Molecular cloning, mRNA expression and enzymatic characterization of cathepsin F from olive flounder (Paralichthys olivaceus)

Cathepsin F is a recently described papain-like cysteine protease of unknown function, and unique among cathepsins due to an elongated N-terminal pro-region, which contains a cystatin domain. In the present study, the cDNA of olive flounder (Paralichthys olivaceus) cathepsin F (PoCtF) was cloned by the combination of homology molecular cloning and rapid amplification of cDNA ends (RACE) approaches. The PoCtF gene was determined to consist of the 1844 bp nucleotide sequence which encodes for a 475-amino acid polypeptide. The results of RT-PCR analysis revealed ubiquitous expression throughout the entirety of healthy flounder tissues; however the PoCtF expressions increased significantly in gill at 3 h post-injection with lipopolysaccharide (LPS). Also, immunostaining using anti-PoCtF antibody was strongest on the epidermal mucus in the fin.The cDNA encoding mature enzyme of PoCtF was expressed in Escherichia coli using the pGEX-4 T-1 expression vector system. Its activity was quantified by cleaving the synthetic peptide Z-Phe-Arg-AMC, a substrate commonly used for functional characterization of cysteine proteinases, and the optimal pH for the protease activity was 7.5. The findings of the present study suggest that PoCtF has a higher optimum pH than mammalian cathepsin F, and PoCtF is an interesting target for future investigations of the role of cathepsin F in the epidermal mucus and fish innate immune system.     

Molecular cloning, expression analysis and enzymatic characterization of cathepsin K from olive flounder (Paralichthys olivaceus)

We assessed the putative physiological roles of cathepsin K from a flatfish, olive flounder. We cloned a cDNA encoding for cathepsin K (PoCtK), a cysteine protease of the papain family from olive flounder, Paralichthys olivaceus. The tissue-specific expression pattern of PoCtK, determined via real-time PCR analysis, revealed ubiquitous expression in normal tissues with high levels of expression in the spleen and bone marrow. However, PoCtK expression was significantly increased in the muscle and gill at 3–24 h post-injection with bacterial lipopolysaccharide (LPS). The cDNA encoding for the mature enzyme of PoCtK was expressed in Escherichia coli using the pGEX-4T-1 expression vector system. Its activity was quantified via the cleavage of the synthetic peptide Z-Gly-Pro-Arg-MCA, zymography, and the collagen degradation assay. The optimum pH for the protease activity was 8, and the recombinant PoCtK enzyme degraded collagen types I, II, III, IV, and VI and acid-soluble collagen from olive flounder muscle in the presence of chondroitin 4-sulphate (C-4S). Therefore, our data indicate that cathepsin K may play a role in the immune system of fish skin and muscle, in addition to its principal bone-specific function as a collagenolytic enzyme.     

Heterologous expression in Pichia pastoris and single-step purification of a cysteine proteinase from northern shrimp

A distinct cysteine proteinase (NsCys) of northern shrimp Pandalus borealis belonging to cathepsin L subgroup of the papain superfamily has been overexpressed as a precursor form (proNsCys) in Pichia pastoris. We adopted a simple and quick procedure to generate an expression cassette by constructing a donor vector harboring proNsCys followed by recombination with an acceptor vector in a way so that the proNsCys gene was placed downstream of the methanol-inducible AOX1 promoter and alpha-mating factor signal sequence gene. In addition, we used glycerol complex medium that supported high growth of yeast before induction while induction was carried out in minimal methanol medium thereby facilitating the secreted protein to be purified with a single size-exclusion chromatography. The recombinant enzyme was purified in two enzymatically active fractions: both corresponding to mature NsCys with, however, the major one comprising two molecular species of NsCys which had their severed prodomain non-covalently attached. The overall yield was about 100 mg of crude or 60 mg of purified recombinant enzyme comprising both mature and prodomain-attached forms of NsCys per liter of yeast culture. The recombinant NsCys was biologically active as observed by gelatin zymography and its ability to cleave Z-Phe-Arg-MCA, a synthetic substrate for cathepsin L. The development of the system reported here provides a cost-effective and easy to manipulate expression system to obtain large quantities of fully functional shrimp enzyme that will enable the functional characterization of this unique enzyme for both research and industrial purposes.     

Isolation and substrate specificities of five chitinases from the hepatopancreas of Northern shrimp, Pandalus borealis

Five enzymes designated chitinase I, IIa, IIb, III, and IV have been isolated from the hepatopancreas of Pandalus borealis in a procedure including column chromatography on Q-Sepharose, Sephacryl S-200, phenyl-Superose and Superdex 75. The isolated enzymes were analysed by SDS PAGE. Chitinase I, III, and IV gave only one major band corresponding to 54–55 kDA. Chitinase IIa showed one major band at 61 kDA and two diminutive bands at 17 and 55 kDa, while chitinase IIb gave two major bands at 17 and 44 kDa. Estimated by gel filtration, the native molecular weights of chitinase I, IIa, IIb, III, and IV were 61, 69, 39, 57, and 54 kDa, respectively. The substrate and reaction specificities of the isolated chitinases were investigated, and the results show that the isolated enzymes are true chitinases. They do not hydrolyse N,N′-diacetylchitobiose or p-Nitrophenyl-N-acetyl-β-D-glucosaminide, but express activities when longer chitooligosaccharides or nitrophenylated chitooligosaccharides are used as substrates. Chitinase I and IIa gave an initial random cleavage pattern and might be classified as endochitinases, while chitinase III and IV released dimeric units from the substrates and might be termed chitobiosidases.     

Molecular cloning of a multidomain cysteine protease and protease inhibitor precursor gene from the tobacco hornworm (Manduca sexta) and functional expression of the cathepsin F-like cysteine protease domain

A Manduca sexta (tobacco hornworm) cysteine protease inhibitor, MsCPI, purified from larval hemolymph has an apparent molecular mass of 11.5 kDa, whereas the size of the mRNA is very large (9 kilobases). MsCPI cDNA consists of a 9,273 nucleotides that encode a polypeptide of 2,676 amino acids, which includes nine tandemly repeated MsCPI domains, four cystatin-like domains and one procathepsin F-like domain. The procathepsin F-like domain protein was expressed in Escherichia coli and processed to its active mature form by incubation with pepsin. The mature enzyme hydrolyzed Z-Leu–Arg–MCA, Z-Phe–Arg–MCA and Boc–Val–Leu–Lys–MCA rapidly, whereas hydrolysis of Suc–Leu–Tyr–MCA and Z-Arg–Arg–MCA was very slow. The protease was strongly inhibited by MsCPI, egg-white cystatin and sunflower cystatin with K_i values in the nanomolar range. When the MsCPI tandem protein linked to two MsCPI domains was treated with proteases, it was degraded by the cathepsin F-like protease. However, tryptic digestion converted the MsCPI tandem protein to an active inhibitory form. These data support the hypothesis that the mature MsCPI protein is produced from the MsCPI precursor protein by trypsin-like proteases. The resulting mature MsCPI protein probably plays a role in the regulation of the activity of endogenous cysteine proteases.     

Purification and characterization of cathepsin B from goat brain

Cathepsin B was purified to an apparent homogeneity from goat brain utilizing the techniques of homogenization, autolysis at pH 4, 30–70% (NH₄)₂SO₄ fractionation, Sephadex G-100 column chromatography, organomercurial afinity chromatography and ion-exchange chromatography on CM-Sephadex C-50. The enzyme had a pH optima of 6 with α-N-benzoyl-D, L-arginine-β-naphthIylamide, benzyloxycarbonyl-arginine-arginme-4-methoxy -β-naphthylamide and azocasein as substrates. TheKm values for the hydrolysis of α-N-benzoyl-D, L-arginine-β-naphthylamide and benzyloxycarbonyl-arginine-arginine-4-methoxy -β-naphthylamide were 2.36 and 0.29 mM respectively in 2.5% dimethylsulphoxide. However, the correspondingKm values for these substrates in 1 % dimethylsulphoxide were 0.51 and 0.09 mM. The enzyme was strongly inhibited by thiol inhibitors and tetrapeptidyl chloromethylketones. Leupeptin inhibited the enzyme competitively withK _i value of 12.5 × l0⁻⁹M. Dithioerythritol was found to be the most potent activator of this sulfhydryl protease. Molecular weight estimations on sodium dodecyl sulphate-polyacrylamide gel electrophoresis and on analytical Sephadex G-75 column were around 27,000 and 29,000 daltons respectively. Cathepsin B was found to reside in the lysosomes of goat brain. The highest percentage of cathepsin B was in cerebrum. However, the specific activity of the enzyme was maximum in pituitary gland.     

Molecular cloning, characterization and expression of a novel jasmonate-dependent defensin gene from Ginkgo biloba

A novel defensin gene was isolated from Ginkgo biloba. The full-length cDNA of G. biloba defensin (designated as Gbd) was 534bp. The cDNA contained a 240-bp open reading frame encoding an 80-amino acid protein of 5.68 kDa with a potential 30 aa signal peptide. The putative GbD mature protein showed striking similarity to other plant defensins, representing low molecular size antimicrobial polypeptides. Eight cysteine sites conserved in plant defensins were also found in GbD at similar positions. Three-dimensional structure modeling showed that GbD strongly resembled defensin from tobacco (NaD1) and consisted of an alpha-helix and a triple-strand antiparallel beta-sheet that were stabilized by four intramolecular disulfide bonds, implying GbD may have functions similar to NaD1. The genomic DNA gel blot indicated that Gbd belonged to a multigene family. Expression analysis revealed that Gbd was up-regulated by wounding and methyl jasmonate treatments, suggesting that Gbd is potentially involved in plant resistance or tolerance to pathogens during wounding.     

Molecular cloning and characterization of O-methyltransferases from the flower buds of Iris hollandica

In plants, O-methyltransferases (OMTs) play an important role in methylation of secondary metabolites, especially flavonoids and other phenylpropanoids, and two cDNA clones, IhOMT1 and IhOMT2 (Iris hollandica OMT), encoding OMTs were successfully isolated from a cDNA library of flower buds of I. hollandica. IhOMT1 encodes an open reading frame (ORF) of 365 amino acids with calculated molecular mass of 40,193Da and isoelectric point (pI) of 5.54, while IhOMT2, which shares 31.5% amino acid sequence identity with IhOMT1, encodes 369 amino acids with calculated molecular mass of 40,385Da and pI of 5.50. In addition, the molecular masses of both recombinant IhOMT1 and IhOMT2 proteins were estimated to be about 40kDa by protein gel blot analysis. Characterization of the enzymatic properties using the recombinant IhOMT1 protein confirmed that IhOMT1 cDNA encodes a S-adenosyl-l-methionine (SAM)-dependent caffeic acid 3-OMT, which catalyzes the transfer of the methyl moiety from SAM to caffeic acid to form ferulic acid. Its optimum activity was observed at pH 7.5-8.0 and at 35 degrees C. This is the first report of the isolation and characterization of a COMT cDNA clone involved in the phenylpropanoid biosynthesis of Iridaceae plants. In contrast, IhOMT2 showed no activity in SAM-dependent assays for various phenylpropanoids.     

Molecular cloning and characterization of a mannose-binding lectin gene from Crinum asiaticum

Full-length cDNA of a mannose-binding lectin or agglutinin gene was cloned from a traditional Chinese medicinal herb Crinum asiaticum var. sinicum through RACE-PCR cloning. The full-length cDNA of C. asiaticum agglutinin (caa) was 820 bp and contained a 528 bp open reading frame encoding a lectin precursor (preproprotein) of 175 amino acid residues with a 22 aa signal peptide. The coding region of the caa gene was high in G/C content. The first 20 bp of the 5' UTR had a dC content of 50%, which was a typical feature of the leader sequence. By cutting away the signal peptide, the CAA proprotein was 15.79 kDa with a pl of 9.27 and contained 3 mannose-binding sites (QDNY). Random coil and extended strand constituted interlaced domination of the main part of the secondary structure. B-lectin conserved domain existed within N24 to G130. Predicted three-dimensional structure of CAA proprotein was very similar to that of GNA (Galanthus nivalis agglutinin). It is significant that besides certain homologies to known monocot mannose-binding lectins from Amaryllidaceae, Orchidaceae, Alliaceae and Liliaceae, caa also showed high similarity to gastrodianin type antifungal proteins. No intron was detected within the region of genomic sequence corresponding to the caa full-length cDNA. Southern blot analysis indicated that the caa gene belonged to a low-copy gene family. Northern blot analysis demonstrated that caa mRNA was constitutively expressed in all the tested tissue types including the root, bulb, leaf, rachise, flower and fruit tissues.     

Molecular cloning and characterization of a calreticulin cDNA from the pinewood nematode Bursaphelenchus xylophilus

The cloning and characterization of a cDNA encoding a calreticulin from the pinewood nematode Bursaphelenchus xylophilus is described herein. The full-length cDNA (Bx-crt-1) contained a 1200 bp open reading frame that could be translated to a 399 amino acid polypeptide. The deduced protein contained highly conserved regions of a calreticulin gene and had 66.2–70.1% amino acid sequence identity to other calreticulin sequences from nematodes. RNAi, RT-PCR amplification, and southern blot suggest that Bx-crt-1 may be important for the development of B. xylophilus.     

Molecular cloning and functional characterization of crustapain: a distinct cysteine proteinase with unique substrate specificity from northern shrimp Pandalus borealis

A cDNA clone encoding a cysteine proteinase of the papain superfamily has been isolated from the hepatopancreas of northern shrimp Pandalus borealis (NsCys). NsCys shares the highest identity of 64% with a cathepsin L-like cysteine proteinase from lobster, and its identity to the well-characterized mammalian cathepsins S, L, and K falls within a narrow range of 54-59%. However, it differs from each of these cathepsins in certain key residues including, for example, the unique occurrence of tryptophan and cysteine residues at the structurally important S2 subsite. Consequently, NsCys produced in Pichia pastoris appears to be distinct in various physicokinetic properties. The recombinant enzyme is active and stable over a wide range of pH values, and its substrate specificity is unusual, as demonstrated by its poor affinity for phenylalanine residues. Instead, it shows the highest specificity for proline residues, a property similar to cathepsin K. Unlike cathepsin K, however, NsCys cleaves valine residues more efficiently than leucine. Similar results were obtained with the natural peptide substrate glucagon. The shrimp proteinase is further distinguished by its potent collagenolytic activity, resulting in a cleavage pattern reminiscent of bacterial collagenase. To distinguish such unique structural and enzymatic properties, we propose the trivial name "crustapain" for the shrimp proteinase, indicating that it is a papain-like cysteine proteinase from a crustacean species.     

Molecular cloning and characterization of a novel mannose-binding lectin gene from Amorphophallus konjac

A new lectin gene was cloned from Amorphophallus konjac. The full-length cDNA of Amorphophallus konjac agglutinin (aka) was 736 bp and contained a 474 bp open reading frame encoding a 158 amino acid protein. Homology analysis revealed that the lectin from this Araceae species belonged to the superfamily of monocot mannose-binding proteins. Molecular modeling of AKA indicated that the three-dimensional structure of AKA strongly resembles that of the snowdrop lectin. Southern blot analysis of the genomic DNA revealed that aka belonged to a low-copy gene family. Northern blot analysis demonstrated that aka expression was tissue-specific with the strongest expression being found in root.     

Molecular cloning of mannose-binding lectins from Clivia miniata

Screening of a cDNA library constructed from total RNA isolated from young developing ovaries of Clivia miniata Regel with the amaryllis lectin cDNA clone resulted in the isolation of four different isolectin clones which clearly differ from each other in their nucleotide sequences and hence also in their deduced amino acid sequences. Apparently the lectin is translated from an mRNA of ca. 800 nucleotides encoding a precursor polypeptide of 163 amino acids. Northern blot analysis of total RNA isolated from different tissues of Clivia miniata has shown that the lectin is expressed in most plant tissues with very high lectin mRNA concentrations in the ovary and the seed endosperm.     

Inhibition of the cathepsin cysteine proteases B and K by square-planar cycloaurated gold(III) compounds and investigation of their anti-cancer activity

Gold(III) compounds have been examined for potential anti-cancer activity. It is proposed that the molecular targets of these compounds are thiol-containing biological molecules such as the cathepsin cysteine proteases. These enzymes have been implicated in many diseases including cancer. The catalytic mechanism of the cathepsin cysteine proteases is dependent upon a cysteine at the active site which is accessible to the interaction of thiophilic metals such as gold. The synthesis and biological activity of square-planar six-membered cycloaurated Au(III) compounds with a pyridinyl-phenyl linked backbone and two monodentate or one bidentate leaving group is described. Gold(III) cycloaurated compounds were able to inhibit both cathepsins B and K. Structure/activity was investigated by modifications to the pyridinyl-phenyl backbone, and leaving groups. Optimal activity was seen with substitution at the 6 position of the pyridine ring. The reversibility of inhibition was tested by reactivation in the presence of cysteine with a bidentate thiosalicylate compound being an irreversible inhibitor. Five compounds were evaluated for in vitro cytotoxicity against a panel of human tumor cell lines. The thiosalicylate compound was tested in vivo against the HT29 human colon tumor xenograft model. A modest decrease in tumor growth was observed compared with the untreated control tumor.     

Molecular cloning and analysis of a cDNA coding for nucleoside diphosphate kinase from ryegrass

A full-length cDNA, LpNDPK, encoding ryegrass nucleoside diphosphate kinase (EC 2.7.4.6) has been cloned and sequenced. The nucleotide sequence of the clone contains an open reading frame of 450 nucleotides encoding a protein of 150 amino acid residues with a calculated molecular mass of 16.5 kDa and a P_i of 6.62. The LpNDPK encoded protein possesses substantial homology with nucleoside diphosphate kinases (NDPKs) isolated and cloned form other sources; the highest identity (86 percnt;) was observed with NDPK from sugarcane (Saccharum officinarum). Amino acid comparisons with other NDPKs show that the presented ryegrass NDPK sequence also contains several motifs and specific residues crucial for catalytic activity which are highly conserved among other NDPKs. RT-PCR expression analysis using primers covering the coding region of LpNDPK revealed that the ryegrass NDPK gene is equally expressed in stem, leaf, and flower tissue.