Subfamily Of Proteases Research Articles

USP7 reduction leads to developmental failure of mouse early embryos

As a member of Ubiquitin-specific protease subfamily, ubiquitin specific protease 7 (USP7) has been reported to participate in a variety of cellular processes, including cell cycle, apoptosis, DNA damage response, and epigenetic modification. However, its function in preimplantation embryos is still obscure. To investigate the functions of USP7 during preimplantation embryo development, we used siRNA to degrade endogenous USP7 messenger RNA. We found that USP7 knockdown significantly decreased the development rate of mouse early embryos. Moreover, depletion of USP7 induced the accumulation of the DNA lesions and apoptotic blastomeres in early embryos. In addition, USP7 knockdown caused an abnormal H3K27me3 modification in 2-cell embryos. Overall, our results indicate that USP7 maintains genome stability perhaps via regulating H3K27me3 and DNA damage, consequently controlling the embryo quality.

UBP12/UBP13-mediated deubiquitination of salicylic acid receptor NPR3 suppresses plant immunity

Salicylic acid (SA), a defense hormone produced after pathogen challenge, is critical for plant immunity. Arabidopsis NONEXPRESSER OF PR GENES 1 (NPR1) and its paralogs NPR3 and NPR4 can bind SA and mediate SA signal transduction. NPR1 functions as a transcriptional co-activator to promote defense gene expression, whereas NPR3 and NPR4 have been shown to function as negative regulators in the SA signaling pathway. Although the mechanism about NPR1 regulation has been well studied, how NPR3/NPR4 proteins are regulated in immune responses remains largely unknown. Here, we show that the stability of NPR3/NPR4 is enhanced by SA. In the absence of pathogen challenge, NPR3/NPR4 are unstable and degraded by the 26S proteasome, whereas the increase in cellular SA levels upon pathogen infection suppresses NPR3/NPR4 degradation. We found that UBP12 and UBP13, two homologous deubiquitinases from a ubiquitin-specific protease subfamily, negatively regulate plant immunity by promoting NPR3/NPR4 stability. Our genetic results further showed that UBP12/UBP13-mediated immunity suppression is partially dependent on NPR3/NPR4 functions. By interacting with NPR3 in the nucleus in an SA-dependent manner, UBP12 and UBP13 remove ubiquitin from polyubiquitinated NPR3 to protect it from being degraded. The stabilization of NPR3/NPR4 promoted by UBP12/UBP13 is essential for negative regulation of basal and SA-induced immunity.

HC-SPH: A conserved serine protease homolog of S1 superfamily in the triangle-shell pearl mussel (Hyriopsis cumingii)

Serine proteases play central roles in immune defense in invertebrates through innate immunity, and are particularly important complement system in molluscs because their susceptibility to infection due to lack of an adaptive immune ability. A gene encoding the serine protease homolog from the triangle - shell pearl mussel (Hyriopsis cumingii) was identified and designated as HC-SPH in this study. Protein sequence analysis revealed that HC-SPH consists of a typical Tryp_SPc functional domain of serine protease of S1 family lead by a signal peptide, and the molecule shares a highly conserved sequence and structural organization with other members, including a cleavage site, 3 enzymatic active sites and 3 substrate binding sites, so that it was clustered into a trypsin-like serine protease subfamily of the S1 superfamily. Semi - quantitative analysis of the amplicons separated on agarose gel by comparing to the β-actin products revealed that the digestive gland had a strong expression while the gonads were seen as weak expression sites. Infected by Aeromonas hydrophila, the gene expression was significantly up - regulated in the kidney at the 6 hours post challenge (hpc), stomach at 12 hpc and gills at 24 hpc while the expression maintained steadily unchanged in the digestive gland. However, up to 48 hpc, the expression levels in all four tissues reached significantly high, and also joined by a high level of expression in intestine that was down - regulated before 24 hpc, to build up an enhanced immune defense. The complementary up - regulation of the gene expression in these tissues suggested a temporal and spatial reinforce model for HC-SPH in immune response.

ADAM and ADAMTS Proteases in Hepatic Disorders

Proteolysis is an irreversible post-translational modification that regulates protein function and signal transduction. This includes remodelling of the extracellular matrix, release of membrane-bound cytokines and receptor ectodomains, as well as the initiation of intracellular signalling cues. Members of the adamalysin protease subfamily, in particular the ADAM (a disintegrin and metalloprotease) and ADAMTS (the ADAM containing thrombospondin motif) families, are involved in these processes. This review presents an overview of how ADAM and ADAMTS proteins are involved in liver physiology and pathophysiology.

Targeting dipeptidyl peptidase 8 genes inhibits proliferation, migration and invasion by inhibition of cyclin D1 and MMP2MMP9 signal pathway in cervical cancer.

DPP8 is a member of the dipeptidyl peptidase IV family, which belongs to the S9b protease subfamily. It regulates cell proliferation, apoptosis, migration and invasion during cancer progression. To investigate the role of DPP8 in cervical cancer, we examined DPP8 levels in cervical cancer tissues and cells. The localization of DPP8 was determined by immunofluorescence staining. Subsequently, SiHa and HeLa cells were treated with small interfering RNA (siRNA)-DPP8. We used cell cycle analysis, an 5-ethyl-2'-deoxyuridine assay proliferation assay and a cellular apoptosis assay to determine the effect of DPP8 on the proliferation and apoptosis of cervical cancer cells. We used a Transwell assay to assess the number of transfection cancer cells migrating through the matrix. A real-time polymerase chain reaction and western blot analysis were used to analyze the expression of related proteins and to determine the phenotype caused by the depletion or overexpression of DPP8 in cervical cancer cells. We observed that DPP8 was highly expressed in cervical cancer tissues and cells. DPP8 expression was observed in the cytosol and in the perinuclear area, as well as in the nuclei of cervical cancer cells. Notably, when cells were treated with siRNA-DPP8, the expression of BAX increased, and the expression of cyclin D1, Bcl-2, MMP2 and MMP9 was downregulated. In cervical cancer cell lines, silencing the expression of DPP8 not only suppressed the proliferation, migration and invasion of the cervical cancer cells, but also promoted cervical cancer cell apoptosis. The data obtained in the present study reveal that DPP8 promotes the progression of cervical cancer.

A systematic study of modulation of ADAM-mediated ectodomain shedding by site-specific O-glycosylation

Regulated shedding of the ectodomain of cell membrane proteins by proteases is a common process that releases the extracellular domain from the cell and activates cell signaling. Ectodomain shedding occurs in the immediate extracellular juxtamembrane region, which is also where O-glycosylation is often found and examples of crosstalk between shedding and O-glycosylation have been reported. Here, we systematically investigated the potential of site-specific O-glycosylation mediated by distinct polypeptide GalNAc-transferase (GalNAc-T) isoforms to coregulate ectodomain shedding mediated by the A Disintegrin And Metalloproteinase (ADAM) subfamily of proteases and in particular ADAM17. We analyzed 25 membrane proteins that are known to undergo ADAM17 shedding and where the processing sites included Ser/Thr residues within ± 4 residues that could represent O-glycosites. We used in vitro GalNAc-T enzyme and ADAM cleavage assays to demonstrate that shedding of at least 12 of these proteins are potentially coregulated by O-glycosylation. Using TNF-α as an example, we confirmed that shedding mediated by ADAM17 is coregulated by O-glycosylation controlled by the GalNAc-T2 isoform both ex vivo in isogenic cell models and in vivo in mouse Galnt2 knockouts. The study provides compelling evidence for a wider role of site-specific O-glycosylation in ectodomain shedding.

Structure of human dipeptidyl peptidase 10 (DPPY): a modulator of neuronal Kv4 channels.

The voltage-gated potassium channel family (Kv) constitutes the most diverse class of ion channels in the nervous system. Dipeptidyl peptidase 10 (DPP10) is an inactive peptidase that modulates the electrophysiological properties, cell-surface expression and subcellular localization of voltage-gated potassium channels. As a consequence, DPP10 malfunctioning is associated with neurodegenerative conditions like Alzheimer and fronto-temporal dementia, making this protein an attractive drug target. In this work, we report the crystal structure of DPP10 and compare it to that of DPP6 and DPP4. DPP10 belongs to the S9B serine protease subfamily and contains two domains with two distinct folds: a β-propeller and a classical α/β-hydrolase fold. The catalytic serine, however, is replaced by a glycine, rendering the protein enzymatically inactive. Difference in the entrance channels to the active sites between DPP10 and DPP4 provide an additional rationale for the lack of activity. We also characterize the DPP10 dimer interface focusing on the alternative approach for designing drugs able to target protein-protein interactions.

GlyGly-CTERM and Rhombosortase: A C-Terminal Protein Processing Signal in a Many-to-One Pairing with a Rhomboid Family Intramembrane Serine Protease

The rhomboid family of serine proteases occurs in all domains of life. Its members contain at least six hydrophobic membrane-spanning helices, with an active site serine located deep within the hydrophobic interior of the plasma membrane. The model member GlpG from Escherichia coli is heavily studied through engineered mutant forms, varied model substrates, and multiple X-ray crystal studies, yet its relationship to endogenous substrates is not well understood. Here we describe an apparent membrane anchoring C-terminal homology domain that appears in numerous genera including Shewanella, Vibrio, Acinetobacter, and Ralstonia, but excluding Escherichia and Haemophilus. Individual genomes encode up to thirteen members, usually homologous to each other only in this C-terminal region. The domain's tripartite architecture consists of motif, transmembrane helix, and cluster of basic residues at the protein C-terminus, as also seen with the LPXTG recognition sequence for sortase A and the PEP-CTERM recognition sequence for exosortase. Partial Phylogenetic Profiling identifies a distinctive rhomboid-like protease subfamily almost perfectly co-distributed with this recognition sequence. This protease subfamily and its putative target domain are hereby renamed rhombosortase and GlyGly-CTERM, respectively. The protease and target are encoded by consecutive genes in most genomes with just a single target, but far apart otherwise. The signature motif of the Rhombo-CTERM domain, often SGGS, only partially resembles known cleavage sites of rhomboid protease family model substrates. Some protein families that have several members with C-terminal GlyGly-CTERM domains also have additional members with LPXTG or PEP-CTERM domains instead, suggesting there may be common themes to the post-translational processing of these proteins by three different membrane protein superfamilies.

Expression and Genetic Loss of Function Analysis of the HAT/DESC Cluster Proteases TMPRSS11A and HAT

Genome mining at the turn of the millennium uncovered a new family of type II transmembrane serine proteases (TTSPs) that comprises 17 members in humans and 19 in mice. TTSPs phylogenetically belong to one of four subfamilies: matriptase, hepsin/TMPRSS, corin and HAT/DESC. Whereas a wealth of information now has been gathered as to the physiological functions of members of the hepsin/TMPRSS, matriptase, and corin subfamilies of TTSPs, comparatively little is known about the functions of the HAT/DESC subfamily of proteases. Here we perform a combined expression and functional analysis of this TTSP subfamily. We show that the five human and seven murine HAT/DESC proteases are coordinately expressed, suggesting a level of functional redundancy. We also perform a comprehensive phenotypic analysis of mice deficient in two of the most widely expressed HAT/DESC proteases, TMPRSS11A and HAT, and show that the two proteases are dispensable for development, health, and long-term survival in the absence of external challenges or additional genetic deficits. Our comprehensive expression analysis and generation of TMPRSS11A- and HAT-deficient mutant mouse strains provide a valuable resource for the scientific community for further exploration of the HAT/DESC subfamily proteases in physiological and pathological processes.

Analysis of binding properties and specificity through identification of the interface forming residues (IFR) for serine proteases in silico docked to different inhibitors

BackgroundEnzymes belonging to the same super family of proteins in general operate on variety of substrates and are inhibited by wide selection of inhibitors. In this work our main objective was to expand the scope of studies that consider only the catalytic and binding pocket amino acids while analyzing enzyme specificity and instead, include a wider category which we have named the Interface Forming Residues (IFR). We were motivated to identify those amino acids with decreased accessibility to solvent after docking of different types of inhibitors to sub classes of serine proteases and then create a table (matrix) of all amino acid positions at the interface as well as their respective occupancies. Our goal is to establish a platform for analysis of the relationship between IFR characteristics and binding properties/specificity for bi-molecular complexes.ResultsWe propose a novel method for describing binding properties and delineating serine proteases specificity by compiling an exhaustive table of interface forming residues (IFR) for serine proteases and their inhibitors. Currently, the Protein Data Bank (PDB) does not contain all the data that our analysis would require. Therefore, an in silico approach was designed for building corresponding complexesThe IFRs are obtained by "rigid body docking" among 70 structurally aligned, sequence wise non-redundant, serine protease structures with 3 inhibitors: bovine pancreatic trypsin inhibitor (BPTI), ecotine and ovomucoid third domain inhibitor. The table (matrix) of all amino acid positions at the interface and their respective occupancy is created. We also developed a new computational protocol for predicting IFRs for those complexes which were not deciphered experimentally so far, achieving accuracy of at least 0.97.ConclusionsThe serine proteases interfaces prefer polar (including glycine) residues (with some exceptions). Charged residues were found to be uniquely prevalent at the interfaces between the "miscellaneous-virus" subfamily and the three inhibitors. This prompts speculation about how important this difference in IFR characteristics is for maintaining virulence of those organisms.Our work here provides a unique tool for both structure/function relationship analysis as well as a compilation of indicators detailing how the specificity of various serine proteases may have been achieved and/or could be altered. It also indicates that the interface forming residues which also determine specificity of serine protease subfamily can not be presented in a canonical way but rather as a matrix of alternative populations of amino acids occupying variety of IFR positions.

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. L ebetina venom having Chymo Trypsin 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.

Processing of the Phalloidin Proprotein by Prolyl Oligopeptidase from the Mushroom Conocybe albipes

The peptide toxins of poisonous Amanita mushrooms are bicyclic octapeptides (amatoxins) or heptapeptides (phallotoxins). In Amanita bisporigera, alpha-amanitin and phallacidin are synthesized as 35- and 34-amino acid proproteins, respectively, in which the amino acid sequences found in the mature toxins are flanked by conserved amino acid sequences. The presence of invariant Pro residues immediately upstream of the toxin regions and as the last predicted amino acid in the toxin regions themselves suggests that a Pro-specific peptidase is responsible for the initial post-translational processing of the Amanita toxin proproteins. We purified an enzyme from the phalloidin-producing mushroom Conocybe albipes that cleaves a synthetic 22-mer phalloidin peptide to release the mature toxin peptide (AWLATCP). Mass spectrometric analysis of the purified protein combined with isolation and sequencing of the encoding gene indicates that the responsible processing enzyme is a member of the prolyl oligopeptidase (POP) subfamily of proteases (EC 3.4.21.26). The processing enzyme was able to use the chromogenic POP substrate benzyloxycarbonyl-Gly-Pro-p-nitroanilide and was inhibited by the specific POP inhibitor benzyloxycarbonyl-Pro-prolinal. Both Pro bonds in the proprotein are cleaved by the same enzyme, with the C-terminal Pro bond cleaved first or much faster than the N-terminal Pro bond. Transient accumulation of the N-terminal intermediate indicates that cleavage is not strongly processive. A synthetic peptide representing the phallacidin proprotein was also cleaved by the POP of C. albipes, but a precursor of amanitin (which is not made by C. albipes) was cleaved inefficiently.

Cloning, expression, and characterization of two novel cuticle-degrading serine proteases from the entomopathogenic fungus Cordyceps sinensis

The entomopathogenic fungus Cordyceps sinensis has been important in traditional Chinese medicine, but is yet to be commercially cultivated. Difficulty in cultivation results in part from the low percentage of fungal infection on artificially inoculated host insects. To better understand the infection mechanism, we cloned two cuticle-degrading serine protease genes ( csp1 and csp2) from C. sinensis. These enzymes are novel members of the S8A subfamily of proteases. Identities of cDNA or amino acid sequences between Csp1 and Csp2 were 72.9% and 68.9%, respectively. After successful expression in the yeast Pichia pastoris, recombinant enzymes were purified and characterized using the synthetic substrate N-suc-AAPF- p-NA. Both were chymotrypsin-like serine proteases with an optimum pH of 7.0 and an optimal temperature of 40 °C (Csp1) or 50 °C (Csp2). Bioassay revealed that Csp1 and Csp2 degraded the cuticle proteins of larval Hepialus sp. in vitro. This is the first report of serine proteases from C. sinensis.

The Ubiquitin-Specific Protease Subfamily UBP3/UBP4 Is Essential for Pollen Development and Transmission in Arabidopsis

Deubiquitinating enzymes are essential to the ubiquitin (Ub)/26S proteasome system where they release Ub monomers from the primary translation products of poly-Ub and Ub extension genes, recycle Ubs from polyubiquitinated proteins, and reverse the effects of ubiquitination by releasing bound Ubs from individual targets. The Ub-specific proteases (UBPs) are one large family of deubiquitinating enzymes that bear signature cysteine and histidine motifs. Here, we genetically characterize a UBP subfamily in Arabidopsis (Arabidopsis thaliana) encoded by paralogous UBP3 and UBP4 genes. Whereas homozygous ubp3 and ubp4 single mutants do not display obvious phenotypic abnormalities, double-homozygous mutant individuals could not be created due to a defect in pollen development and/or transmission. This pollen defect was rescued with a transgene encoding wild-type UBP3 or UBP4, but not with a transgene encoding an active-site mutant of UBP3, indicating that deubiquitination activity of UBP3/UBP4 is required. Nuclear DNA staining revealed that ubp3 ubp4 pollen often fail to undergo mitosis II, which generates the two sperm cells needed for double fertilization. Substantial changes in vacuolar morphology were also evident in mutant grains at the time of pollen dehiscence, suggesting defects in vacuole and endomembrane organization. Even though some ubp3 ubp4 pollen could germinate in vitro, they failed to fertilize wild-type ovules even in the absence of competing wild-type pollen. These studies provide additional evidence that the Ub/26S proteasome system is important for male gametogenesis in plants and suggest that deubiquitination of one or more targets by UBP3/UBP4 is critical for the development of functional pollen.

Role of the α-Helical Linker of the C-Terminal Translocator in the Biogenesis of the Serine Protease Subfamily of Autotransporters

Autotransporters are secreted virulence factors that comprise three domains: an N-terminal signal peptide, an internal passenger domain, and a C-terminal beta-domain. The mechanism of passenger translocation across the outer membrane remains undefined, with four models having been proposed: the "hairpin," the "threading," the "multimeric," and the "Omp85 (YaeT)" models. In an attempt to understand autotransporter biogenesis, we screened the sequences of the serine protease subfamily of autotransporters (SPATEs) for conserved features indicative of a common secretion mechanism. Our analyses revealed a strictly conserved 14-amino-acid motif within the predicted alpha-helical linker region, upstream of the beta-domain of SPATEs. We investigated the function of this motif through a mutagenesis approach using Tsh as a model. Our studies demonstrate that mutations throughout the conserved motif do not block insertion of the beta-domain into the outer membrane. However, nonconservative mutations of four hydrophobic (V1099, L1102, G1107, and L1109) and three polar (N1100, K1104, and R1105) residues of the motif severely decrease or even abolish Tsh biogenesis. Further studies showed that these mutations interfere with passenger transport across the outer membrane. Bioinformatical analyses suggest that the critical polar and hydrophobic amino acids localize on opposite sides of the helix that runs through the beta-barrel pore. Our data indicate that the conserved motif is important for passenger secretion across the outer membrane and that mutations in certain residues severely affect the secretion process. We discuss how these results fit with the four proposed models for autotransporter secretion and potential applications in antimicrobial and vaccine development.

Yeast as a Tractable Genetic System for Functional Studies of the Insulin-degrading Enzyme

We have developed yeast as an expression and genetic system for functional studies of the insulin-degrading enzyme (IDE), which cleaves and inactivates certain small peptide molecules, including insulin and the neurotoxic A beta peptide. We show that heterologously expressed rat IDE is enzymatically active, as judged by the ability of IDE-containing yeast extracts to cleave insulin in vitro. We also show that IDE can promote the in vivo production of the yeast a-factor mating pheromone, a function normally attributed to the yeast enzymes Axl1p and Ste23p. However, IDE cannot substitute for the function of Axl1p in promoting haploid axial budding and repressing haploid invasive growth, activities that require an uncharacterized activity of Axl1p. Particulate fractions enriched for Axl1p or Ste23p are incapable of cleaving insulin, suggesting that the functional conservation of these enzymes may not be bidirectionally conserved. We have made practical use of our genetic system to confirm that residues composing the extended zinc metalloprotease motif of M16A family enzymes are required for the enzymatic activity of IDE, Ste23p, and Axl1p. We have determined that IDE and Axl1p both require an intact C terminus for optimal activity. We expect that the tractable genetic system that we have developed will be useful for investigating the enzymatic and structure/function properties of IDE and possibly for the identification of novel IDE alleles having altered substrate specificity.

Cysteine proteinase activities in the fish pathogen Philasterides dicentrarchi (Ciliophora: Scuticociliatida).

This study investigated protease activities in a crude extract and in vitro excretion/secretion (E/S) products of Philasterides dicentrarchi, a ciliate fish parasite causing economically significant losses in aquaculture. Gelatin/SDS-PAGE analysis (pH 4, reducing conditions) detected 7 bands with gelatinolytic activity (approximate molecular weights 30-63 kDa) in the crude extract. The banding pattern observed in analysis of E/S products was practically identical, except for 1 low-molecular-weight band detected in the crude extract but not in the E/S products. In assays with synthetic peptide p-nitroanilide substrates, the crude extract hydrolysed substrates characteristic of cysteine proteases, namely Z-Arg-Arg pNA, Bz-Phe-Val-Arg pNA and Z-Phe-Arg pNA. These activities were strongly inhibited by the cysteine protease inhibitor E-64 and by Ac-Leu-Val-Lys aldehyde, a potent inhibitor of cysteine proteases of the cathepsin B protease subfamily. The proteases present in the crude extract degraded both type-I collagen and haemoglobin in vitro, consistent with roles in tissue invasion and nutrition respectively. Again, E-64 completely (collagen) or markedly (haemoglobin) inhibited this degradation. Finally, the histolytic activity of the ciliate in turbot fibroblast monolayers was strongly reduced in the presence of E-64, confirming the importance of secreted cysteine proteinases in the biology of Philasterides dicentrarchi.

The α1/2 helical backbone of the prodomains defines the intrinsic inhibitory specificity in the cathepsin L-like cysteine protease subfamily

Proregions of papain-like cysteine proteases are potent and often highly selective inhibitors of their parental enzymes. The molecular basis of their selectivity is poorly understood. For two closely related members of the cathepsin L-like subfamily we established strong selectivity differences. The propeptide of cathepsin S was observed to inhibit cathepsin L with a K i of 0.08 nM, yet cathepsin L propeptide inhibited cathepsin S only poorly. To identify the respective structural correlates we engineered chimeric propeptides and compared their inhibitory specificity with the wild-types. Specificity resided in the N-terminal part, strongly suggesting that the backbone of the prodomain was the underlying structure.

Cathepsin L is an intracellular and extracellular protease in Paramecium tetraurelia. Purification, cloning, sequencing and specific inhibition by its expressed propeptide.

The ciliate Paramecium tetraurelia secretes large amounts of a cysteine protease into the growth medium, presumably for extracellular food digestion. Two endoprotease isozymes (30 and 33 kDa on SDS/PAGE, respectively), both present in cell homogenates and in spent growth medium, were purified to homogeneity. Peptide sequence analysis revealed that these isozymes share identities at the amino acid level but are probably differently processed. Enzymatic characterization of the isolated proteases and sequencing of the cloned cDNA demonstrated that the enzymes belong to the cathepsin-L protease subfamily. Although the identity with mammalian and other protozoan L cathepsins was only around 30%, all important signature sequences for cathepsin L in the preproregion as well as in the catalyst of the enzyme were fully retained. The cDNA of this cysteine protease codes for a preproregion of 108 amino acids. The putative proregion of 86 amino acids which contained the characteristic conserved ERFNIN motif, was fused with a His6 tag, expressed in Escherichia coli, and purified. Both cathepsin L isozymes of Paramecium tetraurelia were inhibited by their cognate propeptide in the nanomolar concentration range. All other cysteine proteases tested (papain and mammalian cathepsin B, G and H) were unaffected by the propeptide up to 10 microM.

Amino acid sequence of the heavy chain of human alpha-factor XIIa (activated Hageman factor).

The amino acid sequence of the heavy chain of human alpha-factor XIIa (activated Hageman factor) was determined by automated Edman degradation using the peptides produced by chemical and enzymatic cleavages of intact factor XII and alpha-factor XIIa. Combining this sequence with the previously determined sequence of beta-factor XIIa (Fujikawa, K., and McMullen, B. A. (1983) J. Biol. Chem. 258, 10924-10933), the complete amino acid sequence of human factor XII has been established. The heavy chain of alpha-factor XIIa is composed of 353 amino acid residues containing one Asn-linked and six probable O-linked carbohydrate chains. The heavy chain of alpha-factor XIIa appears to contain four different domains including a "kringle," a "growth factor" domain, and the "type I" and "type II" domains of fibronectin. The domain organization of factor XII is analogous to those of several fibrinolytic proteins, including tissue plasminogen activator and urokinase, suggesting that factor XII belongs to the same protease subfamily as these two proteins.