Specificity of the dynorphin-processing endoprotease: comparison with prohormone convertases

The cleavage specificity of a monobasic processing dynorphin converting endoprotease is examined with a series of quench fluorescent peptide substrates and compared with the cleavage specificity of prohormone convertases. A dynorphin B-29-derived peptide, Abz-Arg-Arg-Gln-Phe-Lys-Val-Val-Thr-Arg-Ser-Glneddnp (where Abz is o-aminobenzoyl and eddnp is ethylenediamine 2,4-dinitrophenyl), that contains both dibasic and monobasic cleavage sites is efficiently cleaved by the dynorphin converting enzyme and not cleaved by two propeptide processing enzymes, furin and prohormone convertase 1. A shorter prorenin-related peptide, Dnp-Arg-Met-Ala-Arg-Leu-Thr-Leu-eddnp, that contains a monobasic cleavage site is cleaved by the dynorphin converting enzyme and prohormone convertase 1 and not by furin. Substitution of the P1' position by Ala moderately affects cleavage by the dynorphin-processing enzyme and prohormone convertase 1. It is interesting that this substitution results in efficient cleavage by furin. The site of cleavage, as determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry, is N-terminal to the Arg at the P1 position for the dynorphin converting enzyme and C-terminal to the Arg at the P1 position for furin and prohormone convertase 1. Peptides with additional basic residues at the P2 and at P4 positions also serve as substrates for the dynorphin converting enzyme. This enzyme cleaves shorter peptide substrates with significantly lower efficiency as compared with the longer peptide substrates, suggesting that the dynorphin converting enzyme prefers longer peptides that contain monobasic processing sites as substrates. Taken together, these results suggest that the cleavage specificity of the dynorphin converting enzyme is distinct but related to the cleavage specificity of the prohormone convertases and that multiple enzymes could be involved in the processing of peptide hormones and neuropeptides at monobasic and dibasic sites.     

Isolation and identification of a novel mitochondrial metalloprotease (PreP) that degrades targeting presequences in plants

Most of the nuclear encoded mitochondrial precursor proteins contain an N-terminal extension called the presequence that carries targeting information and that is cleaved off after import into mitochondria. The presequences are amphiphilic, positively charged, membrane-interacting peptides with a propensity to form alpha-helices. Here we have investigated the proteolysis of the presequences that have been cleaved off inside mitochondria. A presequence derived from the overexpressed F(1)beta subunit of the ATP synthase and specific synthetic fluorescent peptides (Pep Tag Protease assay) have been shown to undergo rapid degradation catalyzed by a matrix located protease. We have developed a three-step chromatographic procedure including affinity and anion exchange chromatography for isolation of the protease from potato tuber mitochondria. Two-dimensional gel electrophoresis of the isolated proteolytically active fraction followed by electrospray ionization-mass spectrometry/mass spectrometry and data base searches allowed identification of the presequence peptide-degrading protease in Arabidopsis thaliana data base as a novel mitochondrial metalloendoprotease with a molecular mass of 105 kDa. The identified metalloprotease contains an inverted zinc-binding motif and belongs to the pitrilysin family.     

Cleavage specificity of a myofibril-bound serine proteinase from carp (Cyprinus carpio) muscle

Previously, we reported the purification and characterization of a myofibril-bound serine proteinase (MBP) from carp muscle (Osatomi K, Sasai H, Cao M-J, Hara K, Ishihara T. Comp Biochem Physiol 1997;116B:159–66). In the present study, the N-terminal amino acid sequence of the enzyme was determined, which showed high identity with those of other trypsin-like serine proteases. The cleavage specificity of MBP for dibasic and monobasic residues was investigated using various fluorogenic substrates and peptides. Analyses of the cleaved peptide products showed that the enzyme hydrolyzed peptides both at monobasic and dibasic amino acid residues. Monobasic amino acid residues were hydrolyzed at the carboxyl side; dibasic residues were cleaved either at the carboxyl side of the pair or between the two basic residues and the enzyme showed a cleavage preference for the Arg-Arg pair. Unexpectedly, MBP hydrolyzed lysyl-bradykinin and methionyl–lysyl–bradykinin at the carboxyl side of Gly fairly specifically and efficiently displaying a unique cleavage. Because MBP also degraded protein substrates such as casein and myofibrillar proteins, the substrate specificity of MBP appeared not to be strictly specific.     

Sequence of xynC and properties of XynC, a major component of the Clostridium thermocellum cellulosome.

The nucleotide sequence of the Clostridium thermocellum F1 xynC gene, which encodes the xylanase XynC, consists of 1,857 bp and encodes a protein of 619 amino acids with a molecular weight of 69,517. XynC contains a typical N-terminal signal peptide of 32 amino acid residues, followed by a 165-amino-acid sequence which is homologous to the thermostabilizing domain. Downstream of this domain was a family 10 catalytic domain of glycosyl hydrolase. The C terminus separated from the catalytic domain by a short linker sequence contains a dockerin domain responsible for cellulosome assembly. The N-terminal amino acid sequence of XynC-II, the enzyme purified from a recombinant Escherichia coli strain, was in agreement with that deduced from the nucleotide sequence although XynC-II suffered from proteolytic truncation by a host protease(s) at the C-terminal region. Immunological and N-terminal amino acid sequence analyses disclosed that the full-length XynC is one of the major components of the C. thermocellum cellulosome. XynC-II was highly active toward xylan and slightly active toward p-nitrophenyl-beta-D-xylopyranoside, p-nitrophenyl-beta-D-cellobioside, p-nitrophenyl-beta-D-glucopyranoside, and carboxymethyl cellulose. The Km and Vmax values for xylan were 3.9 mg/ml and 611 micromol/min/mg of protein, respectively. This enzyme was optimally active at 80 degrees C and was stable up to 70 degrees C at neutral pHs and over the pH range of 4 to 11 at 25 degrees C.     

Expression of NEP2, a soluble neprilysin-like endopeptidase, during embryogenesis in Drosophila melanogaster

Members of the neprilysin family of neutral endopeptidases (M13) are typically membrane-bound enzymes known to be involved in the extra-cellular metabolism of signalling peptides and have important roles during mammalian embryogenesis. In this study we show that membranes prepared from embryos of Drosophila melanogaster possess neprilysin-like activity that is inhibited by phosphoramidon and thiorphan, both inhibitors of mammalian neprilysin. Unexpectedly, we also found strong neprilysin-like neutral endopeptidase activity in a soluble embryo fraction, which we identify as NEP2 by Western blot and immunoprecipitation experiments using NEP2 specific antibodies. NEP2 is a soluble secreted member of the neprilysin family that has been shown previously to be expressed in larval and adult Malpighian tubules and in the testes of adult males. In situ hybridization studies reveal expression at stage 10-11 in a pattern similar to that previously described for stellate cell progenitors of the caudal visceral mesoderm. In later stages of embryogenesis, some of these cells appear to migrate into the growing Malpighian tubule. Recombinant NEP2 protein is N-glycosylated and displays optimum endopeptidase activity at neutral pH, consistent with a role as an extracellular peptidase. The recombinant enzyme hydrolyses Drosophila tachykinin peptides (DTK) at peptide bonds N-terminal to hydrophobic residues. DTK2, like Locusta tachykinin-1, was cleaved at the penultimate peptide bond (Gly(7)-Leu(8)), whereas the other Drosophila peptides were cleaved centrally at Xxx-Phe bonds. However, the rates of hydrolysis of the latter substrates were much slower than the hydrolysis rates of DTK2 and Locusta tachykinin-1, suggesting that the interaction of the bulky side-chain of phenylalanine at the S'(1) sub-site is less favorable for peptide bond hydrolysis. The secretion of NEP2 from tissues during embryogenesis suggests a possible developmental role for this endopeptidase in peptide signalling in D. melanogaster.     

Two novel targeting peptide degrading proteases, PrePs, in mitochondria and chloroplasts, so similar and still different

Two novel metalloproteases from Arabidopsis thaliana, termed AtPrePI and AtPrePII, were recently identified and shown to degrade targeting peptides in mitochondria and chloroplasts using an ambiguous targeting peptide. AtPrePI and AtPrePII are classified as dually targeted proteins as they are targeted to both mitochondria and chloroplasts. Both proteases harbour an inverted metal binding motif and belong to the pitrilysin subfamily A. Here we have investigated the subsite specificity of AtPrePI and AtPrePII by studying their proteolytic activity against the mitochondrial F(1)beta pre-sequence, peptides derived from the F(1)beta pre-sequence as well as non-mitochondrial peptides and proteins. The degradation products were analysed, identified by MALDI-TOF spectrometry and superimposed on the 3D structure of the F(1)beta pre-sequence. AtPrePI and AtPrePII cleaved peptides that are in the range of 10 to 65 amino acid residues, whereas folded or longer unfolded peptides and small proteins were not degraded. Both proteases showed preference for basic amino acids in the P(1) position and small, uncharged amino acids or serine residues in the P'(1) position. Interestingly, both AtPrePI and AtPrePII cleaved almost exclusively towards the ends of the alpha-helical elements of the F(1)beta pre-sequence. However, AtPrePI showed a preference for the N-terminal amphiphilic alpha-helix and positively charged amino acid residues and degraded the F(1)beta pre-sequence into 10-16 amino acid fragments, whereas AtPrePII did not show any positional preference and degraded the F(1)beta pre-sequence into 10-23 amino acid fragments. In conclusion, despite the high sequence identity between AtPrePI and AtPrePII and similarities in cleavage specificities, cleavage site recognition differs for both proteases and is context and structure dependent.     

Primary structure of the silk fibroin light chain determined by cDNA sequencing and peptide analysis

A cDNA clone, pFL18, carrying a putative full-length fibroin light chain (L-chain) sequence was isolated and its nucleotide sequence was determined. This revealed the presence of an open reading frame corresponding to a polypeptide with 262 amino acid residues. The sequence was concluded to be that of the L-chain with its signal peptide because corresponding amino acid sequences for the seven tryptic and the four chymotryptic peptides from the purified L-chain were all included and an N-terminal region having typical properties of a signal peptide was present. The N terminus of the mature form of L-chain was identified as N-acetyl serine by analyzing the acyl-dansylhydrazide derived from the N-acyl-amino acid which had been released from the N-terminal blocked chymotryptic peptide by the acylamino acid-releasing enzyme. It was suggested that a signal peptide had cleaved between Pro18 and Ser19, yielding a mature L-chain polypeptide consisting of 244 amino acid residues. The molecular weight of the L-chain was calculated to be 25,800 including the N-acetyl group. The L-chain contained three Cys residues, two of which were suggested to form an intramolecular disulfide linkage, leaving the third one at the most C-terminal position and in a relatively hydrophilic region as the most probable site of disulfide linkage with the fibroin heavy chain.     

Heterologous expression, purification, and characterization of human triacylglycerol hydrolase.

Triacylglycerol hydrolase mobilizes stored triacylglycerol some of which is used for very-low-density lipoprotein assembly in the liver. A full-length cDNA coding for a human triacylglycerol hydrolase (hTGH) was isolated from a human liver cDNA library. The cDNA has an open reading frame of 576 amino acids with a cleavable 18-amino-acid signal sequence. The deduced amino acid sequence shows that the protein belongs to the carboxylesterase family. The hTGH was highly expressed in Escherichia coli as a 6xHis-tagged fusion protein, with the tag at the N-terminus in place of the signal peptide. However, the expressed protein was insoluble and inactive. Expression was confirmed by immunoblotting and N-terminal amino acid sequencing of the purified protein. Expression of hTGH with its native signal sequence and a C-terminal 6xHis-tag in Sf9 cells using the baculovirus expression system yielded active enzyme. N-terminal amino acid sequencing of the purified expressed protein showed correct processing of the signal peptide. The enzyme also undergoes glycosylation within the endoplasmic reticulum lumen. The results suggest that hTGH expressed in insect cells is properly folded. Therefore, baculovirus expression of hTGH and facile purification of the His-tagged enzyme will allow detailed characterization of the structure/activity relationship.     

Amyloidogenecity and pitrilysin sensitivity of a lysine-free derivative of amyloid beta-peptide cleaved from a recombinant fusion protein

The progressive cerebral deposition of a 40-42 residues amyloid beta-peptide (Abeta) is regarded as a major factor in the onset of the Alzheimer's disease. It has recently been shown that Abeta(1-40) is cleaved by Escherichia coli pitrilysin, a homologue of insulysin, at a specific site. To facilitate the studies on a recognition mechanism of Abeta by pitrilysin, an overproduction system of Abeta(1-40) as a fusion protein with E. coli RNase HI was constructed. This fusion protein was designed such that an Abeta(1-40) derivative, Abeta(1-40)*, in which Lys16 and Lys28 of Abeta(1-40) are simultaneously replaced by Ala, is attached to the C-terminus of E. coli RNase HI and Abeta(1-40)* is separated from RNase HI upon cleavage with lysyl endopeptidase. The fusion protein was overproduced in E. coli in inclusion bodies, solubilized and purified in the presence of guanidine hydrochloride, and cleaved by lysyl endopeptidase. Abeta(1-40)* was purified from the resultant peptide fragments by reverse-phase HPLC. Measurement of the far-UV CD spectra suggests that Abeta(1-40)* is conformationally similar to Abeta(1-40). However, the thioflavin T binding assay suggests that Abeta(1-40)* is more amyloidogenic than Abeta(1-40). Nevertheless, Abeta(1-40)* was cleaved by pitrilysin at the site identical to that in Abeta(1-40).     

The processing of human mitochondrial leucyl-tRNA synthetase in the insect cells

A His-tagged full-length cDNA of human mitochondrial leucyl-tRNA synthetase was expressed in a baculovirus system. The N-terminal sequence of the enzyme isolated from the mitochondria of insect cells was found to be IYSATGKWTKEYTL, indicating that the mitochondrial targeting signal peptide was cleaved between Ser39 and Ile40 after the enzyme precursor was translocated into mitochondria. The enzyme purified from mitochondria catalyzed the leucylation of Escherichia coli tRNA(1)(Leu)(CAG) and Aquifex aeolicus tRNA(Leu)(GAG) with higher catalytic activity in the leucylation of E. coli tRNA(Leu) than that previously expressed in E. coli without the N-terminal 21 residues.     

cDNA cloning and heterologous expression of coniferin β-glucosidase

Coniferin -glucosidase (CBG) catalyzes the hydrolysis of monolignol glucosides to release the cinnamyl alcohols for oxidative polymerization to lignin. Utilizing the N-terminal amino acid sequence of the purified enzyme, the corresponding full-length cDNA sequence was isolated from a Pinus contorta xylem-specific library. The isolated 1909 nucleotide cDNA was confirmed to be that of CBG on the basis of its high homology to family 1 glycosyl hydrolases, the sequence identity with the N-terminal amino acid residues of the purified enzyme, and the coniferin hydrolytic activity and substrate specificity profile displayed by the recombinant protein when expressed in Escherichia coli. The presence of a 23 amino acid N-terminal signal peptide in the deduced 513 amino acid enzyme suggests that CBG is a secretory protein targeted to the ER. The isolation of CBG cDNA will facilitate the evaluation of the importance of this enzyme in the ultimate stages of lignin biosynthesis and could be a valuable tool in manipulating lignin levels in xylem cell walls.     

A novel mitochondrial and chloroplast peptidasome, PreP

A novel mitochondrial and chloroplast peptidasome, the Presequence Protease (PreP) degrades organellar targeting peptides as well as other unstructured peptides up to 65 amino acid residues in length. PreP belongs to the pitrilysin oligopeptidase family (M16C) containing an inverted zinc-binding motif. The crystal structure of Arabidopsis thaliana PreP, AtPreP, refined at 2.1 ?, revealed a novel mechanism of proteolysis in which two halves of the enzyme connected by a hinge region enclose a large catalytic chamber opening and closing in response to peptide binding. Double knock-out mutant of AtPreP1 and AtPreP2 results in a severe phenotype, including decreased size and growth rate, chlorosis and organellar abnormalities, such as altered chloroplast starch content, partial loss of the integrity of the inner mitochondrial membrane and reduced mitochondrial respiration. PreP homologues are also present in yeast and humans. Interestingly, human PreP has been associated with Alzheimer's disease as it is responsible for degradation of amyloid-β peptide in brain mitochondria.     

Isolation of cDNA clones for human erythrocyte membrane sialoglycoproteins alpha and delta.

We have isolated almost full-length cDNA clones corresponding to human erythrocyte membrane sialoglycoproteins alpha (glycophorin A) and delta (glycophorin B). The predicted amino acid sequence of delta differs at two amino acid residues from the sequence determined by peptide sequencing. The sialoglycoprotein delta clone we have isolated contains an interrupting sequence within the region that gives rise to the cleaved N-terminal leader sequence for the protein and represents a product that is unlikely to be inserted into the erythrocyte membrane. Comparison of the cDNA sequences of alpha and delta shows very strong homology at the DNA level within the coding regions. The two mRNA sequences are closely related and differ by a number of clearly defined insertions and deletions.     

Identification and characterization of hydra metalloproteinase 2 (HMP2): a meprin-like astacin metalloproteinase that functions in foot morphogenesis

Several members of the newly emerging astacin metalloproteinase family have been shown to function in a variety of biological events, including cell differentiation and morphogenesis during both embryonic development and adult tissue differentiation. We have characterized a new astacin proteinase, hydra metalloproteinase 2 (HMP2) from the Cnidarian, Hydra vulgaris. HMP2 is translated from a single mRNA of 1.7 kb that contains a 1488 bp open reading frame encoding a putative protein product of 496 amino acids. The overall structure of HMP2 most closely resembles that of meprins, a subgroup of astacin metalloproteinases. The presence of a transient signal peptide and a putative prosequence indicates that HMP2 is a secreted protein that requires post-translational processing. The mature HMP2 starts with an astacin proteinase domain that contains a zinc binding motif characteristic of the astacin family. Its COOH terminus is composed of two potential protein-protein interaction domains: an "MAM" domain (named after meprins, A-5 protein and receptor protein tyrosine phosphatase mu) that is only present in meprin-like astacin proteinases; and a unique C-terminal domain (TH domain) that is also present in another hydra metalloproteinase, HMP1, in Podocoryne metalloproteinase 1 (PMP1) of jellyfish and in toxins of sea anemone. The spatial expression pattern of HMP2 was determined by both mRNA whole-mount in situ hybridization and immunofluorescence studies. Both morphological techniques indicated that HMP2 is expressed only by the cells in the endodermal layer of the body column of hydra. While the highest level of HMP2 mRNA expression was observed at the junction between the body column and the foot process, immunofluorescence studies indicated that HMP2 protein was present as far apically as the base of the tentacles. In situ analysis also indicated expression of HMP2 during regeneration of the foot process. To test whether the higher levels of HMP2 mRNA expression at the basal pole related to processes underlying foot morphogenesis, antisense studies were conducted. Using a specialized technique named localized electroporation (LEP), antisense constructs to HMP2 were locally introduced into the endodermal layer of cells at the basal pole of polyps and foot regeneration was initiated and monitored. Treatment with antisense to HMP2 inhibited foot regeneration as compared to mismatch and sense controls. These functional studies in combination with the fact that HMP2 protein was expressed not only at the junction between the body column and the foot process, but also as far apically as the base of the tentacles, suggest that this meprin-class metalloproteinase may be multifunctional in hydra.     

Cleavage of various peptides with pitrilysin from Escherichia coli: kinetic analyses using beta-endorphin and its derivatives

Pitrilysin from Escherichia coli was overproduced, purified, and analyzed for enzymatic activity using 14 peptides as a substrate. Pitrilysin cleaved all the peptides, except for two of the smallest, at a limited number of sites, but showed little amino acid specificity. It cleaved beta-endorphin (beta-EP) most effectively, with a K(m) value of 0.36 microM and a k(cat) value of 750 min(-1). beta-EP consists of 31 residues and was predominantly cleaved by the enzyme at Lys(19)-Asn(20). Kinetic analyses using a series of beta-EP derivatives with N and/or C-terminal truncations and with amino acid substitutions revealed that three hydrophobic residues (Leu(14), Val(15), and Leu(17)) and the region 22-26 in beta-EP are responsible for high-affinity recognition by the enzyme. These two regions are located on the N- and C-terminal sides of the cleavage site in beta-EP, suggesting that the substrate binding pocket of pitrilysin spans its catalytic site.     

Consensus sequence for processing of peptide precursors at monobasic sites

Many regulatory peptide precursors undergo post-translational processing at mono- and/or dibasic residues. Comparison of amino acids around the monobasic cleavage sites suggests that these cleavages follow certain sequence motifs and can be described as the rules that govern monobasic cleavages: (i) a basic amino acid it present at either 3, 5, or 7 amino acids N-terminal to the cleavage site, (ii) hydrophobic aliphatic amino acids (leucine, isoleucine, valine, or methionine) are never present in the position C-terminal to the monobasic amino acid at the cleavage site, (iii) a cysteine is never present in the vicinity of the cleavage site, and (iv) an aromatic amino acid is never present at the position N-terminal to the monobasic amino acid at the cleavage site. In addition to these rules, the monobasic cleavages follow certain tendencies: (i) the amino acid at the cleavage site tends to be predominantly arginine, (ii) the amino acid at the position C-terminal to the cleavage site tends to be serine, alanine or glycine in more than 60% of the cases, (iii) the amino acid at either 3, 5, or 7 position N-terminal to the cleavage site tends to be arginine, (iv) aromatic amino acids are rare at the position C-terminal to the monobasic amino acid at the cleavage site, and (v) aliphatic amino acids tend to be in the two positions N-terminal to and the two positions C-terminal to the cleavage site, except as noted above. When compared with a large number of sequence containing single basic amino acids, these rules and tendencies are capable of not only correctly predicting the processing sites, but also are capable of excluding most of the single basic sequences that are known to be uncleaved. Many or these rules can also be applied to correctly predict the dibasic and multibasic cleavage sites suggesting that the rules and tendencies could govern endoproteolytic processing at the monobasic, dibasic and multibasic sites.     

Cloning, expression and characterization of a 46.5-kDa metallopeptidase from Bacillus halodurans H4 sharing properties with the pitrilysin family

A 1242 base pair DNA fragment from Bacillus halodurans H4 isolated from alkaline sediments of Lake Bogoria (Kenya) coding for a potential protease was cloned and sequenced. The hexa-histidine-tagged enzyme was overexpressed in Escherichia coli and was purified in one step by immobilized-metal affinity chromatography (IMAC) on Ni-NTA resin. The protease (ppBH4) presents an inverted zincin motif, HXXEH, which defines the inverzincin family. It shares several biochemical and molecular properties with the clan ME family M16 metallopeptidases (pitrilysins), as well as with database hypothetical proteins that are potential M16 family enzymes. Thus, like insulysin and nardilysin, but contrary to bacterial pitrilysin, ppBH4 is inactivated by sulfhydryl alkylating agents. On the other hand, like bacterial pitrilysin, ppBH4 is sensitive to reducing agents. The enzymatic activity of ppBH4 is limited to substrates smaller than proteins. In contrast to insulin, dynorphin and insulin B-chain are very good substrates for ppBH4 and several cleavage sites are common with those observed with well-characterized pitrilysins. As deduced from amino acid sequence, as well as determined by gel-filtration and SDS-polyacrylamide gel electrophoresis, ppBH4 is an active monomer of 46.5 kDa. This feature distinguishes ppBH4 from all other enzymes of the pitrilysin family so far described whose molecular masses range from 100 to 140 kDa.     

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.