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PubMed1995

Trypanosomatid cysteine protease activity may be enhanced by a kininogen-like moiety from host serum.

African trypanosomes contain cysteine proteases (trypanopains) the activity of which can be measured by in vitro digestion of fibrinogen, after electrophoresis in fibrinogen-containing SDS/polyacrylamide gels. When assessed by this procedure, trypanopain from Trypanosoma brucei (trypanopain-Tb) is estimated to have a molecular mass of 28 kDa. However, two additional bands of trypanopain activity (87 kDa and 105 kDa) are observed if serum is added to the trypanopain before electrophoresis. Formation of the 87 and 105 kDa bands is frequently accompanied by a reduction in the intensity of the 28 kDa activity which suggests that the extra bands are complexes of the 28 kDa trypanopain-Tb and a molecule from rat serum called rat trypanopain moledulator (rTM). The rTM-induced activation of cysteine proteases is not restricted to T. brucei as it is also observed with proteases from other protozoan parasites such as bloodstream forms of Trypanosoma congolense and the mammalian-infective in vitro-derived promastigote forms of Leishmania donovani and Leishmania major. The physical properties of rTM resemble those of the kininogen family of cysteine protease inhibitors. rTM is an acidic (pI 4.7) heat-stable 68 kDa glycoprotein with 15 kDa protease-susceptible domains. This resemblance between rTM and kininogens was confirmed by the positive, albeit weak, immunoreactivity between anti-(human low-molecular-mass kininogen) antibody and rTM as well as anti-rTM antibody and human low-molecular-mass kininogen. Furthermore, commercial preparations of human-low-molecular-mass kininogen and chicken egg white cystatin mimicked rTM by forming extra bands of proteolytic activity in the presence of trypanopain-Tb. In some instances, low-molecular-mass kininogen was also observed to increase the rate of hydrolysis of 7-(benzyloxycarbonyl-phenylalanyl-arginyl-amido)-4- methylcoumarin by live T. brucei. Although this effect was rather erratic, in no instance was significant inhibition observed when this putative cysteine protease inhibitor was used under these conditions. The activation of parasite cysteine proteases by commonly accepted cysteine protease inhibitors is unexpected and may have important pathological repercussions.

Animals↗
PubMed1999

Cloning of a family of serine protease genes from the cat flea Ctenocephalides felis.

Serine protease gene fragments approximately 480 nucleotides in length were amplified from Ctenocephalides felis larval and adult cDNA libraries using degenerate oligonucleotide PCR primers. Partial clones of thirty-eight distinct serine protease encoding sequences were isolated, and nineteen different full-length cDNAs encoding mature serine proteases were subsequently cloned and sequenced. All of the mature proteases contained the histidine, aspartic acid and serine amino acids of the catalytic triad characteristic of serine proteases. The mature C. felis serine proteases had amino acid sequences that were at most 29-53% identical to those known insect and arachnid serine proteases. Two of the C. felis gene sequences had similarity with the Drosophila melanogaster developmental genes snake and stubble. mRNA expression of selected serine protease genes was examined in different life stages, tissues, genders, and in response to bloodfeeding.

Amino Acid Sequence↗
PubMed2001

Molecular characterization of two serine proteases expressed in gut tissue of the African trypanosome vector, Glossina morsitans morsitans.

Serine proteases are major insect gut enzymes involved in digestion of dietary proteins, and in addition they have been implicated in the process of pathogen establishment in several vector insects. The medically important vector, tsetse fly (Diptera:Glossinidiae), is involved in the transmission of African trypanosomes, which cause devastating diseases in animals and humans. Both the male and female tsetse can transmit trypanosomes and both are strict bloodfeeders throughout all stages of their development. Here, we describe the characterization of two putative serine protease-encoding genes, Glossina serine protease-1 (Gsp1) and Glossina serine protease-2 (Gsp2) from gut tissue. Both putative cDNA products represent prepro peptides with hydrophobic signal peptide sequences associated with their 5'-end terminus. The Gsp1 cDNA encodes a putative mature protein of 245 amino acids with a molecular mass of 26 428 Da, while the predicted size of the 228 amino acid mature peptide encoded by Gsp2 cDNA is 24 573 Da. Both deduced peptides contain the Asp/His/Ser catalytic triad and the conserved residues surrounding it which are characteristic of serine proteases. In addition, both proteins have the six-conserved cysteine residues to form the three-cysteine bonds typically present in invertebrate serine proteases. Based on the presence of substrate specific residues, the Gsp1 gene encodes a chymotrypsin-like protease while Gsp2 gene encodes for a protein with trypsin-like activity. Both proteins are encoded by few loci in tsetse genome, being present in one or two copies only. The mRNA expression levels for the genes do not vary extensively throughout the digestive cycle, and high levels of mRNAs can be readily detected in the gut tissue of newly emerged flies. The levels of trypsin and chymotrypsin activities in the gut lumen increase following blood feeding and change significantly in the gut cells throughout the digestion cycle. Hence, the regulation of expression for trypsin and chymotrypsin occurs at the post-transcriptional level in tsetse. Both the coding sequences and patterns of expression of Gsp1 and Gsp2 genes are similar to the serine proteases that have been reported from the bloodfeeding insect Stomoxys calcitrans.

Amino Acid Sequence↗
PubMed2003

Potato virus Y NIa protease activity is not sufficient for elicitation of Ry-mediated disease resistance in potato.

Ry confers extreme resistance (ER) to all strains of potato virus Y (PVY). In previous work, we have shown that the protease domain of the nuclear inclusion a protease (NIaPro) from PVY is the elicitor of the Ry-mediated resistance and that integrity of the protease active site is required for the elicitation of the resistance response. Two possibilities arise from these results: first, the structure of the active protease has elicitor activity; second, NIa-mediated proteolysis is required to elicit the resistance response. To resolve these possibilities, the NIaPro from PVY was randomly mutagenised and the clones obtained were screened for elicitation of cell death as an indicator of resistance and proteolytic activity. We did not find any mutants that had retained the ability to elicit cell death but had lost protease activity, as measured by processing of the NIa cleavage site in the viral genome. This was consistent with the idea that protease activity is necessary for elicitor activity. However, protease activity was not sufficient because we found three elicitor-defective mutants in which there was a high level of protease activity in this assay.

Base Sequence↗
PubMed2002

Isolation of a Microsporum canis gene family encoding three subtilisin-like proteases expressed in vivo.

Microsporum canis is the main agent of dermatophytosis in dogs and cats and is responsible for frequent zoonosis. The pathogenesis of the disease remains largely unknown, however. Among potential fungal virulence factors are secreted keratinolytic proteases, whose molecular characterization would be an important step towards the understanding of dermatophytic infection pathogenesis. M. canis secretes a 31.5 kDa keratinolytic subtilisin-like protease as the major component in a culture medium containing cat keratin as the sole nitrogen source. Using a probe corresponding to a gene's internal fragment, which was obtained by polymerase chain reaction, the entire gene encoding this protease named SUB3 was cloned from a M. canislambdaEMBL3 genomic library. Two closely related genes, termed SUB1 and SUB2, were also cloned from the library using as a probe the gene coding for Aspergillus fumigatus 33 kDa alkaline protease (ALP). Deduced amino acid sequence analysis revealed that SUB1, SUB2, and SUB3 are secreted proteases and show large regions of identity between themselves and with subtilisin-like proteases of other filamentous fungi. Interest ingly, mRNA of SUB1, SUB2, and SUB3 were detected by reverse transcriptase nested-polymerase chain reaction from hair of experimentally infected guinea pigs. These results show that SUB1, SUB2, and SUB3 encode a family of subtilisin-like proteases and strongly suggest that these proteases are produced by M. canis during the invasion of keratinized structures. This is the first report describing the isolation of a gene family encoding potential virulence-related factors in dermatophytes.

Amino Acid Sequence↗
PubMed1998

Antibodies to von Willebrand factor-cleaving protease in acute thrombotic thrombocytopenic purpura.

BACKGROUND: Thrombotic thrombocytopenic purpura is a potentially fatal disease characterized by widespread platelet thrombi in the microcirculation. In the normal circulation, von Willebrand factor is cleaved by a plasma protease. We explored the hypothesis that a deficiency of this protease predisposes patients with thrombotic thrombocytopenic purpura to platelet thrombosis. METHODS: We studied the activity of von Willebrand factor-cleaving protease and sought inhibitors of this protease in plasma from patients with acute thrombotic thrombocytopenic purpura, patients with other diseases, and normal control subjects. We also investigated the effect of shear stress on the ristocetin cofactor activity of purified von Willebrand factor in the cryosupernatant fraction of the plasma samples. RESULTS: Thirty-nine samples of plasma from 37 patients with acute thrombotic thrombocytopenic purpura had severe deficiency of von Willebrand factor-cleaving protease. No deficiency was detected in 16 samples of plasma from patients with thrombotic thrombocytopenic purpura in remission or in 74 plasma samples from normal subjects, randomly selected hospitalized patients or outpatients, or patients with hemolysis, thrombocytopenia, or thrombosis from other causes. Inhibitory activity against the protease was detected in 26 of the 39 plasma samples (67 percent) obtained during the acute phase of the disease. The inhibitors were IgG antibodies. Shear stress increased the ristocetin cofactor activity of von Willebrand factor in the cryosupernatant of plasma samples obtained during the acute phase, but decreased the activity in cryosupernatant of plasma from normal subjects. CONCLUSIONS: Inhibitory antibodies against von Willebrand factor-cleaving protease occur in patients with acute thrombotic thrombocytopenic purpura. A deficiency of this protease is likely to have a critical role in the pathogenesis of platelet thrombosis in this disease.

ADAM Proteins↗
PubMed2004

Gene disruption confirms a critical role for the cysteine protease falcipain-2 in hemoglobin hydrolysis by Plasmodium falciparum.

Erythrocytic malaria parasites degrade hemoglobin in an acidic food vacuole to acquire free amino acids and maintain parasite homeostasis. Hemoglobin hydrolysis appears to be a cooperative process requiring cysteine proteases (falcipains) and aspartic proteases (plasmepsins), but the specific roles of different enzymes in this process are unknown. We previously showed that falcipain-2 is a major trophozoite food vacuole cysteine protease. To characterize the specific role of falcipain-2, we disrupted the falcipain-2 gene and assessed the effect of this alteration. Falcipain-2-knockout trophozoites had markedly diminished cysteine protease activity and swollen, dark staining food vacuoles, consistent with a block in hemoglobin hydrolysis, as caused by cysteine protease inhibitors. However, more mature stages of knockout parasites were indistinguishable from wild-type parasites and developed normally. The knockout parasites had decreased and delayed expression of falcipain-2, which appeared to be directed by increased transcription of a second copy of the gene (falcipain-2'). Expression of other falcipains and plasmepsins was similar in wild-type and knockout parasites. Compared with wild-type, knockout parasites were about 3 times more sensitive to the cysteine protease inhibitors E-64 and leupeptin, and over 50-fold more sensitive to the aspartic protease inhibitor pepstatin. Our results assign a specific function for falcipain-2, the hydrolysis of hemoglobin in trophozoites. In addition, they highlight the cooperative action of cysteine and aspartic proteases in hemoglobin degradation by malaria parasites.

Animals↗
PubMed2006

Entamoeba histolytica cysteine proteases cleave the MUC2 mucin in its C-terminal domain and dissolve the protective colonic mucus gel.

In order for the protozoan parasite Entamoeba histolytica (E.h.) to cause invasive intestinal and extraintestinal infection, which leads to significant morbidity and mortality, it must disrupt the protective mucus layer by a previously unknown mechanism. We hypothesized that cysteine proteases secreted from the amoeba disrupt the mucin polymeric network, thereby overcoming the protective mucus barrier. The MUC2 mucin is the major structural component of the colonic mucus gel. Heavily O-glycosylated and protease-resistant mucin domains characterize gel-forming mucins. Their N- and C-terminal cysteine-rich domains are involved in mucin polymerization, and these domains are likely to be targeted by proteases because they are less glycosylated, thereby exposing their peptide chains. By treating recombinant cysteine-rich domains of MUC2 with proteases from E.h. trophozoites, we showed that the C-terminal domain was specifically targeted at two sites by cysteine proteases, whereas the N-terminal domain was resistant to proteolysis. The major cleavage site is predicted to depolymerize the MUC2 polymers, thereby disrupting the protective mucus gel. The ability of the cysteine proteases to dissolve mucus gels was confirmed by treating mucins from a MUC2-producing cell line with amoeba proteases. These findings suggest a major role for E.h. cysteine proteases in overcoming the protective mucus barrier in the pathogenesis of invasive amoebiasis. In this report, we identify a specific cleavage mechanism used by an enteric pathogen to disrupt the polymeric nature of the mucin gel.

Animals↗
PubMed2000

Predicting protein function from structure: unique structural features of proteases.

We have noted consistent structural similarities among unrelated proteases. In comparison with other proteins of similar size, proteases have smaller than average surface areas, smaller radii of gyration, and higher C(alpha) densities. These findings imply that proteases are, as a group, more tightly packed than other proteins. There are also notable differences in secondary structure content between these two groups of proteins: proteases have fewer helices and more loops. We speculate that both high packing density and low alpha-helical content coevolved in proteases to avoid autolysis. By using the structural parameters that seem to show some separation between proteases and nonproteases, a neural network has been trained to predict protease function with over 86% accuracy. Moreover, it is possible to identify proteases whose folds were not represented during training. Similar structural analyses may be useful for identifying other classes of proteins and may be of great utility for categorizing the flood of structures soon to flow from structural genomics initiatives.

Endopeptidases↗
PubMed2001

Activation of the Mason-Pfizer monkey virus protease within immature capsids in vitro.

For all retroviruses, the completion of the viral budding process correlates with the activation of the viral protease by an unknown mechanism, and, as the structural (Gag) polyproteins are cleaved by the viral protease, maturation of the immature virus-like particle into an infectious virion. Unlike most retroviruses, the Mason-Pfizer monkey virus Gag polyproteins assemble into immature capsids within the cytoplasm of the cell before the viral budding event. The results reported here describe a unique experimental system in which Mason-Pfizer monkey virus immature capsids are removed from the cell, and the protease is activated in vitro by the addition of a reducing agent. The cleavage of the protease from the precursor form is a primary event, which proceeds with a half time of 14 min, and is followed by authentic processing of the Gag polyproteins. Activity of the viral protease in vitro depends on pH, with an increase in catalytic rates at acidic and neutral pH. The initiation of protease activity within immature capsids in vitro demonstrates that viral protease activity is sensitive to oxidation-reduction conditions, and that the viral protease can be activated in the absence of viral budding.

Animals↗
PubMed1990

Different mouse mast cell populations express various combinations of at least six distinct mast cell serine proteases.

Mouse serosal mast cells (SMCs) and Kirsten sarcoma virus-immortalized mast cells store large amounts of mast cell carboxypeptidase A and serine proteases in their secretory granules. Secretory granule proteins from 2.6 x 10(6) purified SMCs were separated by NaDodSO4/PAGE, trans-blotted to poly(vinylidine difluoride) membranes, and subjected to amino-terminal amino acid sequencing. Four distinct mast cell serine proteases were identified. With mast cell carboxypeptidase A, these serine proteases comprise the major proteins of mouse SMC secretory granules. Each of the four SMC serine proteases was distinct from the two serine proteases present in mucosal mast cells in the intestines of helminth-infected mice. The secretory granules of a Kirsten sarcoma virus-immortalized mast cell line contained three of the SMC-derived serine proteases and one of the mucosal mast cell-derived serine proteases. Thus, the family of mouse mast cell secretory granule serine proteases has at least six distinct members that can be expressed in different combinations in different mast cell populations.

Amino Acid Sequence↗
PubMed1992

Molecular polymorphism and epidemiology of Neisseria meningitidis immunoglobulin A1 proteases.

Neisseria meningitidis is one of several important bacterial pathogens that secrete a specific protease capable of cleaving human immunoglobulin A1 (IgA1) in the hinge region. To obtain further information on this putative virulence factor, we examined the IgA1 protease and iga gene region of 133 isolates of N. meningitidis assigned to 88 multilocus enzyme genotypes and representing major epidemics and carrier strains from 19 countries. Of the two IgA1 cleavage specificities previously observed, isolates associated with epidemics of meningococcal disease showed exclusively type 1 IgA1 protease activity. Considerable heterogeneity of the N. meningitidis IgA1 protease was demonstrated at both the protein and gene levels. Thus, five different forms of IgA1 protease were detected with enzyme-neutralizing antibodies raised in rabbits. An antiserum raised against a single type 2 IgA1 protease inhibited the enzyme activity of all strains examined, a finding of potential significance for the possible application of IgA1 protease in a vaccine against meningococcal disease. Examination of the iga gene region with restriction endonucleases revealed a high degree of polymorphism among strains belonging to some multilocus enzyme genotypes. The different iga gene types did not correlate with cleavage type or inhibition of the IgA1 protease. Our findings indicate that horizontal genetic exchange occurs in vivo with considerably different frequency in different clones of meningococci.

Carrier State↗
PubMed1996

Isolation of the human PC6 gene encoding the putative host protease for HIV-1 gp160 processing in CD4+ T lymphocytes.

Production of infectious HIV-1 virions is dependent on the processing of envelope glycoprotein gp160 by a host cell protease. The protease in human CD4+ T lymphocytes has not been unequivocally identified, yet members of the family of mammalian subtilisin-like protein convertases (SPCs), which are soluble or membrane-bound proteases of the secretory pathway, best fulfill the criteria. These proteases are required for proprotein maturation and cleave at paired basic amino acid motifs in numerous cellular and viral glycoprotein precursors, both in vivo and in vitro. To identify the gp160 processing protease, we have used reverse transcription-PCR and Northern blot analyses to ascertain the spectrum of SPC proteases in human CD4+ T cells. We have cloned novel members of the SPC family, known as the human PC6 genes. Two isoforms of the hPC6 protease are expressed in human T cells, hPC6A and the larger hPC6B. The patterns of SPC gene expression in human T cells has been compared with the furin-defective LoVo cell line, both of which are competent in the production of infectious HIV virions. This comparison led to the conclusion that the hPC6 gene products are the most likely candidates for the host cell protease responsible for HIV-1 gp160 processing in human CD4+ T cells.

Amino Acid Sequence↗
PubMed1997

Elevated levels of cysteine protease activity in saliva and salivary glands of the nonobese diabetic (NOD) mouse model for Sjögren syndrome.

Nonobese diabetic (NOD) mice develop an anti-exocrine gland pathology similar to human Sjögren syndrome. Recently, we demonstrated that NOD-scid mice develop severe loss of submandibular acinar cells with concomitant appearance of abnormal isoforms of salivary proteins suggesting de novo enzymatic cleavage. Because these changes may indicate activation of apoptotic proteases, we examined saliva and salivary tissue for cysteine protease activity. Cysteine protease activities were elevated in saliva and gland lysates from 20-week-old NOD and NOD-scid mice as compared with age- and sex-matched BALB/c or 8-week-old NOD mice. This activity appeared in the submandibular glands, but not in the parotid glands. Western blot analyses using antibodies directed against specific apoptotic proteases (interleukin 1beta converting enzyme, Nedd-2, and Apopain/CPP 32) confirmed these findings. Submandibular glands from NOD-scid mice exhibited the greatest increase in proteolytic activity, indicating that infiltrating leukocytes are not responsible for these changes. Western blot analyses also failed to reveal changes in the levels of cystatins (saliva proteins that inhibit protease activity). Thus, increased cysteine protease activity appears to be directly related to submandibular acinar cell loss in NOD-scid mice involving the apoptotic pathway. Additional protease activity in saliva and gland lysates of older NOD and NOD-scid mice, apparently mutually distinct from cysteine proteases, generated an enzymatically cleaved parotid secretory protein. We suggest, therefore, that proteolytic enzyme activity contributes to loss of exocrine gland tolerance by generating abnormally processed protein constituents.

Aging↗
PubMed1995

Protease evolution in Streptomyces griseus. Discovery of a novel dimeric enzymes.

This report describes the cloning and sequencing of a novel protease gene derived from Streptomyces griseus. Also described is the heterologous expression of the gene in Bacillus subtilis and characterization of the gene product. The sprD gene encodes a prepro mature protease of 392 amino acids tentatively named S. griseus protease D (SGPD). A significant component of the enzyme preregion was found to be homologous with the mitochondrial import signal of hsp60. The sprD gene was subcloned into an Escherichia coli/B. subtilis shuttle vector system such that the pro mature portion of SGPD was fused in frame with the promoter, ribosome binding site, and signal sequences of subtilisin. The gene fusion was subsequently expressed in B. subtilis DB104, and active protease was purified. SGPD has a high degree of sequence homology to previously described S. griseus proteases A, B, C, and E and the alpha-lytic protease of Lysobacter enzymogenes, but unlike all previously characterized members of the chymotrypsin superfamily, the recombinant SGPD forms a stable alpha 2 dimer. The amino acid sequence of the protein in the region of the specificity pocket is similar to that of S. griseus proteases A, B, and C. The purified enzyme was found to have a primary specificity for large aliphatic or aromatic amino acids. Nucleotide sequence data were used to construct a phylogenetic tree using a method of maximum parsimony which reflects the relationships and potentially the lineage of the chymotrypsin-like proteases of S. griseus.

Amino Acid Sequence↗
PubMed1998

Requirement of caspase-3(-like) protease-mediated hydrogen peroxide production for apoptosis induced by various anticancer drugs.

Caspase-3(-like) proteases play important roles in controlling mammalian apoptosis. However, the downstream events from the caspase-3(-like) protease activation to death of cells are still unclear. Previously, we reported that hydrogen peroxide (H2O2) was generated by the activation of caspase-3(-like) proteases in the process of tyrosine kinase inhibitor-induced apoptosis in human small cell lung carcinoma Ms-1 cells. In the present study, we examined whether generation of H2O2 is a critical event for the apoptotic pathway downstream of caspase-3(-like) protease activation by various anticancer drugs. Anticancer drugs such as camptothecin, vinblastine, inostamycin, and adriamycin induced activation of caspase-3(-like) proteases and apoptosis. Generation of H2O2 was commonly detected after treatment with each of the four anticancer drugs, and scavenging of H2O2 caused cells to fail to undergo apoptosis. Moreover, anticancer drug-induced H2O2 production was inhibited not only by an inhibitor of caspase-3(-like) proteases but also by diphenyleneiodonium chloride, an inhibitor of flavonoid-containing enzymes such as NADPH oxidase. However, activation of caspase-3(-like) proteases was not inhibited by diphenyleneiodonium chloride. These findings suggest that activation of caspase-3(-like) proteases by various anticancer drugs causes generation of H2O2 presumably through the activation of NADPH oxidase, thereby inducing apoptosis. Therefore, H2O2 may function as a common mediator for apoptosis induced by various anticancer drugs.

Acetylcysteine↗
PubMed2003

Mouse chromosome 17A3.3 contains 13 genes that encode functional tryptic-like serine proteases with distinct tissue and cell expression patterns.

Probing of the mouse EST data base at GenBank trade mark with known tryptase cDNAs resulted in the identification of undiscovered serine protease transcripts whose genes reside at a 1.5-Mb complex on mouse chromosome 17A3.3. Mouse tryptase-5 (mT5), tryptase-6 (mT6), and mast cell protease-11 (mMCP-11) are new members of this serine protease superfamily whose amino acid sequences are 36-54% identical to each other and to their other 10 family members. The 13 functional mouse proteases can be subdivided into two subgroups based on conserved features in their propeptides. Of the three new serine proteases, mT6 is most widely expressed in tissues. mT5 is preferentially expressed in smooth muscle, whereas mMCP-11 is preferentially expressed in the spleen and bone marrow. In contrast to mT5 and mT6, mMCP-11 is also expressed in mast cells. Although mT6 and mMCP-11 are constitutively secreted when expressed in mammalian and insect cells, mT5 remains membrane-associated. The fact that recombinant mT5, mT6, and mMCP-11 possess non-identical expression patterns and substrate specificities suggests that each protease has a unique function in vivo. Of the 13 functional mouse tryptase genes identified at the complex, 12 have orthologs that reside in the syntenic region of human chromosome 16p13.3. The establishment of these ortholog pairs helps clarify the evolutionary relationship of the serine protease locus in the two species. This information provides a useful framework for the functional analysis of each protease using gene targeting and other molecular approaches.

Amino Acid Sequence↗
PubMed2004

Mouse DESC1 is located within a cluster of seven DESC1-like genes and encodes a type II transmembrane serine protease that forms serpin inhibitory complexes.

We report the identification and functional analysis of a type II transmembrane serine protease encoded by the mouse differentially expressed in squamous cell carcinoma (DESC) 1 gene, and the definition of a cluster of seven homologous DESC1-like genes within a 0.5-Mb region of mouse chromosome 5E1. This locus is syntenic to a region of human chromosome 4q13.3 containing the human orthologues of four of the mouse DESC1-like genes. Bioinformatic analysis indicated that all seven DESC1-like genes encode functional proteases. Direct cDNA cloning showed that mouse DESC1 encodes a multidomain serine protease with an N-terminal signal anchor, a SEA (sea urchin sperm protein, enterokinase, and agrin) domain, and a C-terminal serine protease domain. The mouse DESC1 mRNA was present in epidermal, oral, and male reproductive tissues and directed the translation of a membrane-associated 60-kDa N-glycosylated protein with type II topology. Mouse DESC1 was synthesized in insect cells as a zymogen that could be activated by exposure to trypsin. The purified activated DESC1 hydrolyzed synthetic peptide substrates, showing a preference for Arg in the P1 position. DESC1 proteolytic activity was abolished by generic inhibitors of serine proteases but not by other classes of protease inhibitors. Most interestingly, DESC1 formed stable inhibitory complexes with both plasminogen activator inhibitor-1 and protein C inhibitor that are expressed in the same tissues with DESC1, suggesting that type II transmembrane serine proteases may be novel targets for serpin inhibition. Together, these data show that mouse DESC1 encodes a functional cell surface serine protease that may have important functions in the epidermis, oral, and reproductive epithelium.

Amino Acid Sequence↗