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The processing and activation of prolegumain were studied using the recombinant protein synthesized by cells that had been stably transfected with a human legumain cDNA construct. A cell line termed C13 was selected for the high-level expression of prolegumain. C13 cells produced primarily 56kDa prolegumain. The 56kDa form was enzymically inactive but stable at neutral pH, unlike the 35kDa mature pig legumain; it could be converted into a 46kDa active form by incubation at pH 4.5. The 56kDa pro-form and the 46kDa active form were found to have the same N-terminal amino acid sequence, indicating that cleavage at the N-terminus was not necessary for prolegumain activation, and that the decrease in molecular mass was due to a C-terminal cleavage. The C-terminal processing site was identified as Asn323. Replacement of Asn323 at the cleavage site with aspartate, serine, alanine or glutamate abolished the processing and activation of prolegumain. In contrast, mutation of other asparagine and aspartate residues near the cleavage site had no effect. These results demonstrate that Asn323 is essential for prolegumain activation.

Legumain, a recently discovered mammalian cysteine endopeptidase, was found in all mouse tissues examined, but was particularly abundant in kidney and placenta. The distribution in subcellular fractions of mouse and rat kidney showed a lysosomal localization, and activity was detectable only after the organelles were disrupted. Nevertheless, ratios of legumain activity to that of cathepsin B differed considerably between mouse tissues. cDNA encoding mouse legumain was cloned and sequenced, the deduced amino acid sequence proving to be 83% identical to that of the human protein [Chen, Dando, Rawlings, Brown, Young, Stevens, Hewitt, Watts and Barrett (1997) J. Biol. Chem. 272, 8090–8098]. Recombinant mouse legumain was expressed in human embryonic kidney 293 cells by use of a vector containing a cytomegalovirus promoter. The recombinant enzyme was partially purified and found to be an asparagine-specific endopeptidase closely similar to naturally occurring pig kidney legumain.

Zinc metallopeptidases that contain the His-Glu-Xaa-Xaa-His (HEXXH) motif generally have a third ligand of the metal ion that may be either a Glu residue (in clan MA) or a His residue (in clan MB) (Rawlings and Barrett (1995) Methods Enzymol. 248, 183–228). Thimet oligopeptidase has not yet been assigned to either clan, and both Glu and His residues have been proposed as the third ligand. We mutated candidate ligand residues in the recombinant enzyme and identified Glu, His and Asp residues that are important for catalytic activity and/or stability of the protein. However, neither of the Glu and His residues close to the HEXXH motif that have previously been suggested to be ligands is required for the binding of zinc. We conclude that thimet oligopeptidase is not a member of clan MA or clan MB and it is likely that the enzyme possesses a catalytic site and protein fold different from those identified in any metallopeptidase to date. The definitive identification of the third zinc ligand may well require the determination of the crystallographic structure of thimet oligopeptidase or one of its homologues.

Legumain is a cysteine endopeptidase that shows strict specificity for hydrolysis of asparaginyl bonds. The enzyme belongs to peptidase family C13, and is thus unrelated to the better known cysteine peptidases of the papain family, C1 (Rawlings, N. D., and Barrett, A. J. (1994) Methods Enzymol. 244, 461-486). To date, legumain has been described only from plants and a blood fluke, Schistosoma mansoni We now show that legumain is present in mammals. We have cloned and sequenced human legumain and part of pig legumain. We have also purified legumain to homogeneity (2200-fold, 8% yield) from pig kidney. The mammalian sequences are clearly homologous with legumains from non-mammalian species. Pig legumain is a glycoprotein of about 34 kDa, decreasing to 31 kDa on deglycosylation. It is an asparaginyl endopeptidase, hydrolyzing Z-Ala-Ala-Asn-7-(4-methyl)coumarylamide and benzoyl-Asn-p-nitroanilide. Maximal activity is seen at pH 5.8 under normal assay conditions, and the enzyme is irreversibly denatured at pH 7 and above. Mammalian legumain is a cysteine endopeptidase, inhibited by iodoacetamide and maleimides, but unaffected by compound E64 (trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane). It is inhibited by ovocystatin (cystatin from chicken egg white) and human cystatin C with Ki values < 5 nM. We discuss the significance of the discovery of a cysteine endopeptidase of a new family and distinctive specificity in man and other mammals.

Relatively minor perturbations caused by Cp ring substituents in a series of mixed-valence biferrocenium cations have pronounced effects on the electronic structure and rate of intramolecular electron transfer. The X-ray structure of 1',2',1'',2'''-tetraethylbiferrocene has been determined at 298 K: C2/c, a=18.760(3) A, b=9.568(3) A, c=16.441 (3) A, β=130.494(13) o , Z=4, D calcd =1.42 g cm -1 , R f =0.038, and R wf =0.041

Chicken embryo fibroblasts (CEF) transformed by Rous sarcoma virus (RSVCEF) secrete a 70-kDa metallo-gelatinase at elevated levels over that of normal CEF. The 70-kDa enzyme has been purified from RSVCEF conditioned medium and represents 1-3% of the total protein in the RSVCEF conditioned medium. A 22-kDa protein, which appears to be the avian form of the tissue inhibitor of metalloproteases (TIMP), is co-isolated in association with the 70-kDa enzyme and can be separated from the enzyme by gel filtration carried out under denaturing conditions. The isolated 70-kDa species is in the zymogen form. It can be activated by treatment with the organomercurial, p-aminophenylmercuric acetate (APMA), yielding a 62-kDa active species derived by an apparent autoproteolytic cleavage from the 70-kDa proenzyme as determined by both substrate gel analysis and immunoblots using a monospecific antibody to the 70-kDa proenzyme. The proenzyme is poorly activated by trypsin and not activated by plasmin. The APMA-activated enzyme rapidly degrades denatured collagens but under identical conditions is unable to degrade native collagens, including basement membrane type IV collagen. Only at very high enzyme to substrate ratios (1:2) will native type IV collagen be hydrolyzed. Partial N-terminal amino acid sequencing of both the 70-kDa proenzyme and the 62-kDa active enzyme indicates that the avian enzyme is a member of the matrix metalloprotease family (MMP-2). When CEF cultures, infected with a temperature sensitive mutant of RSV, conditional for the expression of the transforming src oncogene, were incubated at the permissive and nonpermissive temperatures, differential levels of the 70-kDa enzyme were produced in direct proportion to the functioning of the src oncogene.

Cultures of transformed fibroblasts actively involved in extracellular matrix degradation have been examined for initial activation of serine and metallo protease cascade systems. Rous sarcoma virus transformed chick embryo fibroblasts (RSVCEF), in contrast to transformed mammalian cells, produce active, two chain urokinase-type plasminogen activator (tcu-PA). Active tcu-PA is found in serum-free, plasmin-free conditioned medium from RSVCEF cultures as determined by two independent methods, immunoprecipitation and differential DFP sensitivity. RSVCEF cultures synthesize and secrete inactive, single chain uPA (scu-PA) which is converted to tcu-PA in a time dependent manner by a catalytic mechanism that appears to involve a functioning uPA receptor on the surface of intact cells. The enzyme activity responsible for this conversion may represent the initiating catalytic event in the PA/plasminogen serine protease cascade system. A 70 kDa prometalloprotease capable of degrading denatured collagen following its activation also is significantly elevated in RSVCEF cultures over that of normal CEF. Trace amounts of the active 62 kDa form of the metalloprotease (gelatinase) is found in the transformed RSVCEF cultures indicating that these cultures produce a natural activator of the prometalloprotease. Plasmin and/or PA do not appear to be the activator of this enzyme as determined by indirect inhibition assays and direct assays employing purified enzymes. The possible central position of pro PA and the 70 kDa prometalloprotease in an interacting, complex protease cascade system involved in extracellular matrix degradation is discussed.

Publisher Summary This chapter discusses the structural chemistry and the biological aspects of dipeptidyl-peptidase III (DPP-III). A recommended assay procedure for DPP III would use as substrate Arg–Arg–NHMee in a Tris–HCl buffer at pH 8.0, the formation of product being monitored continuously by fluorimetry. DPP III purified from mammalian tissues is a monomeric protein of 82–84 kDa with a pI close to 4.5. The deduced sequence shows no evidence of a signal peptide or membrane-spanning regions. The deduced amino acid sequence of DPP III shows a motif HELLGH (residues 450–455) that resembles the HExxH motif that is present in many metallopeptidases, but with the insertion of an extra residue. There is no direct information about the biological functions of DPP III. It is generally found to be a soluble, cytosolic protein, and immunohistochemical studies have shown that rat DPP III is expressed in most tissues, with strongest staining in liver, kidney and adrenal gland. The ubiquitous distribution and broad specificity of action on oligopeptides support the notion that DPP III is primarily a housekeeping protein responsible for intracellular peptide catabolism.

Publisher Summary This chapter elaborates the activity, specificity and structural chemistry of thimet oligopeptidase (TOP). Human TOP cleaved hexa-alanine, but not tetra- or penta-alanine. TOP is an oligopeptidase with a sharply-defined upper limit of substrate size. Heptade-capeptide substrates include γ-endorphin and nociceptin/orphanin FQ. In a study of the specificity of TOP, it is confirmed that all substrates contain 17 or fewer amino acids. The rules governing the substrate specificity of TOP remain unclear, but with both substrates and inhibitors, hydrophobic residues tend to be favored in positions PI and P3′, and Pro in P2′. The enzyme also shows a preference for cleaving bond three to six residues from the C-terminus. TOP is a single-chain protein of about 78.5 kDa that does not contain intramolecular disulfide bonds. There are no indications of post-translational modifications in the biosynthesis of TOP or of a proteolytically activatable proenzyme. Directed mutagenesis has been used to identify amino acid residues important for the catalytic activity of TOP.

We show by site-directed mutagenesis that the catalytic residues of mammalian legumain, a recently discovered lysosomal asparaginycysteine endopeptidase, form a catalytic dyad in the motif His-Gly-spacer-Ala-Cys. We note that the same motif is present in the caspases, aspartate-specific endopeptidases central to the process of apoptosis in animal cells, and also in the families of clostripain and gingipain which are arginyl/lysyl endopeptidases of pathogenic bacteria. We propose that the four families have similar protein folds, are evolutionarily related in clan CD, and have common characteristics including substrate specificities dominated by the interactions of the S1 subsite.

The local degradation of fibronectin substrata by Rous sarcoma virus-transformed chick embryonic fibroblasts requires cell-contact-related metalloendoprotease and serine-protease activities. Using fibronectin-containing SDS gels, two large proteases with apparent molecular weights of 120K and 150K were found only in the membrane fraction of transformed cells and were absent in normal cells. Both 120K and 150K proteases were active at neutral pH, but showed preferential inhibitor sensitivities of serine and metal proteases, respectively. The 150K protease appeared to account for most of the proteolytic activity since metalloendoprotease inhibitors completely blocked proteolytic activity of the 150K in fibronectin gels, more than 80% of the fibronectin-degrading activity of solubilized membranes, and largely suppressed the appearance of fibronectin degradation spots in cultures of transformed cells.