Neutrophils can generate their activator neutrophil-activating peptide 2 by proteolytic cleavage of platelet-derived connective tissue-activating peptide III

We have investigated the conditions that lead to the generation of the neutrophil-activating peptide 2 (NAP-2) from its precursor, the platelet-derived connective tissue-activating peptide III (CTAP-III). Lysed platelets were found to contain predominantly CTAP-III in the cytosolic fraction, but further truncated derivatives, among these NAP-2, occurred tightly bound to the membrane fraction of fresh platelets. NAP-2 biological activity, as measured by the induction of enzyme release in human neutrophils [polymorphonuclear leukocytes (PMN)] was released by stimulated platelets to a low degree. Much higher activities were formed in the presence of peripheral blood leukocytes. Coincubation of CTAP-III with PMN resulted in the almost complete conversion of the precursor to NAP-2, as did incubation of CTAP-III with PMN-conditioned medium. In both situations, the generation of NAP-2 could be prevented by serine-protease inhibitor phenylmethylsulfonyl fluoride but not by inhibitors specific for Ca(2+)-dependent or thiol proteases. From several PMN-derived proteases tested, only cathepsin G had the capacity to cleave CTAP-III into NAP-2 with high specificity and in a relatively short period of time (30 min). Our data indicate that NAP-2, released by platelets in small quantities, could cause PMN to enter into a positive feedback cycle by initiating the secretion of serine proteases, which in turn could convert platelet-derived CTAP-III into biologically active NAP-2.     

Using peptide libraries to identify optimal cleavage motifs for proteolytic enzymes

Proteases and peptidases are involved in a vast array of fundamental cellular processes, including cell growth, survival, motility, death, and differentiation, and can be important players in multicellular systems such as angiogenesis, inflammation, and immunity. Though long considered to be essentially digestive enzymes that mediate complete degradation of their substrates, many proteases are now known to be highly site specific. Knowledge of the cleavage site motif for a protease or peptidase can be useful in the design of substrates and inhibitors for the enzyme, and can also provide insight into its biological function through the identification and characterization of its protein substrates. Here, we describe in detail methodology that allows for the rapid and general determination of optimal recognition sequences for proteolytic enzymes.     

Identification of the amino acid residues essential for proteolytic activity in an archaeal signal peptide peptidase

Signal peptide peptidases (SPPs) are enzymes involved in the initial degradation of signal peptides after they are released from the precursor proteins by signal peptidases. In contrast to the eukaryotic enzymes that are aspartate peptidases, the catalytic mechanisms of prokaryotic SPPs had not been known. In this study on the SPP from the hyperthermophilic archaeon Thermococcus kodakaraensis (SppA(Tk)), we have identified amino acid residues that are essential for the peptidase activity of the enzyme. DeltaN54SppA(Tk), a truncated protein without the N-terminal 54 residues and putative transmembrane domain, exhibits high peptidase activity, and was used as the wild-type protein. Sixteen residues, highly conserved among archaeal SPP homologue sequences, were selected and replaced by alanine residues. The mutations S162A and K214A were found to abolish peptidase activity of the protein, whereas all other mutant proteins displayed activity to various extents. The results indicated the function of Ser(162) as the nucleophilic serine and that of Lys(214) as the general base, comprising a Ser/Lys catalytic dyad in SppA(Tk). Kinetic analyses indicated that Ser(184), His(191) Lys(209), Asp(215), and Arg(221) supported peptidase activity. Intriguingly, a large number of mutations led to an increase in activity levels of the enzyme. In particular, mutations in Ser(128) and Tyr(165) not only increased activity levels but also broadened the substrate specificity of SppA(Tk), suggesting that these residues may be present to prevent the enzyme from cleaving unintended peptide/protein substrates in the cell. A detailed alignment of prokaryotic SPP sequences strongly suggested that the majority of archaeal enzymes, along with the bacterial enzyme from Bacillus subtilis, adopt the same catalytic mechanism for peptide hydrolysis.     

Identification of proteolytic cleavage sites by quantitative proteomics

The identification of natural substrates and their cleavage sites is pivotal to defining proteolytic pathways. Here we report a novel strategy for the identification of the signature of proteolytic cleavage events based on quantitative proteomics. Lysine residues in proteins are blocked by guanidination so that free N-terminals can be labeled with amine-specific iTRAQ reagents. The quantitative nature of iTRAQ reagents allows us to distinguish N-terminals newly formed by proteolytic treatment (neoepitopes) from original N-terminals in proteins. Proteins are digested with trypsin and analyzed using MALDI-TOF/TOF mass spectrometry. Peptides labeled with iTRAQ reagents are distinguished from other peptides by exhibiting intense signature ions in tandem mass spectrometry analysis. A corresponding data acquisition strategy was developed to specifically analyze iTRAQ tagged N-terminal peptides. To validate the procedure, we examined a set of recombinant Escherichia coli proteins that have predicted caspase-3 cleavage motifs. The protein mixture was treated with active or inactive caspase-3 and subsequently labeled with two different iTRAQ reagents. Mass spectrometric analysis located 10 cleavage sites, all corresponding to caspase-3 consensus. Spiking caspase-cleaved substrate into a human cell lysate demonstrated the high sensitivity of the procedure. Moreover, we were able to identify proteolytic cleavage products associated with the induction of cell-free apoptosis. Together, these data reveal a novel application for iTRAQ technology for the detection of proteolytic substrates.     

Identification of MUC1 proteolytic cleavage sites in vivo

Mucins are high molecular weight glycoproteins that provide a protective layer on epithelial surfaces and are involved in cell-cell interactions, signaling, and metastasis. The identification of several membrane-tethered mucins, including MUC1, MUC3, MUC4, and MUC12, has incited interest in the processing of these mucins and the mechanisms that govern their release from the cell surface. MUC1 consists of an extracellular subunit and a membrane-associated subunit. The two moieties are produced from a single precursor polypeptide by an early proteolytic cleavage event but remain associated throughout intracellular processing and transport to the cell surface. We identified the MUC1 proteolytic cleavage site and showed it to be identical in pancreas and colon cell lines and not to be influenced by the presence of heavily glycosylated tandem repeats. The MUC1 cleavage site shows homology with sequences in other cell-surface-associated proteins and may represent a common mechanism for processing of these molecules.     

Identification of proteolytic cleavage sites in the conversion of profilaggrin to filaggrin in mammalian epidermis

Profilaggrin consists of multiple filaggrin domains joined by linker segments which are removed during proteolytic conversion to filaggrin. Analysis of tryptic peptides of filaggrin defined a 26-residue linker segment when aligned on the amino acid sequence of one repeat unit of mouse profilaggrin deduced from a cDNA sequence (Rothnagel, J. A., Mehrel, T., Idler, W. W., Roop, D. R., and Steinert, P. M. (1987) J. Biol. Chem. 262, 15643-15648). Two types of linker segments were distinguished by their different susceptibility to thermolysin and by the presence of a Phe-Tyr-Pro-Val sequence in only one type. These data led to a model of profilaggrin in which the two types of linker segments alternate along the length of profilaggrin. This model provides a structural basis for the two stages of proteolytic processing seen in vivo. In the first stage intermediates accumulate which have several filaggrin domains still joined by linker segments lacking Phe-Tyr-Pro-Val. In the second stage, the other linker segments are cleaved and mature filaggrin domains are released. Proteolytic activity with specificity consistent with first stage cleavage was partially purified from rat epidermis. Chymostatin inhibited both the in vitro enzymatic activity and the processing of profilaggrin in a cultured rat keratinocyte cell line. The products formed in vitro were 3-5 kDa larger than intermediates produced in vivo, suggesting that the linker segments are cleaved at one end only. This implies the existence of a third protease which completes the removal of the linker segments.     

Identification of Pyrococcus furiosus amylopullulanase catalytic residues

Pyrococcus furiosus amylopullulanase (PfAPU) belongs to glycosyl hydrolase family 57. Using sequence alignments of the known family 57 enzymes and site-directed mutagenesis, E291, D394, and E396 were identified as PfAPU putative catalytic residues. The apparent catalytic efficiencies (k_cat/K_m) of PfAPU mutants E291Q and D394N on pullulan were 123.0 and 24.4 times lower, respectively, than that of PfAPU. The activity of mutant E396Q on pullulan was too low to allow reliable determination of its catalytic efficiency. The apparent specific activities of these enzymes on starch also decreased 91.0 times (E291Q), 11.7 times (D394N), and 37.2 times (E396Q). The hydrolytic patterns for pullulan and starch were the same, while the hydrolysis rates differed as reported. Based on sequence alignment and a previous report, E291 is proposed as the catalytic nucleophile.     

Transthyretin protects against A-beta peptide toxicity by proteolytic cleavage of the peptide: a mechanism sensitive to the Kunitz protease inhibitor

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the deposition of amyloid beta-peptide (A-Beta) in the brain. Transthyretin (TTR) is a tetrameric protein of about 55 kDa mainly produced in the liver and choroid plexus of the brain. The known physiological functions of TTR are the transport of thyroid hormone T(4) and retinol, through binding to the retinol binding protein. TTR has also been established as a cryptic protease able to cleave ApoA-I in vitro. It has been described that TTR is involved in preventing A-Beta fibrilization, both by inhibiting and disrupting A-Beta fibrils, with consequent abrogation of toxicity. We further characterized the nature of the TTR/A-Beta interaction and found that TTR, both recombinant or isolated from human sera, was able to proteolytically process A-Beta, cleaving the peptide after aminoacid residues 1, 2, 3, 10, 13, 14,16, 19 and 27, as determined by mass spectrometry, and reversed phase chromatography followed by N-terminal sequencing. A-Beta peptides (1-14) and (15-42) showed lower amyloidogenic potential than the full length counterpart, as assessed by thioflavin binding assay and ultrastructural analysis by transmission electron microscopy. A-Beta cleavage by TTR was inhibited in the presence of an alphaAPP peptide containing the Kunitz Protease Inhibitor (KPI) domain but not in the presence of the secreted alphaAPP derived from the APP isoform 695 without the KPI domain. TTR was also able to degrade aggregated forms of A-Beta peptide. Our results confirmed TTR as a protective molecule in AD, and prompted A-Beta proteolysis by TTR as a protective mechanism in this disease. TTR may prove to be a useful therapeutic agent for preventing or retarding the cerebral amyloid plaque formation implicated in AD pathology.     

Identification of putative active site residues of ACAT enzymes

In this report, we sought to determine the putative active site residues of ACAT enzymes. For experimental purposes, a particular region of the C-terminal end of the ACAT protein was selected as the putative active site domain due to its high degree of sequence conservation from yeast to humans. Because ACAT enzymes have an intrinsic thioesterase activity, we hypothesized that by analogy with the thioesterase domain of fatty acid synthase, the active site of ACAT enzymes may comprise a catalytic triad of ser-his-asp (S-H-D) amino acid residues. Mutagenesis studies revealed that in ACAT1, S456, H460, and D400 were essential for activity. In ACAT2, H438 was required for enzymatic activity. However, mutation of D378 destabilized the enzyme. Surprisingly, we were unable to identify any S mutations of ACAT2 that abolished catalytic activity. Moreover, ACAT2 was insensitive to serine-modifying reagents, whereas ACAT1 was not. Further studies indicated that tyrosine residues may be important for ACAT activity. Mutational analysis showed that the tyrosine residue of the highly conserved FYXDWWN motif was important for ACAT activity. Furthermore, Y518 was necessary for ACAT1 activity, whereas the analogous residue in ACAT2, Y496, was not. The available data suggest that the amino acid requirement for ACAT activity may be different for the two ACAT isozymes.     

Identification of residues important for cleavage of the extracellular signaling peptide CSF of Bacillus subtilis from its precursor protein

Extracellular Phr pentapeptides produced by gram-positive, spore-forming bacteria regulate processes during the transition from exponential- to stationary-phase growth. Phr pentapeptides are produced by cleavage of their precursor proteins. We determined the residues that direct this cleavage for the Bacillus subtilis Phr peptide, CSF, which is derived from the C terminus of PhrC. Strains expressing PhrC with substitutions in residues -1 to -5 relative to the cleavage site had a defect in CSF production. The mutant PhrC proteins retained a functional signal sequence for secretion, as assessed by secretion of PhrC-PhoA fusions. To determine whether the substitutions directly affected cleavage of PhrC to CSF, we tested cleavage of synthetic pro-CSF peptides that corresponded to the C terminus of PhrC and had an amino acid substitution at the -2, -3, or -4 position. The mutant pro-CSF peptides were cleaved less efficiently to CSF than the wild-type pro-CSF peptide whether they were incubated with whole cells, cell wall material, or the processing protease subtilisin or Vpr. To further define the range of amino acids that support CSF production, the amino acid at the -4 position of PhrC was replaced by the 19 canonical amino acids. Only four substitutions resulted in a >2-fold defect in CSF production, indicating that this position is relatively immune to mutational perturbations. These data revealed residues that direct cleavage of CSF and laid the groundwork for testing whether other Phr peptides are processed in a similar manner.     

Asparagine peptide lyases: a seventh catalytic type of proteolytic enzymes

The terms "proteolytic enzyme" and "peptidase" have been treated as synonymous, and all proteolytic enzymes have been considered to be hydrolases (EC 3.4). However, the recent discovery of proteins that cleave themselves at asparagine residues indicates that not all peptide bond cleavage occurs by hydrolysis. These self-cleaving proteins include the Tsh protein precursor of Escherichia coli, in which the large C-terminal propeptide acts as an autotransporter; certain viral coat proteins; and proteins containing inteins. Proteolysis is the action of an amidine lyase (EC 4.3.2). These proteolytic enzymes are also the first in which the nucleophile is an asparagine, defining the seventh proteolytic catalytic type and the first to be discovered since 2004. We have assembled ten families based on sequence similarity in which cleavage is thought to be catalyzed by an asparagine.     

嗜酸热脂环酸杆菌中甘露聚糖酶活性位点的确立

【目的】通过定点突变确定嗜酸热脂环酸杆菌中甘露聚糖酶的活性催化位点。【方法】根据序列比对和GH53家族的结构信息选择可能的催化活性位点,利用重叠PCR法构建定点突变体,采用薄层层析(TLC)法和3,5-二硝基水杨酸(DNS)法检测各酶蛋白活性。【结果】通过重叠PCR法成功构建了7个位点的突变体,其中第150和159位的氨基酸突变对活性改变甚少或几乎没有,而第151和231位谷氨酸的羧基基团的改变以及双位点突变体E2Q则导致其对各种底物催化活性的丧失,说明位于β4和β7折叠的C末端的E151和E231的羧基基团作为功能基团参与了催化反应。【结论】E151和E231分别是新型甘露聚糖酶AaManA的酸碱催化位点和亲核催化位点。     

Identification of the catalytic residues of bifunctional glycogen debranching enzyme

Eukaryotic glycogen debranching enzyme (GDE) possesses two different catalytic activities (oligo-1,4-->1,4-glucantransferase/amylo-1,6-glucosidase) on a single polypeptide chain. To elucidate the structure-function relationship of GDE, the catalytic residues of yeast GDE were determined by site-directed mutagenesis. Asp-535, Glu-564, and Asp-670 on the N-terminal half and Asp-1086 and Asp-1147 on the C-terminal half were chosen by the multiple sequence alignment or the comparison of hydrophobic cluster architectures among related enzymes. The five mutant enzymes, D535N, E564Q, D670N, D1086N, and D1147N were constructed. The mutant enzymes showed the same purification profiles as that of wild-type enzyme on beta-CD-Sepharose-6B affinity chromatography. All the mutant enzymes possessed either transferase activity or glucosidase activity. Three mutants, D535N, E564Q, and D670N, lost transferase activity but retained glucosidase activity. In contrast, D1086N and D1147N lost glucosidase activity but retained transferase activity. Furthermore, the kinetic parameters of each mutant enzyme exhibiting either the glucosidase activity or transferase activity did not vary markedly from the activities exhibited by the wild-type enzyme. These results strongly indicate that the two activities of GDE, transferase and glucosidase, are independent and located at different sites on the polypeptide chain.     

Mutations within the proteolytic cleavage site of the Rous sarcoma virus glycoprotein define a requirement for dibasic residues for intracellular cleavage.

We investigated the amino acid sequence requirements for intracellular cleavage of the Rous sarcoma virus glycoprotein precursor by introducing mutations into the region encoding the cleavage recognition site (Arg-Arg-Lys-Arg). In addition to mutants G1 (Arg-Arg-Glu-Arg) and Dr1 (deletion of all four codons) that we have reported on previously (L. G. Perez and E. Hunter, J. Virol. 61:1609-1614, 1987), we constructed two additional mutants, AR1 (Arg-Arg-Arg-Arg), in which the highly conserved lysine is replaced by an arginine, and S19 (Ser-Arg-Glu-Arg), in which no dibasic pairs remain. The results of these studies demonstrate that when the cleavage sequence is deleted (Dr1) or modified to contain unpaired basic residues (S19), intracellular cleavage of the glycoprotein precursor is completely blocked. This demonstrates that the cellular endopeptidase responsible for cleavage has a stringent requirement for the presence of a pair of basic residues (Arg-Arg or Lys-Arg). Furthermore, it implies that the cleavage enzyme is not trypsinlike, since it is unable to recognize arginine residues that are sensitive to trypsin action. Substitution of the mutated genes into a replication-competent avian retrovirus genome showed that cleavage of the glycoprotein precursor was not required for incorporation into virions but was necessary for infectivity. Treatment of BH-RCAN-S19-transfected turkey cells with low levels of trypsin resulted in the release of infectious virus, demonstrating that exogenous cleavage could generate a biologically active glycoprotein molecule.     

Conserved residues of the leader peptide are essential for cleavage by leader peptidase

Gene 8 of bacteriophage M13 codes for procoat, the precursor of its major coat protein. Gene 8 has been cloned into a plasmid and mutagenized. We have isolated mutants of this gene in which procoat is synthesized but is not processed to coat protein. We now describe mutants in the leader region of procoat, at positions -6, -3, and -1 with respect to the leader peptidase cleavage site. These positions are quite conserved among the leader peptides of various pre-proteins. Each of these mutant procoats is synthesized at a normal rate and inserts correctly into the plasma membrane, as judged by its accessibility to protease in intact spheroplasts. Procoat accumulates, largely in its transmembrane form, and is not cleaved to coat. In detergent extracts, the mutant procoats are very poor substrates for added leader peptidase. We conclude that these 3 residues are not conserved for insertion across the membrane but are part of an essential recognition site for the leader peptidase.