Substrate specificity profiling of heat-sensitive serine protease from the fungus Onygena corvina.

Proteases catalyze hydrolysis of amide bonds within peptides and proteins, therefore they play crucial functions for organism functioning, but also in industry to facilitate numerous processes. Feather-degrading fungus Onygena corvina (O. corvina) is loaded with numerous proteases that can be utilized for variety of applications. The most active species of these enzymes is heat-sensitive serine protease (NHSSP), from O. corvina fungi and due to its potential applications in industry is an alternative to proteinase K. The uniqueness of NHSSP relies on the ability of this enzyme to hydrolyze peptides at neutral to acidic pH values between 5.0 and 8.5, with an optimum of 6.8 and a temperature activity ranging from 15 to 50 °C making NHSSP exceptionally universal enzyme. Thus, we have performed the in-depth characterization of NHSSP substrate specificity by using a positional scanning substrate combinatorial library (PS-SCL). Afterward, we obtained a set of fluorescent substrates hydrolyzed by NHSSP that served as a leading sequence for the first tailored covalent inhibitor of this enzyme, containing a diphenylphosphonate as a warhead and MeOSuc amine protecting group. Our first inhibitor for NHSSP binds potently with target protease and is a tool for future study of this enzyme functions.

An alternative for proteinase K-heat-sensitive protease from fungus Onygena corvina for biotechnology: cloning, engineering, expression, characterization and special application for protein sequencing.

BACKGROUND: A neutral, heat-sensitive serine protease (NHSSP) originating from the feather-degrading fungus Onygena corvina (O. corvina) was described and defined as an alkaline serine protease of the subtilisin type S8 family, exhibiting an enzymatic activity at neutral pH. Generally, broad specificity proteases, such as proteinase K or trypsin, have found numerous applications in research and biotechnology. RESULTS: We report the cloning and expression in the yeast PichiaPink™ system, as well as purification, and characterization of the NHSSP. Recombinant, His6-tagged NHSSP was efficiently expressed from an optimized, synthetic gene and purified using a simple protocol based on ammonium sulfate fractionation and hydrophobic interaction chromatography. The enzyme shows atypical C-terminal processing, the coded preproprotein undergoes signal peptide removal and maturation through the clipping of a propeptide section and 10 amino acids (aa) from the C-terminus, including the His6-tag. The deletion variant has been constructed, devoid of the C-terminal ORF segment, thus eliminating the need for C-terminal processing. Both NHSSP variants exhibit very similar enzymatic characteristics. The purified enzymes were characterized to determine the optimal proteolytic conditions. We revealed that the mature NHSSP is reproducibly active over a wide pH range from neutral to mild acidic (pH of 5.0 to 8.5), with an optimum at pH 6.8, and at temperatures of 15 to 50 °C with an optimum at 38-42 °C. Interestingly, we demonstrated that the protease can be fully deactivated by a moderate increase in temperature of about 15 °C from the optimum to over 50 °C. The protease was partially sensitive to serine protease inhibitors, and not inhibited by chelating or reducing agents and detergents. SDS induced autolysis of NHSSP, which points to a high stimulation of its proteolytic activity. CONCLUSIONS: The NHSSP was produced as a recombinant protein with high efficiency. Compared to proteinase K, the most common serine protease used, NHSSP shows an approx. twofold higher specific activity. Protein sequencing can be a valuable technical application for the protease. The protein coverage is significantly higher in comparison to trypsin and reaches about 84-100% for ß-lactoglobulin (BLG), antibody (mAb) light and heavy chains. Furthermore, the option to perform digestions at neutral to slightly acidic pH-values down to pH 5.0 avoids modification of peptides, e.g. due to deamidation.

Extracellular Alpha-Synuclein Oligomers Induce Parkin S-Nitrosylation: Relevance to Sporadic Parkinson's Disease Etiopathology.

α-Synuclein (ASN) and parkin, a multifunctional E3 ubiquitin ligase, are two proteins that are associated with the pathophysiology of Parkinson's disease (PD). Excessive release of ASN, its oligomerization, aggregation, and deposition in the cytoplasm contribute to neuronal injury and cell death through oxidative-nitrosative stress induction, mitochondrial impairment, and synaptic dysfunction. In contrast, overexpression of parkin provides protection against cellular stresses and prevents dopaminergic neural cell loss in several animal models of PD. However, the influence of ASN on the function of parkin is largely unknown. Therefore, the aim of this study was to investigate the effect of extracellular ASN oligomers on parkin expression, S-nitrosylation, as well as its activity. For these investigations, we used rat pheochromocytoma (PC12) cell line treated with exogenous oligomeric ASN as well as PC12 cells with parkin overexpression and parkin knock-down. The experiments were performed using spectrophotometric, spectrofluorometric, and immunochemical methods. We found that exogenous ASN oligomers induce oxidative/nitrosative stress leading to parkin S-nitrosylation. Moreover, this posttranslational modification induced the elevation of parkin autoubiquitination and degradation of the protein. The decreased parkin levels resulted in significant cell death, whereas parkin overexpression protected against toxicity induced by extracellular ASN oligomers. We conclude that lowering parkin levels by extracellular ASN may significantly contribute to the propagation of neurodegeneration in PD pathology through accumulation of defective proteins as a consequence of parkin degradation.

Intracellular reprogramming of expression, glycosylation, and function of a plant-derived antiviral therapeutic monoclonal antibody.

Plant genetic engineering, which has led to the production of plant-derived monoclonal antibodies (mAb(P)s), provides a safe and economically effective alternative to conventional antibody expression methods. In this study, the expression levels and biological properties of the anti-rabies virus mAb(P) SO57 with or without an endoplasmic reticulum (ER)-retention peptide signal (Lys-Asp-Glu-Leu; KDEL) in transgenic tobacco plants (Nicotiana tabacum) were analyzed. The expression levels of mAb(P) SO57 with KDEL (mAb(P)K) were significantly higher than those of mAb(P) SO57 without KDEL (mAb(P)) regardless of the transcription level. The Fc domains of both purified mAb(P) and mAb(P)K and hybridoma-derived mAb (mAb(H)) had similar levels of binding activity to the FcγRI receptor (CD64). The mAb(P)K had glycan profiles of both oligomannose (OM) type (91.7%) and Golgi type (8.3%), whereas the mAb(P) had mainly Golgi type glycans (96.8%) similar to those seen with mAb(H). Confocal analysis showed that the mAb(P)K was co-localized to ER-tracker signal and cellular areas surrounding the nucleus indicating accumulation of the mAb(P) with KDEL in the ER. Both mAb(P) and mAb(P)K disappeared with similar trends to mAb(H) in BALB/c mice. In addition, mAb(P)K was as effective as mAb(H) at neutralizing the activity of the rabies virus CVS-11. These results suggest that the ER localization of the recombinant mAb(P) by KDEL reprograms OM glycosylation and enhances the production of the functional antivirus therapeutic antibody in the plant.

Recombinant mouse granulocyte-macrophage colony-stimulating factor is glycosylated in transgenic tobacco and maintains its biological activity.

The granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine with many important applications and, due to its immunostimulatory properties, could also be used as a vaccine adjuvant. A simple strategy to produce recombinant mouse GM-CSF (mGM-CSF) in transgenic Nicotiana tabacum plants was used in this study. The mGM-CSF cDNA followed by the sequence encoding endoplasmic reticulum retention signal (KDEL) was cloned into the ImpactVector under the control of the strong promoter from the gene encoding a small subunit of Rubisco. In transgenic plants the accumulation level of recombinant mGM-CSF varied in the individual transformants from 8 to 19 microg/g of fresh leaf tissue, which makes up to 0.22% of total soluble protein. In most analyzed plants, the apparent molecular weight of the recombinant protein was larger than predicted due to its N-glycosylation, presumably in 2 sites. The recombinant plant-produced murine GM-CSF retained its biological activity as confirmed in vitro in proliferation assay using a mouse cell line, which is growth-dependent on GM-CSF.

Plant-produced hepatitis B core protein chimera carrying anthrax protective antigen domain-4.

The hepatitis B core antigen (HBcAg) can generate a strong immune response and is recognized as an effective carrier for foreign epitopes. The domain-4 epitope of the anthrax protective antigen (PA-D4) plays an essential role in generating protective immunity against virulent Bacillus anthracis. Here we report the successful production of a recombinant protein comprised of the antigenic PA-D4 integrated into the c/e1 loop of HBcAg in transgenic low-alkaloid Nicotiana tabacum. Sera of mice injected with the plant-derived purified HB/PA-D4 protein exhibited significant anti-PA- and anti-HBcAg-specific IgG titers; however, formation of virus-like particles (VLP) was not observed. These data support the feasibility of producing complex protein chimeras in plants.

Plant-derived EpCAM antigen induces protective anti-cancer response.

Immunotherapy holds great promise for treatment of infectious and malignant diseases and might help to prevent the occurrence and recurrence of cancer. We produced a plant-derived tumor-associated colorectal cancer antigen EpCAM (pGA733) at high yields using two modern plant expression systems. The full antigenic domain of EpCAM was efficiently purified to confirm its antigenic and immunogenic properties as compared to those of the antigen expressed in the baculovirus system (bGA733). Recombinant plant-derived antigen induced a humoral immune response in BALB/c mice. Sera from those mice efficiently inhibited the growth of SW948 colorectal carcinoma cells xenografted in nude mice, as compared to the EpCAM-specific mAb CO17-1A. Our results support the feasibility of producing anti-cancer recombinant vaccines using plant expression systems.

Smallpox subunit vaccine produced in Planta confers protection in mice.

We report here the in planta production of the recombinant vaccinia virus B5 antigenic domain (pB5), an attractive component of a subunit vaccine against smallpox. The antigenic domain was expressed by using efficient transient and constitutive plant expression systems and tested by various immunization routes in two animal models. Whereas oral administration in mice or the minipig with collard-derived insoluble pB5 did not generate an anti-B5 immune response, intranasal administration of soluble pB5 led to a rise of B5-specific immunoglobulins, and parenteral immunization led to a strong anti-B5 immune response in both mice and the minipig. Mice immunized i.m. with pB5 generated an antibody response that reduced virus spread in vitro and conferred protection from challenge with a lethal dose of vaccinia virus. These results indicate the feasibility of producing safe and inexpensive subunit vaccines by using plant production systems.

Plant-derived anti-Lewis Y mAb exhibits biological activities for efficient immunotherapy against human cancer cells.

Although current demands for therapeutic mAbs are growing quickly, production methods to date, including in vitro mammalian tissue culture and transgenic animals, provide only limited quantities at high cost. Several tumor-associated antigens in tumor cells have been identified as targets for therapeutic mAbs. Here we describe the production of mAb BR55-2 (IgG2a) in transgenic plants that recognizes the nonprotein tumor-associated antigen Lewis Y oligosaccharide overexpressed in human carcinomas, particularly breast and colorectal cancers. Heavy and light chains of mAb BR55-2 were expressed separately and assembled in plant cells of low-alkaloid tobacco transgenic plants (Nicotiana tabacum cv. LAMD609). Expression levels of plant-derived mAb (mAbP) were high (30 mg/kg of fresh leaves) in T1 generation plants. Like the mammalian-derived mAbM, the plant mAbP bound specifically to both SK-BR3 breast cancer cells and SW948 colorectal cancer cells. The Fc domain of both mAbP and mAbM showed the similar binding to FcgammaRI receptor (CD64). Comparable levels of cytotoxicity against SK-BR3 cells were also shown for both mAbs in antibody-dependent cell-mediated cytotoxicity assay. Furthermore, plant-derived BR55-2 efficiently inhibited SW948 tumor growth xenografted in nude mice. Altogether, these findings suggest that mAbP originating from low-alkaloid tobacco exhibit biological activities suitable for efficient immunotherapy.