Bacteria Conference held in Palmerston North, New Zealand [9].)

	MATERIALS AND METHODS

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Organisms. L. lactis subsp. cremoris SK11 (producing CEP_III) and Wg2 (producing CEP_I/III) (14) were used in this study. Recombinant L. lactis MG 1363 strains containing plasmid-located hybrid proteinase genes constructed from the SK11 (ABCD) and Wg2 (abcd) wild-type proteinase genes (34) were obtained from the Netherlands Institute for Dairy Research and from the Department of Genetics, University of Groningen, Groningen, The Netherlands. The hybrid proteinase genes contained one or more interchanged DNA fragments: fragment A or a, encoding amino acid residues 1 to 173; fragment B or b, encoding amino acid residues 174 to 496; fragment C or c, encoding amino acid residues 497 to 1089; and fragment D or d, encoding amino acid residues 1090 to 1775 (1715).

Growth and treatment of cells. Strains were grown in reconstituted skim milk (100 ml) at 25°C until the early stationary phase. In the case of the recombinant MG 1363 strains the milk was supplemented with 1% (wt/vol) glucose and either chloramphenicol (10 µg · ml¹) or erythromycin (5 µg · ml¹) as an additional selection factor (34).

View larger version (15K): [in this window] [in a new window]   FIG. 1.   Influence of treatment of milk-grown cells of L. lactis subsp. cremoris SK11 ( and ) and Wg2 ( and ) with ice-cold Ca2+-free 50 mM imidazole buffer (pH 6.5) on the activity of the cell-bound CEP measured in the absence (solid symbols) or in the presence (open symbols) of 10 mM Ca2+. The activities at zero (no wash) are the activities of cells harvested from the milk culture and resuspended in the Ca-free buffer. Activities are expressed as percentages of the activity of unwashed cells of each strain measured in the presence of Ca2+.

Proteinase assay. To 8 ml of 50 mM imidazole buffer (pH 6.5) containing the appropriate final concentration of Ca²⁺ we added 1 ml of a 10 mM solution in buffer of the substrate succinyl-alanyl-glutamyl-prolyl-phenylalanyl-p-nitroanilide (S-Glu) (Bachem AG, Bubendorf, Switzerland) or methoxysuccinyl-arginyl-prolyl-tyrosyl-p-nitroanilide (MS-Arg) (Chromogenix AB, Mölndal, Sweden). The mixture was warmed to 25°C, and then 1 ml of a cell suspension was added. Hydrolysis was monitored by stopping the reaction with 0.3 ml of 80% acetic acid per ml of incubation mixture. The extinction at 410 nm was measured after filtration through a 0.22-mm-pore-size filter (Millex GV; Millipore, Bedford, Mass.). The progress curve was linear for at least 60 min; all activity assays were performed by using this initial linearity.

Specificity of the CEP. Enzymatic hydrolysis of a_s1-casein fragment f1-23 [_s1-CN(f1-23)] by the (hybrid) CEP in situ (whole cells) at pH 6.5 in 50 mM imidazole containing 0, 0.1, 1, or 10 mM Ca²⁺, Mn²⁺, or Cd²⁺ followed by identification of the products by reversed-phase high-performance liquid chromatography (HPLC) was performed as described previously (12, 13). This peptide has been shown to have excellent characteristics which enable workers to distinguish small changes in the specificity of CEP (14).

	RESULTS

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Loss of CEP activity due to treatment of cells with ice-cold calcium-free buffer. When milk-grown cells of L. lactis SK11 containing CEP_III were harvested and then resuspended (no wash) and washed twice with ice-cold Ca-free imidazole buffer (pH 6.5) (standard procedure), a nearly complete loss of cell-bound CEP activity at 25°C with the substrate S-Glu was observed, which was not accompanied by the appearance of activity in the supernatant.

Thermal stability and release of the Ca-free cell-bound CEP. The Ca-free cell-bound SK11 CEP appeared to be extremely unstable at pH 6.5 and 25°C; within 20 min 99% of its potential activity was lost (Fig. 2a). Even storage on ice resulted in a gradual loss of potential activity (there was a 20% reduction in activity after 4 h) (data not shown). This relatively low rate of inactivation cannot explain the reduction in potential activity after each wash with ice-cold buffer (Fig. 1), indicating that other factors are responsible as well. The instability of the Ca-free CEP may be mainly responsible for the failure to measure activity at 25°C. However, Ca-free CEP activity could be measured at 10°C showing linear initial progress; the rate was 50 to 60% of the potential activity rate at 10°C. Temperature-related changes in relative affinities which result in complete inhibition of the cleavage of S-Glu by competitive autoproteolysis at 25°C and almost no inhibition at 10°C could indeed explain these observations but do not seem very likely. At this stage, therefore, we believe that removal of weakly bound Ca²⁺ destabilizes the enzyme against thermal denaturation rather than against autoproteolytic inactivation and that destabilization is coupled with a decline in specific activity.

View larger version (12K): [in this window] [in a new window]   FIG. 2.   (a) Stability of the cell-bound and released Ca-free SK11 CEP at pH 6.5 and 25°C. Low-density cell suspensions in 50 mM imidazole (pH 6.5) (1:10 dilution of the standard suspension) were incubated at 25°C in the absence of Ca2+ (cells containing the Ca-free CEP) () or in the presence of 10 mM Ca2+ (). At different times residual activities were measured in the presence of 10 mM Ca and expressed as percentages of the initial activity. For comparison, the results obtained for Ca-free cell-bound hybrid proteinases ABCd and Abcd are also shown, as are the results obtained for released Ca-free SK11 CEP in the absence of Ca2+ () or in the presence of 10 mM Ca2+ (). For details see the text. (b) Stability of cell-bound Ca-free Wg2 CEP in 50 mM imidazole (pH 6.5) at 25°C. Residual activities associated with the cell fraction () and in the supernatant () of the cell suspension were assayed in the absence of Ca2+ and were expressed as percentages of the initial activity. For comparison the activities associated with the cell fraction after incubation of a cell suspension in buffer supplemented with 0.2 mM Ca2+ are also shown ().

Effect of divalent cations on the Ca-free cell-bound CEP. Strain SK11 or Wg2 cells that were washed with ice-cold Ca-free buffer in order to obtain the Ca-free form of the cell-bound CEP were resuspended in buffers containing different concentrations of Ca²⁺ ions.

View larger version (19K): [in this window] [in a new window]   FIG. 3.   Restoration of Ca-free cell-bound SK11 CEP as a function of the concentration of Ca2+ (), Cd2+ (), or Mn2+ (). Two different scales were used, 0 to 1 mM (solid lines) and 0 to 10 mM (dashed lines). The dotted lines indicate shifts in the scale at 0.1 mM in the cases of Ca2+ and Mn2+.

View larger version (26K): [in this window] [in a new window]   FIG. 4.   Modulation of cell-bound CEP activity by Ca2+ ions. Plasmid-free strain L. lactis MG 1363 into which the wild-type plasmid or a hybrid proteinase gene-containing plasmid was introduced was used. Activities associated with washed cells containing the Ca-free enzyme were determined at 25°C as a function of the Ca2+ concentration. Symbols: , wild-type SK11 CEP (ABCD); , hybrid CEP Abcd; , hybrid CEP AbCd; , hybrid CEP ABCd; , hybrid CEP ABcD; , hybrid CEP AbCD.

View larger version (17K): [in this window] [in a new window]   FIG. 5.   Stabilization of the Ca-free cell-bound SK11 CEP by Ca2+ ions. Washed cells in 50 mM imidazole (pH 6.5) were incubated at 25°C in the absence of Ca2+ (dashed line) or in the presence of 0.125 mM Ca2+ (), 0.8 mM Ca2+ (), 2 mM Ca2+ (), or 10 mM Ca2+ () (solid lines). At different times residual activities were measured in the presence of 10 mM Ca2+ and expressed as percentages of the initial activity.

Hybrid SK11-Wg2 CEPs. In order to reveal the possible involvement of different segments of the SK11 CEP molecule in the ultimate specific relationships among Ca²⁺ binding, stability, and activity, the activities of Ca-free cell-bound, fragment A-containing hybrid CEPs were measured at 25°C and pH 6.5 as a function of the Ca²⁺ concentration. Highly reproducible, distinct Ca saturation curves were obtained (Fig. 4).

Specificity of the Ca-free cell-bound CEP. The specificities of the Ca-free cell-bound SK11 CEP towards _s1-CN(f1-23) in the presence of different concentrations of Ca²⁺ (corresponding to different phases of activity [Fig. 4]) or in the presence of Cd²⁺ (data not shown) or 10 mM Mn²⁺ were determined (see Materials and Methods) and were found to be identical (Fig. 6). Ca-free CEP activity in the absence of Ca²⁺ could be detected at 25°C (albeit at a very low rate) with this peptide as a substrate and with a relatively dense all suspension. Its specificity was distinguished from that of the Ca-loaded CEP only by a slightly increased relative cleavage rate at bond 17-18 compared to the Ca²⁺-loaded enzyme (Fig. 6a). With the wild-type Wg2 CEP no shift was observed, and with the hybrid CEPs either no shift or only a slight shift in the preference for peptide bonds was observed (data not shown), which again mainly concerned the occurrence of cleavage or an increased relative cleavage rate of bond 17-18 in the absence of Ca²⁺.

View larger version (22K): [in this window] [in a new window]   FIG. 6.   (a) Analytical reversed-phase HPLC of s1-CN(f1-23) (line a) and of the products of degradation of s1-CN(f1-23) by the cell-bound Ca-free CEP of strain SK11 at pH 6.5 in the absence of Ca2+ (120 min, 25°C) (line b) or in the presence of 0.1 mM Ca2+ (30 min, 25°C) (line c). (b) Analytical reversed-phase HPLC of the products of s1-CN(f1-23) degradation (25°C, pH 6.5) by the cell-bound Ca-free CEP of strain SK11 in the absence of Ca2+ (line a), at 10 mM Ca2+ (line b), or at 10 mM Mn2+ (line c).

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

The results of this study suggest that removal of relatively weakly bound calcium in cell-bound CEP initiates a (local) structural rearrangement in the proteinase domain, resulting in an enzyme which not only is susceptible to autoproteolytic release (10, 26) but also has a lower specific activity and, in the case of the SK11 CEP, is significantly destabilized against thermal denaturation, as a result of which no activity can be detected at 25°C and pH 6.5. The Ca-free CEP still can regain its native stable conformation if Ca²⁺ ions are added. Ca-triggered local rearrangements which may be essential to stabilization apparently extend to the substrate-binding site and affect substrate binding and/or the catalytic process itself. The suggestion that destabilization upon Ca²⁺ removal is against denaturation rather than against autoproteolytic attack is supported by results showing that the autoproteolytically released proteinase fraction obtained under conditions in which the enzyme is unstable (pH 6.5, 25°C) migrates under denaturing conditions in a polyacrylamide gel as a major 145-kDa band together with minor 80- to 130-kDa bands. All of these C-terminally truncated products that are larger than 100 kDa in the release fraction of SK11 (4, 11, 31) or the release fractions of other strains (5, 24, 27) are still proteolytically active. These results indicate that no serious autoproteolytic inactivation of the Ca-free cell-bound SK11 CEP occurs; otherwise, an inactive component would have been detected, which, in view of the very small percentage of active CEP released, should have been present at a relatively very high concentration.

Lactococcal CEPs are predicted, on the basis of their sequence similarity to subtilisin and thermitase, to possess at least three Ca-binding sites in their proteinase domains (30) (Fig. 7), and one of these, Ca₁, might be expected to bind Ca²⁺ even more tightly than the corresponding site in thermitase (3, 17, 18) due to an additional negative charge on residue 85 (i.e., D-110 in the CEP). The Ca₂ and Ca₃ binding sites are even weaker than the corresponding weak sites in related proteinases, and Ca₃ is the weakest (9). These binding sites are located in external loops close to the substrate recognition site (Fig. 7). The binding of Ca²⁺, which supposedly occurs first predominantly to Ca₂ and subsequently to Ca₃, may trigger sequential concerted movements within the proteinase domain which exert long-range effects on the geometry of the catalytic triad in particular. The underlying local structural rearrangements at the Ca sites are thus reflected in altered specific activities which are responsible for the biphasic dependence of activity on Ca²⁺ concentration, in the increasing stability of the enzyme, and to some extent in the specificity of the enzyme.

View larger version (43K): [in this window] [in a new window]   FIG. 7.   Model of part of the substrate-binding region and of the calcium-binding sites in CEPIII of L. lactis SK11, predicted on the basis of a close sequence similarity with subtilisin and thermitase. Residues of the catalytic triad (Asp-30, His-94, and Ser-433 in CEP) are stippled. Insertions relative to subtilisin are indicated either by an underlined one-letter code or by the number of residues involved. The numbering is thermitase numbering (roman type) (25) and SK11 CEP numbering (italic type) (33). S'1, S1, S2, S3, and S4 are substrate-binding sites, and Ca1, Ca2, and Ca3 are predicted calcium-binding sites deduced from sequence similarities. Thick lines represent known side chain ligands, and thin lines represent main chain ligands for Ca2+ in subtilisin and thermitase. Dashed arrows indicate stabilizing side chain or main chain interactions. For details see the text.

The association of Ca²⁺ with binding sites in other subtilisinlike serine proteinases has been shown to induce structural changes in the direct surroundings of these sites (18) and in the substrate-binding site as well (1, 2), to protect the enzymes from autoproteolytic degradation, and to contribute to thermal stabilization by reducing the flexibility of the molecules (16, 30, 35). These previously described examples may support the view concerning the impact of Ca²⁺ binding to the predicted sites on CEP described above.

One of the cations tested, Cd²⁺, can replace Ca²⁺ most efficiently, probably because its ion radius (0.97 Å) is very similar to that of Ca²⁺ (0.99 Å). In addition, only Mn²⁺ (ion radius, 0.80 Å) is able to restore the Ca-free enzyme. All other cations have ion radii that are either smaller than 0.80 Å or much larger than 0.99 Å. The inefficacy of these cations may be explained in terms of the rigidity of the area of the Ca site where the protein is not able to either collapse around small ions or expand to admit large ions (32). The relatively poor efficacy of Mn²⁺ could reflect a much lower capacity of the Ca sites involved to bind this small ion and/or a less efficient ultimate effect of binding on stability and activity.

Autoproteolytic release of active CEP at 25°C in a Ca-free buffer at pH 6.5 appeared to be restricted to the Ca-free form. However, the stability of the enzyme against denaturation may determine the relative amount of CEP released and thus, together with a lower specific activity of the released CEP, the total activity in the final soluble fraction. Unlike the release of the relatively stable Ca-free Wg2 CEP, no release of Ca-free CEP from SK11 cells was detected in dilute suspensions, perhaps because of its highly unstable character. An increase in the incidence of intercellular autoproteolytic action may explain the detection of release when cells are treated at very high densities. The released Ca-free SK11 CEP was relatively stable compared to the bound Ca-free enzyme, suggesting that there was conformational stabilization which was independent of Ca²⁺ ions in the weak binding sites.

The binding affinity of a Ca-binding site is determined by the relevant residues delivering the formal ligands, by residues which, owing to their location and charge, can influence the electrostatic potential at the site and thus can affect binding (20, 28), and by the geometry of the site, which determines Ca²⁺ coordination. The binding to the Ca-binding sites in the Ca-free CEP and/or the ultimate consequences of Ca²⁺ binding for the enzyme seem to be influenced by one or more of the substitutions which have been established by comparing the sequences of the SK11 and Wg2 CEPs. All substituted residues within the proteinase domain are outside the predicted Ca-binding regions and are not likely to influence Ca binding directly. Moreover, the present results show that not only substitutions in the proteinase domain but also substitutions in other domains of the molecule are essential for Ca binding and activity. Therefore, if substituted residues have an influence on the affinities of Ca sites, then they do so because the substitutions are connected with modulation of the geometry of these sites. If there is no such influence, apparently only Ca²⁺-triggered specific conformations are responsible for the characteristic Ca²⁺ dependencies of the activities of the hybrid CEPs. Of all of the substitutions, the substitutions in the C-terminal extension (fragments c and d) of the Wg2 CEP, especially the substitutions in fragment c, have the most distinct negative effect on Ca²⁺-dependent restoration of the hybrid proteinase. The results suggest that the C-terminal extension has an impact on the final conformation of the Ca-free proteinase domain and is involved in the folding process which leads to the final, completely Ca²⁺-loaded, optimally active, stable conformation of the proteinase domain. It might guide this domain to adopt an active, Ca₁-loaded specific primary conformation, the Ca-free CEP, with specific additional Ca²⁺-binding characteristics. Only in the case of the wild-type combination abcd (Wg2 CEP) is this conformation relatively stable. The final conformation of the proteinase domain (and thus its specific activity) depends on the continued occupation of Ca sites in the enzyme by Ca²⁺ ions. The proposed identity of the Ca-free CEP, containing a Ca²⁺ ion on Ca₁, was supported by the effect of EDTA treatment at 2°C on this form of the enzyme. A complete loss of potential activity (in the case of the SK11 CEP) or a significant additional irreversible decrease in activity (in the case of the Wg2 CEP) was sustained (15). In this heuristic view the reversibility of a specific Ca²⁺ dependency of the native cell-bound enzyme indicates that the C-terminal extension is permanently involved in maintaining the basic, Ca₁-loaded conformation. Therefore, our hypothesis is that one function of the large C-terminal extension is to act as a template on which the proteinase domain finds and maintains its initial conformation, which is then modulated by Ca²⁺. The extreme C terminus is assumed to function as a membrane anchor which stops translocation of the polypeptide over the membrane; the molecule may then undergo cell wall sorting involving C-terminal processing at the sorting motif which flanks the transmembrane domain at the N-terminal side (9). The sequence preceding this cell wall sorting signal has typical hydrophilic, flexible features of a transwall domain (9); this leaves the middle domain to act, either as a whole or only in part, as the proposed template.