Desmodus Rotundus Salivary Plasminogen Activator Research Articles

Identification, purification, and pharmacological activity analysis of Desmodus rotundus salivary plasminogen activator alpha1 (DSPAα1) expressed in transgenic rabbit mammary glands.

Desmodus rotundus plasminogen activator alpha 1(DSPAα1) is a thrombolytic protein with advantages, such as a long half-life, high accuracy and specificity for thrombolysis, wide therapeutic window, and no neurotoxicity. To date, DSPAα1 has only been expressed in the Chinese hamster ovary, insect cells, transgenic tobacco plants, and Pichia pastoris. To the best of our knowledge, we are the first to report the expression of DSPAα1 in transgenic rabbit mammary glands, extract the product, and analyze its pharmacology activity. An efficient mammary gland-specific expression vector pCL25/DSPAα1 was transferred to prokaryotic zygotes in rabbits by microinjection to generate six DSPAα1 transgenic rabbits. The recombinant DSPAα1 (rDSPAα1) expression in transgenic rabbit milk was 1.19 ± 0.26mg/mL. The rDSPAα1 purification protocol included pretreatment, ammonium sulfate precipitation, benzamidine affinity chromatography, cation exchange chromatography, and Cibacron blue affinity chromatography; approximately 98% purity was achieved using gel electrophoresis. According to sequencing results, the primary structure of rDSPAα1 was consistent with the theoretical design sequence, and its molecular weight was consistent with that of the natural protein. N-terminal sequencing results indicated rDSPAα1 to be a mature protein, as the goat signal peptide sequence of the expression vector was no longer detected. The fibrinolytic activity of rDSPAα1 was estimated to be 773,333IU/mg. Fibrin-agarose plate assay and in vitro rat blood clot degradation assay showed that rDSPAα1 had strong thrombolytic activity. In conclusion, we report recombinant DSPAα1 with high thrombolytic activity expressed in transgenic rabbit mammary glands.

Stirring rate regulates the proliferation and metabolism of microencapsulated recombinant CHO cells.

Stirred tank bioreactors are the most widely used method for the large-scale culture of mammalian cells. However, the scale of stirred tank bioreactors is limited by insufficient oxygen/nutrient mixing and the accumulation of waste products in high cell density cultures. The most effective method to solve these problems is to increase the stirring rate; this usually leads to increased cell proliferation, but can decrease the utilization of nutrients for recombinant protein synthesis. To investigate the effects of stirring rate on the proliferation, metabolism, and recombinant protein yield of microencapsulated recombinant Chinese hamster ovary (rCHO) cells, the cells were cultured under different stirring rates, and cell viability, metabolic activity, and protein yield were measured. Microencapsulation promoted Desmodus rotundus salivary plasminogen activator expression, and higher stirring rates promoted increases in microencapsulated cell density and metabolic activity. However, the maximum yield of recombinant protein was obtained at a moderate stirring rate, whereas protein yield was decreased at the highest tested stirring rate. The stirring rate had a significant impact on the growth and protein expression of microencapsulated rCHO cells, and a specific stirring rate was identified to maximize the yield of recombinant protein.

Growth and production of microencapsulated recombinant CHO in a stirred tank bioreactor.

Microencapsulation supplies cells with a three-dimensional microenvironment enhancing the metabolic activity, cell density and recombinant protein expression in a stirred tank bioreactor which is used widely to culture mammalian cells in many biochemical processes. In this paper, we address the growth and Desmodus rotundus salivary plasminogen activator (DSPA) production of recombinant CHO (rCHO) in a stirred tank bioreactor. Cells were cultured using two different methods--in an unmicroencapsulated versus microencapsulated culture--and compared differences between them in terms of cell reproduction and DSPA protein productivity. Compared to the unmicroencapsulated rCHO, microencapsulated cells got higher cell density and prolonged the plateau phase. Microencapsulated rCHO promoted DSPA production, with a maximum rate that was 4.8 times higher than in unmicroencapsulated cells, and the accumulated production of DSPA was 3.3 higher than in unmicroencapsulated cells. Negative relationship was found between specific growth rate and DSPA production capacity of unit cells. These findings will facilitate the methods for higher DSPA production in stirred tank bioreactors.

Dracula's children: Molecular evolution of vampire bat venom

While vampire bat oral secretions have been the subject of intense research, efforts have concentrated only on two components: DSPA (Desmodus rotundus salivary plasminogen activator) and Draculin. The molecular evolutionary history of DSPA has been elucidated, while conversely draculin has long been known from only a very small fragment and thus even the basic protein class was not even established. Despite the fact that vampire bat venom has a multitude of effects unaccounted by the documented bioactivities of DSPA and draculin, efforts have not been made to establish what other bioactive proteins are secreted by their submaxillary gland. In addition, it has remained unclear whether the anatomically distinct anterior and posterior lobes of the submaxillary gland are evolving on separate gene expression trajectories or if they remain under the shared genetic control. Using a combined proteomic and transcriptomic approach, we show that identical proteins are simultaneously expressed in both lobes. In addition to recovering the known structural classes of DSPA, we recovered a novel DSPA isoform as well as obtained a very large sequence stretch of draculin and thus established that it is a mutated version of the lactotransferrin scaffold. This study reveals a much more complex secretion profile than previously recognised. In addition to obtaining novel versions of scaffolds convergently recruited into other venoms (allergen-like, CRiSP, kallikrein, Kunitz, lysozyme), we also documented novel expression of small peptides related to calcitonin, PACAP, and statherin. Other overexpressed protein types included BPI-fold, lacritin, and secretoglobin. Further, we investigate the molecular evolution of various vampire bat venom-components and highlight the dominant role of positive selection in the evolution of these proteins. Conspicuously many of the proteins identified in the proteome were found to be homologous to proteins with known activities affecting vasodilation and platelet aggregation. We show that vampire bat venom proteins possibly evade host immune response by the mutation of the surface chemistry through focal mutagenesis under the guidance of positive Darwinian selection. These results not only contribute to the body of knowledge regarding haematophagous venoms but also provide a rich resource for novel lead compounds for use in drug design and development. These results have direct implications in understanding the molecular evolutionary history of vampire bat venom. The unusual peptides discovered reinforce the value of studying such neglected taxon for biodiscovery.

Biochemical properties of the matrix metalloproteinase NtMMP1 from Nicotiana tabacum cv. BY-2 suspension cells

A zinc-dependent matrix metalloproteinase (NtMMP1) found in the plasma membrane of Nicotiana tabacum cv. Bright Yellow 2 (BY-2) suspension cells is thought to be responsible for the degradation of recombinant proteins secreted into the culture supernatant. We have characterized the proteolytic activity of NtMMP1 by expressing a recombinant derivative lacking the C-terminal transmembrane domain in yeast. After purifying the protein by affinity chromatography, its autocatalytic activity was analyzed using monoclonal antibodies raised against its N-terminal and C-terminal portions. Both the unprocessed and processed forms of NtMMP1 displayed caseinolytic activity and N-terminal sequencing identified an autocatalytic cleavage site within the sequence motif HFSFFP, which is similar to the corresponding sequences of the human matrix metalloproteinases stromelysin-1 (MMP-3) and stromelysin-2 (MMP-10). Unlike all other matrix metalloproteinases investigated so far, NtMMP1 contains a disulfide bond within its propeptide thus rendering the proenzyme catalytically active. Kinetic analysis of NtMMP1 with a synthetic substrate revealed a K(m) of 10.55 +/- 0.9 microM, a k(cat) of 0.6 +/- 0.01 s(-1) and maximum activity at pH 7.5. We found that NtMMP1 degrades Desmodus rotundus salivary plasminogen activator alpha 1 (DSPAalpha1), a biopharmaceutical protein, that has proven difficult to produce in tobacco BY-2 cells. This provides a likely explanation for the frequent instability of secreted recombinant biopharmaceuticals produced in plant suspension cell cultures. Our data suggest new avenues that can be explored to improve the production of pharmaceutical proteins in plants and plant cells.

Desmoteplase

The high fibrin specificity of Desmodus rotundus salivary plasminogen activator α1 (desmoteplase) renders it a promising candidate for the treatment of acute ischemic stroke. In the DIAS (Desmoteplase in Acute Ischemic Stroke) and DEDAS (Dose Escalation study of Desmoteplase in Acute ischemic Stroke) Phase II studies, doses of 90 μg/kg and 125 μg/kg desmoteplase were reported to have acceptable safety profiles, leading to potentially superior reperfusion compared with placebo, with possible clinical efficacy for up to 9 h after the onset of symptoms in patients with a significant ischemic penumbra selected from magnetic resonance perfusion-diffusion weighted mismatches imaging. However, a Phase III clinical trial (DIAS-2) was unable to detect any benefit from desmoteplase when given 3 – 9 h after stroke onset. In this study with a modest sample size, certain methodological factors may have reduced its potential to detect a desmoteplase effect, as only 30% of these patients had a visible occlusion at presentation, with only small core and mismatched lesion volumes. Indeed, it is surprising that a study testing an occluded vessel ‘reopener’ was conducted in a cohort of stroke patients, the majority of whom was known not to have a detected vessel occlusion. It has also been claimed that the DIAS-2 patients selection using core/penumbra mismatch calculation may not have followed an appropriate mismatch threshold. However, the corrective value of changing the mismatch threshold remains unclear, because the relative mismatch volumes were in fact higher in the ‘negative’ DIAS-2 than in the ‘positive’ DIAS and DEDAS. Two Phase II randomized trials with tPA, Diffusion-weighted imaging Evaluation For Understanding Stroke Evolution (DEFUSE) and Echoplanar Imaging Thrombolytic Evaluation Trial (EPITHET) provided strong biological support for the relation between infarct growth, reperfusion and clinical outcome in the 3 – 6 h time window after onset of stroke using penumbral imaging. In this frame, why exactly desmoteplase should have specific advantages over tPA, is not clear. Taken together, these findings may also lead to the disappointing conclusion that vessel recanalization after 4.5 – 5 h from stroke onset may generally be inefficacious for tissue salvage. Nevertheless, other randomized Phase III clinical trials (DIAS-3 and DIAS-4) are currently under way with a planned sample size of 320 patients having vessel occlusion or high-grade stenosis on MRI or CT-angiography in the proximal cerebral arteries.

Production of Desmodus rotundus salivary plasminogen activator α1 (DSPAα1) in tobacco is hampered by proteolysis

The high fibrin specificity of Desmodus rotundus salivary plasminogen activator alpha1 (DSPAalpha1 or desmoteplase (INN)) makes it a promising candidate for the treatment of acute ischemic stroke. In the current study we explored the use of transgenic tobacco plants and BY-2 suspension cells as alternative production platforms for this drug. Four different N-terminal signal peptides, from plants and animals, were used to translocate the recombinant DSPAalpha1 protein to the endomembrane system. Intact recombinant DSPAalpha1 was produced in transgenic plants and BY-2 cells, although a certain degree of degradation was observed in immunoblotted extracts. The choice of signal peptide had no major influence on the degradation pattern or recombinant protein accumulation, which reached a maximum level of 38 microg/g leaf material. N-terminal sequencing of purified, His6-tagged DSPAalpha1 revealed only minor changes in the position of signal peptide cleavage compared to the same protein expressed in Chinese hamster ovary cells. However, correctly processed recombinant DSPAalpha1 was also detected. The enzymatic activity of the recombinant protein was confirmed using an in vitro assay with unpurified and purified samples, demonstrating that plants are suitable for the production of functional DSPAalpha1. In contrast to whole plant cell extracts, no recombinant DSPAalpha1 was detected in the culture supernatant of transgenic BY-2 cells. Further analysis showed that recombinant DSPAalpha1 is subject to proteolysis and that endogenous secreted BY-2 proteases are responsible for DSPAalpha1 degradation in the culture medium. The addition of a highly concentrated protease inhibitor mixture or 5 mM EDTA reduced DSPAalpha1 proteolysis, improving the accumulation of intact product in the culture medium. Strategies to improve the plant cell suspension system for the production of secreted recombinant proteins are discussed.

Impact of Sample Preparation on Residual Moisture Analyses for a Lyophilized Protein Formulation

This study compares residual moisture data obtained by thermogravimetric (TG) and Karl Fischer (KF) analyses of freeze-dried recombinant Desmodus rotundus salivary plasminogen activator (DSPA) and the protein free placebo. Significant changes in water content were observed depending on the sample type as well as the relative humidity of the exposure environment prior to analysis. The TG and KF analyses gave identical results only when the samples were handled identically. The method of choice for the determination of the true total moisture content of these samples is the KF analysis since it can be performed without any opportunity for water exchange with the environment.

O-Linked L-Fucose Is Present in Desmodus rotundus Salivary Plasminogen Activator

DSPAalpha1 (Desmodus rotundus salivary plasminogen activator), a plasminogen activator from the saliva of the vampire bat Desmodus rotundus, is an effective thrombolytic agent. An unusual type of posttranslational modification, in which L-fucose is O-glycosidically linked to threonine 61 in the epidermal growth factor domain was found for natural DSPAalpha1 and its recombinant form isolated from Chinese hamster ovary cells. In the present study a combination of carbohydrate and amino acid composition analysis, amino acid sequencing, and mass spectrometry revealed that the L-fucose is bound to residues 56-68 of DSPAalpha1. The amino acid sequence of this glycosylation site agreed with the suggested consensus sequence Cys-Xaa-Xaa-Gly-Gly-Ser/Thr-Cys described for other proteins. Anew strategy for the identification of the modified amino acid was established. Direct evidence for the occurrence of fucosyl-threonine was obtained by mass spectrometry after digestion of the glycopeptide with a mixture of peptidases. On the basis of these results, DSPAalpha1 is a suitable model for studying the influence of O-fucosylation on clearance rates, particularly in comparative studies with the identically fucosylated and structurally related tissue plasminogen activator.

Development of an ELISA for the measurement of DSPA α1 ( Desmodus rotundus salivary plasminogen activator) in plasma and its application to investigate pharmacokinetics in monkeys

DSPAα1 is a novel plasminogen activator with high fibrin specificity. A sandwich ELISA-system with affinity purified and peroxidase labelled DSPAα1 antibodies raised in rabbits was developed and exhibited a limit of quantification of 3 ng/ml in undiluted spiked plasma. Accuracy was 98–108% and precision accounted for 3–9.5%. No cross-reactivity with human t-PA or endogenous matrix constituents interfering with assay results was observed. After i.v. administration DSPAα1 at 1 and 3 mg/kg in cynomolgus monkeys antigen levels in plasma were dose-dependent in both gender and exhibited a triphasic disposition profile with half-lives of 0.04–0.26 h, 0.6–3 h and 4–8.5 h. The mean residence time of DSPAα1 ranged from 3 to 9 h and total clearance was approximately 2 ml/min/kg independent of sex and dose. These data obtained in monkeys showed a long systemic circulation of the antigen, linear pharmacokinetics and no sex-specific pharmacokinetic differences in the dose range investigated. In conclusion the present ELISA method is suitable for pharmacokinetic studies of DSPAα1 in animals and man. First investigations in monkeys demonstrated the interesting pharmacokinetic profile of the compound which might be administered by i.v. bolus administration for the treatment of acute myocardial infarction.