Identification and cloning of an aspartyl proteinase from Coccidioides immitis

A 45 kDa protein was isolated from a soluble vaccine prepared from formaldehyde-killed spherules of Coccidioides immitis. From the N-terminal amino acid sequence, the protein yielded a 17-amino-acid peptide that was homologous to sequences of other fungal aspartyl proteinases. The coccidioidal cDNA encoding the proteinase was amplified using oligonucleotide primers designed from the 45 kDa N-terminal amino acid sequence and a fungal aspartyl proteinase consensus amino acid sequence. The PCR product was cloned and sequenced, and the remaining 5' upstream and 3' downstream cDNA was amplified, cloned, and sequenced. The cDNA encoding the coccidioidal aspartyl proteinase open reading frame was cloned and the fusion protein containing a C-terminal His-tag expressed in E. coli. The recombinant aspartyl proteinase was purified by immobilized metal affinity chromatography. This recombinant protein will be used for further studies to evaluate its antigenicity, including protective immunogenicity.     

Isolation and characterization of a cDNA encoding a mammalian cathepsin L-like cysteine proteinase from Acanthamoeba healyi

We have cloned a cDNA encoding a cysteine proteinase of the Acanthamoeba healyi OC-3A strain isolated from the brain of a granulomatous amoebic encephalitis patient. A DNA probe for an A. healyi cDNA library screening was amplified by PCR using degenerate oligonucleotide primers designed on the basis of conserved amino acids franking the active sites of cysteine and asparagine residues that are conserved in the eukaryotic cysteine proteinases. Cysteine proteinase gene of A. healyi (AhCP1) was composed of 330 amino acids with signal sequence, a proposed pro-domain and a predicted active site made up of the catalytic residues. Cys25, His159, and Asn175. Deduced amino acid sequence analysis indicates that AhCP1 belong to ERFNIN subfamily of C1 peptidases. By Northern blot analysis, no direct correlation was observed between AhCP1 mRNA expression and virulence of Acanthamoeba, but the gene was expressed at higher level in amoebae isolated from soil than amoeba from clinical samples. These findings raise the possibility that Ahcp1 protein may play a role in protein metabolism and digestion of phagocytosed bacteria or host tissue debris rather than in invasion of amoebae into host tissue.     

Cloning and characterization of a mouse cysteine proteinase

cDNA clones encoding a mouse cysteine proteinase were isolated from a cDNA library constructed from mRNA derived from the macrophage-like cell line J774. The DNA sequence predicts a protein that is closely related to, but distinct from, the lysosomal enzyme cathepsin H. Alignment of the predicted amino acid sequence with the known protein sequences for seven other cysteine proteinases suggests that the cloned DNA encodes a 334-residue protein containing both a 17-amino acid pre-region and a 96-amino acid pro-region. Consistent with this prediction, antiserum raised to a recombinant fusion protein expressed in Escherichia coli immunoprecipitated multiple forms of the cysteine proteinase in mouse peritoneal macrophages and fibroblasts. In pulse-chase experiments, a 36-kDa precursor, presumedly the pro-form, was converted intracellularly into a 28-kDa protein and subsequently into a 21-kDa protein. Indirect immunofluorescence microscopy results suggested that the cysteine proteinase was localized to lysosomes. Western blot analysis detected significantly more of the proteinase in thioglycolate-elicited peritoneal macrophages than in resident peritoneal macrophages. Northern blot analysis revealed that several cell lines failed to express mouse cysteine proteinase mRNA.     

A novel inhibitor protein for Bombyx cysteine proteinase is homologous to propeptide regions of cysteine proteinases

A cDNA clone for an inhibitor of Bombyx cysteine proteinase was isolated and sequenced. Active inhibitor proteins were expressed in Escherichia coli using the cDNA. The open reading frame of the cDNA encodes a 105 residues protein with 19 residues of a signal sequence. The inhibitor has amino acid sequences homologous to several cysteine proteinases, but only to their propeptide sequences. The results suggest that some cysteine proteinase proregions may have evolved as autonomous modules and become inhibitor proteins for cysteine proteinases.     

马铃薯腐烂茎线虫L 型半胱氨酸蛋白酶新基因(Dd-cpl-1) 的克隆与序列分析

L型半胱氨酸蛋白酶基因 (Cathepsin L-like cysteine proteinase gene) 为与植物寄生线虫寄生能力相关的多功能基因。运用RT-PCR和RACE的方法从马铃薯腐烂茎线虫Ditylenchus destructor中克隆出1个L型半胱氨酸蛋白酶新基因Dd-cpl-1 (GenBank登录号为GQ180107)。该基因Dd-cpl-1 cDNA全长序列含有1个1 131 bp的开放性阅读框 (ORF),编码376个氨基酸残基,其5′末端及3′末端分别含有29 bp和159 bp的非编码区 (UTR)。Dd-cpl-1内含子外显子结构分析结果表明,其基因组序列包含7个内含子,且各内含子两端剪接位点序列遵守GT/AG规则。Dd-cpl-1基因推定的蛋白Dd-CPL-1与松材线虫L型半胱氨酸蛋白酶高度同源,一致性达到77％。以不同物种中L 型半胱氨酸蛋白酶氨基酸序列进行比对分析,推测推定的蛋白 Dd-CPL-1含有L型半胱氨酸蛋白酶基因家族高度保守的催化三联体 (Cys183,His322 和Asn343) 以及ERFNIN基系和GNFD基系。半胱氨酸蛋白酶系统发育分析表明,Dd-cpl-1 属于由L型半胱氨酸蛋白酶组成的进化分支。Dd-cpl-1的这些序列特征进一步表明其为L型半胱氨酸蛋白酶基因。这是首次在马铃薯腐烂茎线虫中克隆到的L型半胱氨酸蛋白酶,为今后在蛋白水平对其进行进一步的功能分析提供基础。     

Cloning and functional expression of a Boophilus microplus cathepsin L-like enzyme

A cysteine proteinase gene homologous to cathepsins L genes was isolated from a B. microplus cDNA library. The precursor protein deduced from the nucleotide sequence contains 332 amino acid residues consisting of a signal sequence (pre-region), a pro-region and a mature proteinase. The DNA fragment coding for the proenzyme was cloned and expressed using the E. coli expression vector pMAL-p. The recombinant protein (MBP+PROCP) once activated is able to hydrolyze synthetic substrates as well as protein substrates like hemoglobin, vitellin and gelatin. Its optimal enzymatic activity on both fluorogenic and protein substrates was found to occur at an acidic pH. Expression of the proteinase gene was tested by RT-PCR with tick larvae RNA. Detection of amplified sequences indicates that the gene is expressed at this stage of the tick life cycle and the molecule is therefore potentially a target for chemotherapy or an immunogen in a vaccine.     

Molecular cloning and structural and functional characterization of human cathepsin F, a new cysteine proteinase of the papain family with a long propeptide domain.

A cDNA encoding a new cysteine proteinase belonging to the papain family and called cathepsin F has been cloned from a human prostate cDNA library. This cDNA encodes a polypeptide of 484 amino acids, with the same domain organization as other cysteine proteinases, including a hydrophobic signal sequence, a prodomain, and a catalytic region. However, this propeptide domain is unusually long and distinguishes cathepsin F from other proteinases of the papain family. Cathepsin F also shows all structural motifs characteristic of these proteinases, including the essential cysteine residue of the active site. Consistent with these structural features, cathepsin F produced in Escherichia coli as a fusion protein with glutathione S-transferase degrades the synthetic peptide benzyloxycarbonyl-Phe-Arg-7-amido-4-methylcoumarin, a substrate commonly used for functional characterization of cysteine proteinases. Furthermore, this proteolytic activity is blocked by trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane, an inhibitor of cysteine proteinases. The gene encoding cathepsin F maps to chromosome 11q13, close to that encoding cathepsin W. Cathepsin F is widely expressed in human tissues, suggesting a role in normal protein catabolism. Northern blot analysis also revealed a significant level of expression in some cancer cell lines opening the possibility that this enzyme could be involved in degradative processes occurring during tumor progression.     

Cloning and expression of functional equistatin

Equistatin is a 199-residue protein composed of three thyroglobulin type-1 domains. It strongly inhibits cysteine proteinases as well as the aspartic proteinase cathepsin D. In order to initiate structure-function studies by protein engineering, a cDNA library from sea anemone, Actinia equina, was screened. A positive clone of 888 nucleotides was shown to encode a protein of 231 amino acids, including the signal sequence. The mature protein region was amplified by PCR, cloned into the pET22b(+)cas expression vector and expressed in Escherichia coli. Isolation of active recombinant equistatin required only one purification step, the His-tag affinity column. The protein displays physical and inhibitory properties closely similar to the native inhibitor.     

A cysteine proteinase cDNA from Trypanosoma brucei predicts an enzyme with an unusual C-terminal extension

A cDNA for a Trypanosoma brucei cysteine proteinase has been cloned and sequenced. The deduced protein can be divided into four domains, based on homologies with other cysteine proteinases: the pre-, pro- and central regions show considerable homology to the cathepsin L class of mammalian enzymes, whilst the long C-terminal extension distinguishes the trypanosome enzyme from all mammalian cysteine proteinases reported. This 108 amino acid extension, which includes 9 contiguous prolines near the junction with the central domain, appears likely to be processed in part to produce the mature enzyme, and may be involved in targeting the protein within the cell. The trypanosome genome contains more than 20 copies of the cysteine proteinase gene arranged in a long tandem array.     

Protein A immunocapture assay detecting antibodies to fluke cysteine proteinases for immunodiagnosis of human paragonimiasis and fascioliasis

Enzyme-linked immunosorbent assays (ELISAs) which detect specific antibodies to fluke cysteine proteinases have provided good sensitivity and specificity for the immunodiagnosis of trematode diseases. To detect specific antibodies without the need for purified proteinase antigens, an immunocapture assay using Protein A was applied for the immunodiagnosis of paragonimiasis and fascioliasis. ELISA plate wells were coated with Protein A, incubated with diluted patient sera, then incubated with a preparation containing fluke cysteine proteinases, excretory-secretory (ES) products of adult Paragonimus westermani or Fasciola sp. The activity of fluke cysteine proteinases bound on the wells was measured by adding fluorogenic peptidyl substrate, Z-Phe-Arg-MCA or Boc-Val-Leu-Lys-MCA. This assay detected specific immunoglobulin G to cysteine proteinases of P. westermani and Fasciola sp. by measuring proteinase activity on the plate wells. Patient sera showed significant high values of proteinase activity when the wells were treated with the respective homologous ES products, whereas the sera had low values after treatment with the heterologous ES products. The sera of patients with other parasitoses and uninfected healthy individuals also showed low values after treatment with the above fluke ES products. Thus, Protein A immunocapture assay, which detected IgG specific for fluke cysteine proteinases, provided a high sensitivity and specificity for immunodiagnosis of paragonimiasis and fascioliasis.     

Identification and characterization of the immunogenic cytotoxic TvCP39 proteinase gene of Trichomonas vaginalis

TvCP39 is a 39 kDa cysteine proteinase (CP) involved in Trichomonas vaginalis cytotoxicity that has been found in vaginal secretions and is immunogenic in patients with trichomonosis. The goal of this work was to identify, clone, express, and characterize the tvcp39 gene. The tvcp39 gene was identified using a proteomic approach, and the complete gene was amplified using PCR, cloned, and sequenced. TvCP39 is encoded by a 915-bp cathepsin L-like CP gene. A fragment corresponding to the mature region (TvCP39r) was expressed, purified, and used to produce rabbit polyclonal antibodies and in functional assays. In one- and two-dimensional western blot assays, the anti-TvCP39r antibody reacted with two protein bands of ~28 and 27 kDa and three spots of ~28, 27, and 24 kDa in trichomonad proteinase-rich extracts that could correspond to the mature and processed fragments of the TvCP39 peptidase. The anti-TvCP39r antibody reacted with the parasitic surface and the native TvCP39 present in vaginal washes from patients with trichomonosis. Moreover, the recombinant TvCP39 protein bound to the surface of HeLa cells and protected HeLa cell monolayers from trichomonal destruction in a concentration-dependent manner. In conclusion, our data support TvCP39 as one of the surface proteinases that is glycosylated and is involved in trichomonal cytotoxicity. Thus, TvCP39 is the first glycosylated cysteine proteinase detected in T. vaginalis.     

Inhibition of the cysteine proteinases cathepsins K and L by the serpin headpin (SERPINB13): a kinetic analysis

Headpin (SERPINB13) is a novel member of the serine proteinase inhibitor (Serpin) gene family that was originally cloned from a keratinocyte cDNA library. Western blot analysis using a headpin-specific antiserum recognized a protein with the predicted M(r) of 44kDa in lysates derived from a transformed keratinocyte cell line known to express headpin mRNA. Similarity of the reactive-site loop (RSL) domain of headpin, notably at the P1-P1(') residues, with other serpins that inhibit cysteine and serine proteinases suggests that headpin may inhibit similar proteinases. This study demonstrates that recombinant headpin indeed inhibits cathepsins K and L, but not chymotrypsin, elastase, trypsin, subtilisin A, urokinase-type plasminogen activator, plasmin, or thrombin. The second-order rate constants (k(a)) for the inhibitory reactions of rHeadpin with cathepsins K and L were 5.1+/-0.6x10(4) and 4.1+/-0.8x10(4)M(-1)s(-1), respectively. Headpin formed SDS-stable complexes with cathepsins K and L, a characteristic property of inhibitory serpins. Interactions of the RSL domain of headpin with cathepsins K and L were indicated by cleavage of headpin near the predicted P1-P1(') residues by these proteinases. These results demonstrate that the serpin headpin possesses specificity for inhibiting lysosomal cysteine proteinases.     

Peptidoglycan inducible expression of a serine proteinase homologue from kuruma shrimp (Marsupenaeus japonicus)

A cDNA encoding a serine proteinase homologue of kuruma shrimp (Marsupenaeus japonicus) was cloned. The 1257 bp cDNA encodes a 339 amino acid putative peptide, with a signal sequence of 16 amino acid residues. The deduced amino acid sequence is 42-67% similar to the immune-related serine proteinases and serine proteinase homologues of arthropods. It contains catalytic triad residues in the putative catalytic domain except for one substitution of Ser by a Gly residue. The six cysteine residues that form three disulphide bridges in most serine proteinases were conserved. The M. japonicus serine proteinase homologue was mainly expressed in haemocytes, in which expression dramatically increased after 3 days feeding with peptidoglycan at 0.2 mg kg(-1) shrimp body weight per day.     

Molecular cloning and characterization of onchocystatin, a cysteine proteinase inhibitor of Onchocerca volvulus.

A cDNA clone designated OV7 encodes a polypeptide that corresponds to a highly antigenic Onchocerca volvulus protein. OV7 has significant amino acid sequence homology to the cystatin superfamily of cysteine proteinase inhibitors. In this report we establish that the OV7 recombinant protein is active as a cysteine proteinase inhibitor, and we have named it onchocystatin. It contains a cystatin-like domain that inhibits the activity of cysteine proteinases at physiological concentrations. Recombinant glutathione S-transferase-OV7 (GST-OV7, 1 microM) and maltose-binding protein-OV7 (MBP-OV7, 4 microM) fusion polypeptides inhibit 50% of the enzymatic activity of the bovine cysteine proteinase cathepsin B. Neither fusion polypeptide inhibits serine or metalloproteinases activity. The Ki for GST-OV7 fusion polypeptide is 170 nM for cathepsin B and 70 pM or 25 nM for cysteine proteinases purified from a protozoan parasite Entamoeba histolytica or the free living nematode Caenorhabditis elegans, respectively. The 5' end of the OV7 clone was isolated by polymerase chain reaction and sequenced, thus extending the previous cDNA clone to 736 base pairs. This represents the complete coding sequence of the mature onchocystatin (130 amino acids). A hydrophobic leader sequence of 32 amino acids was found, indicating a possible extracellular function of the onchocerca cysteine proteinase inhibitor.