Tobacco BY2 cells expressing recombinant cardosin B as an alternative for production of active milk clotting enzymes.

Cynara cardunculus L. or cardoon is a plant that is used as a source of milk clotting enzymes during traditional cheese manufacturing. This clotting activity is due to aspartic proteases (APs) found in the cardoon flower, named cyprosins and cardosins. APs from cardoon flowers display a great degree of heterogeneity, resulting in variable milk clotting activities and directly influencing the final product. Producing these APs using alternative platforms such as bacteria or yeast has proven challenging, which is hampering their implementation on an industrial scale. We have developed tobacco BY2 cell lines as an alternative plant-based platform for the production of cardosin B. These cultures successfully produced active cardosin B and a purification pipeline was developed to obtain isolated cardosin B. The enzyme displayed proteolytic activity towards milk caseins and milk clotting activity under standard cheese manufacturing conditions. We also identified an unprocessed form of cardosin B and further investigated its activation process. The use of protease-specific inhibitors suggested a possible role for a cysteine protease in cardosin B processing. Mass spectrometry analysis identified three cysteine proteases containing a granulin-domain as candidates for cardosin B processing. These findings suggest an interaction between these two groups of proteases and contribute to an understanding of the mechanisms behind the regulation and processing of plant APs. This work also paves the way for the use of tobacco BY2 cells as an alternative production system for active cardosins and represents an important advancement towards the industrial production of cardoon APs.

A surface plasmon resonance-based biosensor with full-length BACE1 in a reconstituted membrane.

A surface plasmon resonance (SPR) biosensor-based assay for membrane-embedded full-length BACE1 (ß-site amyloid precursor protein cleaving enzyme 1), a drug target for Alzheimer's disease, has been developed. It allows the analysis of interactions with the protein in its natural lipid membrane environment. The enzyme was captured via an antibody recognizing a C-terminal His6 tag, after which a lipid membrane was reconstituted on the chip using a brain lipid extract. The interaction between the enzyme and several inhibitors confirmed that the surface was functional. It had slightly different interaction characteristics as compared with a reference surface with immobilized ectodomain BACE1 but had the same inhibitor characteristic pH effect. The possibility of studying interactions with BACE1 under more physiological conditions than assays using truncated enzyme or conditions dictated by high enzyme activity is expected to increase our understanding of the role of BACE1 in Alzheimer's disease and contribute to the discovery of clinically efficient BACE1 inhibitors. The strategy exploited in the current study can be adapted to other membrane-bound drug targets by selecting suitable capture antibodies and lipid mixtures for membrane reconstitution.

Potato aspartic proteases (StAPs) exert cytotoxic activity on bovine and human spermatozoa.

OBJECTIVE: To evaluate the in vitro spermicidal activity of Solanum tuberosum aspartic proteinases (StAPs) on bovine and human sperm. DESIGN: Controlled laboratory study. SETTING: Three research laboratories at a university of biologic science. ANIMAL(S) AND DONOR(S): Frozen semen from five Aberdeen Angus bulls and six proven fertile men volunteers. INTERVENTION(S): The effect of StAPs on sperm motility was studied in vitro by incubation of different concentrations of StAPs with sperm suspensions, and motility was assessed by direct microscopic observation. Membrane integrity was analyzed by SYTOX Green uptake after incubation with different StAP concentrations. The effect of StAPs was evaluated by human erythrocyte lysis, as a control in somatic cells. The StAPs binding was monitored by fluorescence. MAIN OUTCOME MEASURE(S): Total and progressive sperm motility; hypoosmotic swelling test and SYTOX Green uptake as a measure of membrane damage; fluorescein isothiocyanate-labeled StAP binding by an optical microscopy. RESULT(S): The StAPs reduced sperm motility in a dose-dependent manner, and 25 microM of StAP1 and 35 microM of StAP3 completely abolished the progressive motility. The StAPs were able to bind in the postacrosomal and midpiece region only in bovine sperm. Also, StAPs caused spermatozoa agglutination. In vitro cell toxicity was observed by a dose-dependent increase in hypoosmotic swelling negative sperm and SYTOX Green uptake in both human and bovine spermatozoa; however, no toxic effect was observed on erythrocytes. CONCLUSION(S): The spermicidal effect of StAPs involves plasma membrane permeabilization.

Purification and antigenicity of two recombinant forms of Boophilus microplus yolk pro-cathepsin expressed in inclusion bodies.

The tick Boophilus microplus is a bovine ectoparasite present in tropical and subtropical areas of the world and the use of vaccines is a promising method for tick control. BYC is an aspartic proteinase found in eggs that is involved in the embryogenesis of B. microplus and was proposed as an important antigen in the development of an anti-tick vaccine. The cDNA of BYC was amplified by PCR and cloned for expression in two forms with and without thioredoxin fusion protein (Trx), coding recombinant proteins named rBYC-Trx and rBYC, respectively. Expression, solubility, and yields of the two forms were analyzed. The recombinant proteins were expressed in inclusion bodies (IBs) and three denaturant agents (N-lauroyl sarcosine, guanidine hydrochloride, and urea) were tested for IBs solubilization. The N-lauroyl sarcosine solubilized 90.4 and 92.4% of rBYC-Trx and rBYC IBs, respectively, and was the most efficient denaturant. Two recombinant forms were affinity-purified by Ni2+-Sepharose under denaturing conditions. After dialysis, the yield of soluble protein was 84.1% for r-BYC-Trx and 5.9% for rBYC. These proteins were immune-reactive against sera from rabbit, mouse, and bovine previously immunized with native BYC, which confirms the antigenicity of the recombinant BYCs expressed in the Escherichia coli system.

Molecular characterization, enzymatic analysis, and purification of murine proprotein convertase-1/3 (PC1/PC3) secreted from recombinant baculovirus-infected insect cells.

A cDNA coding for the murine proprotein convertase-1 (mPC1 also known as mPC3 or mSPC3) was inserted into the Autographa californica nuclear polyhedrosis virus. Following infection of Spodoptera frugiperda cells, the recombinant N-glycosylated protein is secreted into the cell culture medium from which it can be purified to homogeneity as a fully enzymatically active enzyme. Two major secreted molecular forms of mPC1 with apparent molecular weights of 85 and 71 kDa, respectively, and a minor one of 75 kDa are immunodetected in the medium. Automated NH2-terminal sequencing reveals that all three forms result from processing at the predicted zymogen activation site whereas both the 75- and the 71-kDa forms are truncated at their COOH-terminus. Labeling by an active-site titrant demonstrates that the 85-kDa form is optimally labeled at near neutral pH whereas the COOH-truncated forms are optimally labeled at acidic pH. Additionally it is shown that the 85-kDa mPC1 is transformed into the COOH-truncated forms following in vitro incubation at acidic pH levels and in presence of calcium. Concomitantly, the transformation from 85 to 71 kDa is accompanied by a 10- to 40-fold increase in enzymatic activity upon assaying at pH 6.0. The 71-kDa form can be recovered after purification at a level of 1 to 1.5 mg per liter of cell culture medium and is enzymatically stable only in the pH range from 5.0 to 6.5. Cells treated with tunicamycin show a drastically reduced secretion of the convertase in the medium but are not affected by swainsonine and deoxymannojirimycin. Finally, the 85-kDa secreted mPC1 is shown to be sulfated.

Proteinase A, a storage-globulin-degrading endopeptidase of vetch (Vicia sativa L.) seeds, is not involved in early steps of storage-protein mobilization.

Proteinase A is a papain-like cysteine endopeptidase of vetch (Vicia sativa L.) which was assumed to initiate storage-globulin breakdown just after the onset of seed germination. This enzyme was purified from cotyledons of vetch seedlings. On gelatin-containg SDS gels, active proteinase A migrated with an apparent molecular mass of 21 kDa, whereas after heat denaturation its molecular size on SDS/PAGE was 29 kDa. Although proteinase A is capable of hydrolyzing storage globulins in vitro it could not be localized in the protein-body fraction of cotyledons from germinating seeds. cDNA clones encoding proteinase A precursor have been obtained by PCR. The precursor is composed of an N-terminal signal sequence followed by a propeptide, the region encoding mature proteinase A, and a C-terminal KDEL sequence. Mature proteinase A with a derived molecular mass of 25,244 Da does not have the KDEL sequence. The derived amino acid sequence of the proteinase A precursor is 78.2% identical to sulfhydryl-endopeptidase (SH-EP), a cysteine endopeptidase from germinating Vigna mungo seedlings. Northern blot analysis indicated that proteinase A mRNA appears de novo in cotyledons of 1-day-germinated vetch seeds, where its amount increases up to day 6. No proteinase A mRNA was detected in other vetch organs, not even in the embryo axis, which contains stored globulins. By means of antibodies raised against the purified and against recombinantly produced proteinase A, the 29-kDa bands of mature proteinase A were detected in cotyledon extracts of 6-day-germinated seeds when globulin degradation has already far proceeded. The reported data do not agree with the proposed triggering role of proteinase A in storage-globulin breakdown during germination.

Molecular characterization of human and bovine endothelin converting enzyme (ECE-1).

A membrane-bound protease activity that specifically converts Big endothelin-1 has been purified from bovine endothelial cells (FBHE). The enzyme was cleaved with trypsin and the peptide sequencing analysis confirmed it to be a zinc chelating metalloprotease containing the typical HEXXH (HELTH) motif. RT-PCR and cDNA screens were employed to isolate the complete cDNAs of the bovine and human enzymes. This human metalloprotease was expressed heterologously in cell culture and oocytes. The catalytic activity of the recombinant enzyme is the same as that determined for the natural enzyme. The data suggest that the characterized enzyme represents the functional human endothelin converting enzyme ECE-1.