Activation of interleukin-1 beta by a co-induced protease

The proteolytic generation of mature interleukin-1 beta (IL-1 beta) from its inactive precursor does not proceed by a conventional pathway for hormonal processing. Pro-IL-1 beta is found dispersed in the cytoplasm, and there are no basic amino acid residues or other commonly recognized processing sites adjoining the mature N-terminus. Processing appears to occur during release of the hormone. In the present study, we have identified a specific protease that generates mature IL-1 beta from the precursor. This enzyme is co-induced with the hormone, and it differs in its cleavage specificity and inhibitor sensitivity from all known proteases.     

A pre-aspartate-specific protease from human leukocytes that cleaves pro-interleukin-1 beta

Interleukin-1 beta is a 17.4-kilodalton hormone derived from a 33-kilodalton inactive precursor produced by monocytes. We used the precursor as a substrate to detect proteolytic activities in peripheral blood mono-nuclear cell-conditioned medium that might be involved in interleukin-1 beta processing. We found that the conditioned medium, following passage through DEAE-Sephacel, generates a biologically active fragment from the precursor that runs slightly higher than the mature hormone in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The responsible activity behaved as a single protein in ion exchange chromatography. It was completely inhibited by metal ion chelators and not by inhibitors of serine, cysteine, or aspartate proteases, and it was dependent on both calcium (or magnesium) and zinc. The enzyme was not inhibited by three substrate-based metalloprotease inhibitors, phosphoramidon, benzyloxycarbonyl-Gly-Leu-NH2, and N-(2-carboxy-3-phenylpropionyl)-Leu. NH2-terminal sequence analysis showed that cleavage of the precursor occurred between a histidine and an aspartate residue, and digestion of synthetic peptides indicated that the protease is specific for pre-aspartate cleavages.     

Generation of biologically active interleukin-1beta by meprin B

Interleukin-1beta (IL-1beta) is a proinflammatory cytokine that is synthesized as an inactive precursor molecule that must be proteolytically processed to generate the biologically active form. Maturation of the precursor is primarily performed by caspase-1, an intracellular cysteine protease; however, processing by other proteases has been described. Meprins are cell surface and secreted metalloproteases expressed by renal and intestinal brush-border membranes, leukocytes, and cancer cells. In this study we show that purified recombinant meprin B can process the interleukin-1beta precursor to a biologically active form. Amino-terminal sequencing and mass spectrometry analysis of the product of digestion by activated meprin B determined that proteolytic cleavage resulted in an additional six amino acids relative to the site utilized by caspase-1. The biological activity of the meprin B-cleaved cytokine was confirmed by measuring the proliferative response of helper T-cells. These results suggest that meprin may play an important role in activation of this proinflammatory cytokine in various pathophysiological conditions.     

Hetero- and autoprocessing of the extracellular metalloprotease (Mpr) in Bacillus subtilis

Most proteases are synthesized as inactive precursors which are processed by proteolytic cleavage into a mature active form, allowing regulation of their proteolytic activity. The activation of the glutamic-acid-specific extracellular metalloprotease (Mpr) of Bacillus subtilis has been examined. Analysis of Mpr processing in defined protease-deficient mutants by activity assay and Western blotting revealed that the extracellular protease Bpr is required for Mpr processing. pro-Mpr remained a precursor form in bpr-deficient strains, and glutamic-acid-specific proteolytic activity conferred by Mpr was not activated in bpr-deficient strains. Further, purified pro-Mpr was processed to an active form by purified Bpr protease in vitro. We conclude that Mpr is activated by Bpr in vivo, and that heteroprocessing, rather than autoprocessing, is the major mechanism of Mpr processing in vivo. Exchange of glutamic acid for serine in the cleavage site of Mpr (S93E) allowed processing of Mpr into its mature form, regardless of the presence of other extracellular proteases, including Bpr. Thus, a single amino acid change is sufficient to convert the Mpr processing mechanism from heteroprocessing to autoprocessing.     

Carboxyl-terminal processing of the D1 protein and photoactivation of water-splitting in photosystem II. Partial purification and characterization of the processing enzyme from Scenedesmus obliquus and Pisum sativum.

The D1 polypeptide of photosystem II (PSII) is synthesized as a precursor that is processed by cleavage at the carboxyl terminus during assembly of the active PSII complex. A mutant of the green alga Scenedesmus obliquus, LF-1, inactive in water-splitting, lacks the D1 processing activity but assembles otherwise normal PSII complexes containing the precursor D1 molecule. We have isolated and partially purified a soluble protease from sonicated thylakoids of both wild-type S. obliquus and Pisum sativum which will process the precursor D1 molecule in PSII-enriched membranes from the LF-1 mutant to the mature size. After processing (but not before), photoactivation of these PSII membranes in the presence of manganese restores water-splitting to levels seen after photoactivation of PSII membranes from dark-grown, wild-type, cells. The protease is unable to process D1 in intact thylakoids from the LF-1 mutant but processes D1 if present during sonication of the thylakoids, indicating that processing of the carboxyl-terminal extension of D1 occurs in the lumen of the thylakoid. The processing protease from both S. obliquus and P. sativum is a single subunit enzyme of native molecular mass 33-35 kDa. Processing rate is optimal at pH 6.5. Processing in vitro is evident within 5 min and is markedly inhibited by millimolar concentrations of divalent cations (Cu, Zn greater than Mn greater than Ca, Mg) but not by any known inhibitors of the major classes of proteases. The protease is inactive against the precursors of other thylakoidal proteins and is thus distinct from the thylakoidal amino-terminal processing enzyme involved in the removal of transit peptides from cytoplasmically-synthesised proteins imported into the thylakoid lumen.     

IL-1-converting enzyme requires aspartic acid residues for processing of the IL-1 beta precursor at two distinct sites and does not cleave 31-kDa IL-1 alpha

IL-1 converting enzyme (ICE) specifically cleaves the human IL-1 beta precursor at two sequence-related sites: Asp27-Gly28 (site 1) and Asp116-Ala117 (site 2). Cleavage at Asp116-Ala117 results in the generation of mature, biologically active IL-1 beta. ICE is unusual in that preferred cleavage at Asp-X bonds (where X is a small hydrophobic residue), has not been described for any other eukaryotic protease. To further examine the substrate specificity of ICE, proteins that contain Asp-X linkages including transferrin, actin, complement factor 9, the murine IL-1 beta precursor, and human and murine IL-1 alpha precursors, were assayed for cleavage by 500-fold purified ICE. The human and murine IL-1 beta precursors were the only proteins cleaved by ICE, demonstrating that ICE is an IL-1 beta convertase. Analysis of human IL-1 beta precursor mutants containing amino acid substitutions or deletions within each processing site demonstrated that omission or replacement of Asp at site 1 or site 2 prevented cleavage by ICE. To quantitatively assess the substrate requirements of ICE, a peptide-based cleavage assay was established using a 14-mer spanning site 2. Cleavage between Asp [P1] and Ala [P1']2 was demonstrated. Replacement of Asp with Ala, Glu, or Asn resulted in a greater than 100-fold reduction in cleavage activity. The rank order in position P1' was Gly greater than Ala much greater than Leu greater than Lys greater than Glu. Substitutions at P2'-P4' and P6' had relatively little effect on cleavage activity. These results show that ICE is a highly specific IL-1 beta convertase with absolute requirements for Asp in P1 and a small hydrophobic amino acid in P1'.     

Mutational Analysis of the Pseudomonas aeruginosa Myovirus ?KZ Morphogenetic Protease gp175

Pseudomonas aeruginosa myovirus ϕKZ has a 270-kb genome within a T=27 icosahedral capsid that contains a large, unusual, and structurally well-defined protein cylindrical inner body (IB) spanning its interior. Proteolysis forms a pivotal stage in ϕKZ head and IB morphogenesis, with the protease gp175 cleaving at least 19 of 49 different head proteins, including the major capsid protein and five major structural IB proteins. Here we show that the purified mature form of gp175 is active and cleaves purified IB structural proteins gp93 and gp89. Expression vector synthesis and purification of the zymogen/precursor yielded an active, mature-length protease, showing independent C-terminal gp175 self-cleavage autoactivation. Mutation of either the predicted catalytic serine or histidine inactivated mature gp175, supporting its classification as a serine protease and representing the first such direct biochemical demonstration with purified protease and substrate proteins for any phage protease. These mutations also blocked self-cleavage of the precursor while allowing intermolecular gp175 processing. To confirm the cleavage specificity of gp175, we mutated three cleavage sites in gp93, which blocked proteolysis at these sites. The N-terminal propeptide of gp93 was shown to undergo more extensive proteolysis than previously identified. We found that proteolysis in gp93 progressed from the N to C terminus, while blocking cleavage sites slowed but did not eliminate downstream proteolysis. These findings were shown by informatics to be relevant to the head morphogenesis of numbers of other related IB-containing giant phages as well as to T4 and herpesviruses, which have homologous proteases.     

Processing of precursor interleukin 1 beta and inflammatory disease

The processing of precursor interleukin 1 beta (IL1 beta) by elastase, cathepsin G, and collagenase, the major proteases released at sites of inflammation, was investigated using recombinant pro-IL1 beta. Each of these proteases cleaved the 31-kDa inactive precursor to a form similar in size and specific activity (greater than 10(8) units/mg) to the 17-kDa mature protein isolated from activated monocytes. Elastase, collagenase, and cathepsin G cleaved the IL1 beta precursor at distinct sites which are amino-terminal to the monocyte-processing site, Ala-117 (Cameron, P., Lumjuco, G., Rodkey, J., Bennett, C., and Schmidt, J. A. (1985) J. Exp. Med. 162, 790-801). Amino-terminal sequencing of the products of digestion by elastase and cathepsin G determined that resultant active IL1 beta proteins contained an additional 13 or 3 amino acids relative to mature IL1 beta. Synovial fluid collected from patients with inflammatory polyarthritis and bronchoalveolar lavage fluid from patients with sarcoidosis supplied similar processing activity(s). Control fluids from patients who had no symptoms of inflammatory disease did not exhibit processing activity. Lavage fluids that processed precursor IL1 beta were demonstrated to contain cathepsin G and/or elastase activity, whereas controls were negative. Because a significant fraction of IL1 beta may be secreted from monocytes as the inactive 31-kDa precursor (Hazuda, D. J., Lee, J. C., and Young, P. R. (1988) J. Biol. Chem. 263, 8473-8479, Bomford, R., Absull, E., Hughes-Jenkins, C., Simpkin, D., and Schmidt, J. (1987) Immunology 62, 543-549, and Mizel, S. B. (1988) in Cellular and Molecular Aspects of Inflammation Poste, G., and Crooke, S., eds) pp. 75-93, Plenum Publishing Corp., New York), these results suggest that in vivo the IL1 beta precursor can be processed after secretion by any of several proteases released at inflammatory sites.     

Structure-function mapping of interleukin 1 precursors. Cleavage leads to a conformational change in the mature protein

The two interleukin 1 (IL-1) genes (IL-1 alpha and beta) encode 31-kDa precursor molecules, which are cleaved upon secretion to generate the mature, active, carboxyl-terminal 17-kDa proteins. The IL-1 beta precursor is inactive, whereas the IL-1 alpha precursor is as active as the mature IL-1 alpha. In this report, we demonstrate that when either of the recombinant precursors is processed to the mature form, the mature region undergoes a conformational change from a proteinase K-sensitive structure to one that is proteinase K-insensitive. In addition, cysteine residues that are exposed to solvent in the IL-1 beta precursor become buried in the mature protein. Limited structure-activity mapping of the IL-1 beta precursor indicates that the amino-terminal 76 residues are responsible for the conformational change, whereas the most dramatic change in biological activity occurs after further removal of residues 77-94. These findings suggest that the altered structure of the mature region in precursor IL-1s has been conserved for some function. Denaturation/renaturation experiments implicate the precursor domain in protein folding, and by analogy with signal-directed secretory proteins, the unique conformation of the precursors may play a role in IL-1 secretion.     

Purification and characterization of a novel thiol protease involved in processing the enkephalin precursor

Proteolytic processing enzymes are required to convert the enkephalin precursor to active opioid peptides. In this study, a novel 33-kDa thiol protease that cleaves complete precursor in the form of [35S]methionine preproenkephalin was purified from bovine adrenal medullary chromaffin granules. Chromatography on concanavalin A-Sepharose and Sephacryl S-200, chromatofocusing, and chromatography on thiopropyl-Sepharose resulted in an 88,000-fold purification with a recovery of 35% of enzyme activity. The thiol protease is a glycoprotein with a pI of 6.0. It cleaves [35S]methionine preproenkephalin with a pH optimum of 5.5, indicating that it is functional at the intragranular pH of 5.5-6.0. Interestingly, production of trichloroacetic acid-soluble products was optimal at pH 4.0, suggesting that processing of initial precursor and intermediates may require slightly different pH conditions. The protease requires dithiothreitol for activity and is inhibited by the thiol protease inhibitors iodoacetate, p-hydroxymercuribenzoate, mercuric chloride, and cystatin. These properties distinguish it from other thiol proteases (cathepsins B, H, L, N, and S), indicating that a unique thiol protease has been identified. The enzyme converted [35S]cysteine preproenkephalin (possessing [35S]cysteine residues specifically within the precursor's NH2-terminal segment) to 22.1-, 21.6-, 17.7-, 17.3-, and 15.0-kDa intermediates that contain the precursor's NH2-terminal segment; proenkephalin in vivo is converted to similar intermediates. The enzyme cleaves peptide F at Lys-Arg and Lys-Lys dibasic amino acid sites to generate methionine enkephalin and intermediates. The appropriate vesicular localization, pH optimum, proteolytic products, and cleavage site specificity suggest that this thiol protease may be involved in enkephalin precursor processing. Most interestingly, [35S]methionine beta-preprotachykinin, a precursor of substance P, is minimally cleaved, suggesting that the thiol protease may possess some selectivity for the enkephalin precursor.     

Analysis and characterisation of IL-1beta processing in rainbow trout, Oncorhynchus mykiss

Mammalian IL-1beta is produced as a biologically inactive 31 kDa precursor, which is converted to the active 18 kDa form by proteolytic processing. Synthesis and processing of native piscine IL-1beta is poorly understood. In the present study, the native IL-1beta precursor or mature peptides were detected at sizes of approx. 29 kDa and 24 kDa in cell lysates of a rainbow trout macrophage cell line RTS-11, with or without LPS stimulation, by Western blot analysis using a polyclonal antibody against the putative trout mature IL-1beta (rmIL-1beta) produced in Escherichia coli. Processing of the 29 kDa precursor into a 24 kDa mature peptide was confirmed by analysis of such proteins using a monoclonal conjugate (Ni-NTA-HRP) against 6 histidines in lysates of the RTS-11 cells transfected with an expression plasmid containing the IL-1beta precursor molecule tagged with 6 histidines at its C terminus. Only the recombinant mature 24 kDa) IL-1beta/HIS protein was purified from the culture supernatants of the transfected cells, indicating the molecule is cleaved to be secreted. These findings strongly suggest that the trout IL-1beta molecule is processed in trout macrophages in an analogous way to the situation with mammalian IL-1beta despite the lack of a clear ICE cut site.     

Contradictory functions (activation/termination) of neutrophil proteinase 3 enzyme (PR3) in interleukin-33 biological activity

IL-1 family ligand does not possess a typical hydrophobic signal peptide and needs a processing enzyme for maturation. The maturation process of IL-33 (IL-1F11), a new member of the IL-1 family ligand, remains unclear. Precursor IL-33 ligand affinity column isolates neutrophil proteinase 3 (PR3) from human urinary proteins. PR3 is a known IL-1 family ligand-processing enzyme for IL-1β (IL-1F2) and IL-18 (IL-1F4), including other inflammatory cytokines. We investigated PR3 in the maturation process of precursor IL-33 because we isolated urinary PR3 by using the precursor IL-33 ligand affinity column. PR3 converted inactive human and mouse precursor IL-33 proteins to biological active forms; however, the increase of PR3 incubation time abrogated IL-33 activities. Unlike caspase-1-cleaved precursor IL-18, PR3 cut precursor IL-33 and IL-18 at various sites and yielded multibands. The increased incubation period of PR3 abated mature IL-33 in a time-dependent manner. The result is consistent with the decreased bioactivity of IL-33 along with the increased PR3 incubation time. Six different human and mouse recombinant IL-33 proteins were expressed by the predicted consensus amino acid sequence of PR3 cleavage sites and tested for bioactivities. The human IL-33/p1 was highly active, but human IL-33/p2 and p3 proteins were inactive. Our results suggest the dual functions (activation/termination) of PR3 in IL-33 biological activity.     

Extracellular autoprocessing of a metalloprotease from Streptomyces cacaoi.

We have previously demonstrated that the extracellular neutral metalloprotease (Npr) of Streptomyces cacaoi is synthesized as a 60-kDa preproenzyme (P60), then processed to the 35-kDa mature form (P35) (Chang, P. C., Kuo, T.-C., Tsugita, A., and Lee, Y.-H. W. (1990) Gene (Amst.) 88, 87-95). In this study, we investigated the active site and the mechanism involved in the maturation of the protease. Site-specific mutations at the putative zinc-binding ligands and active site of Npr at His202, Glu203, His206, and Glu240 led to complete abolishment of Npr activity and concomitant accumulation of a 57-kDa inactive protein (P57) which was secreted. Sequence analysis of the NH2 terminus indicated that P57 was derived from P60 after removal of the signal peptide and represented the proenzyme form of Npr (pro-Npr). Analysis of the zinc content of purified mutant P57 proteins revealed a dramatic loss of zinc atom as compared with the wild-type P35 protein. In vitro with the aid of exogenous active Npr, the mutant P57 protein could be converted to the mature inactive P35 with an identical NH2-terminal sequence and a molecular mass the same as that of the wild-type P35. From these studies, we conclude that these highly conserved residues (His202, Glu203, His206, and Glu240) are indispensable for zinc binding and protease activity, as well as processing of Npr. In addition, we have clearly demonstrated that maturation of Npr occurs extracellularly via an autocatalytic cleavage of the pro-Npr propeptide. This is the first report of such a maturation mechanism for an extracellular protease in streptomycetes which can serve as a model for further studies on the mechanism of secretion and processing of proteases from Gram-positive bacteria.     

Extracellular proteolytic cleavage by urokinase is required for activation of hepatocyte growth factor/scatter factor.

The extracellular protease urokinase is known to be crucially involved in morphogenesis, tissue repair and tumor invasion by mediating matrix degradation and cell migration. Hepatocyte growth factor/scatter factor (HGF/SF) is a secretory product of stromal fibroblasts, sharing structural motifs with enzymes of the blood clotting cascade, including a zymogen cleavage site. HGF/SF promotes motility, invasion and growth of epithelial and endothelial cells. Here we show that HGF/SF is secreted as a single-chain biologically inactive precursor (pro-HGF/SF), mostly found in a matrix-associated form. Maturation of the precursor into the active alpha beta heterodimer takes place in the extracellular environment and results from a serum-dependent proteolytic cleavage. In vitro, pro-HGF/SF was cleaved at a single site by nanomolar concentrations of pure urokinase, generating the active mature HGF/SF heterodimer. This cleavage was prevented by specific urokinase inhibitors, such as plasminogen activator inhibitor type-1 and protease nexin-1, and by antibodies directed against the urokinase catalytic domain. Addition of these inhibitors to HGF/SF responsive cells prevented activation of the HGF/SF precursor. These data show that urokinase acts as a pro-HGF/SF convertase, and suggest that some of the growth and invasive cellular responses mediated by this enzyme may involve activation of HGF/SF.     

Partial characterization of an enzyme fraction with protease activity which converts the spore peptidoglycan hydrolase (SleC) precursor to an active enzyme during germination of Clostridium perfringens S40 spores and analysis of a gene cluster involved in the activity

A spore cortex-lytic enzyme of Clostridium perfringens S40 which is encoded by sleC is synthesized at an early stage of sporulation as a precursor consisting of four domains. After cleavage of an N-terminal presequence and a C-terminal prosequence during spore maturation, inactive proenzyme is converted to active enzyme by processing of an N-terminal prosequence with germination-specific protease (GSP) during germination. The present study was undertaken to characterize GSP. In the presence of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), a nondenaturing detergent which was needed for the stabilization of GSP, GSP activity was extracted from germinated spores. The enzyme fraction, which was purified to 668-fold by column chromatography, contained three protein components with molecular masses of 60, 57, and 52 kDa. The protease showed optimum activity at pH 5.8 to 8.5 in the presence of 0.1% CHAPS and retained activity after heat treatment at 55 degrees C for 40 min. GSP specifically cleaved the peptide bond between Val-149 and Val-150 of SleC to generate mature enzyme. Inactivation of GSP by phenylmethylsulfonyl fluoride and HgCl(2) indicated that the protease is a cysteine-dependent serine protease. Several pieces of evidence demonstrated that three protein components of the enzyme fraction are processed forms of products of cspA, cspB, and cspC, which are positioned in a tandem array just upstream of the 5' end of sleC. The amino acid sequences deduced from the nucleotide sequences of the csp genes showed significant similarity and showed a high degree of homology with those of the catalytic domain and the oxyanion binding region of subtilisin-like serine proteases. Immunochemical studies suggested that active GSP likely is localized with major cortex-lytic enzymes on the exterior of the cortex layer in the dormant spore, a location relevant to the pursuit of a cascade of cortex hydrolytic reactions.     

Proteolytic processing of Ty3 proteins is required for transposition.

Ty3 is a retroviruslike element found in Saccharomyces cerevisiae. It encodes GAG3 and GAG3-POL3 polyproteins which are processed into mature proteins found in the Ty3 viruslike particle. In this study, the region encoding a protease that is homologous to retroviral aspartyl proteases was identified and shown to be required for production of mature Ty3 proteins and transposition. The Ty3 protease has the Asp-Ser-Gly consensus sequence found in copia, Ty1, and Rous sarcoma virus proteases, rather than the Asp-Thr-Gly found in most retroviral proteases. The Asp-Ser-Gly consensus is flanked by residues similar to those which flank the active sites of cellular aspartyl proteases. Mutations were made in the Ty3 active-site sequence to examine the role of the protease in Ty3 particle maturation and to test the functional significance of the Ser active-site variant in the consensus sequence. Mutation of the active-site Asp blocked processing of Gag3 and Gag3-Pol3 and allowed identification of a GAG3-POL3 polyprotein. This protein was turned over rapidly in cells expressing the mutant Ty3. Changing the active-site Ser to Thr caused only a modest reduction in the levels of certain Ty3 proteins. Five putative cleavage sites of this protease in Ty3 GAG3 and GAG3-POL3 polyproteins were defined by amino-terminal sequence analysis. The existence of an additional protein(s) of unknown function, encoded downstream of the protease-coding region, was deduced from the positions of these amino termini and the sizes of known Ty3 proteins. Although Ty3 protease cleavage sites do not correspond exactly to known retroviral protease cleavage sites, there are similarities. Residues P3 through P2' in the regions encompassing each of the five sites are uncharged, and no P1 position is occupied by an amino acid with a branched beta carbon.     

Characterization of a mitochondrial matrix protease catalyzing the processing of adrenodoxin precursor

Adrenodoxin (Ad) is synthesized as a larger precursor (preAd) by cytoplasmic polysomes and then transported into mitochondria concomitant with its proteolytic processing to the mature form. The protease in bovine adrenal cortex mitochondria, which converts preAd to the mature form, is a metalloprotease in the matrix (Sagara, Y., Ito, A. & Omura, T. (1984) J. Biochem. 96, 1743-1752). In this study, the protease was purified about 100-fold from the matrix fraction of bovine adrenal cortex mitochondria. The partially purified protease converted not only preAd, but also the precursors of malate dehydrogenase (MDH) and 27 kDa protein (P-27) to the corresponding mature forms. However, it was inactive toward the precursors of P-450(SCC) and of P-450(11 beta). Since isolated rat liver mitochondria can import and process preAd as efficiently as bovine adrenal cortex mitochondria, we partially purified a preAd-processing protease from rat liver mitochondria and compared its properties with those of the bovine adrenal cortex enzyme. The properties of the rat liver protease were indistinguishable from those of the bovine adrenal cortex enzyme in molecular weight determined from Sephadex G-150 gel filtration, metal requirement and ability to process preMDH and preP-27. The rat liver enzyme was also inactive toward the precursors of P-450(SCC) and P-450(11 beta). These results indicate the presence in both adrenal cortex and liver mitochondria of the same type of processing protease, which processes preAd and also the precursors of some other mitochondrial proteins.     

Proteolysis of ProPTHrP(1-141) by "prohormone thiol protease" at multibasic residues generates PTHrP-related peptides: implications for PTHrP peptide production in lung cancer cells

The parathyroid hormone-related protein (PTHrP) precursor requires proteolytic processing to generate PTHrP-related peptide products that possess regulatory functions in the control of PTH-like (parathyroid-like) actions and cell growth, calcium transport, and osteoclast activity. Biologically active peptide domains within the PTHrP precursor are typically flanked at their NH2- and COOH-termini by basic residue cleavage sites consisting of multibasic, dibasic, and monobasic residues. These basic residues are predicted to serve as proteolytic cleavage sites for converting the PTHrP precursor into active peptide products. The coexpression of the prohormone processing enzyme PTP ("prohormone thiol protease") in PTHrP-containing lung cancer cells, and the lack of PTP in cell lines that contain little PTHrP, implicate PTP as a candidate processing enzyme for proPTHrP. Therefore, in this study, PTP cleavage of recombinant proPTHrP(1-141) precursor was evaluated by MALDI mass spectrometry to identify peptide products and cleavage sites. PTP cleaved the PTHrP precursor at the predicted basic residue cleavage sites to generate biologically active PTHrP-related peptides that correspond to the NH2-terminal domain (residues 1-37) that possesses PTH-like and growth regulatory activities, the mid-region domain (residues 38-93) that regulates calcium transport, and the COOH-terminal domain (residues 102-141) that modulates osteoclast activity. Lack of cleavage at other types of amino acids demonstrated the specificity of PTP processing at basic residue cleavage sites. Overall, these results demonstrate the ability of PTP to cleave the PTHrP precursor at multibasic, dibasic, and monobasic residue cleavage sites to generate active PTHrP-related peptides. The presence of PTP immunoreactivity in PTHrP-containing lung cancer cells suggests PTP as a candidate processing enzyme for the PTHrP precursor.