Fate of peptides in peptidase mutants of Lactococcus lactis

The utilization of exogenous peptides was studied in mutants of Lactococcus lactis in which combinations of the peptidase genes pepN pepC pepO pepX and pepT were deleted. Multiple mutants lacking PepN, PepC, PepT plus PepX could not grow on peptides such as Leu–Gly–Gly, Gly–Phe–Leu, Leu–Gly–Pro, Ala–Pro–Leu and Gly–Leu–Gly–Leu, respectively, indicating that no other peptidases are present to release the essential amino acid Leu. In these mutants, peptides accumulate intracellularly, demonstrating that peptides are translocated as whole entities prior to degradation. The mutant lacking all five peptidases could still grow on Gly–Leu and Tyr–Gly–Gly–Phe–Leu, which confirmed the presence of a dipeptidase and led to the identification of an unknown PepO-like endopeptidase. These studies have also shown that the general aminopeptidases PepN, PepC and PepT have overlapping but not identical specificities and differ in their overall activity towards individual peptides. In contrast, PepX has an unique specificity, because it is the only enzyme which can efficiently degrade Ala–Pro–Leu. The concerted action of peptidases in the breakdown of particular peptides revealed how these substrates are utilized as sources of nitrogen.     

Multiple-peptidase mutants of Lactococcus lactis are severely impaired in their ability to grow in milk.

To examine the contribution of peptidases to the growth of lactococcus lactis in milk, 16 single- and multiple-deletion mutants were constructed. In successive rounds of chromosomal gene replacement mutagenesis, up to all five of the following peptidase genes were inactivated (fivefold mutant): pepX, pepO, pepT, pepC, and pepN. Multiple mutations led to slower growth rates in milk, the general trend being that growth rates decreased when more peptidases were inactivated. The fivefold mutant grew more than 10 times more slowly in milk than the wild-type strain. In one of the fourfold mutants and in the fivefold mutant, the intracellular pools of amino acids were lower than those of the wild type, whereas peptides had accumulated inside the cell. No significant differences in the activities of the cell envelope-associated proteinase and of the oligopeptide transport system were observed. Also, the expression of the peptidases still present in the various mutants was not detectably affected. Thus, the lower growth rates can directly be attributed to the inability of the mutants to degrade casein-derived peptides. These results supply the first direct evidence for the functioning of lactococcal peptidases in the degradation of milk proteins. Furthermore, the study provides critical information about the relative importance of the peptidases for growth in milk, the order of events in the proteolytic pathway, and the regulation of its individual components.     

Peptidase-deficient mutants of Escherichia coli.

Mutant derivatives of Escherichia coli K-12 deficient in several peptidases have been obtained. Mutants lacking a naphthylamidase, peptidase N, were isolated by screening for colonies unable to hydrolyze L-alanine beta-naphthylamide. Other mutants were isolated using positive selections for resistance to valine peptides. Mutants lacking peptidase A, a broad-specificity aminopeptidase, were obtained by selection for resistance to L-valyl-L-leucine amide. Mutants lacking a dipeptidase, peptidase D, were isolated from a pepN pepA strain by selection for resistance to L-valyl-glycine. Starting with a pepN pepA pepD strain, selection for resistance to L-valyl-glycyl-glycine or several other valine peptides produced mutants deficient in another aminopeptidase, peptidase B. Mutants resistant to L-valyl-L-proline lack peptidase Q, an activity capable of rapid hydrolysis of X-proline dipeptides. Using these selection procedures, a strain (CM89) lacking five different peptidases has been isolated. Although still sensitive to valine, this strain is resistant to a variety of valine di- and tripeptides. The ability of this strain to use peptides as sources of amino acids is much more restricted than that of wild-type E. coli strains. Strains containing only one of the five peptidases missing in CM89 have been constructed by transduction. The peptide utilization profiles of these strains show that each of the five peptidases can function during growth in the catabolism of peptides.     

Degradation of proline peptides in peptidase-deficient strains of Salmonella typhimurium.

A mutant strain of Salmonella typhimurium that lacks two proline-specific peptidases (peptidases P and Q) could not complete the degradation of proline peptides formed as intermediates in starvation-induced protein breakdown. The wild-type strain produced free proline as the product of degradation of proline-labeled proteins. The pepP pepQ mutant, however, produced a mixture of small proline peptides. In the absence of peptidase Q only, peptidase P could complete the degradation of most of the proline peptide intermediates formed. In the absence of peptidase P only, about 50% of the proline-labeled, acid-soluble products were proline peptides. These results are consistent with in vitro specificity data indicating that peptidase Q hydrolyzes X-Pro dipeptides only, whereas peptidase P attacks both X-Pro dipeptides and longer peptides with X-Pro at their N-termini. A mutant strain lacking four broad-specificity peptidases (peptidases N, A, B, and D), but containing peptidases P and Q, also produced proline peptides as products of protein breakdown. This observation suggests that broad-specificity peptidases are required to generate the X-Pro substrates of peptidases P and Q. A strain lacking six peptidases (N, A, B, D, P, and Q) was constructed and produced less free proline from protein breakdown than either the pepP pepQ strain or the pepN pepA pepB pepD strain. These observations suggest that the degradation of peptide intermediates involves the sequential removal of N-terminal amino acids and requires both broad-specificity aminopeptidases (peptidases N, A, and B) and the X-Pro-specific aminopeptidase, peptidase P.     

Peptidases and amino acid catabolism in lactic acid bacteria

The conversion of peptides to free amino acids and their subsequent utilization is a central metabolic activity in prokaryotes. At least 16 peptidases from lactic acid bacteria (LAB) have been characterized biochemically and/or genetically. Among LAB, the peptidase systems of Lactobacillus helveticus and Lactococcus lactis have been examined in greatest detail. While there are homologous enzymes common to both systems, significant differences exist in the peptidase complement of these organisms. The characterization of single and multiple peptidase mutants indicate that these strains generally exhibit reduced specific growth rates in milk compared to the parental strains. LAB can also catabolize amino acids produced by peptide hydrolysis. While the catabolism of amino acids such as Arg, Thr, and His is well understood, few other amino acid catabolic pathways from lactic acid bacteria have been characterized in significant detail. Increasing research attention is being directed toward elucidating these pathways as well as characterizing their physiological and industrial significance.     

Peptidase mutants of Salmonella typhimurium

Six peptidase activities have been distinguished electrophoretically in cell extracts of Salmonella typhimurium with the aid of a histochemical stain. The activities can also be partially separated by chromatography on diethylaminoethyl-cellulose. These peptidases show overlapping substrate specificities. Mutants (pepN) of the parent strain leu-485 lacking one of these enzymes (peptidase N) were obtained by screening for colonies that do not hydrolyze the chromogenic substrate l-alanyl-beta-naphthylamide. The absence of this broad-specificity peptidase in leu-485 pepN(-) mutants allowed the selection of mutants unable to use l-leucyl-l-alaninamide as a leucine source. These mutants (leu-485 pepN(-)pepA(-)) lack a broad-specificity peptidase (peptidase A) similar to aminopeptidase I previously described in Escherichia coli. Mutants (pepD) lacking a dipeptidase (peptidase D) have been isolated from a leu-485 pepN(-)pepA(-) parent by penicillin selection for mutants unable to use l-leucyl-l-glycine as a leucine source. Mutants (pepB) lacking a fourth peptidase (peptidase B) have been isolated from a leu-485 pepN(-)pepA(-)pepD(-) strain by penicillin selection for failure to utilize l-leucyl-l-leucine as a source of leucine. Single recombinants were obtained by transduction for each of the peptidases missing in a leu-485 pepN(-)pepA(-)pepD(-)pepB(-) strain. The growth response of these recombinants to leucine peptides shows that all of these peptidases can function in the catabolism of peptides and that they display overlapping substrate specificities in vivo.     

Amino acid requirements and peptidase activities of Streptococcus salivarius subsp. thermophilus

The aim of this work was to investigate the relationship between amino acid requirements and peptidase enzyme systems in three Streptococcus salivarius subsp. thermophilus strains. A synthetic medium without nitrogen components and a milk (RD milk) without its non-protein nitrogen fraction were prepared with different mixtures of amino acids. The strains showed different amino acid requirements. Some amino acids proved to be essential, some were required, while others did not affect growth. In the synthetic medium, only leucine and glutamic acid were essential for growth. In RD milk, the amino acid requirements were found to be lower, with only the absence of glutamic acid causing complete inhibition of growth. Relationships between aminopeptidase activities of the strains and their amino acid requirements were observed. Strains with higher amino acid requirements were also found to express a wider range of peptidases.     

Soluble di- and aminopeptidases in Escherichia K-12. Dispensible enzymes.

As part of a study of the peptidase content of Escherichia coli K-12, two peptidase-deficient amino acid auxotrophs isolated and characterized by Miller as pepD- (strain CM17) and pepD- pepN- pepA- pepB- pepQ- (strain CM89) were examined for the presence of several peptidases previously obtained from strain K-12 in this laboratory. The soluble fraction of each mutant was found to lack the broad-specificity strain K-12 dipeptidase DP and the strain CM89 fraction also lacked activity characteristic of the strain K-12 aminopeptidases AP, L, and OP; like strain CM17, strain CM89 contained the tripeptide-specific aminopeptidase TP. Strain CM89 (but not CM17) appeared to contain little if any activity attributable to the ribosome-bound aminopeptidase I of strain K-12. Whereas loss of DP, AP, OP, and aminopeptidase I activity may be attributed to the pepD-, pepB-, pepN-, and pepA- mutations, respectively, the reason for the loss of L activity remains uncertain. Grown responses of strain CM89 in liquid media containing di- or tripeptides were in accord with absence of enzymes catalyzing rapid hydrolysis of dipeptides. In synthetic liquid media supplemented with the required amino acids per se or with peptone, cultures of both CM strains grew more slowly than strain K-12 and produced smaller cell-yields than those produced by strain K-12.     

Expression of six peptidases from Lactobacillus helveticus in Lactococcus lactis

For development of novel starter strains with improved proteolytic properties, the ability of Lactococcus lactis to produce Lactobacillus helveticus aminopeptidase N (PepN), aminopeptidase C (PepC), X-prolyl dipeptidyl aminopeptidase (PepX), proline iminopeptidase (PepI), prolinase (PepR), and dipeptidase (PepD) was studied by introducing the genes encoding these enzymes into L. lactis MG1363 and its derivatives. According to Northern analyses and enzyme activity measurements, the L. helveticus aminopeptidase genes pepN, pepC, and pepX are expressed under the control of their own promoters in L. lactis. The highest expression level, using a low-copy-number vector, was obtained with the L. helveticus pepN gene, which resulted in a 25-fold increase in PepN activity compared to that of wild-type L. lactis. The L. helveticus pepI gene, residing as a third gene in an operon in its host, was expressed in L. lactis under the control of the L. helveticus pepX promoter. The genetic background of the L. lactis derivatives tested did not affect the expression level of any of the L. helveticus peptidases studied. However, the growth medium used affected both the recombinant peptidase profiles in transformant strains and the resident peptidase activities. The levels of expression of the L. helveticus pepD and pepR clones under the control of their own promoters were below the detection limit in L. lactis. However, substantial amounts of recombinant pepD and PepR activities were obtained in L. lactis when pepD and pepR were expressed under the control of the inducible lactococcal nisA promoter at an optimized nisin concentration.     

Presence of additional peptidases in Streptococcus thermophilus CNRZ 302 compared to Lactococcus lactis

Streptococcus thermophilus is widely used in the dairy industry but little is known about its peptidase system. The aim of this study was to determine the biochemical and genetic characteristics of this system, and to compare it to the well known system of Lactococcus lactis . We separated the intracellular proteins of Strep. thermophilus CNRZ 302 and L. lactis NCDO 763 by ion-exchange chromatography and we detected the activity of the different types of peptidases. In both L. lactis and Strep. thermophilus strains, we showed 13 different peptidase activities with biochemical homologies between both species. Streptococcus thermophilus also possessed two peptidases which we did not find in L. lactis : an aminopeptidase and an oligopeptidase. We performed Southern blot experiments and among the eight peptidase genes tested, only the genes encoding the general aminopeptidases, pepC and pepN , were homologous between the L. lactis and Strep. thermophilus strains. Besides biochemical and genetic similarities, the peptidase systems of Strep. thermophilus and L. lactis thus differed by the presence of additional peptidases in Strep. thermophilus .     

Hydrolysis of casein-derived peptides alpha(S1)-casein(f1-9) and beta-casein(f193-209) by Lactobacillus helveticus peptidase deletion mutants indicates the presence of a previously undetected endopeptidase

Peptides derived from hydrolysis of alpha(S1)-casein(f1-9) [alpha(S1)-CN(f1-9)] and beta-CN(f193-209) with cell extracts of Lactobacillus helveticus CNRZ32 and single-peptidase mutants (Delta pepC, Delta pepE, Delta pepN, Delta pepO, and Delta pepX) were isolated by using reverse-phase high-performance liquid chromatography and were characterized by mass spectrometry. The peptides identified suggest that there was activity of an endopeptidase, distinct from previously identified endopeptidases (PepE and PepO), with specificity for peptide bonds C terminal to Pro residues. Identification of hydrolysis products derived from a carboxyl-blocked form of beta-CN(f193-209) confirmed that the peptides were derived from the activity of an endopeptidase.     

Salmonella typhimurium peptidase active on carnosine.

Wild-type Salmonella typhimurium can use carnosine (beta-alanyl-L-histidine) as a source of histidine, but carnosine utilization is blocked in particular mutants defective in the constitutive enzyme peptidase D, the product of the pepD gene. Biochemical evidence for assigning carnosinase activity to peptidase D (a broad-specificity dipeptidase) includes: (i) coelution of carnosinase and dipeptidase activity from diethylaminoethyl-cellulose and Bio-Gel P-300 columns; (ii) coelectrophoresis of carnosinase and dipeptidase on polyacrylamide gels; and (iii) inactivation of carnosinase and dipeptidase activities at identical rates at both 4 and 42 degrees C. Genetic evidence indicates that mutations leading to loss of carnosinase activity map at pepD. Several independent pepD mutants have been isolated by different selection procedures, and the patterns of peptide utilization of strains carrying various pepD alleles have been studied. Many pepD mutations lead to the production of partially active peptidase D enzymes with substrate specificities that differ strikingly from those of the wild-type enzyme. The growth yields of carnosinase-deficient strains growing in Difco nutrient broth indicate that carnosine is the major utilizable source of histidine in this medium.     

The efficiency of human cytomegalovirus pp65(495-503) CD8+ T cell epitope generation is determined by the balanced activities of cytosolic and endoplasmic reticulum-resident peptidases

Control of human CMV (HCMV) infection depends on the cytotoxic activity of CD8(+) CTLs. The HCMV phosphoprotein (pp)65 is a major CTL target Ag and pp65(495-503) is an immunodominant CTL epitope in infected HLA-A*0201 individuals. As immunodominance is strongly determined by the surface abundance of the specific epitope, we asked for the components of the cellular Ag processing machinery determining the efficacy of pp65(495-503) generation, in particular, for the proteasome, cytosolic peptidases, and endoplasmic reticulum (ER)-resident peptidases. In vitro Ag processing experiments revealed that standard proteasomes and immunoproteasomes generate the minimal 9-mer peptide epitope as well as N-terminal elongated epitope precursors of different lengths. These peptides are largely degraded by the cytosolic peptidases leucine aminopeptidase and tripeptidyl peptidase II, as evidenced by increased pp65(495-503) epitope presentation after leucine aminopeptidase and tripeptidyl peptidase II knockdown. Additionally, with prolyl oligopeptidase and aminopeptidase B we identified two new Ag processing machinery components, which by destroying the pp65(495-503) epitope limit the availability of the specific peptide pool. In contrast to cytosolic peptidases, silencing of ER aminopeptidases 1 and 2 strongly impaired pp65(495-503)-specific T cell activation, indicating the importance of ER aminopeptidases in pp65(495-503) generation. Thus, cytosolic peptidases primarily interfere with the generation of the pp65(495-503) epitope, whereas ER-resident aminopeptidases enhance such generation. As a consequence, our experiments reveal that the combination of cytosolic and ER-resident peptidase activities strongly shape the pool of specific antigenic peptides and thus modulate MHC class I epitope presentation efficiency.     

Degradation of abnormal proteins in peptidase-deficient mutants of Salmonella typhimurium.

The degradation of abnormal proteins produced as a result of incorporation of the arginine analog L-canavanine or generated by exposure to puromycin was studied in wild-type and multiply peptidase-deficient strains of Salmonella typhimurium. Both types of abnormal protein were rapidly degraded during growth of Pep+ strains of this organism. Peptidase--deficient mutants (lacking peptidases N, A, B, and D) could also degrade these abnormal proteins, although the rate of production of trichloroacetic acid-soluble degradation products was slower in the mutant strain than in a strain carrying a normal complement of peptidases. Analysis of these trichloroacetic acid-soluble degradation products of ion-exchange chromatography showed that free amino acid was the major breakdown product produced by the wild-type strain. The acid-soluble degradation product produced by the mutant strain, however, was a complex mixture that contained a variety of small peptides as well as free amino acids. These results indicate that the same group of peptidases shown previously to function in the degradation of exogenously supplied peptides and in protein turnover during carbon starvation also lie on the pathway by which abnormal proteins are degraded.     

Utilization of dipeptides by Lactococcus lactis ssp. cremoris

Different strains of Lactococcus lactis ssp. cremoris hydrolyze peptides at different rates while the cell-free extracts of these strains all show the same or much higher rates of hydrolysis. These observations indicate that the uptake of peptides is the rate-limiting step in peptide hydrolysis. Utilization of leucyl-leucine by non-growing cells is competitively inhibited by the structurally related dipeptide alanyl-alanine. After hydrolysis of peptides, the amino acids are released into the medium and only a small fraction is accumulated and/or incorporated. This hydrolysis is independent of the synthesis of proteases indicating that the synthesis of proteases and peptidases are regulated differently. The specific growth rate of L. lactis ssp. cremoris E8 depends upon the amino acid source in the medium. No significant differences have been observed in the intracellular peptidase activities and the rates of peptide uptake between L. lactis ssp. cremoris E8 cells grown in different media, indicating that this growth rate is determined by the availability of amino acids in free amino acids or peptides.     

Localization of Peptidases in Lactococci

The localization of two aminopeptidases, an X-prolyl-dipeptidyl aminopeptidase, an endopeptidase, and a tripeptidase in Lactococcus lactis was studied. Polyclonal antibodies raised against each purified peptidase are specific and do not cross-react with other peptidases. Experiments were performed by immunoblotting after cell fractionation and by electron microscopy of immunogold-labeled peptidases. All peptidases were found to be intracellular. However, immunogold studies showed a peripheral labeling of the X-prolyl-dipeptidyl aminopeptidase, the tripeptidase, and the endopeptidase. This peripheral location was further supported by the detection of these three enzymes in cell membrane fractions in which none of the two aminopeptidases was present.     

An X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis: cloning, expression in Escherichia coli, and application for removal of N-terminal Pro-Pro from recombinant proteins

A novel pepX gene was cloned from isolated DNA of Lactococcus lactis by PCR. The deduced amino acid sequence of the 89-kDa protein showed 94, 93, 65, and 44% identity with the pepX protein from Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis, Lactobacillus delbruecki subsp. bulgaricus, and Lactobacillus helveticus, respectively, and contained a serine protease G-K-S-Y-L-G consensus motif. The pepX gene has been cloned into pET17b and was expressed at a high level in Escherichia coli BL21 (DE3) LysS. PepX was purified to approximate homogeneity with ammonium sulfate precipitation and DEAE Sephadex A-50 chromatography. Optimal pepX activity was observed at pH 8.0 and 37 degrees C. According to SDS-PAGE analysis, pepX has a molecular mass of approximately 89 kDa. The peptidase can remove completely the unwanted X-Pro from the N-terminal of the target protein, releasing the naturally active protein and peptide, revealing a prospective application of pepX in large-scale production of pharmaceutical protein and peptide products.     

Genetic and Environmental Effects on the Expression of Peptidases and Larval Viability in Drosophila Melanogaster

The peptidase system in Drosophila melanogaster, consisting of dipeptidase-A, dipeptidase-B, dipeptidase-C and the leucine aminopeptidases, was used as a model to study the adaptive significance of enzyme activity variation. The involvement of the peptidases in osmoregulation has been suggested from the ubiquitous distribution of peptidase activities in nearly all tissues and the high concentration of amino acids and oligopeptides in the hemolymph. Under this hypothesis, larvae counteract increases in environmental osmotic stress by hydrolyzing peptides into amino acids both intra- and extracellularly to increase physiological osmotic concentration. The expression of the peptidases was studied by assaying for peptidase activities in third instar larvae of isogenic lines, which were reared under increasing levels of environmental osmotic stress using either D-mannitol or NaCl. Second and third chromosome substitution isogenic lines were used to assess the relative contribution of regulatory and structural genes in enzyme activity variation. Results indicate that: (1) genetic variation exists for peptidase activities, (2) the effect of osmotic stress is highly variable among peptidases, (3) changes in peptidase activities in response to osmotic stress depend on both genetic background and osmotic effector and (4) peptidase activities are correlated with each other, but these phenotypic correlations depend on genetic background, osmotic effector, and level of osmotic stress. Osmotic concentration in the larval hemolymph is correlated with leucine aminopeptidase activity, but changes in hemolymph osmotic concentration in response to environmental osmotic stress depend on the osmotic effector in the environment. Although these findings suggest that genetic and environmental factors contribute significantly toward the expression of enzymes with similar functions, a relative larval viability study of genotypes that differed significantly in dipeptidase-B (DIP-B) activity revealed that low DIP-B activity did not confer any measurable reduction in larval viability under increasing levels of environmental osmotic stress. These negative results suggest that, either DIP-B does not play a major role in osmoregulation or differential osmoregulation is not related to egg to adult viability in these tests.     

Mechanism of proline-specific proteinases: (I) Substrate specificity of dipeptidyl peptidase IV from pig kidney and proline-specific endopeptidase from Flavobacterium meningosepticum

The substrate specificity of dipeptidyl peptidase IV (dipeptidyl peptide hydrolase, EC 3.4.14.5) from pig kidney and proline-specific endopeptidase from Flavobacterium meningosepticum, was investigated with a series of N-terminal unprotected (dipeptidyl peptidases IV) and succinylated dipeptidyl-p-nitroanilides (proline-specific endopeptidase). Both enzymes are specific for the S configuration of the amino-acid residue in P1 and P2 position if the penultimate residue is proline. In the case of alanine substrates (Ala in P1, dipeptidyl peptidase IV hydrolyzes such compounds where the configuration of the P2 residue is R. The penultimate residue with dipeptidyl peptidase IV can be, beside proline and alanine, dehydroproline, hydroxyproline and pipecolic acid. Proline substrates (Pro in P1) with an R configuration in P2 are inhibitors of the hydrolysis of proline substrates with an S,S configuration in an uncompetitive (dipeptidyl peptide IV) or mixed inhibition type (proline-specific endopeptidase). Derivatives of Gly-Pro-pNA where the N-terminal amino group is methylated are hydrolyzed by dipeptidyl peptidase IV.