Active-site residues in the type IV prepilin peptidase homologue PibD from the archaeon Sulfolobus solfataricus

Archaeal preflagellin peptidases and bacterial type IV prepilin peptidases belong to a family of aspartic acid proteases that cleave the leader peptides of precursor proteins with type IV prepilin signal sequences. The substrate repertoire of PibD from the crenarchaeon Sulfolobus solfataricus is unusually diverse. In addition to flagellin, PibD cleaves three sugar-binding proteins unique to this species and a number of proteins with unknown function. Here we demonstrate that PibD contains two aspartic acid residues that are essential for cleavage activity. An additional pair of aspartic acids in a large cytoplasmic loop is also important for function and is possibly involved in leader peptide recognition. Combining the results of transmembrane segment predictions and cysteine-labeling experiments, we suggest a membrane topology model for PibD with the active-site aspartic acid residues exposed to the cytosol.     

Archaeal homolog of bacterial type IV prepilin signal peptidases with broad substrate specificity

A large number of secretory proteins in the thermoacidophile Sulfolobus solfataricus are synthesized as a precursor with an unusual leader peptide that resembles bacterial type IV prepilin signal sequences. This set of proteins includes the flagellin subunit but also various solute binding proteins. Here we describe the identification of the S. solfataricus homolog of bacterial type IV prepilin peptidases, termed PibD. PibD is an integral membrane protein that is phylogenetically related to the bacterial enzymes. When heterologously expressed in Escherichia coli, PibD is capable of processing both the flagellin and glucose-binding protein (GlcS) precursors. Site-directed mutagenesis of the GlcS signal peptide shows that the substrate specificity of PibD is consistent with the variations found in proteins with type IV prepilin-like signal sequences of S. solfataricus. We conclude that PibD is responsible for the processing of these secretory proteins in S. solfataricus.     

Type I signal peptidase: an overview

The signal hypothesis suggests that proteins contain information within their amino acid sequences for protein targeting to the membrane. These distinct targeting sequences are cleaved by specific enzymes known as signal peptidases. There are various type of signal peptidases known such as type I, type II, and type IV. Type I signal peptidases are indispensable enzymes, which catalyze the cleavage of the amino-terminal signal-peptide sequences from preproteins, which are translocated across biological membranes. These enzymes belong to a novel group of serine proteases, which generally utilize a Ser-Lys or Ser-His catalytic dyad instead of the prototypical Ser-His-Asp triad. Despite having no distinct consensus sequence other than a commonly found 'Ala-X-Ala' motif preceding the cleavage site, signal sequences are recognized by type I signal peptidase with high fidelity. Type I signal peptidases have been found in bacteria, archaea, fungi, plants, and animals. In this review, I present an overview of bacterial type I signal peptidases and describe some of their properties in detail.     

Regulated intramembrane proteolysis of Bri2 (Itm2b) by ADAM10 and SPPL2a/SPPL2b

Presenilin, the catalytic component of the gamma-secretase complex, type IV prepilin peptidases, and signal peptide peptidase (SPP) are the founding members of the family of intramembrane-cleaving GXGD aspartyl proteases. SPP-like (SPPL) proteases, such as SPPL2a, SPPL2b, SPPL2c, and SPPL3, also belong to the GXGD family. In contrast to gamma-secretase, for which numerous substrates have been identified, very few in vivo substrates are known for SPP and SPPLs. Here we demonstrate that Bri2 (Itm2b), a type II-oriented transmembrane protein associated with familial British and Danish dementia, undergoes regulated intramembrane proteolysis. In addition to the previously described ectodomain processing by furin and related proteases, we now describe that the Bri2 protein, similar to gamma-secretase substrates, undergoes an additional cleavage by ADAM10 in its ectodomain. This cleavage releases a soluble variant of Bri2, the BRICHOS domain, which is secreted into the extracellular space. Upon this shedding event, a membrane-bound Bri2 N-terminal fragment remains, which undergoes intramembrane proteolysis to produce an intracellular domain as well as a secreted low molecular weight C-terminal peptide. By expressing all known SPP/SPPL family members as well as their loss of function variants, we demonstrate that selectively SPPL2a and SPPL2b mediate the intramembrane cleavage, whereas neither SPP nor SPPL3 is capable of processing the Bri2 N-terminal fragment.     

Degradation of a signal peptide by protease IV and oligopeptidase A.

The degradation of the prolipoprotein signal peptide in vitro by membranes, cytoplasmic fraction, and two purified major signal peptide peptidases from Escherichia coli was followed by reverse-phase liquid chromatography (RPLC). The cytoplasmic fraction hydrolyzed the signal peptide completely into amino acids. In contrast, many peptide fragments accumulated as final products during the cleavage by a membrane fraction. Most of the peptides were similar to the peptides formed during the cleavage of the signal peptide by the purified membrane-bound signal peptide peptidase, protease IV. Peptide fragments generated during the cleavage of the signal peptide by protease IV and a cytoplasmic enzyme, oligopeptidase A, were identified from their amino acid compositions, their retention times during RPLC, and knowledge of the amino acid sequence of the signal peptide. Both enzymes were endopeptidases, as neither dipeptides nor free amino acids were formed during the cleavage reactions. Protease IV cleaved the signal peptide predominantly in the hydrophobic segment (residues 7 to 14). Protease IV required substrates with hydrophobic amino acids at the primary and the adjacent substrate-binding sites, with a minimum of three amino acids on either side of the scissile bond. Oligopeptidase A cleaved peptides (minimally five residues) that had either alanine or glycine at the P'1 (primary binding site) or at the P1 (preceding P'1) site of the substrate. These results support the hypothesis that protease IV is the major signal peptide peptidase in membranes that initiates the degradation of the signal peptide by making endoproteolytic cuts; oligopeptidase A and other cytoplasmic enzymes further degrade the partially degraded portions of the signal peptide that may be diffused or transported back into the cytoplasm from the membranes.     

The potential active site of the lipoprotein-specific (type II) signal peptidase of Bacillus subtilis.

Type II signal peptidases (SPase II) remove signal peptides from lipid-modified preproteins of eubacteria. As the catalytic mechanism employed by type II SPases was not known, the present studies were aimed at the identification of their potential active site residues. Comparison of the deduced amino acid sequences of 19 known type II SPases revealed the presence of five conserved domains. The importance of the 15 best conserved residues in these domains was investigated using the type II SPase of Bacillus subtilis, which, unlike SPase II of Escherichia coli, is not essential for viability. The results showed that only six residues are important for SPase II activity. These are Asp-14, Asn-99, Asp-102, Asn-126, Ala-128, and Asp-129. Only Asp-14 was required for stability of SPase II, indicating that the other five residues are required for catalysis, the active site geometry, or the specific recognition of lipid-modified preproteins. As Asp-102 and Asp-129 are the only residues invoked in the known catalytic mechanisms of proteases, we hypothesize that these two residues are directly involved in SPase II-mediated catalysis. This implies that type II SPases belong to a novel family of aspartic proteases.     

Amino acid substitutions in pilin of Pseudomonas aeruginosa. Effect on leader peptide cleavage, amino-terminal methylation, and pilus assembly.

A total of 37 separate mutants containing single and multiple amino acid substitutions in the leader and amino-terminal conserved region of the Type IV pilin from Pseudomonas aeruginosa were generated by oligonucleotide-directed mutagenesis. The effect of these substitutions on the secretion, processing, and assembly of the pilin monomers into mature pili was examined. The majority of substitutions in the highly conserved amino-terminal region of the pilin monomer had no effect on piliation. Likewise, substitution of several of the residues within the six amino acid leader sequence did not affect secretion and leader cleavage (processing), including replacement of one or both of the positively charged lysine residues with uncharged or negatively charged amino acids. One characteristic of the Type IV pili is the presence of an amino-terminal phenylalanine after leader peptide cleavage which is N-methylated prior to assembly of pilin monomers into pili. Substitution of the amino-terminal phenylalanine with a number of other amino acids, including polar, hydrophobic, and charged residues, did not affect proper leader cleavage and subsequent assembly into pili. Amino-terminal sequencing showed that the majority of substitute residues were also methylated. Substitution of the glycine residue at the -1 position to the cleavage site resulted in the inability to cleave the prepilin monomers and blocked the subsequent assembly of monomers into pili. These results indicate that despite the high degree of conservation in the amino-terminal sequences of the Type IV pili, N-methylphenylalanine at the +1 position relative to the leader peptide cleavage site is not strictly required for pilin assembly. N-Methylation of the amino acids substituted for phenylalanine was shown to have taken place in four of the five mutants tested, but it remains unclear as to whether pilin assembly is dependent on this modification. Recognition and proper cleavage of the prepilin by the leader peptidase appears to be dependent only on the glycine residue at the -1 position. Cell fractionation experiments demonstrated that pilin isolated from mutants deficient in prepilin processing and/or assembly was found in both inner and outer membrane fractions, indistinguishable from the results seen with the wild type.     

Signal peptidases and signal peptide hydrolases

Signal peptidases, the endoproteases that remove the amino-terminal signal sequence from many secretory proteins, have been isolated from various sources. Seven signal peptidases have been purified, two fromE. coli, two from mammalian sources, and three from mitochondrial matrix. The mitochondrial enzymes are soluble and function as a heterogeneous dimer. The mammalian enzymes are isolated as a complex and share a common glycosylated subunit. The bacterial enzymes are isolated as monomers and show no sequence homology with each other or the mammalian enzymes. The membrane-bound enzymes seem to require a substrate containing a consensus sequence following the –3, –1 rule of von Heijne at the cleavage site; however, processing of the substrate is strongly influenced by the hydrophobic region of the signal peptide. The enzymes appear to recognize an unknown three-dimensional motif rather than a specific amino acid sequence around the cleavage site. The matrix mitochondrial enzymes are metallo-endopeptidases; however, the other signal peptidases may belong to a unique class of proteases as they are resistant to chelators and most protease inhibitors. There are no data concerning the substrate binding site of these enzymes. In vivo, the signal peptide is rapidly degraded. Three different enzymes inEscherichia coli that can degrade a signal peptidein vitro have been identified. The intact signal peptide is not accumulated in mutants lacking these enzymes, which suggests that these peptidases individually are not responsible for the degredation of an intact signal peptidein vivo. It is speculated that signal peptidases and signal peptide hydrolases are integral components of the secretory pathway and that inhibition of the terminal steps can block translocation.     

Processing and methylation of PulG, a pilin-like component of the general secretory pathway of Klebsiella oxytoca

The signal sequence of the Klebsiella oxytoca pulG gene product, which is required for extracellular secretion of the enzyme pullulanase, is similar in many respects to the corresponding segment of the precursors of type IV (me-Phe) pilins. The significance of this similarity is confirmed by the observation that the pulO gene product processes prePulG at the consensus type IV prepilin peptidase cleavage site at the amino-terminal end of the PulG signal sequence. Like most type IV pilins, processed PuiG was found to have a methylated amino-terminal phenylaianine residue. Site-directed mutagenesis was used to replace amino acids in prePulG that correspond to residues shown by others to be essential for processing, methylation and assembly of type IV pilins. The glycine residue on the amino-terminal side of the prePulG cleavage site is absolutely required for processing and for pullulanase secretion. The glutamate residue at position 11 (+5) is also required for pullulanase secretion but not for processing or methylation. This result contrasts with that reported for corresponding variants of Pseudomonas aeruginosa type IV prepilin, which were processed but only inefficiently IV-methylated. Cleavage of prePulG and pullulanase secretion were both unaffected by replacement of the phenylalanine residue on the car-boxy-terminal side of the cleavage site by leucine, isoleucine or valine, by a conservative substitution within the hydrophobic core of the prePulG signal sequence, or by a glutamine to proline substitution within the processed segment. However, replacement of the same glutamine residue by arginine abolished secretion without affecting either processing or methylation.     

Intramembrane Proteolysis by Signal Peptide Peptidases: A Comparative Discussion of GXGD-type Aspartyl Proteases

Intramembrane-cleaving proteases are required for reverse signaling and membrane protein degradation. A major class of these proteases is represented by the GXGD-type aspartyl proteases. GXGD describes a novel signature sequence that distinguishes these proteases from conventional aspartyl proteases. Members of the family of the GXGD-type aspartyl proteases are the Alzheimer disease-related γ-secretase, the signal peptide peptidases and their homologs, and the bacterial type IV prepilin peptidases. We will describe the major biochemical and functional properties of the signal peptide peptidases and their relatives. We then compare these properties with those of γ-secretase and discuss common mechanisms but also point out a number of substantial differences.During the last years, a number of intramembrane-cleaving proteases termed I-CLiPs³ have been identified (1). I-CLiPs are generally involved in regulated intramembrane proteolysis (2). Upon shedding of a large part of the ectodomain of membrane proteins, the remaining membrane-retained stub is cleaved by specialized proteases within the hydrophobic lipid membrane. Generally, this cleavage can have two predominant biological functions: first, signaling via the liberated ICD within the substrate-expressing cell (reverse signaling) (2); and second, degradation of membrane-retained stubs, which are not required for any further biological function (3). I-CLiPs of three protease classes, metalloproteases, serine proteases, and aspartyl proteases, have been discovered so far (see accompanying minireview by Wolfe (44)).Intramembrane-cleaving aspartyl proteases are represented by the class of the GXGD-type proteases (4). These are unconventional aspartyl proteases that, like the conventional aspartyl proteases, utilize two critical aspartyl residues for peptide bond cleavage. However, in contrast to the conventional proteases, the critical aspartyl residues are located within two TMDs (Fig. 1A). Moreover, these aspartyl residues are embedded in active-site motifs that are completely different from those of conventional aspartyl proteases. The class of GXGD-type aspartyl proteases is currently represented by three different protease families, the most prominent of which is the PS family, providing the catalytically active subunit of γ-secretase (Fig. 1A) (4). PS/γ-secretase is the I-CLiP that liberates amyloid β-peptide, the major component of senile plaques in Alzheimer disease patients (5). In addition, the bacterial type IV prepilin peptidases also belong to the class of the GXGD-type proteases (6). Besides these two protease families, two additional subfamilies of related proteases that also belong to the GXGD-type aspartyl protease family have been identified. These include SPP as well as the SPP homologs, the SPP-like (SPPL) proteases (Fig. 1A) (7, 8).Open in a separate windowFIGURE 1.A, schematic representation of SPPL2a/b, a member of the SPP/SPPL family, and PS, the catalytic core of theγ-secretase. Note the opposite topology of the active sites (indicated by arrows) of the two proteases and their substrates, APP for PS and TNFα for SPPL2a/b. B, proteolytic processing of APP and TNFα. Shedding releases the extracellular part of APP (APPs) and TNFα (TNFα soluble). In the case of APP, a C-terminal fragment (APP CTF), and in case of TNFα, an N-terminal fragment (TNFα NTF) are produced. These membrane-bound fragments are substrate to intramembrane cleavage by PS or SPPL2a/b, respectively, releasing small peptides to the extracellular space (Aβ and TNFα C-domain, respectively) and to the cytosol (APP intracellular domain (AICD) and TNFα ICD), respectively). TNFα FL, full-length TNFα.We will first describe the biochemical, functional, and structural properties of SPP family members. By comparison of these properties, we will then identify common mechanisms of intramembrane proteolysis by GXGD-type proteases but also point out some fundamental differences.     

Kinetics and sequence specificity of processing of prepilin by PilD, the type IV leader peptidase of Pseudomonas aeruginosa.

PilD, originally isolated as an essential component for the biogenesis of the type IV pili of Pseudomonas aeruginosa, is a unique endopeptidase responsible for processing the precursors of the P. aeruginosa pilin subunits. It is also required for the cleavage of the leader peptides from the Pdd proteins, which are essential components of an extracellular secretion pathway specific for the export of a number of P. aeruginosa hydrolytic enzymes and toxins. Substrates for PilD are initially synthesized with short, i.e., 6- to 8-amino-acid-long, leader peptides with a net basic charge and share a high degree of amino acid homology through the first 16 to 30 residues at the amino terminus. In addition, they all have a phenylalanine residue at the +1 site relative to the cleavage site, which is N methylated prior to assembly into the oligomeric structures. In this study, the kinetics of leader peptide cleavage from the precursor of the P. aeruginosa pilin subunit by PilD was determined in vitro. The rates of cleavage were compared for purified enzyme and substrate as well as for enzyme and substrate contained within total membranes extracted from P. aeruginosa strains overexpressing the cloned pilD or pilA genes. Optimal conditions were obtained only when both PilD and substrate were contained within total membranes. PilD catalysis of P. aeruginosa prepilin followed normal Michaelis-Menten kinetics, with a measured apparent Km of approximately 650 microM, and a kcat of 180 min-1. The kinetics of PilD processing of another type IV pilin precursor, that from Neisseria gonorrhoeae with a 7-amino-acid-long leader peptide, were essentially the same as that measured for wild-type P. aeruginosa prepilin. Quite different results were obtained for a number of prepilin substrates containing substitutions at the conserved phenylalanine at the +1 position relative to the cleavage site, which were previously shown to be well tolerated in vivo. Substitutions of methionine, serine, and cysteine for phenylalanine show that Km values remain close to that measured for wild-type substrate, while kcat and kcat/Km values were significantly decreased. This indicates that while the affinity of enzyme for substrate is relatively unaffected by the substitutions, the maximum rate of catalysis favors a phenylalanine at this position. Interesting, PilD cleavage of one mutated pillin (asparagine) resulted in a lower Km value of 52.5 microM, which indicates a higher affinity for the enzyme, as well as a lower kcat value of 6.1 min m(-1). This suggests that it may be feasible to design peptide inhibitors of PilD.     

Different Minimal Signal Peptide Lengths Recognized by the Archaeal Prepilin-Like Peptidases FlaK and PibD

In Archaea, the preflagellin peptidase (a type IV prepilin-like peptidase designated FlaK in Methanococcus voltae and Methanococcus maripaludis) is the enzyme that cleaves the N-terminal signal peptide from preflagellins. In methanogens and several other archaeal species, the typical flagellin signal peptide length is 11 to 12 amino acids, while in other archaea preflagellins possess extremely short signal peptides. A systematic approach to address the signal peptide length requirement for preflagellin processing is presented in this study. M. voltae preflagellin FlaB2 proteins with signal peptides 3 to 12 amino acids in length were generated and used as a substrate in an in vitro assay utilizing M. voltae membranes as an enzyme source. Processing by FlaK was observed in FlaB2 proteins containing signal peptides shortened to 5 amino acids; signal peptides 4 or 3 amino acids in length were unprocessed. In the case of Sulfolobus solfataricus, where the preflagellin peptidase PibD has broader substrate specificity, some predicted substrates have predicted signal peptides as short as 3 amino acids. Interestingly, the shorter signal peptides of the various mutant FlaB2 proteins not processed by FlaK were processed by PibD, suggesting that some archaeal preflagellin peptidases are likely adapted toward cleaving shorter signal peptides. The functional complementation of signal peptidase activity by FlaK and PibD in an M. maripaludis ΔflaK mutant indicated that processing of preflagellins was detected by complementation with either FlaK or PibD, yet only FlaK-complemented cells were flagellated. This suggested that a block in an assembly step subsequent to signal peptide removal occurred in the PibD complementation.The bacterial type IV prepilin peptidase (TFPP) is a well-characterized enzyme belonging to a family of novel aspartic acid proteases (20). It is responsible for the cleavage of N-terminal signal peptides from prepilins and pseudopilins, prior to their incorporation into the type IV pilus structure (22, 30, 31). The prepilin peptidase is also responsible for the processing of prepilin-like proteins needed for type II secretion (22). In Archaea, the existence of bacterial TFPP-like enzymes has also been reported, and they have been most extensively studied in relation to the assembly of the archaeal flagellum. In the euryarchaeotes Methanococcus maripaludis and Methanococcus voltae, the preflagellin peptidase FlaK was demonstrated to be responsible for cleaving the N-terminal signal peptide from the preflagellin prior to its incorporation into the growing flagellar filament, a step essential to flagellar assembly (6, 7, 26). In Sulfolobus solfataricus, an acidophilic crenarchaeote, the equivalent enzyme, PibD, was also shown to process preflagellins (4). Site-directed mutagenesis of FlaK and PibD demonstrated that both aspartic acid residues that aligned with aspartic acid residues essential for bacterial TFPP activity were also essential in the archaeal enzymes (6, 32), indicating that the two archaeal peptidases belong with the bacterial TFPPs in this novel family of aspartic acid proteases (20). More recently, an additional archaeal TFPP was found to be required for cleavage of the prepilin substrates (33) that are assembled into the unique pili of M. maripaludis (37).The substrate specificity of the archaeal preflagellin peptidase remains an open question. Like prepilin peptidases, FlaK in M. voltae has stringent requirements for the amino acids surrounding the cleavage site of the substrate, especially the −1 glycine, −2 and −3 lysines, and the +3 glycine (numbers given relative to the cleavage site) (35); the last position was conserved in all archaeal flagellins (25). Upon N-terminal sequence alignment of all available archaeal flagellin amino acid sequences at the predicted cleavage site, it was found that most archaeal preflagellin signal peptides are quite conserved in length, with the typical flagellin signal peptide being 11 to 12 amino acids in length (Table ​(Table1).1). It is speculated that while a certain amount of flexibility might exist, some optimum and minimum length probably exists that is crucial for the juxtaposition of the signal peptide and signal peptidase with respect to each other and the membrane (18). A recent study examining possible type IV pilin-like substrates in archaea using the FlaFind program indicated that such substrates may be more widespread than initially thought (33). Since in Methanococcus the pilins are processed by a second TFPP (EppA) (33), it is very possible that the preflagellins might be the only substrates of FlaK in these archaea.

TABLE 1.

N-terminal amino acid alignment of selected archaeal flagellin sequences^a

Proline residues in the maturation and degradation of peptide hormones and neuropeptides

The proteases involved in the maturation of regulatory peptides like those of broader specificity normally fail to cleave peptide bonds linked to the cyclic amino acid proline. This generates several mature peptides with N-terminal X-Pro-sequences. However, in certain non-mammalian tissues repetitive pre-sequences of this type are removed by specialized dipeptidyl (amino)peptidases during maturation. In mammals, proline-specific proteases are not involved in the biosynthesis of regulatory peptides, but due to their unique specificity they could play an important role in the degradation of them. Evidence exists that dipeptidyl (amino)peptidase IV at the cell surface of endothelial cells sequesters circulating peptide hormones which are then susceptible to broader aminopeptidase attack. The cleavage of several neuropeptides by prolyl endopeptidase has been demonstrated in vitro, but its role in the brain is questionable since the precise localization of the protease is not clarified.     

Can the topological distribution of membrane spanning amino acid residues be responsible for the recognition of signal peptides by signal peptide peptidases?

Signal peptides are selectively recognized and degraded by membrane associated proteases called as signal peptide peptidases. The hydrolysis of the signal peptide occurs only after its cleavage from the precursor. The possible reasons for this selectivity have been investigated. The results indicate that in signal peptides, leucine residues are clustered to a large extent on the same side of the membrane spanning alpha helix as the polar residues, but are distinctly separated along the length of the axis. Such topological differences in the distribution of amino acids on the surface of the membrane spanning alpha helix may play a crucial role in selective degradation of signal peptides.     

A new tyrosine-specific chymotrypsin-like and angiotensin-degrading serine proteinase from Vipera lebetina snake venom

Vipera lebetina venom contains different metallo- and serine proteinases that affect coagulation and fibrin(ogen)olysis. A novel serine proteinase from V. Lebetina venom having ChymoTrypsin Like Proteolytic activity (VLCTLP) was purified to homogeneity from the venom using Sephadex G-100sf, DEAE-cellulose, heparin-agarose and FPLC on Superdex 75 chromatographies. VLCTLP is a glycosylated serine proteinase with a molecular mass of 41926 Da. It reacts with N-acetyl-l-tyrosine ethyl ester (ATEE) but not with Suc-Ala-Ala-Pro-Phe-pNA or Suc-Ala-Ala-Pro-Leu-pNA. The complete amino acid sequence of the VLCTLP is deduced from the nucleotide sequence of the cDNA encoding this protein. The full-length cDNA sequence of the VLCTLP encodes open reading frame of 257 amino acid residues that includes a putative signal peptide of 18 amino acids, a proposed activation peptide of six amino acid residues and serine proteinase of 233 amino acid residues. VLCTLP belongs to the S1 (chymotrypsin) subfamily of proteases. The multiple alignment of its deduced amino acid sequence showed structural similarity with other serine proteases from snake venoms. The protease weakly hydrolyses azocasein, Aα-chain and more slowly Bβ-chain of fibrinogen. VLCTLP does not cleave fibrin and has no gelatinolytic activity. Specificity studies against peptide substrates (angiotensin I and II, oxidized insulin B-chain, glucagon, fibrinogen fragments etc.) showed that VLCTLP catalysed the cleavage of peptide bonds after tyrosine residues. VLCTLP is the only purified and characterized serine proteinase from snake venoms that catalyses ATEE hydrolysis. We detected ATEE-hydrolysing activities also in 9 different Viperidae and Crotalidae venoms.     

Expression of the Bacillus subtilis TasA signal peptide leads to cell death in Escherichia coli due to inefficient cleavage by LepB

Bacillus subtilis has five type I signal peptidases, one of these, SipW, is an archaeal-like peptidase. SipW is expressed in an operon (tapA-sipW-tasA) and is responsible for removing the signal peptide from two proteins: TapA and TasA. It is unclear from the signal peptide sequence of TasA and TapA, why an archaeal-like signal peptidase is required for their processing. Bioinformatic analysis of TasA and TapA indicates that both contain highly similar signal peptide cleavage sites, both predicted to be cleaved by Escherichia coli signal peptidase I, LepB. We show that expressing full length TasA in E. coli is toxic and leads to cell death. To determine if this phenotype is due to the inability of the E. coli LepB to process the TasA signal peptide, we fused the TasA signal peptide and two amino acids of mature TasA (up to P2′) to both maltose binding protein (MBP) and β-lactamase (Bla). We observed a defect in secretion, indicated by an abundance of unprocessed protein with both TasA-MBP and TasA-Bla fusions. A series of mutations in both TasA-MBP and TasA-Bla were made around the junction of the TasA signal peptide and the fusion protein. Both of these studies indicate that residues around the predicted TasA signal sequence cleavage site, particularly the sequence from P3 to P2′, inhibit processing by LepB. The cell death observed when TasA and TasA signal sequence fusion proteins are expressed is likely due to the TasA signal peptide blocking LepB and thereby the general secretion pathway.     

Processing of the dual targeted precursor protein of glutathione reductase in mitochondria and chloroplasts

Pea glutathione reductase (GR) is dually targeted to mitochondria and chloroplasts by means of an N-terminal signal peptide of 60 amino acid residues. After import, the signal peptide is cleaved off by the mitochondrial processing peptidase (MPP) in mitochondria and by the stromal processing peptidase (SPP) in chloroplasts. Here, we have investigated determinants for processing of the dual targeting signal peptide of GR by MPP and SPP to examine if there is separate or universal information recognised by both processing peptidases. Removal of 30 N-terminal amino acid residues of the signal peptide (GRDelta1-30) greatly stimulated processing activity by both MPP and SPP, whereas constructs with a deletion of an additional ten amino acid residues (GRDelta1-40) and deletion of 22 amino acid residues in the middle of the GR signal sequence (GRDelta30-52) could be cleaved by SPP but not by MPP. Numerous single mutations of amino acid residues in proximity of the cleavage site did not affect processing by SPP, whereas mutations within two amino acid residues on either side of the processing site had inhibitory effect on processing by MPP with a nearly complete inhibition for mutations at position -1. Mutation of positively charged residues in the C-terminal half of the GR targeting peptide inhibited processing by MPP but not by SPP. An inhibitory effect on SPP was detected only when double and triple mutations were introduced upstream of the cleavage site. These results indicate that: (i) recognition of processing site on a dual targeted GR precursor differs between MPP and SPP; (ii) the GR targeting signal has similar determinants for processing by MPP as signals targeting only to mitochondria; and (iii) processing by SPP shows a low level of sensitivity to single mutations on targeting peptide and likely involves recognition of the physiochemical properties of the sequence in the vicinity of cleavage rather than a requirement for specific amino acid residues.     

The gene encoding the prepilin peptidase involved in biosynthesis of pilus colonization factor antigen III (CFA/III) of human enterotoxigenic Escherichia coli.

The assembly of pilus colonization factor antigen III (CFA/III) of human enterotoxigenic Escherichia coli requires the processing of CFA/III major pilin (CofA) by a peptidase, likely another type IV pilus formation system. Western blot analysis of CofA reveals that CofA is produced initially as a 26.5-kDa preform pilin (prepilin) and then processed to 20.5-kDa mature pilin by a prepilin peptidase. This processing is essential for exportation of the CofA from the cytoplasm to the periplasm. In this experiment, the structural gene, cofP, encoding CFA/III prepilin peptidase which cleavages at the Gly-30-Met-31 junction of CofA was identified, and the nucleotide sequence of the gene was determined. CofP consists of 819 bp encoding a 273-amino acid protein with a relative molecular mass of 30,533 Da. CofP is predicted to be localized in the inner membrane based on its hydropathy index. The amino acid sequence of CofP shows a high degree of homology with other prepilin peptidases which play a role in the assembly of type IV pili in several gram-negative bacteria.     

Amino acid sequence of 37 residues surrounding Nxi-pyridoxyllysine in mitochondrial aspartate aminotransferase.

Following reduction with NaBH4, carboxymethylation and cleavage with cyanogen bromide, a peptide of thirty-seven amino acid residues containing N^?-pyridoxyllysine (coenzyme binding lysine) was isolated from the mitochondrial aspartate aminotransferase of pig heart by Sephadex G-75 column chromatography and then preparative polyacrylamide gel electrophoresis. The primary structure of this peptide was determined to be Ala-Tyr-Gln-Gly-Phe-Ala-Ser-Gly-Asp-Gly-Asn-Lys-Asp-Ala-Trp-Ala-Val-Arg-His-Phe-Ile-Glu-Gln-Gly-Ile-Asn-Val-Cys-Leu-Cys-Gln-Ser-Tyr-Ala-(Pxy) Lys-Asn-Met. Its structure showed a high degree of homology with the corresponding part of the cytoplasmic isozyme.     

Mutational Analysis of Plasmid R64 Thin Pilus Prepilin: the Entire Prepilin Sequence Is Required for Processing by Type IV Prepilin Peptidase

The thin pili of IncI1 plasmid R64, which is required for conjugation in liquid media, belong to the type IV pilus family. They consist of a major subunit, the pilS product, and a minor component, one of the seven pilV products. The pilS product is first synthesized as a 22-kDa prepilin, processed to a 19-kDa mature pilin by the function of the pilU product, and then secreted outside the cell. The mature pilin is assembled to form a thin pilus with the pilV product. To reveal the relationship between the structure and function of the pilS product, 27 missense mutations, three N-terminal deletions, and two C-terminal deletions were constructed by PCR and site-directed mutagenesis. The characteristics of 32 mutant pilS products were analyzed. Four pilS mutant phenotype classes were identified. The products of 10 class I mutants were not processed by prepilin peptidase; the extracellular secretion of the products of two class II mutants was inhibited; from 11 class III mutants, thin pili with reduced activities in liquid mating were formed; from 9 class IV mutants, thin pili with mating activity similar to that of the wild-type pilS gene were formed. The point mutations of the class I mutants were distributed throughout the prepilin sequence, suggesting that processing of the pilS product requires the entire prepilin sequence.Type IV pili are flexible, rod-like, polarly inserted surface appendages protruding from the cell surface of gram-negative bacteria including Pseudomonas aeruginosa, Bacteroides nodosus, Neisseria gonorrhoeae, Moraxella bovis, Vibrio cholerae, and enteropathogenic and enterotoxigenic Escherichia coli (9, 19, 20, 23, 27, 32). Type IV pili promote the attachment of bacterial pathogens to receptors of host cells during colonization, and they mediate the bacterial locomotion called twitching motility of P. aeruginosa (35) and the social gliding motility of Myxococcus xanthus (36). In addition, they act as receptors for pilus-specific bacteriophage (6).Type IV pili are polymers of type IV pilin subunits (23, 27), which are produced from type IV prepilins by the function of prepilin peptidases (18). In many cases, the N-terminal amino acid of mature pilin is phenylalanine and is N-methylated. In P. aeruginosa, both processing of prepilin and N-methylation of mature pilin are catalyzed by a single bifunctional enzyme, the PilD protein (28). Among all type IV pilins, the N-terminal region including the cleavage site is highly conserved. Particularly, the C-terminal amino acid of the prepeptide is invariantly glycine, and the fifth amino acid of mature pilin is always glutamic acid. The C-terminal one-third of mature pilin forms a disulfide loop between two conserved cysteine residues (21, 25).During bacterial conjugation, the donor cells harboring self-transmissible plasmids synthesize sex pili encoded by the genes on the plasmids (6). Sex pili of donor cells create a specific contact with recipient cells, leading to the formation of a mating pair. IncI1 plasmids such as R64 and ColIb-P9 form two types of sex pili, a thick rigid pilus and a thin flexible one (1, 2). Thick rigid pili are required for both surface and liquid mating, while thin flexible pili are required only for liquid mating. Cells producing R64 thin pili become sensitive to bacteriophages Iα and PR64FS, which adsorb to the shaft and tip of IncI1 thin pilus, respectively (4, 5).DNA sequence analysis of the R64 pil region responsible for thin-pilus formation revealed that the pil region consists of 14 genes, pilI through pilV, and that several pil products contain amino acid sequence homology with proteins involved in type IV pilus biogenesis (11) (Fig. ​(Fig.1A).1A). Thus, the R64 thin pilus was shown to belong to the type IV family, specifically group IVB, of pili. Open in a separate windowFIG. 1(A) Organization of the tra-pil region of plasmid R64. The horizontal bold line represents a restriction map. B, BglII; E, EcoRI; H, HindIII. The open bar above the map represents the extent of movement of the EcoRI site through DNA rearrangement of the shufflon. Below the map, the open reading frames are represented by open bars. tra, transfer; pil, formation of thin pilus; shf, shufflon; rci, recombinase for the shufflon. DNA regions of pKK641 and pKK692 are indicated above the map. The cross on pKK641 marks the location of the pilS1 mutation. (B) Amino acid sequence of the PilS protein. The downward arrow indicates the type IV prepilin cleavage site. The conserved glycine, glutamic acid, and two cysteine residues are indicated by the outline letters.R64 and ColIb-P9 thin pili were sedimented by ultracentrifugation from the culture medium, in which E. coli cells harboring R64- and ColIb-P9-derived plasmids had grown, and purified by CsCl density gradient centrifugation (13, 37). In negatively stained thin-pilus samples, long rods with a diameter of 6 nm, characteristic of type IV pili, were observed under an electron microscope. R64 and ColIb-P9 thin pili consist of a major 19-kDa pilin protein, the product of the pilS gene, and a minor 45-kDa protein, the product of the pilV gene. The amino acid sequence of the pilS product contains residues characteristic of a type IV prepilin, although its prepeptide is unusually long (Fig. ​(Fig.1B).1B). The pilS product is first synthesized as a 22-kDa prepilin and then cleaved between Gly²³ and Trp²⁴ to produce a 19-kDa protein via the function of the pilU product, prepilin peptidase. The N-terminal amino group of the processed PilS protein appears to be modified. The C-terminal segments of the pilV gene are under the control of shufflon DNA rearrangement mediated by the rci product (15, 16). The shufflon determines the recipient specificity in liquid mating by converting seven C-terminal segments of the pilV product (13, 14). The pilV product also carries a type IV prepilin cleavage site. Formation of PilV-specific cell aggregates by ColIb-P9 and R64 thin pili was shown and suggested to play an important role in liquid mating (37).Recently, the three-dimensional structure of the N. gonorrhoeae pilin was determined by X-ray crystallography (21). The monomer structure was an α-β-roll fold with an 85-Å N-terminal α-helical spine. The gross monomer structure resembles a ladle with the N-terminal half of the α-helical spine forming the handle. From the monomer structure, a model of fiber structure with a parameter of five turns per helix, 41-Å pitch, and 60-Å diameter (34) was proposed. In the model, the N-terminal α helices gather in the center of the fiber, forming a core of coiled α helices banded by a β sheet. Slight similarities including two conserved cysteine residues are noted between the amino acid sequences of R64 and N. gonorrhoeae pilins, suggesting that the two proteins fold similarly and then assemble to form similar fibers.In N. gonorrhoeae, P. aeruginosa, and V. cholerae, amino acid substitutions were introduced into the prepeptide and highly conserved N-terminal regions of prepilin genes (3, 22, 26). The mutant genes were analyzed with respect to processing, secretion, and function. The importance of the conserved glycine in the prepeptide and some hydrophobic amino acids in the N-terminal region has been established.This work was performed to reveal the relationship between the structure and function of the pilS product. Thirty-two missense and deletion mutations were introduced throughout the entire sequence of the pilS product by PCR and site-directed mutagenesis. The characteristics of the mutant pilS products were analyzed in terms of processing, secretion, and assembly to active thin pili with the pilV product. The activities of the thin pili composed of the mutant pilS genes were determined as the transfer frequency in liquid mating and the sensitivity to IncI1-specific phages.