Deletion of various carboxy-terminal domains of Lactococcus lactis SK11 proteinase: effects on activity, specificity, and stability of the truncated enzyme

The Lactococcus lactis SK11 cell envelope proteinase is an extracellular, multidomain protein of nearly 2,000 residues consisting of an N-terminal serine protease domain, followed by various other domains of largely unknown function. Using a strategy of deletion mutagenesis, we have analyzed the function of several C-terminal domains of the SK11 proteinase which are absent in cell envelope proteinases of other lactic acid bacteria. The various deletion mutants were functionally expressed in L. lactis and analyzed for enzyme stability, activity, (auto)processing, and specificity toward several substrates. C-terminal deletions of first the cell envelope W (wall) and AN (anchor) domains and then the H (helix) domain leads to fully active, secreted proteinases of unaltered specificity. Gradually increasing the C-terminal deletion into the so-called B domain leads to increasing instability and autoproteolysis and progressively less proteolytic activity. However, the mutant with the largest deletion (838 residues) from the C terminus and lacking the entire B domain still retains proteolytic activity. All truncated enzymes show unaltered proteolytic specificity toward various substrates. This suggests that the main role played by these domains is providing stability or protection from autoproteolysis (B domain), spacing away from the cell (H domain), and anchoring to the cell envelope (W and AN domains). In addition, this study allowed us to more precisely map the main C-terminal autoprocessing site of the SK11 proteinase and the epitope for binding of group IV monoclonal antibodies.     

The Autoproteolysis of Lactococcus lactis Lactocepin III Affects Its Specificity towards β-Casein

The effect of autoproteolysis of Lactococcus lactis lactocepin III on its specificity towards β-casein was investigated. β-Casein degradation was performed by using either an autolysin-defective derivative of L. lactis MG1363 carrying the proteinase genes of L. lactis SK11, which was unable to transport oligopeptides, or autoproteolyzed enzyme purified from L. lactis SK11. Comparison of the peptide pools by high-performance liquid chromatography analysis revealed significant differences. To analyze these differences in more detail, the peptides released by the cell-anchored proteinase were identified by on-line coupling of liquid chromatography to mass spectrometry. More than 100 oligopeptides were released from β-casein by the cell-anchored proteinase. Analysis of the cleavage sites indicated that the specificity of peptide bond cleavage by the cell-anchored proteinase differed significantly from that of the autoproteolyzed enzyme.     

Role of Calcium in Activity and Stability of the Lactococcus lactis Cell Envelope Proteinase

The mature lactococcal cell envelope proteinase (CEP) consists of an N-terminal subtilisin-like proteinase domain and a large C-terminal extension of unknown function whose far end anchors the molecule in the cell envelope. Different types of CEP can be distinguished on the basis of specificity and amino acid sequence. Removal of weakly bound Ca²⁺ from the native cell-bound CEP of Lactococcus lactis SK11 (type III specificity) is coupled with a significant reversible decrease in specific activity and a dramatic reversible reduction in thermal stability, as a result of which no activity at 25°C (pH 6.5) can be measured. The consequences of Ca²⁺ removal are less dramatic for the CEP of strain Wg2 (mixed type I-type III specificity). Autoproteolytic release of CEP from cells concerns this so-called “Ca-free” form only and occurs most efficiently in the case of the Wg2 CEP. The results of a study of the relationship between the Ca²⁺ concentration and the stability and activity of the cell-bound SK11 CEP at 25°C suggested that binding of at least two Ca²⁺ ions occurred. Similar studies performed with hybrid CEPs constructed from SK11 and Wg2 wild-type CEPs revealed that the C-terminal extension plays a determinative role with respect to the ultimate distinct Ca²⁺ dependence of the cell-bound CEP. The results are discussed in terms of predicted Ca²⁺ binding sites in the subtilisin-like proteinase domain and Ca-triggered structural rearrangements that influence both the conformational stability of the enzyme and the effectiveness of the catalytic site. We argue that distinctive primary folding of the proteinase domain is guided and maintained by the large C-terminal extension.     

Role of calcium in activity and stability of the Lactococcus lactis cell envelope proteinase

The mature lactococcal cell envelope proteinase (CEP) consists of an N-terminal subtilisin-like proteinase domain and a large C-terminal extension of unknown function whose far end anchors the molecule in the cell envelope. Different types of CEP can be distinguished on the basis of specificity and amino acid sequence. Removal of weakly bound Ca2+ from the native cell-bound CEP of Lactococcus lactis SK11 (type III specificity) is coupled with a significant reversible decrease in specific activity and a dramatic reversible reduction in thermal stability, as a result of which no activity at 25 degrees C (pH 6.5) can be measured. The consequences of Ca2+ removal are less dramatic for the CEP of strain Wg2 (mixed type I-type III specificity). Autoproteolytic release of CEP from cells concerns this so-called "Ca-free" form only and occurs most efficiently in the case of the Wg2 CEP. The results of a study of the relationship between the Ca2+ concentration and the stability and activity of the cell-bound SK11 CEP at 25 degrees C suggested that binding of at least two Ca2+ ions occurred. Similar studies performed with hybrid CEPs constructed from SK11 and Wg2 wild-type CEPs revealed that the C-terminal extension plays a determinative role with respect to the ultimate distinct Ca2+ dependence of the cell-bound CEP. The results are discussed in terms of predicted Ca2+ binding sites in the subtilisin-like proteinase domain and Ca-triggered structural rearrangements that influence both the conformational stability of the enzyme and the effectiveness of the catalytic site. We argue that distinctive primary folding of the proteinase domain is guided and maintained by the large C-terminal extension.     

Autolysis of Lactococcus lactis Is Influenced by Proteolysis

The autolysin AcmA of Lactococcus lactis was shown to be degraded by the extracellular lactococcal proteinase PrtP. Autolysis, as evidenced by reduction in optical density of a stationary-phase culture and concomitant release of intracellular proteins, was greatly reduced when L. lactis MG1363 cells expressed the cell wall-anchored lactococcal proteinase PrtP of the PI-type caseinolytic specificity (PI). On the other hand, lactococcal strains that did not produce the proteinase showed a high level of autolysis, which was also observed when the cells produced the secreted form of PI or a cell wall-anchored proteinase with PIII-type specificity. Autolysis was also increased when MG1363 expressed the cell wall-anchored hybrid PI/PIII-type proteinase PIac. Zymographic analysis of AcmA activity during stationary phase showed that AcmA was quickly degraded by PI and much more slowly by PrtP proteinases with PIII-type and intermediate specificities. Autolysis of L. lactis by AcmA was influenced by the specificity, amount, and location of the lactococcal proteinase. No autolysis was observed when the various proteinases were expressed in an L. lactis acmA deletion mutant, indicating that PrtP itself did not cause lysis of cells. The chain length of a strain was significantly shortened when the strain expressed a cell wall-anchored active proteinase.     

Autoproteolysis of the Extracellular Serine Proteinase of Lactococcus lactis subsp. cremoris Wg2

The molecular masses of purified extracellular serine proteinase of a number of Lactococcus lactis strains vary significantly, and these molecular mass values do not correspond to the values estimated on the basis of genetic data. The discrepancies can only partially be explained by N-terminal processing during maturation of the precursor enzyme and by C-terminal cleaving during the release from the cell envelope. With a monoclonal antibody that binds in the active site region of the L. lactis proteinase, the processing of the released proteinase was followed. At 30°C the proteinase was degraded with a concomitant loss of β-casein hydrolytic activity. In the presence of CaCl₂, proteinase degradation was inhibited, and new degradation products were detected. The specific serine proteinase inhibitors phenylmethylsulfonyl fluoride and diisopropylfluorophosphate also inhibited proteinase degradation. Two major high-molecular-mass proteinase fragments (165 and 90 kDa) were found to have the same N-terminal amino acid sequence as the mature proteinase, i.e., [Asp-1-Ala-2-Lys-3-Ala-4-Asn-5-Ser-6, indicating that both fragments were formed by cleavage at the C terminus. The N terminus of a proteinase fragment with low molecular mass (58 kDa) started with Gln-215. In this fragment part of the active site region was eliminated, suggesting that it is proteolytically inactive. Unlike larger fragments, this 58-kDa fragment remained intact after prolonged incubations. These results indicate that autoproteolysis of the L. lactis subsp. cremoris Wg2 proteinase ultimately leads to inactivation of the proteinase by deletion of the active site region.     

Engineering of the Lactococcus lactis serine proteinase by construction of hybrid enzymes

Plasmids containing wild-type and hybrid proteinase genes were constructed from DNA fragments of the prtP genes of Lactococcus lactis strains Wg2 and SK11. These plasmids were introduced into the plasmid-free strain L. lactis MG1363. The serine proteinases produced by these L. lactis strains were isolated, and their cleavage specificity and rate towards alpha s1- and beta-casein was investigated. The catalytic properties of both the SK11 and Wg2 proteinases, which differ in 44 out of 1902 amino acid residues, could be changed dramatically by the reciprocal exchange of specific fragments between the two enzymes. As a result, various L. lactis strains were constructed having new proteolytic properties that differ from those of the parental strains. Furthermore, two segments in the proteinase could be identified that contribute significantly to the cleavage specificity towards casein; within these two segments, several amino acid residues were identified that are important for substrate cleavage rate and specificity. The results also indicate that the lactococcal proteinase has an additional domain involved in substrate binding compared with the related subtilisins. This suggests that the 200 kd L. lactis proteinase may be the representative of a new subclass of subtilisin-like enzymes.     

Streptococcus thermophilus cell wall-anchored proteinase: release, purification, and biochemical and genetic characterization

Streptococcus thermophilus CNRZ 385 expresses a cell envelope proteinase (PrtS), which is characterized in the present work, both at the biochemical and genetic levels. Since PrtS is resistant to most classical methods of extraction from the cell envelopes, we developed a three-step process based on loosening of the cell wall by cultivation of the cells in the presence of glycine (20 mM), mechanical disruption (with alumina powder), and enzymatic treatment (lysozyme). The pure enzyme is a serine proteinase highly activated by Ca(2+) ions. Its activity was optimal at 37 degrees C and pH 7.5 with acetyl-Ala-Ala-Pro-Phe-paranitroanilide as substrate. The study of the hydrolysis of the chromogenic and casein substrates indicated that PrtS presented an intermediate specificity between the most divergent types of cell envelope proteinases from lactococci, known as the PI and PIII types. This result was confirmed by the sequence determination of the regions involved in substrate specificity, which were a mix between those of PI and PIII types, and also had unique residues. Sequence analysis of the PrtS encoding gene revealed that PrtS is a member of the subtilase family. It is a multidomain protein which is maturated and tightly anchored to the cell wall via a mechanism involving an LPXTG motif. PrtS bears similarities to cell envelope proteinases from pyogenic streptococci (C5a peptidase and cell surface proteinase) and lactic acid bacteria (PrtP, PrtH, and PrtB). The highest homologies were found with streptococcal proteinases which lack, as PrtS, one domain (the B domain) present in cell envelope proteinases from all other lactic acid bacteria.     

Functional Specialization and Evolution of Leader Proteinases in the Family Closteroviridae

Members of the Closteroviridae and Potyviridae families of the plant positive-strand RNA viruses encode one or two papain-like leader proteinases. In addition to a C-terminal proteolytic domain, each of these proteinases possesses a nonproteolytic N-terminal domain. We compared functions of the several leader proteinases using a gene swapping approach. The leader proteinase (L-Pro) of Beet yellows virus (BYV; a closterovirus) was replaced with L1 or L2 proteinases of Citrus tristeza virus (CTV; another closterovirus), P-Pro proteinase of Lettuce infectious yellows virus (LIYV; a crinivirus), and HC-Pro proteinase of Tobacco etch virus (a potyvirus). Each foreign proteinase efficiently processed the chimeric BYV polyprotein in vitro. However, only L1 and P-Pro, not L2 and HC-Pro, were able to rescue the amplification of the chimeric BYV variants. The combined expression of L1 and L2 resulted in an increased RNA accumulation compared to that of the parental BYV. Remarkably, this L1-L2 chimera exhibited reduced invasiveness and inability to move from cell to cell. Similar analyses of the BYV hybrids, in which only the papain-like domain of L-Pro was replaced with those derived from L1, L2, P-Pro, and HC-Pro, also revealed functional specialization of these domains. In subcellular-localization experiments, distinct patterns were observed for the leader proteinases of BYV, CTV, and LIYV. Taken together, these results demonstrated that, in addition to a common proteolytic activity, the leader proteinases of closteroviruses possess specialized functions in virus RNA amplification, virus invasion, and cell-to-cell movement. The phylogenetic analysis suggested that functionally distinct L1 and L2 of CTV originated by a gene duplication event.     

Cell-Wall-Bound Proteinase of Lactobacillus delbrueckii subsp. lactis ACA-DC 178: Characterization and Specificity for β-Casein

Lactobacillus delbrueckii subsp. lactis ACA-DC 178, which was isolated from Greek Kasseri cheese, produces a cell-wall-bound proteinase. The proteinase was removed from the cell envelope by washing the cells with a Ca²⁺-free buffer. The crude proteinase extract shows its highest activity at pH 6.0 and 40°C. It is inhibited by phenylmethylsulfonyl fluoride, showing that the enzyme is a serine-type proteinase. Considering the substrate specificity, the enzyme is similar to the lactococcal P_I-type proteinases, since it hydrolyzes β-casein mainly and α- and κ-caseins to a much lesser extent. The cell-wall-bound proteinase from L. delbrueckii subsp. lactis ACA-DC 178 liberates four main peptides from β-casein, which have been identified.     

Primary structure and organization of the gene for a procaryotic, cell envelope-located serine proteinase

We have determined the complete nucleotide sequence of the gene for the cell envelope-located proteinase of Lactococcus lactis SK11. The gene contains a very AT-rich promoter region followed by the coding sequence of a protein of 1962 amino acids. Comparison of the NH2-terminal amino acid sequence of the mature proteinase and the expected primary translation product of the proteinase gene indicates that the enzyme is probably synthesized as a pre-pro-protein. This is confirmed by expression studies of the proteinase gene in Escherichia coli. The amino acid sequence of the proteinase shows significant homology to a number of serine proteinases of the subtilisin family. Compared with the related proteinase of L. lactis Wg2, the proteinase of L. lactis SK11 contains a 60-amino acids duplication and a total of 44-amino acid substitutions, some of which may account for the different cleavage specificity of both enzymes. Furthermore, a region was identified in the Lactococcus proteinase, which shows homology to the membrane-anchoring domains of a number of proteins from other Gram-positive bacteria.     

The autoproteolysis of Lactococcus lactis lactocepin III affects its specificity towards beta-casein

The effect of autoproteolysis of Lactococcus lactis lactocepin III on its specificity towards beta-casein was investigated. beta-Casein degradation was performed by using either an autolysin-defective derivative of L. lactis MG1363 carrying the proteinase genes of L. lactis SK11, which was unable to transport oligopeptides, or autoproteolyzed enzyme purified from L. lactis SK11. Comparison of the peptide pools by high-performance liquid chromatography analysis revealed significant differences. To analyze these differences in more detail, the peptides released by the cell-anchored proteinase were identified by on-line coupling of liquid chromatography to mass spectrometry. More than 100 oligopeptides were released from beta-casein by the cell-anchored proteinase. Analysis of the cleavage sites indicated that the specificity of peptide bond cleavage by the cell-anchored proteinase differed significantly from that of the autoproteolyzed enzyme.     

An Ancient Autoproteolytic Domain Found in GAIN,ZU5 and Nucleoporin98

A large family of G protein-coupled receptors (GPCRs) involved in cell adhesion has a characteristic autoproteolysis motif of HLT/S known as the GPCR proteolysis site (GPS). GPS is also shared by polycystic kidney disease proteins and it precedes the first transmembrane segment in both families. Recent structural studies have elucidated the GPS to be part of a larger domain named GPCR autoproteolysis inducing (GAIN) domain. Here we demonstrate the remote homology relationships of GAIN domain to ZU5 domain and Nucleoporin98 (Nup98) C-terminal domain by structural and sequence analysis. Sequence homology searches were performed to extend ZU5-like domains to bacteria and archaea, as well as new eukaryotic families. We found that the consecutive ZU5-UPA-death domain domain organization is commonly used in human cytoplasmic proteins with ZU5 domains, including CARD8 (caspase recruitment domain-containing protein 8) and NLRP1 (NACHT, LRR and PYD domain-containing protein 1) from the FIIND (Function to Find) family. Another divergent family of extracellular ZU5-like domains was identified in cartilage intermediate layer proteins and FAM171 proteins. Current diverse families of GAIN domain subdomain B, ZU5 and Nup98 C-terminal domain likely evolved from an ancient autoproteolytic domain with an HFS motif. The autoproteolytic site was kept intact in Nup98, p53-induced protein with a death domain and UNC5C-like, deteriorated in many ZU5 domains and changed in GAIN and FIIND. Deletion of the strand after the cleavage site was observed in zonula occluden-1 and some Nup98 homologs. These findings link several autoproteolytic domains, extend our understanding of GAIN domain origination in adhesion GPCRs and provide insights into the evolution of an ancient autoproteolytic domain.     

Specificity of hydrolysis of bovine kappa-casein by cell envelope-associated proteinases from Lactococcus lactis strains.

The cell envelope-associated proteinases from Lactococcus lactis subsp. cremoris H2 (a PI-type proteinase-producing strain) and SK11 (a PIII-type proteinase-producing strain) both actively hydrolyze the kappa-casein component of bovine milk but with significant differences in the specificity of peptide bond hydrolysis. The peptide bonds Ala-23-Lys-24, Leu-32-Ser-33, Ala-71-Gln-72, Leu-79-Ser-80, Met-95-Ala-96, and Met-106-Ala-107 were cleaved by both proteinase types, although the relative rates of hydrolysis at some of these sites were quite different for the two proteinases. Small histidine-rich peptides were formed as early products of the action of the cell envelope-associated proteinases on kappa-casein, implicating this casein as a possible significant source of histidine, which is essential for starter growth. The major difference between the two proteinase types in their action on kappa-casein was in their ability to cleave bonds near the C-terminal end of the molecule. The bond Asn-160-Thr-161 and, to a lesser extent, the bond Glu-151-Val-152 were very rapidly cleaved by the PIII-type proteinase, whereas hydrolysis of these bonds by the PI-type proteinase was barely detectable (even after 24 h of digestion). Differential hydrolysis of kappa-casein at these sites by the two different proteinase types resulted in the formation of distinctive, high-M(r) products detectable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)     

SK3 Trafficking in Hippocampal Cells: The Role of Different Molecular Domains

The regulative steps that control trafficking of ion channels are fundamental determinants of their qualitative and quantitative expression on the cell membrane. In this work the trafficking of the small conductance calcium-activated potassium channel, SK3 was studied in neurons in order to identify relevant molecular domains involved in this process. Hippocampal cell cultures were transfected with fusion proteins of green fluorescent protein (GFP) and different SK3 subunit truncations. The differential distribution of the mutants was analyzed by confocal microscopy and compared to the localization of the control fusion protein with full length SK3. The transport of chimeric proteins was quantified from fluorescence images by developing a morphometric analytical method. We found that the full length SK3 was distributed in cell body, axon and dendrites, whereas the deleted forms GFPΔ578–736 (deletion of the entire C-terminal domain), GFPΔCaMBD (deletion of the calmodulin-binding site) and GFPΔN (deletion of the N-terminal domain) were not transported into cell processes but accumulated in the cell body. The GFPΔ640–736 (deletion of the distal C-terminal domain) showed a distribution similar to control. The quantification and statistical analysis confirmed the differences in distribution across the three groups. In conclusion, the current work provides evidence for a fundamental role of the N-terminal domain and the calmodulin binding domain in SK3 trafficking in neurons.     

Effects of High Pressure on the Viability, Morphology, Lysis, and Cell Wall Hydrolase Activity of Lactococcus lactis subsp. cremoris

Viability, morphology, lysis, and cell wall hydrolase activity of Lactococcus lactis subsp. cremoris MG1363 and SK11 were determined after exposure to pressure. Both strains were completely inactivated at pressures of 400 to 800 MPa but unaffected at 100 and 200 MPa. At 300 MPa, the MG1363 and SK11 populations decreased by 7.3 and 2.5 log cycles, respectively. Transmission electron microscopy indicated that pressure caused intracellular and cell envelope damage. Pressure-treated MG1363 cell suspensions lysed more rapidly over time than did non-pressure-treated controls. Twenty-four hours after pressure treatment, the percent lysis ranged from 13.0 (0.1 MPa) to 43.3 (300 MPa). Analysis of the MG1363 supernatants by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) confirmed pressure-induced lysis. Pressure did not induce lysis or membrane permeability of SK11. Renaturing SDS-PAGE (zymogram analysis) revealed two hydrolytic bands from MG1363 cell extracts treated at all pressures (0.1 to 800 MPa). Measuring the reducing sugars released during enzymatic cell wall breakdown provided a quantitative, nondenaturing assay of cell wall hydrolase activity. Cells treated at 100 MPa released significantly more reducing sugar than other samples, including the non-pressure-treated control, indicating that pressure can activate cell wall hydrolase activity or increase cell wall accessibility to the enzyme. The cell suspensions treated at 200 and 300 MPa did not differ significantly from the control, whereas cells treated at pressures greater than 400 MPa displayed reduced cell wall hydrolase activity. These data suggest that high pressure can cause inactivation, physical damage, and lysis in L. lactis. Pressure-induced lysis is strain dependent and not solely dependent upon cell wall hydrolase activity.     

The proteolytic system of Lactobacillus sanfrancisco CB1: purification and characterization of a proteinase, a dipeptidase, and an aminopeptidase.

A cell envelope 57-kDa proteinase, a cytoplasmic 65-kDa dipeptidase, and a 75-kDa aminopeptidase were purified from Lactobacillus sanfrancisco CB1 sourdough lactic acid bacterium by sequential fast protein liquid chromatography steps. All of the enzymes are monomers. The proteinase was most active at pH 7.0 and 40 degrees C, while aminopeptidase and dipeptidase had optima at pH 7.5 and 30 to 35 degrees C. Relatively high activities were observed at the pH and temperature of the sourdough fermentation. The proteinase is a serine enzyme. Urea-polyacrylamide gel electrophoresis of digest of alpha s1- and beta-caseins showed differences in the pattern of peptides released by the purified proteinase and those produced by crude preparations of the cell envelope proteinases of Lactobacillus delbrueckii subsp. bulgaricus B397 and Lactococcus lactis subsp. lactis SK11. Reversed-phase fast protein liquid chromatography of gliadin digests showed a more-complex peptide pattern produced by the proteinase of Lactobacillus sanfrancisco CB1. The dipeptidase is a metalloenzyme with high affinity for dipeptides containing hydrophobic amino acids but had no activity on tripeptides or larger peptides. The aminopeptidase was also inhibited by metal-chelating agents, and showed a broad N-terminal hydrolytic activity including di- and tripeptides. Km values of 0.70 and 0.44 mM were determined for the dipeptidase on Leu-Leu and the aminopeptidase on Leu-p-nitroanilide, respectively.     

Purification and characterization of the mature,membrane-associated cell-envelope proteinase of Lactobacillus helveticus L89

Lactobacillus helveticus L89 possesses a cell-envelope proteinase (Lb-CEP) that is biochemically and genetically related to that of the lactococci (Lc-CEP). The in-situ proteinase is resistant to autoproteolysis and remains associated with the membrane during lysozyme treatment of cells and subsequent mechanical disruption of the treated cells. The proteinase was purified from isolated membranes by a procedure that preserves the complete in-situ proteinase (mature proteinase) assumed to be the N-terminally processed translation product including the membrane anchor: its monomer molecular mass is approximately 180 kDa. The purified enzyme appeared to be more stable towards heat than hitherto known related, but C-terminally truncated cell-envelope proteinases of lactobacilli and lactococci, which were released from the cells by autoproteolysis. On the basis of its specificity towards caseins, towards the _sl-casein-(1-23)-fragment and towards two differently charged chromophoric peptides, the proteinase was recognized as an (Lb-)CEP_I/III mixed-type variant different from those identified so far among the lactococcal proteinases. Correspondence to: F. A. Exterkate